Associate Professor Doug Smith

Associate Professor Doug Smith

Associate Professor

School of Biomedical Sciences and Pharmacy (Anatomy)

The age of reason

Dr Doug Smith is investigating the impacts of ageing on the nervous system, with the aim of identifying interventions that will extend healthspan.

Lifespan encapsulates the number of years you are alive, whereas healthspan refers to the number of years you maintain mental and physical health, without serious disease, during your lifespan.

Dr Doug Smith wants you to live better for longer. That doesn't necessarily mean living for longer, although that may be a benefit of good health. He isn't interested in finding the elixir of youth, just the key to maximum healthspan. 

"Some people want to live forever, I have no interest in extending lifespan, I want to extend healthspan, it's a very different thing," he explains.

With life expectancy increasing and family sizes decreasing, our national population is ageing fast, prompting urgency in research in this field.

"In about 20 years from now, there are going to be more people living in Australia who are over the age of 65 then under the age of 15," Doug asserts.

"If more people reach their latter years still in pretty good shape, physically and mentally, then presumably we can lessen the health care cost burden."

"And more importantly, those people will have a better quality of life."

NERVOUS SYSTEMS

With a background in neurobiology, Doug is focusing his study on the impacts of ageing on the nervous system.

The central nervous system (CNS) includes the brain and spinal cord, whilst the peripheral nervous system (PNS) comprises all the body's nerve pathways outside of the CNS. Both the CNS and PNS are affected by ageing.

For example, declining senses, slowing of messages controlling movements, a loss of clarity in cognitive processes and failing memory are all nervous system specific changes that have been attributed to ageing.   

Using an animal model to study the ageing brain, spinal cord and vestibular (balance) system, Doug and his collaborators hope to further understand these changes on a cellular and molecular level.

Together with his team, Doug uses modern genomics approaches, such as next generation sequencing, to obtain a global picture of age-related gene expression changes to inform their directions in the lab.

"There is much more that we don't know than we do know, so doing a discovery driven approach first, that can then can direct us, is very powerful," he says.

Doug and his team then use more traditional hypothesis driven approaches when looking to confirm or refute possible truths suggested by the broader genomics data.

MICRODISSECTION

Utilising laser-capture microdissection (LCMD) technology, the team are able to identify and extract specific cell types from the highly complex nervous system for study.

For example, LCMD extraction allows the team to investigate the effects of ageing on dopamine neurons, the degeneration of which causes Parkinson's disease. They also investigate the breakdown of the blood brain barrier, which is seen in vascular dementia.

Another study saw the team comparing levels of mitochondrial DNA mutation in young and old tissue. Mitochondria are the power generators of a cell and they have their own very small genome. Age-related mutations in this genome are thought to compromise the ability of mitochondria to generate energy for the myriad of cell activities needed for proper nervous system function.

"Based on our genomics findings we are now doing a lipidomics investigation," Doug explains. 

"The CNS is chock full of different types of lipids, and we know they change with ageing, so we are trying to understand those changes in greater detail."

"Once we align these various approaches, it will give us a really good indication of where to head in terms of our more focused or directed studies."

THE NEXT GENERATION SEQUENCERS

Doug credits PhD and undergrad students with undertaking the bulk of the laborious lab work.

"We are always looking for more students who are passionate about biology and who would like to work with us," Doug says and adds, laughing, "Bring your own money!"

A senior lecturer in the School of Biomedical Sciences and Pharmacy, Doug admits that although teaching drains research time and energy, it actually enhances his outcomes.

"I actually believe that teaching is good for your research because when you teach you have to stand back and take a bigger picture view of things," he says.

"And when you do that, sometimes it gives you a different perspective for your research."

Doug was a post-doctoral scientist when a fascination with the biological phenomenon of ageing drew him to this field of study.

"I have always been curious, I have always wanted to know how things work," he conveys.

"Age related degeneration just doesn't make much sense. A lot of energy and effort goes into creating a sexually mature organism. So, why not then just maintain it? I find it very interesting."

AGEING GRACEFULLY

Although studies across species and domains show that ageing has a major impact on both the animal and human body, some individuals seem somewhat resilient to the effects of ageing.

"Everybody has a grandma or elderly uncle that still seems to be running around like a sprightly 30 or 40 year old, both mentally and physically they are doing really well," Doug acknowledges.

"We are trying to determine what it is about these individuals that allows them to have such a good healthspan."

"A potentially disappointing outcome of our work would be that we find out what the cause or causes of ageing are, but we can't find an intervention."

"However, there are many examples of people ageing well, and if you look at what they do, they are all pretty active, not just physically but mentally as well."

FREEDOM OF CHOICE

Due to the mounting evidence, Doug suggests it is more realistic to earn a longer healthspan, than win one in the unexplained resilience to ageing jackpot.

He refers to several existing intervention studies that show vastly different outcomes for participants relative to different diets and levels of exercise.

But don't wait too long. Evidence emerging from Doug's lab suggests the ageing process begins much earlier than we would like to believe.

"Preliminary data is certainly indicating that if you are going to live well, you can't wait until you are 75 and then decide to make healthier choices."

In the end, the solution to retarding the ageing process may simply involve choices around modifying behaviour.

"Once we characterise some of the processes of normal ageing, we can then go and see if a high fat, high sugar, Omega 3, or resveratrol diet, and/or exercise, are going to change the ageing process at the molecular, cellular, organ and whole body levels," Doug explains.

"The next step would be to design interventions that can be applied to human populations. Humans won't like some of the dietary restrictions, or the exercise. The red wine they might go for," he smiles.

"People will always have free will. We are just trying to figure out ways that are acceptable to the majority to help them to age well and stay active for longer."

The age of reason

Dr Doug Smith is investigating the impacts of ageing on the nervous system, with the aim of identifying interventions that will extend healthspan.

Read more

Career Summary

Biography

The main aim of my lab's research is to better understand the effects of ageing on nervous system function. It is well known that the world's population is ageing and soon there will be more people over the age of 65 than there are children. If we are to improve the quality of life of the aged, then we must first understand how ageing affects the body's various physiological systems. As the nervous system has an important role in most functions, its preservation with ageing is paramount.

We are primarily using molecular approaches and focusing on ageing-related changes in the cell's two genomes - the nuclear and mitochondrial genomes. It is well-established that both genomes accumulate mutations with ageing, although it is not completely clear whether these mutations are more detrimental to cell function if they accumulate more so in one of the genomes as opposed to the other. It is also not known whether the mutation accumulations occur in a cell-specific manner. This is particularly important for the nervous system with its highly heterogeneous cell population. A number of nervous system functions appear more susceptible to the ageing process than others. For example, the special senses of hearing, vision and balance are particularly prone, although it remains to be determined whether the peripheral components of these systems (cochlea, retina, and vestibular apparatus, respectively) are more affected than their central nervous system connections and processes.

Cognitive function and motor control are two more nervous system functions that are affected by the ageing process as can be readily observed in the aged population. We are characterising the genomic changes in specific populations of cells within these ageing-affected systems. For example, using state-of-the-art, laser based microdissection, we can collect midbrain dopamine neurons, which play an important role in motor control as can be appreciated from Parkinson's disease, at different ages and determine changes in mitochondrial DNA and the expression level of various genes.

Similar approaches are being used for spinal cord motor neurons and inner ear vestibular hair cells. Knowing how ageing affects the nervous system is important, but we also need to be able to intervene if we are to improve the quality of life of aged individuals. Calorie restriction, whereby the daily calorie intake is reduced by 20-40% (but without malnutrition), is the only presently known intervention that retards the ageing process, at least in animal models. We are establishing CR to determine whether the ageing-related changes we see in the various cell populations of interest are modified by this intervention. If this is the case, we will then set out to determine how CR achieves this.

Research methods used in the lab include: Animal models Laser based microdissection Immunocytochemistry RNA and DNA techniques Real-time and end-point PCR Immunofluorescence microscopy Cryosectioning


Qualifications

  • Doctor of Philosophy, University of Queensland

Keywords

  • Aging and Brain Function
  • Anatomy
  • Animal Models
  • Cell Culture Models
  • Developmental Neurobiology
  • Ergonomics
  • Gene Expression
  • Immunocytochemistry
  • Mitochondrial genomics
  • Modern Techniques
  • Molecular Biology
  • Neuroanatomy
  • Neurobiology
  • Physiology
  • Single Cell Genomics
  • Viral Vectors

Fields of Research

Code Description Percentage
060199 Biochemistry and Cell Biology not elsewhere classified 25
110399 Clinical Sciences not elsewhere classified 50
170199 Psychology not elsewhere classified 25

Professional Experience

UON Appointment

Title Organisation / Department
Associate Professor University of Newcastle
School of Biomedical Sciences and Pharmacy
Australia

Academic appointment

Dates Title Organisation / Department
1/03/2005 - 21/06/2015 Academic University of Newcastle
School of Biomedical Sciences and Pharmacy
Australia
1/07/1997 - 1/04/2004 Research Scientist University of California, San Diego
Department of Pediatrics, School of Medicine UCSD
United States

Awards

Research Award

Year Award
1995 CJ Martin Fellowship
Unknown
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Publications

For publications that are currently unpublished or in-press, details are shown in italics.


Journal article (121 outputs)

Year Citation Altmetrics Link
2017 Tu L, Poppi L, Rudd J, Cresswell ET, Smith DW, Brichta A, Nalivaiko E, 'Alpha-9 nicotinic acetylcholine receptors mediate hypothermic responses elicited by provocative motion in mice.', Physiol Behav, 174 114-119 (2017)
DOI 10.1016/j.physbeh.2017.03.012
Co-authors Eugene Nalivaiko
2017 Khan SI, Hübner PP, Brichta AM, Smith DW, Migliaccio AA, 'Aging reduces the high-frequency and short-term adaptation of the vestibulo-ocular reflex in mice', Neurobiology of Aging, 51 122-131 (2017)

© 2016 Elsevier Inc.Prevailing evidence indicates a relatively late life decline in human vestibulo-ocular reflex (VOR) function. Although mice are commonly used in mechanistic s... [more]

© 2016 Elsevier Inc.Prevailing evidence indicates a relatively late life decline in human vestibulo-ocular reflex (VOR) function. Although mice are commonly used in mechanistic studies of vestibular function, it remains unclear whether aging produces a corresponding decline in VOR function in mice. We sought to determine how the baseline VOR and its short-term adaptation were affected by aging. We tested 8 young (3-month old) and 8 aged (30-month old¿equivalent to a ~80-year old human) C57BL/6 mice. We measured their VOR response to whole-body static tilts and during 0.1¿10¿Hz whole-body sinusoidal and transient rotations before and after VOR adaptation training. Our data revealed minimal differences in static counter-tilt response between young and aged mice, but a significant deficit in baseline VOR gain in aged mice during transient rotations. Moreover, aged mice had a significant decrease in short-term VOR adaptation, particularly for training that sought to decrease the VOR response.

DOI 10.1016/j.neurobiolaging.2016.12.007
Citations Scopus - 1Web of Science - 1
Co-authors Alan Brichta
2016 Parkinson GM, Dayas CV, Smith DW, 'Perturbed cholesterol homeostasis in aging spinal cord', Neurobiology of Aging, 45 123-135 (2016) [C1]

© 2016 Elsevier Inc.The spinal cord is vital for the processing of sensorimotor information and for its propagation to and from both the brain and the periphery. Spinal cord func... [more]

© 2016 Elsevier Inc.The spinal cord is vital for the processing of sensorimotor information and for its propagation to and from both the brain and the periphery. Spinal cord function is affected by aging, however, the mechanisms involved are not well-understood. To characterize molecular mechanisms of spinal cord aging, microarray analyses of gene expression were performed on cervical spinal cords of aging rats. Of the metabolic and signaling pathways affected, cholesterol-associated pathways were the most comprehensively altered, including significant downregulation of cholesterol synthesis-related genes and upregulation of cholesterol transport and metabolism genes. Paradoxically, a significant increase in total cholesterol content was observed-likely associated with cholesterol ester accumulation. To investigate potential mechanisms for the perturbed cholesterol homeostasis, we quantified the expression of myelin and neuroinflammation-associated genes and proteins. Although there was minimal change in myelin-related expression, there was an increase in phagocytic microglial and astrogliosis markers, particularly in the white matter. Together, these results suggest that perturbed cholesterol homeostasis, possibly as a result of increased inflammatory activation in spinal cord white matter, may contribute to impaired spinal cord function with aging.

DOI 10.1016/j.neurobiolaging.2016.05.017
Citations Scopus - 1Web of Science - 1
Co-authors Christopher Dayas
2016 James MH, Quinn RK, Ong LK, Levi EM, Smith DW, Dickson PW, Dayas CV, 'Rapamycin reduces motivated responding for cocaine and alters GluA1 expression in the ventral but not dorsal striatum', European Journal of Pharmacology, 784 147-154 (2016) [C1]

© 2016 Published by Elsevier B.V. All rights reserved.The mechanistic target of rapamycin complex 1 (mTORC1) regulates synaptic protein synthesis and therefore synaptic function ... [more]

© 2016 Published by Elsevier B.V. All rights reserved.The mechanistic target of rapamycin complex 1 (mTORC1) regulates synaptic protein synthesis and therefore synaptic function and plasticity. A role for mTORC1 has recently been demonstrated for addiction-related behaviors. For example, central or intra-accumbal injections of the mTORC1 inhibitor rapamycin attenuates several indices of cocaine-seeking including progressive ratio (PR) responding and reinstatement. These behavioral effects are associated with decreased mTORC1 activity and synaptic protein translation in the nucleus accumbens (NAC) and point to a possible therapeutic role for rapamycin in the treatment of addiction. Currently, it is unclear whether similar behavioral and biochemical effects can be achieved by administering rapamycin systemically, which represents a more clinically-appropriate route of administration. Here, we assessed the effects of repeated, systemic administration of rapamycin (10 mg/kg, i.p.) on PR responding for cocaine. We also assessed whether systemic rapamycin was associated with changes in measures of mTORC1 activity and GluA1 expression in the ventral and dorsal striatum. We report that systemic rapamycin treatment reduced PR breakpoints to levels comparable to intra-NAC rapamycin. Systemic rapamycin treatment also reduced phosphorylated p70S6K and GluA1 AMPARs within the NAC but not dorsal striatum. Thus, systemic administration of rapamycin is as effective at reducing drug seeking behavior and measures of mTORC1 activity compared to direct accumbal application and may therefore represent a possible therapeutic option in the treatment of addiction. Possible caveats of this treatment approach are discussed.

DOI 10.1016/j.ejphar.2016.05.013
Citations Scopus - 1Web of Science - 1
Co-authors Phil Dickson, Christopher Dayas, Linkooi Ong
2016 Murray HC, Maltby VE, Smith DW, Bowden NA, 'Nucleotide excision repair deficiency in melanoma in response to UVA', Experimental Hematology and Oncology, 5 (2016) [C1]

© 2016 Murray et al.Background: The causative link between UV exposure and melanoma development is well known, however the mechanistic relationship remains incompletely character... [more]

© 2016 Murray et al.Background: The causative link between UV exposure and melanoma development is well known, however the mechanistic relationship remains incompletely characterised. UVA and UVB components of sunlight are implicated in melanomagenesis; however the majority of studies have focused on the effects of UVB and UVC light. Interestingly, melanoma tumour sequencing has revealed an overrepresentation of mutations signature of unrepaired UV-induced DNA damage. Repair of UVA-induced DNA damage is thought to occur primarily through the Nucleotide Excision Repair (NER) pathway, which recognises and repairs damage either coupled to transcription (Transcription Coupled Repair; TCR), or through global genome scanning (Global Genome Repair; GGR). Current literature suggests NER is deficient in melanoma, however the cause of this remains unknown; and whether reduced NER activity in response to UVA may be involved in melanoma development remains uncharacterised. In this study we aimed to determine if melanoma cells exhibit reduced levels of NER activity in response to UVA. Methods: Melanocyte and melanoma cell lines were UVA-irradiated, and DNA damage levels assessed by immunodetection of Cyclobutane Pyrimidine Dimer (CPD) and (6-4) Photoproduct [(6-4)PP] lesions. Expression of NER pathway components and p53 following UVA treatment was quantified by qPCR and western blot. Results: UVA did not induce detectable induction of (6-4)PP lesions, consistent with previous studies. Repair of CPDs induced by UVA was initiated at 4 h and complete within 48 h in normal melanocytes, whereas repair initiation was delayed to 24 h and >40 % of lesions remained in melanoma cell lines at 48 h. This was coupled with a delayed and reduced induction of GGR component XPC in melanoma cells, independent of p53. Conclusion: These findings support that NER activity is reduced in melanoma cells due to deficient GGR. Further investigation into the role of NER in UVA-induced melanomagenesis is warranted and may have implications for melanoma treatment.

DOI 10.1186/s40164-016-0035-4
Citations Scopus - 1Web of Science - 1
Co-authors Vicki E Maltby, Nikola Bowden
2015 McIlroy DJ, Bigland M, White AE, Hardy BM, Lott N, Smith DW, Balogh ZJ, 'Cell necrosis-independent sustained mitochondrial and nuclear DNA release following trauma surgery', JOURNAL OF TRAUMA AND ACUTE CARE SURGERY, 78 282-288 (2015) [C1]
DOI 10.1097/TA.0000000000000519
Citations Scopus - 12Web of Science - 11
Co-authors Zsolt Balogh
2015 Quinn RK, Brown AL, Goldie BJ, Levi EM, Dickson PW, Smith DW, et al., 'Distinct miRNA expression in dorsal striatal subregions is associated with risk for addiction in rats', Translational Psychiatry, 5 (2015) [C1]

Recently, we published data using an animal model that allowed us to characterize animals into two groups, addiction vulnerable and addiction resilient, where we identified that a... [more]

Recently, we published data using an animal model that allowed us to characterize animals into two groups, addiction vulnerable and addiction resilient, where we identified that addiction/relapse vulnerability was associated with deficits in synaptic plasticityassociated gene expression in the dorsal striatum (DS). Notable was the strong reduction in expression for activity-regulated cytoskeleton-associated protein (Arc) considered a master regulator of synaptic plasticity. In the present study, we confirmed that Arc messenger RNA was significantly decreased in the DS, but importantly, we identified that this reduction was restricted to the dorsomedial (DMS) and not dorsolateral striatum (DLS). There is recent evidence of microRNA (miRNA)-associated posttranscriptional suppression of Arc and animal models of addiction have identified a key role for miRNA in the regulation of addiction-relevant genes. In further support of this link, we identified several differentially expressed miRNA with the potential to influence addiction-relevant plasticity genes, including Arc. A key study recently reported that miR-212 expression is protective against compulsive cocaine-seeking. Supporting this hypothesis, we found that miR-212 expression was significantly reduced in the DMS but not DLS of addiction-vulnerable animals. Together, our data provide strong evidence that miRNA promote ongoing plasticity deficits in the DS of addiction-vulnerable animals.

DOI 10.1038/tp.2014.144
Citations Scopus - 7Web of Science - 7
Co-authors Christopher Dayas, Phil Dickson, Murray Cairns
2015 Parkinson GM, Dayas CV, Smith DW, 'Age-related gene expression changes in substantia nigra dopamine neurons of the rat.', Mech Ageing Dev, 149 41-49 (2015) [C1]
DOI 10.1016/j.mad.2015.06.002
Citations Scopus - 1Web of Science - 1
Co-authors Christopher Dayas
2015 Thomas J, Garg ML, Smith DW, 'Effects of dietary supplementation with docosahexaenoic acid (DHA) on hippocampal gene expression in streptozotocin induced diabetic C57Bl/6 mice', Journal of Nutrition and Intermediary Metabolism, 2 2-7 (2015) [C1]

© 2015 The Authors.A body of evidence has accumulated indicating diabetes is associated with cognitive impairments. Effective strategies are therefore needed that will delay or p... [more]

© 2015 The Authors.A body of evidence has accumulated indicating diabetes is associated with cognitive impairments. Effective strategies are therefore needed that will delay or prevent the onset of these diabetes-related deficits. In this regard, dietary modification with the naturally occurring compound, docosahexaenoic acid (DHA), holds significant promise as it has been shown to have anti-inflammatory, anti-oxidant, and anti-apoptotic properties. The hippocampus, a limbic structure involved in cognitive functions such as memory formation, is particularly vulnerable to the neurotoxic effects related to diabetes, and we have previously shown that streptozotocin-induced diabetes alters hippocampal gene expression, including genes involved in synaptic plasticity and neurogenesis. In the present study, we explored the effects of dietary supplementation with DHA on hippocampal gene expression in C57Bl/6 diabetic mice. Diabetes was established using streptozotocin (STZ) and once stable, the dietary intervention group received AIN93G diet supplemented with DHA (50 mg/kg/day) for 6 weeks. Microarray based genome-wide expression analysis was carried out on the hippocampus of DHA supplemented diabetic mice and confirmed by real time polymerase chain reaction (RT-qPCR). Genome-wide analysis identified 353 differentially expressed genes compared to non-supplemented diabetic mice. For example, six weeks of dietary DHA supplementation resulted in increased hippocampal expression of Igf II and Sirt1 and decreased expression of Tnf-a, Il6, Mapkapk2 and ApoE, compared to non-supplemented diabetic mice. Overall, DHA supplementation appears to alter hippocampal gene expression in a way that is consistent with it being neuroprotective in the context of the metabolic and inflammatory insults associated with diabetes.

DOI 10.1016/j.jnim.2015.04.001
Co-authors Manohar Garg
2014 James MH, Quinn RK, Ong LK, Levi EM, Charnley JL, Smith DW, et al., 'mTORC1 inhibition in the nucleus accumbens 'protects' against the expression of drug seeking and 'relapse' and is associated with reductions in GluA1 AMPAR and CAMKIIa levels.', Neuropsychopharmacology, 39 1694-1702 (2014) [C1]
DOI 10.1038/npp.2014.16
Citations Scopus - 7Web of Science - 7
Co-authors Phil Dickson, Linkooi Ong, Christopher Dayas
2014 McIlroy DJ, Jarnicki AG, Au GG, Lott N, Smith DW, Hansbro PM, Balogh ZJ, 'Mitochondrial DNA neutrophil extracellular traps are formed after trauma and subsequent surgery', Journal of Critical Care, 29 1133.e1-1133.e5 (2014) [C1]

© 2014 The Authors.Introduction: Neutrophil extracellular traps (NETs) have not been demonstrated after trauma and subsequent surgery. Neutrophil extracellular traps are formed f... [more]

© 2014 The Authors.Introduction: Neutrophil extracellular traps (NETs) have not been demonstrated after trauma and subsequent surgery. Neutrophil extracellular traps are formed from pure mitochondrial DNA (mtDNA) under certain conditions, which is potently proinflammatory. We hypothesized that injury and orthopedic trauma surgery would induce NET production with mtDNA as a structural component. Methods: Neutrophils were isolated 8 trauma patients requiring orthopedic surgery postinjury and up to 5 days postoperatively. Four healthy volunteers provided positive and negative controls. Total hip replacement patients acted as an uninjured surgical control group. Neutrophil extracellular traps were visualized with DNA (Hoechst 33342TM/Sytox Green/MitoSox/MitoTracker) stains using live cell fluorescence microscopy with downstream quantitative polymerase chain reaction analysis of DNA composition. Results: Neutrophil extracellular traps were present after injury in all 8 trauma patients. They persisted for 5 days postoperatively. Delayed surgery resulted in NET resolution, but they reformed postoperatively. Total hip replacement patients developed NETs postoperatively, which resolved by day 5. Quantitative polymerase chain reaction analysis of NET-DNA composition revealed that NETs formed after injury and surgery were made of mtDNA with no detectable nuclear DNA component. Conclusions: Neutrophil extracellular traps formed after major trauma and subsequent surgery contain mtDNA and represent a novel marker of heightened innate immune activation. They could be considered when timing surgery after trauma to prevent systemic NET-induced inflammatory complications.

DOI 10.1016/j.jcrc.2014.07.013
Citations Scopus - 15Web of Science - 9
Co-authors Zsolt Balogh, Gough Au, Philip Hansbro
2014 Thomas J, Garg ML, Smith DW, 'Dietary resveratrol supplementation normalizes gene expression in the hippocampus of streptozotocin-induced diabetic C57Bl/6 mice', Journal of Nutritional Biochemistry, 25 313-318 (2014) [C1]

Diabetes is associated with cognitive impairment and brain aging, with alterations in hippocampal neurogenesis and synaptic plasticity implicated in these changes. As the prevalen... [more]

Diabetes is associated with cognitive impairment and brain aging, with alterations in hippocampal neurogenesis and synaptic plasticity implicated in these changes. As the prevalence of diabetes continues to rise, readily implemented strategies are increasingly needed in order to protect the brain's cognitive functions. One possibility is resveratrol (RES) (3,5,4- trihydroxystilbene), a polyphenol of the phytoalexin family that has been shown to be protective in a number of neuropathology paradigms. In the present study, we sought to determine whether dietary supplementation with RES has potential for the protection of cognitive functions in diabetes. Diabetes was induced using streptozotocin, and once stable, animals received AIN93G rodent diet supplemented with RES for 6 weeks. Genome-wide expression analysis was conducted on the hippocampus and genes of interest were confirmed by quantitative, real-time polymerase chain reaction. Genome-wide gene expression analysis of the hippocampus revealed that RES supplementation of the diabetic group resulted in 481differentially expressed genes compared to non-supplemented diabetic mice. Intriguingly, gene expression that was previously found significantly altered in the hippocampus of diabetic mice, and that is implicated in neurogenesis and synaptic plasticity (Hdac4, Hat1, Wnt7a, ApoE), was normalized following RES supplementation. In addition, pathway analysis revealed Jak-Stat signaling was the most significantly enriched pathway. The Jak-Stat pathway induces a pro-inflammatory signaling cascade, and we found most genes involved in this cascade (e.g. Il15, Il22, Socs2, Socs5) had significantly lower expression following RES supplementation. These data indicate RES could be neuroprotective and beneficial for the maintenance of cognitive function in diabetes. © 2014 Elsevier Inc.

DOI 10.1016/j.jnutbio.2013.11.005
Citations Scopus - 12Web of Science - 11
Co-authors Manohar Garg
2014 Parkinson GM, Dayas CV, Smith DW, 'Increased mitochondrial DNA deletions in substantia nigra dopamine neurons of the aged rat.', Current aging science, 7 155-160 (2014) [C2]
Citations Scopus - 2
Co-authors Christopher Dayas
2014 James MH, Campbell EJ, Walker FR, Smith DW, Richardson HN, Hodgson DM, Dayas CV, 'Exercise reverses the effects of early life stress on orexin cell reactivity in male but not female rats', Frontiers in Behavioral Neuroscience, 8 (2014) [C1]
DOI 10.3389/fnbeh.2014.00244
Citations Scopus - 9Web of Science - 8
Co-authors Rohan Walker, Deborah Hodgson, Christopher Dayas
2013 Balogh ZJ, McIlroy DJ, Smith DW, Hansbro PM, 'The origin and the role of mitochondrial DNA in postinjury inflammation', Journal of Critical Care, 28 1099-1100 (2013) [C3]
DOI 10.1016/j.jcrc.2013.08.027
Citations Scopus - 3Web of Science - 3
Co-authors Philip Hansbro, Zsolt Balogh
2013 Thomas J, Garg ML, Smith DW, 'Altered expression of histone and synaptic plasticity associated genes in the hippocampus of streptozotocin-induced diabetic mice', Metabolic Brain Disease, 28 613-618 (2013) [C1]
DOI 10.1007/s11011-013-9418-y
Citations Scopus - 10Web of Science - 7
Co-authors Manohar Garg
2013 Thomas J, Garg ML, Smith DW, 'Dietary supplementation with resveratrol and/or docosahexaenoic acid alters hippocampal gene expression in adult C57Bl/6 mice', Journal of Nutritional Biochemistry, 24 1735-1740 (2013) [C1]

The hippocampus is an important brain structure for multiple cognitive functions, including memory formation. It is particularly sensitive to insults, such as stress, ischemia, an... [more]

The hippocampus is an important brain structure for multiple cognitive functions, including memory formation. It is particularly sensitive to insults, such as stress, ischemia, and aging; all of these can affect hippocampal and therefore cognitive function. To understand the potential of diet for the preservation of hippocampal function, we investigated the effects of dietary supplementation with resveratrol (RES) or docosahexaenoic acid (DHA), or their combination, on hippocampal gene expression in adult C57BL/6 mice. Animals in the supplemented group received either 50 mg/kg/day of RES or DHA, while the combination group received 50 mg/kg/day of each supplement. Dietary supplements were mixed with the AIN93G diet, and supplementation lasted 6 weeks. The control group received AIN93G diet alone for the same period. At the end of the experiment, the hippocampi were processed for genome-wide gene expression and pathway analyses. Most of the genes that were significantly altered were associated with inflammatory responses as determined by pathway analysis. RES-supplemented animals showed decreased expression of IL-6 ( P=001), MAPKapk2 ( P=015), and increased expression for PI3. KR2 ( P=034) and Wnt7a ( P=004) expression. DHA-supplemented animals showed a decreased IL-6 ( P=003) and an increased Wnt7a ( P=003) expression. Animals on the combination diet showed a decreased IL-6 ( P=005) and Apolipoprotien E ( ApoE) ( P=035) expression. Our findings demonstrate that hippocampal gene expression is significantly altered by all three dietary supplementation regimes. Moreover, our analysis indicates that RES and DHA likely exert their beneficial effects through antiinflammatory mechanisms. © 2013 Elsevier Inc.

DOI 10.1016/j.jnutbio.2013.03.002
Citations Scopus - 8Web of Science - 5
Co-authors Manohar Garg
2013 Cahif A, Parkinson GM, Dayas CV, Smith DW, 'Characterisation of mitochondrial DNA deletions by long-PCR in central nervous system regions of young, middle- and old-aged rats.', Current Aging Science, 6 232-238 (2013) [C1]
Citations Scopus - 2
Co-authors Christopher Dayas
2013 Brown AL, Day TA, Dayas CV, Smith DW, 'Purity and Enrichment of Laser-Microdissected Midbrain Dopamine Neurons', BIOMED RESEARCH INTERNATIONAL, (2013) [C1]
DOI 10.1155/2013/747938
Citations Scopus - 6Web of Science - 5
Co-authors Christopher Dayas
2012 Barreto RDA, Walker FR, Dunkley PR, Day TA, Smith DW, 'Fluoxetine prevents development of an early stress-related molecular signature in the rat infralimbic medial prefrontal cortex. Implications for depression?', BMC Neuroscience, 13 1-12 (2012) [C1]
Citations Scopus - 19Web of Science - 18
Co-authors Rohan Walker, Peter Dunkley
2012 Dayas CV, Smith DW, Dunkley PR, 'An emerging role for the mammalian Target of Rapamycin (mTOR) in 'pathological' protein translation: Relevance to cocaine addiction', Frontiers in Pharmacology, 3 1-12 (2012) [C1]
Citations Scopus - 12Web of Science - 9
Co-authors Peter Dunkley, Christopher Dayas
2011 James MH, Charnley JL, Flynn JR, Smith DW, Dayas CV, 'Propensity to 'relapse' following exposure to cocaine cues is associated with the recruitment of specific thalamic and epithalamic nuclei', Neuroscience, 199 235-242 (2011) [C1]
Citations Scopus - 27Web of Science - 26
Co-authors Christopher Dayas, Jamie Flynn
2011 James MH, Charnley JL, Levi EM, Jones E, Yeoh JW, Smith DW, Dayas CV, 'Orexin-1 receptor signalling within the ventral tegmental area, but not the paraventricular thalamus, is critical to regulating cue-induced reinstatement of cocaine-seeking', International Journal of Neuropsychopharmacology, 14 684-690 (2011) [C1]
DOI 10.1017/s1461145711000423
Citations Scopus - 56Web of Science - 53
Co-authors Christopher Dayas
2011 Brown AL, Flynn JR, Smith DW, Dayas CV, 'Down-regulated striatal gene expression for synaptic plasticity-associated proteins in addiction and relapse vulnerable animals', International Journal of Neuropsychopharmacology, 14 1099-1110 (2011) [C1]
Citations Scopus - 17Web of Science - 14
Co-authors Jamie Flynn, Christopher Dayas
2010 James MH, Charnley JL, Jones E, Levi E, Yeoh JW, Flynn JR, et al., 'Cocaine- and amphetamine-regulated transcript (CART) signaling within the paraventricular thalamus modulates cocaine-seeking behaviour', Plos One, 5 e12980 (2010) [C1]
DOI 10.1371/journal.pone.0012980
Citations Scopus - 40Web of Science - 35
Co-authors Jamie Flynn, Christopher Dayas
2009 McInerny SC, Brown AL, Smith DW, 'Region-specific changes in mitochondrial D-loop in aged rat CNS', Mechanisms of Ageing and Development, 130 343-349 (2009) [C1]
DOI 10.1016/j.mad.2009.01.008
Citations Scopus - 17Web of Science - 13
2009 Rana BK, Wessel J, Mahboubi V, Rao F, Haeller J, Gayen JR, et al., 'Natural variation within the neuronal nicotinic acetylcholine receptor cluster on human chromosome 15q24: Influence on heritable autonomic traits in twin pairs', Journal of Pharmacology and Experimental Therapeutics, 331 419-428 (2009)

Nicotinic acetylcholine receptors (nAChRs) are combinations of subunits arranged as pentamers encircling a central cation channel. At least nine a and four ß subunits are express... [more]

Nicotinic acetylcholine receptors (nAChRs) are combinations of subunits arranged as pentamers encircling a central cation channel. At least nine a and four ß subunits are expressed in the central and peripheral nervous systems; their presence in autonomic ganglia, the adrenal medulla, and central nervous system, with accompanying responses elicited by nicotinic agonists, point to their involvement in cardiovascular homeostasis. nAChRs formed by a3, a5, and ß4 subunits may regulate blood pressure (BP) by mediating release of catestatin, the endogenous nicotinic antagonist fragment of chromogranin A (CHGA) and potent inhibitor of catecholamine secretion. Genes encoding these subunits (CHRNA3, CHRNA5, and CHRNB4) are clustered on human chromosome 15q24. Because variation in this cluster may alter autonomic regulation of BP, we sequenced ~15 kilobase pairs in 15q24 containing their coding and 5'- and 3'-untranslated regions in 80 individuals. We identified 63 variants: 25 in coding regions of CHRNA3, CHRNA5, and CHRNB4 and 48 noncoding single-nucleotide polymorphisms (SNPs). Haplotype frequencies varied across ethnic populations. We assessed the contribution of six SNPs in the putative catestatin binding region of CHRNA3 and CHRNB4 to autonomic traits. In twins, catestatin and BP were heritable. CHRNA3 SNPs and haplotypes containing K95K (G285A) associated with circulating plasma catestatin, epinephrine levels, as well as systolic BP, suggesting altered coupling of the nAChRs to BP. Studies of chromaffin cells in vitro reveal that nicotinic agonist stimulation releases catecholamines and CHGA, a process augmented by overexpression of CHRNA3 and blocked by catestatin. These cellular events suggest a homeostatic mechanism underlying the pleiotropic actions of CHRNA3 genetic variation on autonomic function observed in twins.

DOI 10.1124/jpet.109.157271
Citations Scopus - 8
2009 Zhang K, Rao F, Rana BK, Gayen JR, Calegari F, King A, et al., 'Autonomic function in hypertension role of genetic variation at the catecholamine storage vesicle protein chromogranin B', Circulation: Cardiovascular Genetics, 2 46-56 (2009)

Background: Hypertension is a complex trait, with deranged autonomic control of circulation. Chromogranin B (CHGB) is the most abundant core protein in human catecholamine secreto... [more]

Background: Hypertension is a complex trait, with deranged autonomic control of circulation. Chromogranin B (CHGB) is the most abundant core protein in human catecholamine secretory vesicles, playing an important role in their biogenesis. Does common interindividual variation at the CHGB locus contribute to phenotypic variation in CHGB and catecholamine secretion, autonomic stability of circulation, or blood pressure (BP) in the population? Methods and Results: To probe interindividual variability in CHGB, we systematically studied polymorphism across the locus by resequencing CHGB (¿6 kbp footprint spanning the promoter, 5 exons, exon/intron borders, untranslated regions) in 160 subjects (2n=320 chromosomes) of diverse biogeographic ancestries. We identified 53 single-nucleotide polymorphisms, of which 22 were common. We then studied 1182 subjects drawn from the most extreme BP values in the population (highest and lowest 5th percentiles), typing 4 common polymorphisms spanning the ¿14 kbp locus. Sliding-window haplotype analysis indicated BP associations peaking in the 5'/promoter region, most prominent in men, and a peak effect in the proximal promoter at variant A-261T (A>T), accounting for ¿8/¿6 mm Hg BP in males. The promoter allele (A-261) that was a predictor of higher diastolic BP and systolic BP was also associated with lower circulating/plasma CHGB concentration (CHGB439to451 epitope) in twin pairs. In twins, the same CHGB variants that were predictors of lower basal CHGB secretion were also associated with exaggerated catecholamine secretion and BP response to environmental (cold) stress; likewise, women displayed increased plasma CHGB439to451 but decreased catecholamine secretion as well as BP response to environmental stress. The effect of A-261T on CHGB expression was confirmed in chromaffin cells by site-directed mutagenesis on transfected CHGB promoter/luciferase reporter activity, and the allelic effects of A-261T on gene expression were directionally coordinate in cella and in vivo. To confirm these clinical associations experimentally, we undertook targeted homozygous (-/-) ablation of the mouse CHGB gene; knockout mice displayed substantially increased BP, by ¿20/¿18 mm Hg, confirming the mechanistic basis of our findings in humans. Conclusion-Common genetic variation at the CHGB locus, especially in the proximal promoter, influences CHGB expression and later catecholamine secretion and the early heritable responses to environmental stress, eventuating in changes in resting/basal BP in the population. Both the early (gene expression) and late (population BP) consequences of CHGB variation are sex dependent. These results point to new molecular strategies for probing autonomic control of circulation and, ultimately, the susceptibility to and pathogenesis of cardiovascular disease states such as hypertension. © 2009 American Heart Association, Inc.

DOI 10.1161/CIRCGENETICS.108.785659
Citations Scopus - 17
2009 Brown AL, Smith DW, 'Improved RNA preservation for immunolabeling and laser microdissection', RNA: A Publication of the RNA Society, 15 2364-2374 (2009) [C1]
DOI 10.1261/rna.1733509
Citations Scopus - 17Web of Science - 16
2008 Salem RM, Cadman PE, Chen Y, Rao F, Wen G, Hamilton BA, et al., 'Chromogranin A polymorphisms are associated with hypertensive renal disease', Journal of the American Society of Nephrology, 19 600-614 (2008)

Chromogranin A is released together with epinephrine and norepinephrine from catecholaminergic cells. Specific endopeptidases cleave chromogranin A into biologically active peptid... [more]

Chromogranin A is released together with epinephrine and norepinephrine from catecholaminergic cells. Specific endopeptidases cleave chromogranin A into biologically active peptide fragments, including catestatin, which inhibits catecholamine release. Previous studies have suggested that a deficit in this sympathetic "braking" system might be an early event in the pathogenesis of human hypertension. Whether chromogranin A (CHGA) polymorphisms predict end-organ complications of hypertension, such as end-stage renal disease, is unknown. Among blacks, we studied common genetic variants spanning the CHGA locus in 2 independent case-control studies of hypertensive ESRD. Two haplotypes were significantly more frequent among subjects with hypertensive ESRD: 1) in the promoter (5') region, G-462A¿T-415C¿C-89A, haplotype ATC (adjusted odds ratio = 2.65; P = 0.037), and 2) at the 3'-end, C11825T (3'-UTR, C+87T)¿G12602C, haplotype TC (adjusted odds ratio = 2.73, P = 0.0196). Circulating levels of catestatin were lower among those with hypertensive ESRD than controls, an unexpected finding given that peptide levels are usually elevated in ESRD because of reduced renal elimination. We found that the 3'-UTR + 87T variant decreased reporter gene expression, providing a possible mechanistic explanation for diminished catestatin. In summary, common variants in chromogranin A associate with the risk of hypertensive ESRD in blacks. Copyright © 2008 by the American Society of Nephrology.

DOI 10.1681/ASN.2007070754
Citations Scopus - 43
2007 Smith DW, Jinnah HA, 'Role of neuronal nitric oxide in the dopamine deficit of HPRT-deficient mice', Metabolic Brain Disease, 22 39-43 (2007) [C1]
DOI 10.1007/s11011-007-9044-7
Citations Scopus - 3Web of Science - 3
2007 Lillie EO, Mahata M, Khandrika S, Rao F, Bundey RA, Wen G, et al., 'Heredity of endothelin secretion: Human twin studies reveal the influence of polymorphism at the chromogranin A locus, a novel determinant of endothelial function', Circulation, 115 2282-2291 (2007)

BACKGROUND - Endothelial dysfunction predisposes to vascular injury in association with hypertension. Endothelin (ET-1) is a potent vasoactive peptide that is synthesized and rele... [more]

BACKGROUND - Endothelial dysfunction predisposes to vascular injury in association with hypertension. Endothelin (ET-1) is a potent vasoactive peptide that is synthesized and released by the vascular endothelium and is a marker of endothelial function. Chromogranin A (CHGA) regulates the storage and release of catecholamines and may have direct actions on the microvasculature. CHGA, a candidate gene for intermediate phenotypes that contribute to hypertension, shows a pattern of single nucleotide polymorphism variations that alter the expression and function of this gene both in vivo and in vitro. METHODS AND RESULTS - In a study of twins (n=238 pairs), plasma ET-1 was 58±5% (P<0.0001) heritable. Plasma ET-1 was both correlated and associated with chromogranin fragment levels, and the 2 were influenced by shared genetic determination (pleiotropy [¿G]; for the CHGA precursor, ¿G=0. 318±0.105; P=0.0032). We therefore hypothesized that variation in the CHGA gene may influence ET-1 secretion. Carriers of the CHGA promoter -988G, -462A, and -89A minor alleles showed significantly higher mean plasma ET-1 than their major allele homozygote counterparts (P=0.02, P=0.006, P=0.03, respectively). Analysis of a linkage disequilibrium block that spans these 3 single nucleotide polymorphisms showed a significant association between the GATACA haplotype and plasma ET-1 (P=0.0075). In cultured human umbilical vein endothelial cells, CHGA caused dose-dependent secretion of ET-1 over a brief (<1 hour) time course at relatively low concentrations of CHGA (10 to 100 nmol/L) with a threshold concentration (10 nmol/L) in the range found circulating in humans in vivo. CONCLUSIONS - These results suggest that common, heritable variation in expression of the human CHGA gene influences endothelial ET-1 secretion in vivo, explained by a CHGA stimulus/ET-1 secretion coupling in endothelial cells in vitro. The findings document a previously unsuspected interaction between the sympathochromaffin system and the endothelium and suggest novel genetic and cell biological approaches to the prediction, diagnosis, and mechanism of endothelial dysfunction in human disease. © 2007 American Heart Association, Inc.

DOI 10.1161/CIRCULATIONAHA.106.648345
Citations Scopus - 13
2007 Rao F, Wen G, Gayen JR, Das M, Vaingankar SM, Rana BK, et al., 'Catecholamine release-inhibitory peptide catestatin (chromogranin A352-372): Naturally occurring amino acid variant Gly364Ser causes profound changes in human autonomic activity and alters risk for hypertension', Circulation, 115 2271-2281 (2007)

BACKGROUND - Chromogranin A, coreleased with catecholamines by exocytosis, is cleaved to the catecholamine release-inhibitory fragment catestatin. We identified a natural nonsynon... [more]

BACKGROUND - Chromogranin A, coreleased with catecholamines by exocytosis, is cleaved to the catecholamine release-inhibitory fragment catestatin. We identified a natural nonsynonymous variant of catestatin, Gly364Ser, that alters human autonomic function and blood pressure. METHODS AND RESULTS - Gly364Ser heterozygotes and controls underwent physiological and biochemical phenotyping, including catecholamine production, chromogranin A precursor, and its catestatin product. Case-control studies replicated effects of the gene on blood pressure in the population. Gly364Ser displayed diminished inhibition of catecholamine secretion from cultured neurons. Gly/Ser heterozygotes displayed increased baroreceptor slope during upward deflections (by ¿47%) and downward deflections (by ¿44%), increased cardiac parasympathetic index (by ¿2.4-fold), and decreased cardiac sympathetic index (by ¿26%). Renal norepinephrine excretion was diminished by ¿26% and epinephrine excretion by ¿34% in Gly/Ser heterozygotes. The coalescent dated emergence of the variant to ¿70 000 years ago. Gly364Ser was in linkage disequilibrium with 1 major Chromogranin A promoter haplotype, although promoter haplotypes did not predict autonomic phenotypes. The 364Ser variant was associated with lower diastolic blood pressure in 2 independent/confirmatory groups of patients with hypertension; genotype groups differed by ¿5 to 6 mm Hg, and the polymorphism accounted for ¿1.8% of population diastolic blood pressure variance, although a significant gene-by-sex interaction existed, with an enhanced effect in men. CONCLUSIONS - The catestatin Gly364Ser variant causes profound changes in human autonomic activity, both parasympathetic and sympathetic, and seems to reduce risk of developing hypertension, especially in men. A model for catestatin action in the baroreceptor center of the nucleus of the tractus solitarius accounts for these actions. © 2007 American Heart Association, Inc.

DOI 10.1161/CIRCULATIONAHA.106.628859
Citations Scopus - 78
2007 Rao F, Zhang L, Wessel J, Zhang K, Wen G, Kennedy BP, et al., 'Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis: Discovery of common human genetic variants governing transcription, autonomic activity, and blood pressure in vivo', Circulation, 116 993-1006 (2007)

BACKGROUND - Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Does common genetic variation at human TH alter autonomic activity and predispose... [more]

BACKGROUND - Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Does common genetic variation at human TH alter autonomic activity and predispose to cardiovascular disease? We undertook systematic polymorphism discovery at the TH locus and then tested variants for contributions to sympathetic function and blood pressure. METHODS AND RESULTS - We resequenced 80 ethnically diverse individuals across the TH locus. One hundred seventy-two twin pairs were evaluated for sympathetic traits, including catecholamine production, reflex control of the circulation, and environmental (cold) stress responses. To evaluate hypertension, we genotyped subjects selected from the most extreme diastolic blood pressure percentiles in the population. Human TH promoter haplotype/reporter plasmids were transfected into chromaffin cells. Forty-nine single-nucleotide polymorphisms were discovered, but coding region polymorphism did not account for common phenotypic variation. A block of linkage disequilibrium spanned 4 common variants in the proximal promoter. Catecholamine secretory traits were significantly heritable (h), as were stress-induced blood pressure changes. In the TH promoter, significant associations were found for urinary catecholamine excretion and for blood pressure response to stress. TH promoter haplotype 2 (TGGG) showed pleiotropy, increasing both norepinephrine excretion and blood pressure during stress. Coalescent simulations suggest that TH haplotype 2 likely arose ¿380 000 years ago. In hypertension, 2 independent case-control studies (1266 subjects with 53% women and 927 subjects with 24% women) replicated the effect of C-824T in the determination of blood pressure. CONCLUSIONS - We conclude that human catecholamine secretory traits are heritable, displaying joint genetic determination (pleiotropy) with autonomic activity and finally with blood pressure in the population. Catecholamine secretion is influenced by genetic variation in the adrenergic pathway encoding catecholamine synthesis, especially at the classically rate-limiting step, TH. The results suggest novel pathophysiological links between a key adrenergic locus, catecholamine metabolism, and blood pressure and suggest new strategies to approach the mechanism, diagnosis, and treatment of systemic hypertension. © 2007 American Heart Association, Inc.

DOI 10.1161/CIRCULATIONAHA.106.682302
Citations Scopus - 61
2007 Zhang L, Rao F, Zhang K, Khandrika S, Das M, Vaingankar SM, et al., 'Discovery of common human genetic variants of GTP cyclohydrolase 1 (GCH1) governing nitric oxide, autonomic activity, and cardiovascular risk', Journal of Clinical Investigation, 117 2658-2671 (2007)

GTP cyclohydrolase 1 (GCH1) is rate limiting in the provision of the cofactor tetrahydrobiopterin for biosynthesis of catecholamines and NO. We asked whether common genetic variat... [more]

GTP cyclohydrolase 1 (GCH1) is rate limiting in the provision of the cofactor tetrahydrobiopterin for biosynthesis of catecholamines and NO. We asked whether common genetic variation at GCH1 alters transmitter synthesis and predisposes to disease. Here we undertook a systematic search for polymorphisms in GCH1, then tested variants' contributions to NO and catecholamine release as well as autonomic function in twin pairs. Renal NO and neopterin excretions were significantly heritable, as were baroreceptor coupling (heart rate response to BP fluctuation) and pulse interval (1/heart rate). Common GCH1 variant C+243T in the 3'-untranslated region (3'-UTRs) predicted NO excretion, as well as autonomic traits: baroreceptor coupling, maximum pulse interval, and pulse interval variability, though not catecholamine secretion. In individuals with the most extreme BP values in the population, C+243T affected both diastolic and systolic BP, principally in females. In functional studies, C+243T decreased reporter expression in transfected 3'-UTRs plasmids. We conclude that human NO secretion traits are heritable, displaying joint genetic determination with autonomic activity by functional polymorphism at GCH1. Our results document novel pathophysiological links between a key biosynthetic locus and NO metabolism and suggest new strategies for approaching the mechanism, diagnosis, and treatment of risk predictors for cardiovascular diseases such as hypertension.

DOI 10.1172/JCI31093
Citations Scopus - 58
2007 Rao F, Wessel J, Wen G, Zhang L, Rana BK, Kennedy BP, et al., 'Renal albumin excretion: Twin studies identify influences of heredity, environment, and adrenergic pathway polymorphism', Hypertension, 49 1015-1031 (2007)

Albumin excretion marks early glomerular injury in hypertension. This study investigated heritability of albumin excretion in twin pairs and its genetic determination by adrenergi... [more]

Albumin excretion marks early glomerular injury in hypertension. This study investigated heritability of albumin excretion in twin pairs and its genetic determination by adrenergic pathway polymorphism. Genetic associations used single nucleotide polymorphisms at adrenergic pathway loci spanning catecholamine biosynthesis, storage, catabolism, receptor action, and postreceptor signal transduction. We studied 134 single nucleotide polymorphisms at 46 loci for a total of >51 000 genotypes. Albumin excretion heritability was 45.2±7.4% (P=2×10), and the phenotype aggregated significantly with adrenergic, renal, metabolic, and hemodynamic traits. In the adrenergic system, excretions of both norepinephrine and epinephrine correlated with albumin. In the kidney, albumin excretion correlated with glomerular and tubular traits (Na and K excretion; fractional excretion of Na and Li). Albumin excretion shared genetic determination (genetic covariance) with epinephrine excretion, and environmental determination with glomerular filtration rate and electrolyte intake/excretion. Albumin excretion associated with polymorphisms at multiple points in the adrenergic pathway: catecholamine biosynthesis (tyrosine hydroxylase), catabolism (monoamine oxidase A), storage/release (chromogranin A), receptor target (dopamine D1 receptor), and postreceptor signal transduction (sorting nexin 13 and rho kinase). Epistasis (gene-by-gene interaction) occurred between alleles at rho kinase, tyrosine hydroxylase, chromogranin A, and sorting nexin 13. Dopamine D1 receptor polymorphism showed pleiotropic effects on both albumin and dopamine excretion. These studies establish new roles for heredity and environment in albumin excretion. Urinary excretions of albumin and catecholamines are highly heritable, and their parallel suggests adrenergic mediation of early glomerular permeability alterations. Albumin excretion is influenced by multiple adrenergic pathway genes and is, thus, polygenic. Such functional links between adrenergic activity and glomerular injury suggest novel approaches to its prediction, prevention, diagnosis, and treatment. © 2007 American Heart Association, Inc.

DOI 10.1161/HYPERTENSIONAHA.106.081679
Citations Scopus - 30
2007 Wessel J, Moratorio G, Rao F, Mahata M, Zhang L, Greene W, et al., 'C-reactive protein, an 'intermediate phenotype' for inflammation: Human twin studies reveal heritability, association with blood pressure and the metabolic syndrome, and the influence of common polymorphism at catecholaminergic/ß-adrenergic pathway loci', Journal of Hypertension, 25 329-343 (2007)

BACKGROUND: C-reactive protein (CRP) both reflects and participates in inflammation, and its circulating concentration marks cardiovascular risk. Here we sought to understand the ... [more]

BACKGROUND: C-reactive protein (CRP) both reflects and participates in inflammation, and its circulating concentration marks cardiovascular risk. Here we sought to understand the role of heredity in determining CRP secretion. METHODS: CRP, as well as multiple facets of the metabolic syndrome, were measured in a series of 229 twins, both monozygotic (MZ) and dizygotic (DZ), to estimate trait heritability (h). Single nucleotide polymorphism (SNP) genotyping was done at adrenergic pathway loci. Haplotypes were inferred from genotypes by likelihood methods. Association of CRP with hypertension and the metabolic syndrome was studied in a larger series of 732 individuals, including 79 with hypertension. RESULTS: MZ and DZ twin variance components indicated substantial h for CRP, at ~56 ± 7% (P < 0.001). CRP was significantly associated (P < 0.05) with multiple features of the metabolic syndrome in twins, including body mass index (BMI), blood pressure (BP), leptin and lipids. In established hypertension, elevated CRP was associated with increased BP, BMI, insulin, HOMA (index of insulin resistance), leptin, triglycerides and norepinephrine. Twin correlations indicated pleiotropy (shared genetic determination) for CRP with BMI (P = 0.0002), leptin (P < 0.001), triglycerides (P = 0.002) and systolic blood pressure (SBP) (P = 0.042). Approximately 9800 genotypes (43 genetic variants at 17 loci) were scored within catecholaminergic pathways: biosynthetic, receptor and signal transduction. Plasma CRP concentration in twins was predicted by polymorphisms at three loci in physiological series within the catecholamine biosynthetic/ß-adrenergic pathway: TH (tyrosine hydroxylase), ADRB1 (ß1-adrenergic receptor) and ADRB2 (ß2-adrenergic receptor). In the TH promoter, common allelic variation accounted for up to ~6.6% of CRP inter-individual variance. At ADRB1, variation at Gly389Arg predicted ~2.8% of CRP, while ADRB2 promoter variants T-47C and T-20C also contributed. Particular haplotypes and diplotypes at TH and ADRB1 also predicted CRP, though typically no better than single SNPs alone. Epistasis (gene-by-gene interaction) was demonstrated for particular combinations of TH and ADRB2 alleles, consistent with their actions in a pathway in series. In an illustration of pleiotropy, not only CRP but also plasma triglycerides were predicted by polymorphisms at TH (P = 0.0053) and ADRB2 (P = 0.027). CONCLUSIONS: CRP secretion is substantially heritable in humans, demonstrating pleiotropy (shared genetic determination) with other features of the metabolic syndrome, such as BMI, triglycerides or BP. Multiple, common genetic variants in the catecholaminergic/ß-adrenergic pathway contribute to CRP, and these variants (especially at TH and ADRB2) seem to interact (epistasis) to influence the trait. The results uncover novel pathophysiological links between the adrenergic system and inflammation, and suggest new strategies to probe the role and actions of inflammation within this setting. © 2007 Lippincott Williams & Wilkins, Inc.

DOI 10.1097/HJH.0b013e328011753e
Citations Scopus - 68
2007 Bhatnagar V, O'Connor DT, Schork NJ, Salem RM, Nievergelt CM, Rana BK, et al., 'Angiotensin-converting enzyme gene polymorphism predicts the time-course of blood pressure response to angiotensin converting enzyme inhibition in the AASK trial', Journal of Hypertension, 25 2082-2092 (2007)

OBJECTIVE: It has yet to be determined whether genotyping at the angiotensin-converting enzyme (ACE) locus is predictive of blood pressure response to an ACE inhibitor. METHODS: P... [more]

OBJECTIVE: It has yet to be determined whether genotyping at the angiotensin-converting enzyme (ACE) locus is predictive of blood pressure response to an ACE inhibitor. METHODS: Participants from the African American Study of Kidney Disease and Hypertension trial randomized to the ACE inhibitor ramipril (n = 347) were genotyped at three polymorphisms on ACE, just downstream from the ACE insertion/deletion polymorphism (Ins/Del): G12269A, C17888T, and G20037A. Time to reach target mean arterial pressure (=107 mmHg) was analyzed by genotype and ACE haplotype using Kaplan-Meier survival curves and Cox proportional hazard models. RESULTS: Individuals with a homozygous genotype at G12269A responded significantly faster than those with a heterozygous genotype; the adjusted (average number of medications and baseline mean arterial pressure) hazard ratio (homozygous compared to heterozygous genotype) was 1.86 (95% confidence limits 1.32-3.23; P < 0.001 for G12269A genotype). The adjusted hazard ratio for participants with homozygous ACE haplotypes compared to those heterozygous ACE haplotypes was 1.40 (1.13-1.75; P = 0.003 for haplotype). The ACE genotype effects were specific for ACE inhibition (i.e., not seen among those randomized to a calcium channel blocker), and were independent of population stratification. CONCLUSIONS: African-Americans with a homozygous genotype at G12269A or homozygous ACE haplotypes responded to ramipril significantly faster than those with a heterozygous genotype or heterozygous haplotypes, suggesting that heterosis may be an important determinant of responsiveness to an ACE inhibitor. These associations may be a result of biological activity of this polymorphism, or of linkage disequilibrium with nearby variants such as the ACE Ins/Del, perhaps in the regulation of ACE splicing. © 2007 Lippincott Williams & Wilkins, Inc.

DOI 10.1097/HJH.0b013e3282b9720e
Citations Scopus - 26
2006 Wilkinson TG, Kedar GC, Lee C, Guzmán EC, Smith DW, Zyskind JW, 'The synchrony phenotype persists after elimination of multiple GATC sites from the dnaA promoter of Escherichia coli', Journal of Bacteriology, 188 4573-4576 (2006)

To examine whether methylation of the GATC sites present in the dnaA promoter region is responsible for the strict temporal coordination of initiation events at oriC as measured b... [more]

To examine whether methylation of the GATC sites present in the dnaA promoter region is responsible for the strict temporal coordination of initiation events at oriC as measured by the synchrony of initiation, we introduced point mutations eliminating three (TGW1) and five (TGW2) of the six GATC sites present in the dnaA promoter region. All of the strains containing these mutations, including the one with five GATC sites eliminated, initiated chromosomal replication synchronously. Copyright © 2006, American Society for Microbiology. All Rights Reserved.

DOI 10.1128/JB.00089-06
Citations Scopus - 4
2006 Seasholtz TM, Wessel J, Rao F, Rana BK, Khandrika S, Kennedy BP, et al., 'Rho kinase polymorphism influences blood pressure and systemic vascular resistance in human twins: Role of heredity', Hypertension, 47 937-947 (2006)

The Rho/Rho kinase (ROCK) pathway is implicated in experimental hypertension. We, therefore, explored the role of ROCK2 genetic variation in human blood pressure (BP) regulation, ... [more]

The Rho/Rho kinase (ROCK) pathway is implicated in experimental hypertension. We, therefore, explored the role of ROCK2 genetic variation in human blood pressure (BP) regulation, exploiting the advantages of a human twin sample to probe heritability. The focus of this work is the common nonsynonymous variant at ROCK2: Thr431Asn. Cardiovascular and autonomic traits displayed substantial heritability (from ¿33% to 71%; P<0.05). The Asn/Asn genotype (compared with Asn/Thr or Thr/Thr) was associated with greater resting systolic (P<0.001), diastolic (P<0.0001), and mean BP (P<0.0001); allelic variation at ROCK2 accounted for up to ¿5% of BP variation (P<0.0001). Systemic vascular resistance was higher in Asn/Asn individuals (P=0.049), whereas cardiac output, large artery compliance, and vasoactive hormone secretion were not different. Coupling of the renin-angiotensin system to systemic resistance and BP was diminished in Asn/Asn homozygotes, suggesting genetic pleiotropy of Thr431Asn, confirmed by bivariate genetic analyses. The Asn/Asn genotype also predicted higher BP after environmental (cold) stress. The rise in heart rate after cold was less pronounced in Asn/Asn individuals, consistent with intact baroreceptor function, and baroreceptor slope was not influenced by genotype. Common genetic variation (Thr431Asn) at ROCK2 predicts increased BP, systemic vascular resistance (although not large artery compliance), and resistance in response to the endogenous renin-angiotensin system, indicating a resistance vessel-based effect on elevated BP. The results suggest that common variation in ROCK2 exerts systemic resistance-mediated changes in BP, documenting a novel mechanism for human circulatory control, and suggesting new possibilities for diagnostic profiling and treatment of subjects at risk of developing hypertension. © 2006 American Heart Association, Inc.

DOI 10.1161/01.HYP.0000217364.45622.f0
Citations Scopus - 62
2005 Bao X, Mills PJ, Rana BK, Dimsdale JE, Schork NJ, Smith DW, et al., 'Interactive effects of common ß2-adrenoceptor haplotypes and age on susceptibility to hypertension and receptor function', Hypertension, 46 301-307 (2005)

Few studies have examined to what extent genetic variants of the ß2-adrenoceptor (ADRB2) are involved in the development of hypertension with age, although ß2-adrenergic recepto... [more]

Few studies have examined to what extent genetic variants of the ß2-adrenoceptor (ADRB2) are involved in the development of hypertension with age, although ß2-adrenergic receptor responsiveness declines in older subjects. To investigate this. 10 common single-nucleotide polymorphisms (SNPs) in the promoter and coding regions of the ADRB2 gene were genotyped in an unrelated population consisting of 2 ethnic groups: European American (EA; n=610) and African American (AA; n=420). ADRB2 haplotypes were estimated by expectation maximization (EM) algorithm-based methods. In the general population for EAs and AAs, the variants of the ADRB2 gene, including the individual SNPs and their haplotypes, were not associated with hypertension. However, there was a significant interaction between age and one of the common haplotypes (haplotypc 1) in EAs (P=0.01). Haplotype 1 was associated with protection against hypertension in young (>50 years of age) but not in old (>50 years of age) EAs (odds ratio. 0.5; 95% confidence interval. 0.27 to 0.91; P=0.02). This age-specific effect was further supported by the observations that young subjects carrying =1 copy of haplotype 1 had significantly lower diastolic blood pressure and nearly 2-fold higher ADRB2 binding density than the noncarriers (P<0.05). With aging, their ADRB2 numbers decreased to the level of the noncarriers. along with increased body mass index (7%; P<0.05) and decreased heart rate (7%; P<0.001). Our study suggests that age is an important modifier for the effects of ADRB2 polymorphisms on ADRB2 function and the development of hypertension. © 2005 American Heart Association, Inc.

DOI 10.1161/01.HYP.0000175842.19266.95
Citations Scopus - 36
2004 Smith DW, Friedmann T, 'Discrepant effects of culture conditions on survival and function of dopaminergic neurons.', Neuroreport, 15 1025-1028 (2004) [C1]
DOI 10.1097/00001756-200404290-00018
Citations Scopus - 5
2004 Wen G, Mahata SK, Cadman P, Mahata M, Ghosh S, Mahapatra NR, et al., 'Both Rare and Common Polymorphisms Contribute Functional Variation at CHGA, a Regulator of Catecholamine Physiology', American Journal of Human Genetics, 74 197-207 (2004)

The chromogranin/secretogranin proteins are costored and coreleased with catecholamines from secretory vesicles in chromaffin cells and noradrenergic neurons. Chromogranin A (CHGA... [more]

The chromogranin/secretogranin proteins are costored and coreleased with catecholamines from secretory vesicles in chromaffin cells and noradrenergic neurons. Chromogranin A (CHGA) regulates catecholamine storage and release through intracellular (vesiculogenic) and extracellular (catecholamine release-inhibitory) mechanisms. CHGA is a candidate gene for autonomic dysfunction syndromes, including intermediate phenotypes that contribute to human hypertension. Here, we show a surprising pattern of CHGA variants that alter the expression and function of this gene, both in vivo and in vitro. Functional variants include both common alleles that quantitatively alter gene expression and rare alleles that qualitatively change the encoded product to alter the signaling potency of CHGA-derived catecholamine release-inhibitory catestatin peptides.

DOI 10.1086/381399
Citations Scopus - 72
2004 Le Corre P, Parmer RJ, Kailasam MT, Kennedy BP, Skaar TP, Ho H, et al., 'Human sympathetic activation by a 2-adrenergic blockade with yohimbine: Bimodal, epistatic influence of cytochrome P450-mediated drug metabolism', Clinical Pharmacology and Therapeutics, 76 139-153 (2004)

Background a 2-Adrenergic blockade responses suggest adrenergic dysfunction in hypertension. a 2-Blockade is also used to treat autonomic dysfunction. However, pharmacokinetic det... [more]

Background a 2-Adrenergic blockade responses suggest adrenergic dysfunction in hypertension. a 2-Blockade is also used to treat autonomic dysfunction. However, pharmacokinetic determinants of yohimbine disposition are not understood. Methods We evaluated a 2-blockade with intravenous yohimbine in 172 individuals. Specific cytochrome P450 (CYP) isoform-mediated metabolism was investigated. Results were evaluated by ANOVA and by maximum likelihood analysis for bimodality of response distributions. Results Yohimbine metabolism to 11-hydroxy-yohimbine displayed greater than 1000-fold variability, with 17 individuals showing no metabolism. Nonmetabolizers differed from others in ethnicity but not in age, sex, body habitus, blood pressure, heart rate, or family history of hypertension. Bimodality of metabolism was suggested by frequency histogram, as well as maximum likelihood and cluster analysis. Among ethnic groups, subjects of European ancestry had the highest frequency of nonmetabolism. In vitro oxidation suggested that the major route of metabolism (lowest Michaelis-Menten constant and greatest intrinsic clearance) was likely via CYP2D6 to 11-hydroxy-yohimbine. In vivo genotypes at both CYP2D6 and CYP3A4 were necessary to predict metabolism (overall F = 3.03, P = .005); an interaction of alleles at these 2 loci (interaction F = 3.05, P = .033) suggested an epistatic effect on drug metabolism in vivo. Nonmetabolizers had greater activation of sympathetic nervous system activity. Yohimbine increased blood pressure, an effect mediated hemodynamically by elevation of cardiac output rather than systemic vascular resistance. Blood pressure and cardiac output responses did not differ by metabolizer group. Conclusions We conclude that heterogeneous, bimodally distributed yohimbine metabolism depends on common genetic variation in both CYP2D6 and CYP3A4 and contributes to differences in sympathetic neuronal response to a 2-blockade. These results have implications for both diagnostic and therapeutic uses of this a 2-antagonist.

DOI 10.1016/j.clpt.2004.04.010
Citations Scopus - 30
2004 Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, Scherer S, et al., 'Genome sequence of the Brown Norway rat yields insights into mammalian evolution', NATURE, 428 493-521 (2004)
DOI 10.1038/nature02426
Citations Scopus - 1288Web of Science - 1241
2004 Nievergelt CM, Smith DW, Kohlenberg JB, Schork NJ, 'Large-scale integration of human genetic and physical maps', Genome Research, 14 1199-1205 (2004)

Genetic maps are used routinely in family-based linkage studies to identify the rough location of genes that influence human traits and diseases. Unlike physical maps, genetic map... [more]

Genetic maps are used routinely in family-based linkage studies to identify the rough location of genes that influence human traits and diseases. Unlike physical maps, genetic maps are based on the amount of recombination occurring between adjacent loci rather than the actual number of bases separating them. Genetic maps are constructed by statistically characterizing the number of crossovers observed in parental meioses leading to the transmission of alleles to their offspring. Considerations such as the number of meioses observed, the heterozygosity and physical distance between the loci studied, and the statistical methods used can impact the construction and reliability of a genetic map. As is well known, poorly constructed genetic maps can have adverse effects on linkage mapping studies. With the availability of sequence-based maps, as well as genetic maps generated by different researchers (such as those generated by the Marshfield and deCODE groups), one can investigate the compatibility and properties of different maps. We have integrated information from the most current human genome sequence data (UCSC genome assembly Human July 2003) as well as 8399 microsatellite markers used in the Marshfield and deCODE maps to reconcile the these maps. Our efforts resulted in updated sex-specific genetic maps. ©2004 by Cold Spring Harbor Laboratory Press.

DOI 10.1101/gr.1475304
Citations Scopus - 35
2003 Smith DW, Day TA, 'Catecholamine and oxytocin cells respond to hypovolaemia as well as hypotension', NEUROREPORT, 14 1493-1495 (2003)
DOI 10.1097/01.wnr.0000080100.80506.d2
Citations Web of Science - 12
2003 Kerk D, Bulgrien J, Smith DW, Gribskov M, 'Arabidopsis proteins containing similarity to the universal stress protein domain of bacteria', Plant Physiology, 131 1209-1219 (2003)

We have collected a set of 44 Arabidopsis proteins with similarity to the USPA (universal stress protein A of Escherichia coli) domain of bacteria. The USPA domain is found either... [more]

We have collected a set of 44 Arabidopsis proteins with similarity to the USPA (universal stress protein A of Escherichia coli) domain of bacteria. The USPA domain is found either in small proteins, or it makes up the N-terminal portion of a larger protein, usually a protein kinase. Phylogenetic tree analysis based upon a multiple sequence alignment of the USPA domains shows that these domains of protein kinases 1.3.1 and 1.3.2 form distinct groups, as do the protein kinases 1.4.1. This indicates that their USPA domain structures have diverged appreciably and suggests that they may subserve distinct cellular functions. Two USPA fold classes have been proposed: one based on Methanococcus jannaschii MJ0577 (1MJH) that binds ATP, and the other based on the Haemophilus influenzae universal stress protein (1JMV), highly similar to E. coli UspA, which does not bind ATP. A set of common residues involved in ATP binding in 1MJH and conserved in similar bacterial sequences is also found in a distinct cluster of Arabidopsis sequences. Threading analysis, which examines aspects of secondary and tertiary structure, confirms this Arabidopsis sequence cluster as highly similar to 1MJH. This structural approach can distinguish between the characteristic fold differences of 1MJH-like and 1JMV-like bacterial proteins and was used to assign the complete set of candidate Arabidopsis proteins to one of these fold classes. It is clear that all the plant sequences have arisen from a 1MJH-like ancestor.

DOI 10.1104/pp.102.016006
Citations Scopus - 52
2003 Tchieu JH, Fana F, Fink JL, Harper J, Nair TM, Niedner RH, et al., 'The PlantsP and PlantsT functional genomics databases', Nucleic Acids Research, 31 342-344 (2003)

PlantsP and PlantsT allow users to quickly gain a global understanding of plant phosphoproteins and plant membrane transporters, respectively, from evolutionary relationships to b... [more]

PlantsP and PlantsT allow users to quickly gain a global understanding of plant phosphoproteins and plant membrane transporters, respectively, from evolutionary relationships to biochemical function as well as a deep understanding of the molecular biology of individual genes and their products. As one database with two functionally different web interfaces, PlantsP and PlantsT are curated plant-specific databases that combine sequence-derived information with experimental functional-genomics data. PlantsP focuses on proteins involved in the phosphorylation process (i.e., kinases and phosphatases), whereas PlantsT focuses on membrane transport proteins. Experimentally, PlantsP provides a resource for information on a collection of T-DNA insertion mutants (knockouts) in each kinase and phosphatase, primarily in Arabidopsis thaliana, and PlantsT uniquely combines experimental data regarding mineral composition (derived from inductively coupled plasma atomic emission spectroscopy) of mutant and wild-type strains. Both databases provide extensive information on motifs and domains, detailed information contributed by individual experts in their respective fields, and descriptive information drawn directly from the literature. The databases incorporate a unique user annotation and review feature aimed at acquiring expert annotation directly from the plant biology community. PlantsP is available at http://plantsp.sdsc.edu and PlantsT is available at http://plantst.sdsc.edu.

DOI 10.1093/nar/gkg025
Citations Scopus - 46
2003 Tokumine J, Kakinohana O, Cizkova D, Smith DW, Marsala M, 'Changes in spinal GDNF, BDNF and NT-3 expression after transient spinal cord ischemia in the rat.', Neuroscience Research, 74 552-561 (2003) [C1]
DOI 10.1002/jnr.10760
Citations Scopus - 54
2003 Smith DW, Day TA, 'Catecholamine and oxytocin cells respond to hypovolaemia as well as hypotension', Neuroreport, 14 1493-1495 (2003) [C1]
DOI 10.1097/00001756-200308060-00018
Citations Scopus - 13
2002 Kerk D, Bulgrien J, Smith DW, Barsam B, Veretnik S, Gribskov M, 'The complement of protein phosphatase catalytic subunits encoded in the genome of Arabidopsis', Plant Physiology, 129 908-925 (2002)

Reversible protein phosphorylation is critically important in the modulation of a wide variety of cellular functions. Several families of protein phosphatases remove phosphate gro... [more]

Reversible protein phosphorylation is critically important in the modulation of a wide variety of cellular functions. Several families of protein phosphatases remove phosphate groups placed on key cellular proteins by protein kinases. The complete genomic sequence of the model plant Arabidopsis permits a comprehensive survey of the phosphatases encoded by this organism. Several errors in the sequencing project gene models were found via analysis of predicted phosphatase coding sequences. Structural sequence probes from aligned and unaligned sequence models, and all-against-all BLAST searches, were used to identify 112 phosphatase catalytic subunit sequences, distributed among the serine (Ser)/threonine (Thr) phosphatases (STs) of the protein phosphatase P (PPP) family, STs of the protein phosphatase M (PPM) family (protein phosphatases 2C [PP2Cs] subfamily), protein tyrosine (Tyr) phosphatases (PTPs), low-Mr protein Tyr phosphatases, and dual-specificity (Tyr and Ser/Thr) phosphatases (DSPs). The Arabidopsis genome contains an abundance of PP2Cs (69) and a dearth of PTPs (one). Eight sequences were identified as new protein phosphatase candidates: five dual-specificity phosphatases and three PP2Cs. We used phylogenetic analyses to infer clustering patterns reflecting sequence similarity and evolutionary ancestry. These clusters, particularly for the largely unexplored PP2C set, will be a rich source of material for plant biologists, allowing the systematic sampling of protein function by genetic and biochemical means.

DOI 10.1104/pp.004002
Citations Scopus - 164
2002 Visser JE, Smith DW, Fried T, Rothstein JD, Jinnah HA, 'Oxidative stress and dopamine deficiency in a genetic mouse model of Lesch-Nyhan disease.', Brain Res Dev Brain Res, (2002) [C1]
Citations Scopus - 35
2001 Zhai Y, Heijne WHM, Smith DW, Saier MH, 'Homologues of archaeal rhodopsins in plants, animals and fungi: Structural and functional predications for a putative fungal chaperone protein', Biochimica et Biophysica Acta - Biomembranes, 1511 206-223 (2001)

The microbial rhodopsins (MR) are homologous to putative chaperone and retinal-binding proteins of fungi. These proteins comprise a coherent family that we have termed the MR fami... [more]

The microbial rhodopsins (MR) are homologous to putative chaperone and retinal-binding proteins of fungi. These proteins comprise a coherent family that we have termed the MR family. We have used modeling techniques to predict the structure of one of the putative yeast chaperone proteins, YRO2, based on homology with bacteriorhodopsins (BR). Availability of the structure allowed depiction of conserved residues that are likely to be of functional significance. The results lead us to predict an extracellular protein folding function and a transmembrane proton transport pathway. We suggest that protein folding is energized by a novel mechanism involving the proton motive force. We further show that MR family proteins are distantly related to a family of fungal, animal and plant proteins that include the human lysosomal cystine transporter (LCT) of man (cystinosin), mutations in which cause cystinosis. Sequence and phylogenetic analyses of both the MR family and the LCT family are reported. Proteins in both families are of the same approximate size, exhibit seven putative transmembrane a-helical spanners (TMSs) and show limited sequence similarity. We show that the LCT family arose by an internal gene duplication event and that TMSs 1-3 are homologous to TMSs 5-7. Although the same could not be demonstrated statistically for MR family members, homology with the LCT family suggests (but does not prove) a common evolutionary pathway. Thus, TMSs 1-3 and 5-7 in both LCT and MR family members may share a common origin, accounting for their shared structural features. © 2001 Elsevier Science B.V.

DOI 10.1016/S0005-2736(00)00389-8
Citations Scopus - 49
2001 Gribskov M, Fana F, Harper J, Hope DA, Harmon AC, Smith DW, et al., 'PlantsP: A functional genomics database for plant phosphorylation', Nucleic Acids Research, 29 111-113 (2001)

The PlantsP database is a curated database that combines information derived from sequences with experimental functional genomics information. PlantsP focuses on plant protein kin... [more]

The PlantsP database is a curated database that combines information derived from sequences with experimental functional genomics information. PlantsP focuses on plant protein kinases and protein phosphatases. The database will specifically provide a resource for information on a collection of T-DNA insertion mutants (knockouts) in each protein kinase and phosphatase in Arabidopsis thaliana. PlantsP also provides a curated view of each protein that includes a comprehensive annotation of functionally related sequence motifs, sequence family definitions, alignments and phylogenetic trees, and descriptive information drawn directly from the literature. PlantsP is available at http://PlantsP.sdsc.edu.

Citations Scopus - 49
2000 Kedar GC, Ozcan F, Guzmán EC, Smith DW, Newman VG, Zyskind JW, 'Role of DNA methylation at GATC sites in the dnaA promoter, dnaAp2', Journal of Molecular Microbiology and Biotechnology, 2 301-310 (2000)

DnaA protein is required for the initiation of DNA replication at the bacterial chromosomal origin, oriC, and at the origins of many plasmids. The concentration of DnaA protein is... [more]

DnaA protein is required for the initiation of DNA replication at the bacterial chromosomal origin, oriC, and at the origins of many plasmids. The concentration of DnaA protein is an important factor in determining when initiation occurs during the cell cycle. Methylation of GATC sites in the DnaAp2 promoter, two of which are in the -35 and -10 sequences, has been predicted to play an important role in regulating DnaA gene expression during the cell cycle because the promoter is sequestered from methylation immediately following replication. Mutations that eliminate these two GATC sites but do not substantially change the activity of the promoter were introduced into a reporter gene fusion and into the chromosome. The chromosomal mutants are able to initiate DNA replication synchronously at both moderately slow and fast growth rates, demonstrating that GATC methylation at these two sites is not directly involved in providing the necessary amount of DnaA for precise timing of initiation during the cell cycle. Either sequestration does not involve these GATC sites, or cell cycle control of DnaA expression is not required to supply the concentration necessary for correct timing of initiation.

Citations Scopus - 6
2000 O'Connor DT, Insel PA, Ziegler MG, Hook VY, Smith DW, Hamilton BA, et al., 'Heredity and the autonomic nervous system in human hypertension', Current Hypertension Reports, 2 16-22 (2000)

Because the complex phenotype of human hypertension is at least in part genetically determined, how individual genes ultimately contribute to the disease is not well understood. B... [more]

Because the complex phenotype of human hypertension is at least in part genetically determined, how individual genes ultimately contribute to the disease is not well understood. By contrast, intermediate phenotypes are traits associated with complex disease, but which may display simpler genetic properties such as greater heritability, more consistent and earlier penetrance and bimodality, and may suggest particular candidate susceptibility genes. Because autonomic nervous system activity is altered in hypertension, we examined biochemical, physiologic, and pharmacologic autonomic traits that fulfill at least some of these properties. Such biochemical, physiologic, or pharmacologic autonomic traits may be especially valuable as phenotypic anchor points in linkage or association studies probing the genetic basis of human hypertension. Copyright © 2000 by Current Science Inc.

DOI 10.1007/s11906-000-0053-8
Citations Scopus - 52
2000 Smith DW, Friedmann T, 'Characterization of the dopamine defect in primary cultures of dopaminergic neurons from hypoxanthine phosphoribosyltransferase knockout mice', Molecular Therapy, 1 486-491 (2000)

Lesch-Nyhan disease (LND) is an X-linked metabolic disorder caused by lack of activity of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) and characterized... [more]

Lesch-Nyhan disease (LND) is an X-linked metabolic disorder caused by lack of activity of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT) and characterized by hyperuricemia and debilitating neurological manifestations. The mechanisms underlying the neuropathology are not well understood and the principal neurochemical lesion characterized to date is a deficiency of the dopamine system in the basal ganglia. To facilitate the study of mechanism(s) by which HPRT deficiency causes the dopamine defect, we have compared the survival and dopamine phenotype of primary cultures of dopamine neurons derived from HPRT-deficient mice with the dopaminergic neurons from wild-type mice. The survival of dopaminergic neurons from both sources was promoted to an equal extent by glial cell line-derived neurotrophic factor (GDNF), a potent survival factor for dopamine neurons in vitro. Although the survival of the HPRT-deficient neurons was indistinguishable from that of cells derived from wild-type counterparts, the HPRT-deficient cells demonstrated a persistent deficiency of dopamine content and dopamine uptake with increasing neuritic differentiation, indicating that GDNF does not restore the normal phenotype in HPRT-deficient dopamine neurons despite its well-known protective and regenerative properties in several neurodegeneration models. Nevertheless, the demonstration that GDNF trophic support promotes the survival of these dopaminergic neurons will facilitate gaining a better understanding of the neuropathological mechanisms of LND by allowing a more extensive analysis of the cells central to the Lesch-Nyhan phenotype, the dopaminergic neurons of the basal ganglia.

Citations Scopus - 21
1999 Buller KM, Smith DW, Day TA, 'Differential recruitment of hypothalamic neuroendocrine and ventrolateral medulla catecholamine cells by non-hypotensive and hypotensive hemorrhages', BRAIN RESEARCH, 834 42-54 (1999)
DOI 10.1016/S0006-8993(99)01539-5
Citations Scopus - 62Web of Science - 61
1999 Young GB, Jack DL, Smith DW, Saier MH, 'The amino acid/auxin: Proton symport permease family', Biochimica et Biophysica Acta - Biomembranes, 1415 306-322 (1999)

Amino acids and their derivatives are transported into and out of cells by a variety of permease types which comprise several distinct protein families. We here present a systemat... [more]

Amino acids and their derivatives are transported into and out of cells by a variety of permease types which comprise several distinct protein families. We here present a systematic analysis of a group of homologous transport proteins which together comprise the eukaryotic-specific amino acid/auxin permease (AAAP) family (TC #2.18). In characterizing this family, we have (1) identified all sequenced members of the family, (2) aligned their sequences, (3) identified regions of striking conservation, (4) derived a family-specific signature sequence, and (5) proposed a topological model that appears to be applicable to all members of the family. We have also constructed AAAP family phylogenetic trees and dendrograms using six different programs that allow us to trace the evolutionary history of the family, estimate the relatedness of proteins from dissimilar organismal phyla, and evaluate the reliability of the different programs available for phylogenetic studies. The TREE and neighbor-joining programs gave fully consistent results while CLUSTAL W gave similar but non-identical results. Other programs gave less consistent results. The phylogenetic analyses reveal (1) that many plant AAAP family proteins arose recently by multiple gene duplication events that occurred within a single organism, (2) that some plant members of the family with strikingly different specificities diverged early in evolutionary history, and (3) that AAAP family proteins from fungi and animals diverged from the plant proteins long ago, possibly when animals, plants and fungi diverged from each other. The Neurospora protein nevertheless exhibits overlapping specificity with those found in plants. Preliminary evidence is presented suggesting that proteins of the AAAP family are distantly related to proteins of the large ubiquitous amino acid/polyamine/choline family (TC #2.3) as well as to those of two small bacterial amino acid transporter families, the ArAAP family (TC #2.42) and the STP family (TC #2.43). Copyright (C) 1999 Elsevier Science B.V.

DOI 10.1016/S0005-2736(98)00196-5
Citations Scopus - 99
1999 Rashidi HH, Bauer M, Patterson J, Smith DW, 'Sequence motifs determine structure and Ca++-binding by EF-hand proteins', Journal of Molecular Microbiology and Biotechnology, 1 175-182 (1999)

Prediction of protein structural and functional characteristics based on specific motif interactions could serve as a powerful tool in many facets of the biological sciences. Such... [more]

Prediction of protein structural and functional characteristics based on specific motif interactions could serve as a powerful tool in many facets of the biological sciences. Such improvements in protein modeling will be instrumental in the enhancement of drug design. A new approach to a sequence description of EF-hand motifs with more than one EF-hand domain is presented here; this permits precise insight into the structural and functional properties of many members of the EF-hand superfamily of calcium-binding proteins. Three separate regular expressions, or signatures, are used to describe an EF-hand motif, and specific relationships must exist between the two sequence motifs for the two neighboring EF-hands in a given calcium-binding domain. Specifically, each of the sequence motifs has a conserved phenylalanine. These two phenylalanine residues are separated by 57±10 amino acid residues but interact closely with each other in the tertiary structure of the calcium-binding domain. Changes in conserved residues in the sequence motifs have been shown experimentally to decrease or eliminate the ability of the protein to bind calcium. This new approach of use of multiple sequence motifs, with motif interrelationships, yields a highly specific and robust tool for the prediction of structural and functional properties of new and novel proteins.

Citations Scopus - 15
1999 Buller KM, Smith DW, Day TA, 'NTS catecholamine cell recruitment by hemorrhage and hypoxia', NEUROREPORT, 10 3853-3856 (1999)
DOI 10.1097/00001756-199912160-00024
Citations Scopus - 30Web of Science - 27
1998 Laub MT, Smith DW, 'Finding intron/exon splice junctions using INFO, INterruption finder and organizer', Journal of Computational Biology, 5 307-321 (1998)

INFO, INterruption Finder and Organizer, has been used to find coding sequence intron-exon splice junctions in human and other DNA by comparing the six conceptual translations of ... [more]

INFO, INterruption Finder and Organizer, has been used to find coding sequence intron-exon splice junctions in human and other DNA by comparing the six conceptual translations of the input DNA sequence with sequences in protein databanks using a similarity matrix and windowing algorithm. Similarities detected both delineate position of the gene and provide clues as to the function of the gene product. In addition to use of a standard similarity matrix and windowing algorithm, INFO uses two novel steps, the MiniLibrary and Reverse Sequence steps, to enhance identification of small exons and to improve precision of junction nucleotide delineation. Exons as small as about 30 bases can be reliably found, and >90% of junctions are precisely identified when canonical splice junction information is used. With the MiniLibrary and Reverse Sequence steps, INFO parameters need not be optimized by the user. In comparative test runs using 19 human DNA sequences, INFO found 108 of 111 exons, with 0 reported false positives, compared with 111 exons and 51 false positives for BLASTX, 99 exons and 6 false positives for GRAIL II, 77 exons and 24 false positives for GeneMark, 61 exons and 9 false positives for GeneID, and 105 exons and 6 false positives for PROCRUSTES. The correlation coefficient for finding and positioning these 111 exons was greater than 98% for INFO. Comparable results were obtained in test runs of 13 nonhuman DNA sequences. INFO is applicable to DNA from any species, will become more robust as sequence databanks expand, and complements other heuristic approaches.

Citations Scopus - 12
1995 SMITH DW, SIBBALD JR, KHANNA S, DAY TA, 'RAT VASOPRESSIN CELL RESPONSES TO SIMULATED HEMORRHAGE - STIMULUS-DEPENDENT ROLE FOR A1 NORADRENERGIC NEURONS', AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY, 268 R1336-R1342 (1995)
Citations Web of Science - 41
1995 Loomis WF, Smith DW, 'Consensus phylogeny of Dictyostelium', Experientia, 51 1110-1115 (1995)

The evolutionary relationship of Dictyostelium discoideum to the yeasts, fungi, plants, and animals is considered on the basis of physiological, morphological and molecular charac... [more]

The evolutionary relationship of Dictyostelium discoideum to the yeasts, fungi, plants, and animals is considered on the basis of physiological, morphological and molecular characteristics. Previous analyses of five proteins indicated that Dictyostelium diverged after the yeasts but before the metazoan radiation. However, analyses of the small ribosomal subunit RNA indicated divergence prior to the yeasts. We have extended the molecular phylogenetic analyses to six more proteins and find consistent evidence for a more recent common ancestor with metazoans than yeast. A consensus phylogeny generated from these new results by both distance matrix and parsimony analyses establishes Dictyostelum's place in evolution between the yeasts Saccharomyces cerevisiae and Schizzosaccharomyces pombe and the worm Caenorhabditis elegans. © 1995 Birkhäuser Verlag Basel.

DOI 10.1007/BF01944728
Citations Scopus - 44
1995 Smith DW, Sibbald JR, Khanna S, Day TA, 'Rat vasopressin cell responses to simulated hemorrhage: Stimulus-dependent role for A1 noradrenergic neurons', American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 268 (1995)

c-fos expression mapping and electrophysiological recording experiments were done to clarify the role of the A1 noradrenergic cell group in the vasopressin response to hypotensive... [more]

c-fos expression mapping and electrophysiological recording experiments were done to clarify the role of the A1 noradrenergic cell group in the vasopressin response to hypotensive hemorrhage. In pentobarbital-anesthetized rats, moderate and severe hypotensive hemorrhages were simulated by brief occlusion of the inferior vena cava sufficient to reduce mean arterial pressure to ~50 or 30 mmHg, respectively. Both stimuli significantly increased the number of A1 region catecholamine cells displaying Fos-like immunoreactivity, this effect being most prominent at the level of the area postrema. Both stimuli also increased the number of supraoptic nucleus vasopressin cells displaying Fos-like immunoreactivity. Accordingly, electrophysiological studies involving separate animals confirmed that both moderate and severe caval occlusion significantly increased the firing of functionally identified vasopressin cells recorded in the supraoptic nucleus. However, although interruption of A1 region neuronal function by injection of ¿-aminobutyric acid at the level of the area postrema eliminated the increase in vasopressin cell firing elicited by moderate caval occlusion, it did not block the response to severe caval occlusion. These findings suggest that, in the rat, the vasopressin response to an acute reduction in central blood volume, such as that produced by hemorrhage, depends on the A1 projection only if the stimulus is of moderate intensity. Severe stimuli appear to involve activation of both the A1 projection and an additional vasopressin-stimulatory pathway that bypasses the A1 region.

Citations Scopus - 44
1995 SMITH DW, DAY TA, 'HYPOVOLEMIC AND OSMOTIC STIMULI INDUCE DISTINCT PATTERNS OF C-FOS EXPRESSION IN THE RAT SUBFORNICAL ORGAN', BRAIN RESEARCH, 698 232-236 (1995)
DOI 10.1016/0006-8993(95)00975-V
Citations Scopus - 26Web of Science - 23
1995 Smith DW, Buller KM, Day TA, 'Role of ventrolateral medulla catecholamine cells in hypothalamic neuroendocrine cell responses to systemic hypoxia', JOURNAL OF NEUROSCIENCE, 15 7979-7988 (1995)
Citations Scopus - 81Web of Science - 76
1994 Khanna S, Sibbald JR, Smith DW, Day TA, 'Initiation of rat vasopressin cell responses to simulated hypotensive hemorrhage', AM.J.PHYSIOL., 267 (1994)
Citations Scopus - 7
1994 Hultner M, Smith DW, Wills C, 'Similarity landscapes: A way to detect many structural and sequence motifs in both introns and exons', Journal of Molecular Evolution, 38 188-203 (1994)

When investigators undertake searches of DNA databases, they normally discard large numbers of alignments that demonstrate very weak resemblances to each other, retaining only tho... [more]

When investigators undertake searches of DNA databases, they normally discard large numbers of alignments that demonstrate very weak resemblances to each other, retaining only those that show statistically significant levels of resemblance. We show here that a great deal of information can be extracted from these weak alignments by examining them en masse. This is done by building three-dimensional similarity landscapes from the alignments, landscapes that reveal whether an unusual number of individually nonsignificant alignments tend to match up to a particular region of the query sequence being searched. The power of the search is increased by the use of libraries consisting entirely of introns or of exons. We show that (1) similarity landscapes with a variety of features can be generated from both intron and exon libraries, using introns or exons as query sequences; (2) the landscape features are real and not a statistical artifact; (3) well-known protein motifs used as query sequences can generate various landscape features; and (4) there is some evidence for resemblances between short regions of sequence carried by introns and exons. One possible interpretation of these results is that both introns and exons may have been built up during their evolution from short regions of sequence that as a result are now widely distributed throughout eukaryotic genomes. Such an interpretation would imply that these short regions have common ancestry. Alternatively, the wide sharing of short pieces of DNA may reflect regions with particular structural properties that have arisen through convergent evolution. The similarity-landscape approach can be used to detect such widespread structural motifs and sequence motifs in the genome that might be missed by less-global searches. It can also be used in conjunction with algorithms developed for detecting significant multiple alignments by isolating promising subsets of the databases that can be examined in more detail. © 1994, Springer-Verlag New York Inc. All rights reserved.

DOI 10.1007/BF00166165
Citations Scopus - 5
1994 Van der Vliet A, Smith D, O'Neill CA, Kaur H, Darley-Usmar V, Cross CE, Halliwell B, 'Interactions of peroxynitrite with human plasma and its constituents: Oxidative damage and antioxidant depletion', Biochemical Journal, 303 295-301 (1994)

Endothelial cells and activated phagocytes produce both nitric oxide ((¿)No) and superoxide (O2(¿-)), which react to form peroxynitrite. Peroxynitrite has been suggested to be d... [more]

Endothelial cells and activated phagocytes produce both nitric oxide ((¿)No) and superoxide (O2(¿-)), which react to form peroxynitrite. Peroxynitrite has been suggested to be directly cytotoxic and also to decompose into other toxic species. In order to understand the consequences of peroxynitrite generation in vivo, we examined its reaction with human blood plasma. Peroxynitrite decreased the total peroxyl-trapping capacity of plasma. In terms of specific antioxidants, addition of peroxynitrite to plasma leads to rapid oxidation of ascorbic acid, uric acid and plasma SH groups. The oxidation of plasma SH groups was enhanced in dialysed plasma and returned to control levels by the addition of physiological levels of bicarbonate. Evidence was found for formation of nitro-adducts to aromatic side chains in plasma proteins by peroxynitrite. Peroxynitrite also leads to depletion of ubiquinol and formation of traces of lipid hydroperoxides in plasma, although a-tocopherol levels were only slightly decreased. Peroxynitrite formation in human body fluids is likely to cause antioxidant depletion and oxidative damage.

Citations Scopus - 259
1994 Khanna S, Sibbald JR, Smith DW, Day TA, 'Initiation of rat vasopressin cell responses to simulated hypotensive hemorrhage', American Journal of Physiology - Regulatory Integrative and Comparative Physiology, 267 (1994)

Hypotensive hemorrhage is a major stimulus for vasopressin (VP) release, but in rats it is uncertain which receptors initiate this response. We have investigated this issue using ... [more]

Hypotensive hemorrhage is a major stimulus for vasopressin (VP) release, but in rats it is uncertain which receptors initiate this response. We have investigated this issue using transient occlusion of the inferior vena cava to simulate hypotensive hemorrhage. Single-unit recording experiments done in the supra-optic nucleus of pentobarbital-anesthetized rats demonstrated that severe caval occlusion, sufficient to drop mean arterial pressure (MAP) below 30 mmHg, excited 88% of putative VP neurosecretory cells and a similar proportion of putative oxytocin (OT) cells. Responsive VP cells increased their firing by 8.5 ± 0.6 spikes/s within 11.2 ± 0.8 s of the fall in MAP. This response was unrelated to the size of the fall in MAP and was unchanged by combined sinoaortic denervation (SAD) and vagal denervation, by T1 spinal section, or by administration of the angiotensin-converting enzyme inhibitor captopril, except that spinal section decreased the response latency. Moderate caval occlusion, sufficient to drop MAP to ~50 mmHg, did not excite any of the OT cells tested but did excite 65% of VP cells, causing a 3.8 ± 0.3 spikes/s increase in firing after a delay of 9.0 ± 1.3 s. This response was proportional to the size of the preceding fall in MAP, and after combined SAD and vagal denervation only 20% of VP cells still responded. Elimination of sinoaortic or vagal afferents alone had no effect on VP cell responses to moderate caval occlusion, except that SAD significantly increased the response latency. These data suggest that in rat the mechanisms that initiate the VP response to hypotensive hemorrhage depend on stimulus intensity. The response to moderately hypotensive hemorrhage (MAP 50 mmHg) can be initiated by sinoaortic or vagal afferents while severe hypotensive hemorrhage (MAP <30 mmHg) triggers a VP response that is independent of peripheral cardiovascular receptors and may be secondary to cerebral ischemia.

Citations Scopus - 11
1994 SMITH DW, DAY TA, 'C-FOS EXPRESSION IN HYPOTHALAMIC NEUROSECRETORY AND BRAIN-STEM CATECHOLAMINE CELLS FOLLOWING NOXIOUS SOMATIC STIMULI', NEUROSCIENCE, 58 765-775 (1994)
DOI 10.1016/0306-4522(94)90453-7
Citations Scopus - 49Web of Science - 49
1994 KHANNA S, SIBBALD JR, SMITH DW, DAY TA, 'INITIATION OF RAT VASOPRESSIN CELL RESPONSES TO SIMULATED HYPOTENSIVE HEMORRHAGE', AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY, 267 R1142-R1149 (1994)
Citations Web of Science - 20
1993 SMITH DW, DAY TA, 'NEUROCHEMICAL IDENTIFICATION OF FOS-POSITIVE NEURONS USING 2-COLOR IMMUNOPEROXIDASE STAINING', JOURNAL OF NEUROSCIENCE METHODS, 47 73-83 (1993)
DOI 10.1016/0165-0270(93)90023-K
Citations Scopus - 39Web of Science - 41
1992 DAY TA, SIBBALD JR, SMITH DW, 'A1-NEURONS AND EXCITATORY AMINO-ACID RECEPTORS IN RAT CAUDAL MEDULLA MEDIATE VAGAL EXCITATION OF SUPRAOPTIC VASOPRESSIN CELLS', BRAIN RESEARCH, 594 244-252 (1992)
DOI 10.1016/0006-8993(92)91131-W
Citations Scopus - 42Web of Science - 45
1992 Romling U, Duchene M, Essar DW, Galloway D, Guidi-Rontani C, Hill D, et al., 'Localization of alg, opr, phn, pho, 4.5S RNA, 6S RNA, tox, trp, and xcp genes, rrn operons, and the chromosomal origin on the physical genome map of Pseudomonas aeruginosa PAO', Journal of Bacteriology, 174 327-330 (1992)

The genes encoding the rrn operons, the 4.5S and 6S RNAs, elements of protein secretion, and outer membrane proteins F and I, and regulatory as well as structural genes for exotox... [more]

The genes encoding the rrn operons, the 4.5S and 6S RNAs, elements of protein secretion, and outer membrane proteins F and I, and regulatory as well as structural genes for exotoxin A, alkaline phosphatase, and alginate and tryptophan biosynthesis, were assigned on the SpeI/DpnI macrorestriction map of the Pseudomonas aeruginosa PAO chromosome. The zero point of the map was relocated to the chromosomal origin of replication.

Citations Scopus - 20
1992 Smith DW, Stine WB, Svitil AL, Bakker A, Zyskind JW, 'Escherichia coli cells lacking methylation-blocking factor (leucine- responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle', Journal of Bacteriology, 174 3078-3082 (1992)

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown ... [more]

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.

Citations Scopus - 4
1992 Zyskind JW, Smith DW, 'DNA replication, the bacterial cell cycle, and cell growth', Cell, 69 5-8 (1992)
DOI 10.1016/0092-8674(92)90112-P
Citations Scopus - 61
1992 Zyskind JW, Svitil AL, Stine WB, Biery MC, Smith DW, 'RecA protein of Escherichia coli and chromosome partitioning', Molecular Microbiology, 6 2525-2537 (1992)

Escherichia coli cells deficient in RecA protein frequently contain an abnormal number of chromosomes after completion of ongoing rounds of DNA replication. This suggests that Rec... [more]

Escherichia coli cells deficient in RecA protein frequently contain an abnormal number of chromosomes after completion of ongoing rounds of DNA replication. This suggests that RecA protein may be required for correct timing of initiation of DNA replication; however, we show here that initiation of DNA replication is properly timed in recA mutants. We also find that more than 10% of recA mutant cells contain no DNA. These anucleate cells appear to arise from partitioning of all the DNA into one daughter cell and no DNA into the other daughter cell. Based on these and previously published results, we propose that RecA protein is required for equal partitioning of chromosomes into the two daughter cells. Copyright © 1992, Wiley Blackwell. All rights reserved

DOI 10.1111/j.1365-2958.1992.tb01429.x
Citations Scopus - 36
1991 Sundstrom P, Irwin M, Smith D, Sypherd PS, 'Both genes for EF-1a in Candida albicans are translated', Molecular Microbiology, 5 1703-1706 (1991)

In previous work, we showed that Candida albicans has two genes, TEF-1 and TEF-2, which encode identical polypeptides for the highly conserved, essential, protein synthesis factor... [more]

In previous work, we showed that Candida albicans has two genes, TEF-1 and TEF-2, which encode identical polypeptides for the highly conserved, essential, protein synthesis factor EF-1a (Breviario et al., 1988). This result prompted questions as to whether C. albicans preferentially uses one of the genes over the other and whether both genes are actually translated into protein. Gene-specific sequence differences in the untranslated portion of each gene made it possible to prepare gene-specific oligonucleotide hybridization probes. Results with the probes showed that the relative steady-state mRNA levels of the two genes were equivalent and that the mRNA for each gene was present in active translation complexes. Copyright © 1991, Wiley Blackwell. All rights reserved

DOI 10.1111/j.1365-2958.1991.tb01918.x
Citations Scopus - 3
1991 Smith DW, Yee TW, Baird C, Krishnapiilai V, 'Pseudomonad replication origins: a paradigm for bacterial origins?', Molecular Microbiology, 5 2581-2587 (1991)

Structural features of three analysed bacterial DNA replication origin classes (six enteric origins, three pseudomonad origins, and the Bacillus subtilis origin region) are compar... [more]

Structural features of three analysed bacterial DNA replication origin classes (six enteric origins, three pseudomonad origins, and the Bacillus subtilis origin region) are compared in order to deduce characteristics common to all bacterial origins and characteristics that distinguish the three origin classes. The two Pseudomonas aeruginosa origins are shown to map within 10 kb of each other, and correlations are drawn with four potential origin regions in B. subtilis. The enteric origin class is further distinguished from the other two classes by its genetic organization, the presence of GATC sites, and the role of Dam methylation in enteric initiation. The pseudomonad origin class has the most features that are common to all of these bacterial origins, and hence may be the paradigm bacterial origin class. Copyright © 1991, Wiley Blackwell. All rights reserved

DOI 10.1111/j.1365-2958.1991.tb01966.x
Citations Scopus - 33
1990 Sundstrom P, Smith D, Sypherd PS, 'Sequence analysis and expression of the two genes for elongation factor 1a from the dimorphic yeast Candida albicans', Journal of Bacteriology, 172 2036-2045 (1990)

Two Candida albicans genes that encode the protein synthesis factor elongation factor 1a (EF-1a) were cloned by using a heterologous TEF1 probe from Mucor racemosus to screen libr... [more]

Two Candida albicans genes that encode the protein synthesis factor elongation factor 1a (EF-1a) were cloned by using a heterologous TEF1 probe from Mucor racemosus to screen libraries of C. albicans genomic, DNA. Sequence analysis of the clones showed that regions of DNA flanking the coding regions of the two genes were not homologous, verifying the presence of two genes, called TEF1 and TEF2, for EF-1a in C. albicans. The coding regions of TEF1 and TEF2 differed by only five nucleotides and encoded identical EF-1a proteins of 458 amino acids. Both genes were transcribed into mRNA in vivo, as shown by hybridization of oligonucleotide probes, which found specifically to the 3' nontranslated regions of TEF1 and TEF2, respectively, to C. albicans total RNA in Northern (RNA) blot analysis. The predicted EF-1a protein of C. albicans was more similar to Saccharomyces cerevisiae EF-1a than to M. racemosus EF-1a. Furthermore, codon bias and the promoter and termination signals of the C. albicans EF-1a proteins were remarkably similar to those of S. cerevisiae EF-1a. Taken together, these results suggest that C. albicans is more closely related to the ascomycete S. cerevisiae than to the zygomycete M. racemosus.

Citations Scopus - 41
1990 Yee TW, Smith DW, 'Pseudomonas chromosomal replication origins: A bacterial class distinct from Escherichia coli-type origins', Proceedings of the National Academy of Sciences of the United States of America, 87 1278-1282 (1990)

The bacterial origins of DNA replication have been isolated from Pseudomonas aeruginosa and Pseudomonas putida. These origins comprise a second class of bacterial origins distinct... [more]

The bacterial origins of DNA replication have been isolated from Pseudomonas aeruginosa and Pseudomonas putida. These origins comprise a second class of bacterial origins distinct from enteric-type origins: both origins function in both Pseudomonas species, and neither functions in Escherichia coli; enteric origins do not function in either pseudomonad. Both cloned sequences hybridize to chromosomal fragments that show properties expected of replication origins. These origin plasmids are highly unstable, are present at low copy number, and show mutual incompatibility properties. DNA sequence analysis shows that both origins contain several 9-base-pair (bp) E. coli DnaA protein binding sites; four of these are conserved in position and orientation, two of which resemble the R1 and R4 sites of the E. coli origin. Conserved 13-bp direct repeats adjacent to the analogous R1 site are also found. No GATC sites are in the P. aeruginosa origin and only four are in the P. putida origin; no other 4-bp sequence is present in high abundance. Both origins are found between sequences similar to the E. coli and Bacillus subtilis dnaA, dnaN, rpmH, and rnpA genes, a gene organization identical to that for B. subtilis and unlike that of E. coli. A second autonomously replicating sequence was obtained from P. aeruginosa that has some properties of bacterial origins. (.

Citations Scopus - 65
1990 Loomis WF, Smith DW, 'Molecular phylogeny of Dictyostelium discoideum by protein sequence comparison', Proceedings of the National Academy of Sciences of the United States of America, 87 9093-9097 (1990)

Comparison of the amino acid sequences of eight proteins from the soil amoeba Dictyostelium discoideum to those of their homologs in bacteria, yeast, and other eukaryotes indicate... [more]

Comparison of the amino acid sequences of eight proteins from the soil amoeba Dictyostelium discoideum to those of their homologs in bacteria, yeast, and other eukaryotes indicates that Dictyostelium diverged from the line leading to mammals at about the same time as the plant/animal divergence. Yeast appear to have diverged considerably earlier. It is argued that previous analyses indicating that D. discoideum diverged before yeast were misleading because of the nature of the small ribosomal subunit rRNA sequences used in these studies. We suggest that amino acid sequences may be more reliable than untranslated nucleic acid sequences for evolutionary comparisons, especially among organisms with significant skewing of their A+T content.

Citations Scopus - 95
1990 Seely O, Feng DF, Smith DW, Sulzbach D, Doolittle RF, 'Construction of a facsimile data set for large genome sequence analysis', Genomics, 8 71-82 (1990)

A test was devised for exploring the question of whether it will be possible to identify genes in largescale genome studies solely by sequence comparison with current sequence col... [more]

A test was devised for exploring the question of whether it will be possible to identify genes in largescale genome studies solely by sequence comparison with current sequence collections. To this end, a facsimile data set was constructed by dividing GenBank Release 56 randomly into two halves, one to serve as a reference set and the other intended to simulate raw data anticipated from large genome sequence projects. All supplementary information and identifying marks were removed from the test set after assignment of random identification numbers to each entry and their encryption. Because noncoding intervening sequences (introns) are underrepresented in GenBank, a program that introduced (simulated) introns into mRNA and prokaryotic sequences was devised. In a further attempt to make the problem of identification more realistic, random base substitutions and single-base deletions were also incorporated. The randomly ordered entries were concatenated, along with random intergenic flanking sequences, into a single long "chromosome" 33 Mb in length and then cut into "cosmids" 50-100 kb long. The chopping process was conducted in such a way that terminal overlaps would allow the order of the entries in the chromosome to be reconstituted. Finally, the sequences of a substantial fraction of the cosmids were converted to their complements. Preliminary searching of 10 test cosmids revealed that more than two-thirds of the entries in the test set should be readily identifiable by type of gene product solely on the basis of comparison with the reference set. These preliminary results suggest that existing computer regimens and sequence collections would be able to identify the majority of eukaryotic genes in any new raw data set, the existence of introns notwithstanding. Moreover, the analysis can be conducted in pace with the data collection so that the search results and summary identifications will be instantly available to the research community at large. © 1990.

DOI 10.1016/0888-7543(90)90227-L
Citations Scopus - 7
1989 Jonczyk P, Hines R, Smith DW, 'The Escherichia coli dam gene is expressed as a distal gene of a new operon', MGG Molecular & General Genetics, 217 85-96 (1989)

DNA containing the Escherichia coli dam gene and sequences upstream from this gene were cloned from the Clarke-Carbon plasmids pLC29-47 and pLC13-42. Promoter activity was localiz... [more]

DNA containing the Escherichia coli dam gene and sequences upstream from this gene were cloned from the Clarke-Carbon plasmids pLC29-47 and pLC13-42. Promoter activity was localized using pKO expression vectors and galactokinase assays to two regions, one 1650-2100 bp and the other beyon 2400 bp upstream of the dam gene. No promoter activity was detected immediately in front of this gene; plasmid pDam118, from which the nucleotide sequence of the dam gene was determined, is shown to contain the pBR322 promoter for the primer RNA from the pBR322 rep region present on a 76 bp Sau3A fragment inserted upstream of the dam gene in the correct orientation for dam expression. The nucleotide sequence upstream of dam has been determined. An open reading frame (ORF) is present between the nearest promoter region and the dam gene. Codon usage and base frequency analysis indicate that this is expressed as a protein of predicted size 46 kDa. A protein of size close to 46 kDa is expressed from this region, detected using minicell analysis. No function has been determined for this protein, and no significant homology exist between it and sequences in the PIR protein or GenBank DNA databases. This unidentified reading frame (URF) is termed urf-74.3, since it is an URF located at 74.3 min on the E. coli chromosome. Sequence comparisons between the regions upstream of urf-74.3 and the aroB gene show that the aroB gene is located immediately upstream of urf-74.3, and that the promoter activity nearest to dam is found within the aroB structural gene. This activity is relatively weak (about 15% of that of the E. coli gal operon promoter). The promoter activity detected beyond 2400 bp upstream of dam is likely to be that of the aroB gene, and is 3 to 4 times stronger than that found within the aroB gene. Three potential DnaA binding sites, each with homology of 8 of 9 bp, are present, two in the aroB promoter region and one just upstream of the dam gene. Expression through the site adjacent to the dam gene is enhanced 2-to 4-fold in dnaA mutants at 38°C. Restriction site comparisons map these regions precisely on the Clarke-Carbon plasmids pLC13-42 and pLC29-47, and show that the E. coli ponA (mrcA) gene resides about 6 kb upstream of aroB. © 1989 Springer-Verlag.

DOI 10.1007/BF00330946
Citations Scopus - 32
1989 Botschner J, Smith DW, Simell O, Scriver CR, 'Comparison of ornithine metabolism in hyperornithinaemia-hyperammonaemia-homocitrullinuria syndrome, lysinuric protein intolerance and gyrate atrophy fibroblasts', Journal of Inherited Metabolic Disease, 12 33-40 (1989)

We measured l-ornithine oxidation in cultured skin fibroblasts from seven patients with hyperornithinaemia-hyperammonaemia-homocitrullinuria (HHH) syndrome (McKusick 23897), and c... [more]

We measured l-ornithine oxidation in cultured skin fibroblasts from seven patients with hyperornithinaemia-hyperammonaemia-homocitrullinuria (HHH) syndrome (McKusick 23897), and compared it with oxidation by ornithine aminotransferase deficient gyrate atrophy (McKusick 25887) cells and lysinuric protein intolerance (McKusick 22270) cells in which there is an ornithine transport abnormality at the plasma membrane. Net uptake of ornithine is not abnormal in intact HHH cells. Ornithine oxidation was depressed in HHH and gyrate atrophy cells but not in lysinuric protein intolerance cells; the latter finding suggests there is no significant mitochondrial defect in lysinuric protein intolerance cells. Since HHH cells have intact ornithine aminotransferase, impaired oxidation is compatible with deficient penetration of ornithine into mitochondria in this disease. We could not demonstrate a gene dosage effect in oxidation values. © 1989 SSIEM and Kluwer Academic Publishers.

DOI 10.1007/BF01805528
Citations Scopus - 3
1988 Smith DW, 'A complete, yet flexible, system for DNA/protein sequence analysis using VAX/VMS computers', Computer Applications in the Biosciences, 4 212 (1988)

A complete, flexible, and highly integrated system of computer programs and libraries have been implemented on VAX/VMS computers in San Diego. This system performs all commonly de... [more]

A complete, flexible, and highly integrated system of computer programs and libraries have been implemented on VAX/VMS computers in San Diego. This system performs all commonly desired tasks involving structure and function of DNA, RNA, and protein molecules at the primary and secondary structure level, including sequence analysis use of databases, and experimental data analysis. In designing this system, we wanted (i) to use familiar, well proven, inexpensive, and powerful programs, and (ii) to have more than one program present to perform a given task, thereby permitting direct comparison of algorithms and estimating reliability of results. System integration in the multiuser VAX environment and ease of use is solved by a combination of VAX/VMS features (e. g. , logical name assignments, command language files, DNAHELP help library, and utility programs) and of program and system documentation.

Citations Scopus - 18
1988 Revie D, Smith DW, Yee TW, 'Kinetic analysis for optimization of DNA ligation reactions', Nucleic Acids Research, 16 10301-10321 (1988)

Kinetic equations describing ligation of DNA to circular recombinant forms were developed and solved for tour types of reactions: (a) a homogeneous population of singly restricted... [more]

Kinetic equations describing ligation of DNA to circular recombinant forms were developed and solved for tour types of reactions: (a) a homogeneous population of singly restricted DNA fragments, (b) Insertion of singly restricted Insert into vector, (C) forced directional Insertion of doubly restricted insert into vector, and (d) Insertion of singly restricted insert Into pliosphatased vector. The effects of varying vector and Insert sizes, starting concentrations, and phosphatase treatment on the yield of cIrcular 1:1 recombinants were analyzed. Selected theoretical predictions were experimentally tested and verified. Our suggestions on optimizing ligation reactions in several cases are at variance with common practice. For example, optimum conditions in case (b) and (d) ligatlons are best specified as Individual insert and vector concentrations rather than as insert/vector molar ratios, except in case (d) ligations Involving very small Insert size. In case (c) ligatlons, highest efficiencies are obtained when both vector and Insert are at relatively iow concentration. © 1988 IRL Press Ltd.

DOI 10.1093/nar/16.21.10301
Citations Scopus - 5
1988 Smith DW, Scriver CR, Simell O, 'Lysinuric protein intolerance mutation is not expressed in the plasma membrane of erythrocytes', Human Genetics, 80 395-396 (1988)

We measured mediated fluxes of l-lysine and l-ornithine across the plasma membrane of erythrocytes from control subjects and patients homozygous for the lysinuric protein intolera... [more]

We measured mediated fluxes of l-lysine and l-ornithine across the plasma membrane of erythrocytes from control subjects and patients homozygous for the lysinuric protein intolerance (LPI) mutation. We found no differences in net uptake or efflux of cationic amino acids in control and LPI cells, contrary to our findings in cultured skin fribroblasts. We conclude that expression of the LPI (y+) transport system for cationic amino acids varies between tissues and that measurements of fluxes in erythrocytes cannot be used for diagnosis of LPI. © 1988 Springer-Verlag.

DOI 10.1007/BF00273660
Citations Scopus - 11
1987 Smith DW, Scriver CR, Tenenhouse HS, Simell O, 'Lysinuric protein intolerance mutation is expressed in the plasma membrane of cultured skin fibroblasts.', Proceedings of the National Academy of Sciences of the United States of America, 84 7711-7715 (1987)

Lysinuric protein intolerance (LPI) is an autosomal recessive phenotype consistent with impaired transport of cationic amino acids at the basolateral membrane of intestinal and re... [more]

Lysinuric protein intolerance (LPI) is an autosomal recessive phenotype consistent with impaired transport of cationic amino acids at the basolateral membrane of intestinal and renal epithelia. On the assumption that the basolateral membrane of epithelial cells and plasma membrane of parenchymal cells are functional analogues, we studied transport of cationic amino acids by cultured skin fibroblasts from LPI and control subjects matched for age, sex, and site of biopsy. We measured Na+-independent transport of radiolabeled lysine, arginine, ornithine, and homoarginine on system y+, the carrier with preference for cationic amino acids, and leucine transport on system L (as the internal control). LPI cells had increased net uptake of cationic amino acids (nmol/mg of protein) relative to leucine. LPI cells also maintained increased steady-state intracellular pools of cationic amino acids. Neither increased metabolic utilization nor increased pool size were responsible for high uptake of cationic amino acids in LPI cells. We then measured trans-stimulated efflux of homoarginine as a specific test of system y+ activity. Homoarginine efflux was significantly impaired in LPI cells (P less than 0.05), whereas leucine efflux was similar in LPI and control cells. Percent trans-stimulation of homoarginine efflux was 1.0 +/- 0.5% in homozygous LPI cells, 10 +/- 0.5% in heterozygous cells, and 22 +/- 0.5% in control cells indicating a gene-dosage effect. The LPI mutation affects system y+ asymmetrically, selectively impairing efflux in fibroblast plasma membrane. To our knowledge, this appears to be the first demonstration that the skin fibroblast can be used to study a corresponding transport defect in intestinal and renal membranes.

Citations Scopus - 27
1986 Enns RE, Garland AM, Smith DW, 'M13-oriC cloning vehicles: Use for amplification of high copy lethal (HCL) genetic elements', Plasmid, 15 147-155 (1986)

M13 cloning vehicles have been constructed which contain the Escherichia coli origin for DNA replication (oriC), with and without selectable antibiotic-resistance genes. Since the... [more]

M13 cloning vehicles have been constructed which contain the Escherichia coli origin for DNA replication (oriC), with and without selectable antibiotic-resistance genes. Since the M13 viral strand origin requires a functional rep gene product, using oriC these vehicles propagate as low-copy-number plasmids in E. coli rep mutants. This property is exploited to amplify cloned "high copy lethal" (HCL) DNA fragments, those containing genetic elements which kill the E. coli host when present at multiple copies in the cell. Following cloning of such fragments in these vehicles and initial selection in E. coli rep cells, the M13-oriC chimeric plasmid DNA is used to transfect appropriate E. coli rep+ cells. The chimeric DNA propagates as M13 viral DNA, yielding double-stranded and single-stranded DNA products and phage particles prior to killing of the host via expression of the HCL element; these events mimic a lytic phage infection. Such amplification will greatly facilitate both DNA "library" constructions (HCL elements are absent a priori from libraries using high-copy-number cloning vehicles) and studies of HCL elements including restriction mapping, DNA sequencing, and physiological studies. © 1986.

DOI 10.1016/0147-619X(86)90050-8
Citations Scopus - 1
1986 Motulsky HJ, Smith D, Terman BI, Feldman R, 'Regulation of hormone-stimulated cyclic AMP accumulation in intact human mononuclear leukocytes by blood plasma', Journal of Cyclic Nucleotide and Protein Phosphorylation Research, 11 329-343 (1986)

Several hormones, including catecholamines, histamine, and prostaglandin E1, regulate the function of human mononuclear leukocytes (MNL) by stimulating the accumulation of cAMP. I... [more]

Several hormones, including catecholamines, histamine, and prostaglandin E1, regulate the function of human mononuclear leukocytes (MNL) by stimulating the accumulation of cAMP. Isoproterenol-stimulated cAMP accumulation in MNL isolated and washed at 4° is five times greater than in cells prepared at ambient temperature. The current study was aimed at understanding this difference. cAMP accumulation in MNL prepared at ambient temperature could not be increased by chilling the cells for 4 hours. Warming MNL prepared at 4° for 30 min, however, reduced later isoproterenol-, histamine-, and PGE1-stimulated cAMP accumulation by 65-85% without altering forskolin-stimulated cAMP accumulation and without altering cellular viability or ATP content. In broken cell preparations, there was no difference in either adenylate cyclase or phosphodiesterase activities, and no difference in the binding of isoproterenol to the beta-adrenergic receptors. The reduction in isoproterenol-stimulated cAMP accumulation in warmed intact cells was reversed when the MNL were incubated with autologous leukocyte-depleted blood or with plasma. These data suggest the presence of one or more factors in plasma that enhances hormone-stimulated adenylate cyclase activity in intact MNL.

Citations Scopus - 12
1986 Zyskind JW, Smith DW, 'The bacterial origin of replication, oriC', Cell, 46 489-490 (1986)
Citations Scopus - 54
1985 Smith DW, Garland AM, Herman G, Enns RE, Baker TA, Zyskind JW, 'Importance of state of methylation of oriC GATC sites in initiation of DNA replication in Escherichia coli.', EMBO Journal, 4 1319-1326 (1985)

In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis. Transfo... [more]

In vivo and in vitro evidence is presented implicating a function of GATC methylation in the Escherichia coli replication origin, oriC, during initiation of DNA synthesis. Transformation frequencies of oriC plasmids into E. coli dam mutants, deficient in the GATC-specific DNA methylase, are greatly reduced compared with parental dam+ cells, particularly for plasmids that must use oriC for initiation. Mutations that suppress the mismatch repair deficiency of dam mutants do not increase these low transformation frequencies, implicating a new function for the Dam methylase. oriC DNA isolated from dam- cells functions 2- to 4-fold less well in the oriC-specific in vitro initiation system when compared with oriC DNA from dam+ cells. This decreased template activity is restored 2- to 3-fold if the DNA from dam- cells is first methylated with purified Dam methylase. Bacterial origin plasmids or M13-oriC chimeric phage DNA, isolated from either base substitution or insertion dam mutants of E. coli, exhibit some sensitivity to digestion by DpnI, a restriction endonuclease specific for methylated GATC sites, showing that these dam mutants retain some Dam methylation activity. Sites of preferred cleavage are found within the oriC region, as well as in the ColE1-type origin.

Citations Scopus - 85
1983 Zyskind JW, Cleary JM, Brusilow WSA, Harding NE, Smith DW, 'Chromosomal replication origin from the marine bacterium Vibrio harveyi functions in Escherichia coli: OriC consensus sequence', Proceedings of the National Academy of Sciences of the United States of America, 80 1164-1168 (1983)
Citations Scopus - 105
1982 Cleary JM, Smith DW, Harding NE, Zyskind JW, 'Primary structure of the chromosomal origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae: Comparisons and evolutionary relationships', Journal of Bacteriology, 150 1467-1471 (1982)

The nucleotide sequences of the E. aerogenes and K. pneumoniae DNA replication origins (oriC) were determined and compared with those of Escherichia coli and Salmonella typhimuriu... [more]

The nucleotide sequences of the E. aerogenes and K. pneumoniae DNA replication origins (oriC) were determined and compared with those of Escherichia coli and Salmonella typhimurium. Four interrelated, 9-base-pair repeats were identified from the conserved regions within the minimal origin. Evolutionary rates calculated from the minimal origin sequences yielded a quantitative phylogenic tree which agreed with the taxonomic classification of these genera.

Citations Scopus - 18
1982 Harding NE, Cleary JM, Smith DW, Michon JJ, Brusilow WS, Zyskind JW, 'Chromosomal replication origins (oriC) of Enterobacter aerogenes and Klebsiella pneumoniae are functional in Escherichia coli', Journal of Bacteriology, 152 983-993 (1982)

The chromosomal DNA replication origins (oriC) from 2 members of the family Enterobacteriaceae, E. aerogenes and K. pneumoniae, have been isolated as functional replication origin... [more]

The chromosomal DNA replication origins (oriC) from 2 members of the family Enterobacteriaceae, E. aerogenes and K. pneumoniae, have been isolated as functional replication origins in E. coli. The origins in the SalI restriction fragments of 17.5 and 10.2 kilobase pairs, cloned from E. aerogenes and K. pneumoniae, respectively, were found to be between the asnA and uncB genes, as are the origins of the E. coli and Salmonella typhimurium chromosomes. Plasmids containing oriC from E. aerogenes, K. pneumoniae, and S. typhimurium replicate in the E. coli cell-free enzyme system and this replication is dependent on dnaA protein activity. These SalI fragments from E. aerogenes and K. pneumoniae carry a region which is lethal to E. coli when many copies are present. It is shown that this region is also carried on the E. coli 9.0-kilobase-pair EcoRI restriction fragment containing oriC. The F 0 genes of the atp or unc operon, when linked to the unc operon promoter, are apparently responsible for the lethality.

Citations Scopus - 17
1982 Takeda Y, Harding NE, Smith DW, Zyskind JW, 'The chromosomal origin of replication (oriC) of Erwinia carotovora', Nucleic Acids Research, 10 2639-2650 (1982)

The chromosomal DNA replication origin (oriC) of the plant pathogen Erwinia carotovora has been isolated and sequenced. The minimal E. carotovora oric region functional in Escheri... [more]

The chromosomal DNA replication origin (oriC) of the plant pathogen Erwinia carotovora has been isolated and sequenced. The minimal E. carotovora oric region functional in Escherichia coli is a 374 base pair region located on a 7.9 kilobase pair SalI fragment which also contains a functional asnA gene. Differences between the nucleotide sequences of the minimal origin regions of E. carotovora and those of E. coli and Salmonella typhimurium are clustered nucleotide substitutions, with regions of complete homology, up to 19 base pairs long, between the three origins. Nine GATC sites are found in the minimal origin, and all are conserved. In contrast, the region toward asnA from the minimal origin shows little clustering and the differences occur mainly every third nucleotide, suggesting that this region is a protein coding region. © 1982 IRL Press Limited.

DOI 10.1093/nar/10.8.2639
Citations Scopus - 13
1981 Helfman WB, Hendler SS, Smith DW, 'Divalent cation-activated RNA synthesis in toluene-treated Escherichia coli', Canadian Journal of Biochemistry, 59 511-518 (1981)
1980 Zyskind JW, Smith DW, 'Nucleotide sequence of the Salmonella typhimurium origin of DNA replication', Proceedings of the National Academy of Sciences of the United States of America, 77 2460-2464 (1980)
Citations Scopus - 28
1979 Zyskind JW, Deen LT, Smith DW, 'Isolation and mapping of plasmids containing the Salmonella typhimurium origin of DNA replication', Proceedings of the National Academy of Sciences of the United States of America, 76 3097-3101 (1979)
Citations Scopus - 13
1979 Fujimura FK, Zyskind JW, Smith DW, 'The Escherichia coli dnaB protein is required for initiation of chromosomal DNA replication', Cold Spring Harbor Symposia on Quantitative Biology, 43 559-562 (1979)
Citations Scopus - 4
1978 Helfman WB, Hendler SS, Shannahoff DH, Smith DW, 'Escherichia coli DNA polymerases II and III: Substrate specificity', Biochemistry, 17 1607-1611 (1978)

Escherichia coli DNA polymerases II and III have a greater specificity for the 2'-deoxy configuration of substrate than has been found previously for DNA polymerase I. Neither rib... [more]

Escherichia coli DNA polymerases II and III have a greater specificity for the 2'-deoxy configuration of substrate than has been found previously for DNA polymerase I. Neither ribosyl nor arabinosyl derivatives appreciably substitute in DNA polymerase II and III reactions, even in the presence of Mn2+ ions. The derivative 2'-fluorodeoxycytidine 5'-triphosphate partially substitutes for dCTP in DNA polymerase II reactions, and not at all in DNA polymerase HI catalyzed reactions. The corresponding dUTP analogue, 2'-fluorodeoxyuridine 5'-triphosphate, has negligible substrate activity for either enzyme. Both DNA polymerases II and III catalyze considerable synthesis in the absence of one of the four deoxyribonucleoside triphosphates. This activity is inhibited by the respective arabinonucleoside triphosphate in the case of DNA polymerase II, but there is no inhibition of DNA polymerase III. Reactions catalyzed by both enzymes, either Mg2+- or Mn2+-activated, show a sigmoidal dependence on dCTP concentration, but a nonsigmoidal dependence on the concentrations of the other dNTPs, when each concentration is varied singly. DNA polymerases II and III have an absolute specificity for the 5'-triphosphate derivative of substrate, since neither deoxyribonucleoside 5'-monophosphates nor diphosphates serve as substrates even in the presence of ATP. Both enzymes apparently require the anti conformation of nucleotide, since the syn derivative, 8-bromodeoxyguanosine 5'-triphosphate, does not support DNA polymerase II or III activity. In contrast, the base analogues dUTP and dITP, derivatives having an anti conformation, substitute nearly completely for dTTP and dGTP, respectively, in the DNA polymerase II and III reactions. © 1978 American Chemical Society.

Citations Scopus - 5
1977 Zyskind JW, Smith DW, 'Novel Escherichia coli dnaB mutant: direct involvement of the dnaB252 gene product in the synthesis of an origin ribonucleic acid species during initiation of a round of deoxyribonucleic acid replication', Journal of Bacteriology, 129 1476-1486 (1977)
Citations Scopus - 32
1977 Zyskind JW, Deen LT, Smith DW, 'Temporal sequence of events during the initiation process in Escherichia coli deoxyribonucleic acid replication: roles of the dnaA and dnaC gene products and ribonucleic acid polymerase', Journal of Bacteriology, 129 1466-1475 (1977)

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of E. coli exposed to the restrictive temperature for one to two generations were examined for the ability to ... [more]

Three thermosensitive deoxyribonucleic acid (DNA) initiation mutants of E. coli exposed to the restrictive temperature for one to two generations were examined for the ability to reinitiate DNA replication after returning to the permissive temperature in the presence of rifampin, chloramphenicol, or nalidixic acid. Reinitiation in the dnaA mutant was inhibited by rifampin but not by chloramphenicol, whereas reinitiation was not inhibited in two dnaC mutants by either rifampin or chloramphenicol. To observe the rifampin inhibition, the antibiotic must be added at least 10 min before return to the permissive temperature. The rifampin inhibition of reinitiation was not observed when a rifampin-resistant ribonucleic acid (RNA) polymerase gene was introduced into the dnaA mutant, demonstrating that RNA polymerase synthesizes one or more RNA species required for the initiation of DNA replication (origin-RNA). Reinitiation at 30°C was not inhibited by streptolydigin in a streptolydigin-sensitive dnaA mutant. Incubation in the presence of nalidixic acid prevented subsequent reinitiation in the dnaC28 mutant but did not inhibit reinitiation in the dnaA5 mutant. These results demonstrate that the dnaA gene product acts before or during the synthesis of an origin-RNA, RNA polymerase synthesizes this origin-RNA, and the dnaC gene product is involved in a step after this RNA synthesis event. Furthermore, these results suggest that the dnaC gene product is involved in the first deoxyribonucleotide polymerization event whereas the dnaA gene product acts prior to this event. A model is presented describing the temporal sequence of events tht occur during initiation of a round of DNA replication, based on results in this and the accompanying paper.

Citations Scopus - 40
1976 Helfman WB, Hendler SS, Smith DW, 'Escherichia coli DNA polymerases II and III: Activation by magnesium or by manganous ions', BBA Section Nucleic Acids And Protein Synthesis, 447 175-187 (1976)

Escherichia coli DNA polymerases II and III have been extensively studied in vitro when activated with Mg2+. The Mn2+-activated polymerization reactions are considered here, and s... [more]

Escherichia coli DNA polymerases II and III have been extensively studied in vitro when activated with Mg2+. The Mn2+-activated polymerization reactions are considered here, and shown to differ from the Mg2+-activated reactions. The Mn2+-activated DNA polymerase II reaction requires K+ or spermidine, and the effects of monovalent cation and polyamine are additive. In contrast, the Mg2+-activated reaction does not require, but is stimulated by, K+ or spermidine, in a non-additive manner. Under optimal conditions, DNA polymerase II is activated better with Mn2+ than it is with Mg2+, suggesting a physiological role for the Mn2+-activated enzyme. The observed preference for Mn2+ over Mg2+ in reaction kinetics and at high DNA template concentrations suggest that Mg2+ may preferentially activate the associated exonuclease activity. At 29°C, the Mn2+-activated DNA polymerase III reaction is stimulated by K+ and inhibited by ethanol or phosphatidylethanolamine. In contrast, the latter compounds and Triton X-100 increase the initial rate of the Mg2+-activated reaction, where-as K+ inhibits this reaction at all concentrations. The K+ inhibition is reduced at low Mg2+ concentrations when Mn2+ is also present. After stimulating the initial reaction rate, ethanol causes a rapid decrease in the rate of the Mg2+-activated reaction during incubation at 20°C. At 27°C, all surface-active compounds inhibit the Mg2+-activated reaction. Preincubation of the enzyme at 30°C or below with DNA template and divalent cation increases the initial reaction rate, suggesting that formation of an enzyme-divalent cation-DNA template complex occurs as the first step in DNA polymerase III catalysis. The apparent Km at 21°C for gapped calf thymus DNA was 25 µM with Mn2+ and 125 µM with Mg2+ for DNA polymerase III, and 18 µM at 30°C for DNA polymerase II with either Mn2+ or Mg2+. Reactions with poly[d(A-T)] were enhanced by Mn2+ relative to Mg2+, and activity with poly(rA) · poly(dT) was Mn2+ dependent for both enzymes. © 1976.

DOI 10.1016/0005-2787(76)90341-5
Citations Scopus - 2
1975 Smith DW, Boerner P, 'Stimulation of adenosine 5' triphosphate dependent in vitro deoxyribonucleic acid replication by factors from the periplasmic space of Escherichia coli', Journal of Bacteriology, 122 159-170 (1975)
1975 Smith DW, Tait RC, Harris AL, 'DNA repair in DNA-polymerase-deficient mutants of Escherichia coli.', Basic life sciences, 5 B 473-481 (1975)

Escherichia coli mutants deficient in DNA polymerase I, in DNA polymerases I and II, or in DNA polymerase III can efficiently and completely execute excision-repair and postreplic... [more]

Escherichia coli mutants deficient in DNA polymerase I, in DNA polymerases I and II, or in DNA polymerase III can efficiently and completely execute excision-repair and postreplication repair of the UV-damaged DNA at 30 degrees C and 43 degrees C when assayed by alkaline sucrose gradients. Repair by Pol I- and Pol I-, Pol II- cells is inhibited by 1-beta-D-arabinofuranosylcytosine (araC) at 43 degrees C but not at 30 degrees C, whereas that by Pol III- cells is insensitive to araC at any temperature. Thus, either Pol I or Pol III is required for complete and efficient repair, and in their absence Pol II mediates a limited, incomplete dark repair of UV-damaged DNA.

Citations Scopus - 1
1975 Ryder OA, Smith DW, 'Properties of membrane-associated folded chromosomes of E. coli related to initiation and termination of DNA replication', Cell, 4 337-345 (1975)

Analysis of folded chromosomes prepared from amino acid-starved E. coli cells or from a dnaC initiation mutant indicates that a unique structure is associated with completion or n... [more]

Analysis of folded chromosomes prepared from amino acid-starved E. coli cells or from a dnaC initiation mutant indicates that a unique structure is associated with completion or near completion of rounds of chromosome replication in E. coli. Chromosomes remain associated with portions of the bacterial cell envelope throughout the DNA replication cycle, but become more rapidly sedimenting as replication proceeds in the absence of reinitiation. Before reinitiation of chromosome replication occurs after restoring required amino acids to amino acid-starved cells or after lowering the temperature in a thermosensitive dnaC mutant, sedimentation velocities of the membrane-associated folded chromosomes decrease substantially. The decrease in sedimentation velocity does not depend on renewed DNA synthesis, but does require the activity of at least the dnaC gene product. © 1975.

DOI 10.1016/0092-8674(75)90154-3
Citations Scopus - 2
1974 Ryder OA, Smith DW, 'Isolation of membrane-associated folded chromosomes from Escherichia coli: effect of protein synthesis inhibition.', Journal of Bacteriology, 120 1356-1363 (1974)
Citations Scopus - 15
1974 Tait RC, Harris AL, Smith DW, 'DNA repair in Escherichia coli mutants deficient in DNA polymerases I, II, and/or III', Proceedings of the National Academy of Sciences of the United States of America, 71 675-679 (1974)

E. coli mutants deficient in DNA polymerase I, in DNA polymerase I and II, or in DNA polymerase III, can efficiently and completely execute excision repair and post replication re... [more]

E. coli mutants deficient in DNA polymerase I, in DNA polymerase I and II, or in DNA polymerase III, can efficiently and completely execute excision repair and post replication repair of UV damaged DNA at 43° when assayed by alkaline sucrose gradients. Repair by cells deficient in polymerase I and in polymerases I and II is inhibited by 1 ß D arabinofuranosylcytosine at 43°, whereas that by cells deficient in polymerase III is insensitive to the inhibitor. When both DNA polymerases I and III are deficient, both excision repair and post replication repair are greatly reduced at 43°, and the residual repair capability is inhibited by 1 ß D arabinofuranosylcytosine. Vry little dark repair is observed in cells deficient in DNA polymerase I, II and III, and the DNA is extensively degraded. These results suggest that either DNA polymerase I or DNA polymerase III is required for complete and efficient repair, and that when both DNA polymerases I and III are deficient, DNA polymerase II mediates a limited, incomplete dark repair of UV damaged DNA. DNA polymerases Iand III thus appear to be important enzymes in both DNA replication and DNA dark repair.

Citations Scopus - 32
1974 Tait RC, Smith DW, 'Roles for E coli DNA polymerases I, II, and III in DNA replication', Nature, 249 116-119 (1974)

Chain elongation during E. coli DNA replication proceeds in three distinct stages. Participatory roles of the three E. coli DNA polymerases in each of these stages has been determ... [more]

Chain elongation during E. coli DNA replication proceeds in three distinct stages. Participatory roles of the three E. coli DNA polymerases in each of these stages has been determined. © 1974 Nature Publishing Group.

DOI 10.1038/249116a0
Citations Scopus - 11
1973 Smith DW, 'DNA synthesis in prokaryotes: Replication', Progress in Biophysics and Molecular Biology, 26 321-408 (1973)
DOI 10.1016/0079-6107(73)90022-9
Citations Scopus - 4
1970 Smith DW, Schaller HE, Bonhoeffer FJ, 'DNA synthesis in vitro', Nature, 226 711-713 (1970)

In a new in vitro system, DNA is synthesized semiconservatively at rates of chain growth comparable with replication in vivo. This DNA synthesis is also observed with a strain of ... [more]

In a new in vitro system, DNA is synthesized semiconservatively at rates of chain growth comparable with replication in vivo. This DNA synthesis is also observed with a strain of E. coli, which lacks DNA polymerase activity in vitro. © 1970 Nature Publishing Group.

DOI 10.1038/226711a0
Citations Scopus - 86
1969 Smith DW, 'DNA replication in Mycoplasma laidlawii B', BBA Section Nucleic Acids And Protein Synthesis, 179 408-421 (1969)

The DNA of a strain of the pleuropneumonia-like organism Mycoplasma laidlawii B, adapted to grow in a semi-defined medium, was density-labeled for various periods of time by subst... [more]

The DNA of a strain of the pleuropneumonia-like organism Mycoplasma laidlawii B, adapted to grow in a semi-defined medium, was density-labeled for various periods of time by substituting 5-bromodeoxyuridine for thymidine in the growth medium. Analysis of extracted DNA fragments by CsCl equilibrium sedimentation demonstrated semi-conservative replication, proceeding sequentially along the chromosome. The kinetics of formation of density-labeled DNA showed that 5-bromodeoxyuridine probably effects a premature initiation of a round of replication. By lysing the cells directly in the centrifuge tube, the M. laidlawii B chromosome was isolated in one piece in the CsCl gradient. Comparison of the buoyant-density distribution of the intact chromosomes with that of very mildly sheared chromosomes (shear yielding pieces at least 1 8 to 1 4 the size of the total chromosome) eliminated nearly all dispersive replication mechanisms and suggested that replication probably proceeds from only a few growing points. The buoyant-density distributions of intact chromosomes isolated after different times of density labeling are shown to be consistent with theory. Some physicochemical properties of the hybrid (density-labeled) and normal M. laidlawii B DNA are presented. These properties, and similarities and differences of DNA replication in M. laidlawii B and in Escherichia coli, are discussed. © 1969.

Citations Scopus - 2
1969 Smith DW, Hanawalt PC, 'Repair replication of DNA in ultraviolet irradiated Mycoplasma laidlawii B', Journal of Molecular Biology, 46 57-72 (1969)

Cesium chloride density-gradient techniques were used to detect repair replication in the pleuropneumonia-like organism Mycoplasma laidlawii B following ultraviolet irradiation. T... [more]

Cesium chloride density-gradient techniques were used to detect repair replication in the pleuropneumonia-like organism Mycoplasma laidlawii B following ultraviolet irradiation. Two methods of analysis were used, the first assaying repair in parental DNA shortly after u.v. irradiation, the second assaying repair in the parental DNA only after one round of semi-conservative replication. The second method assays repair only in DNA which has been rendered functional with respect to normal replication following the u.v. irradiation. For a dose of 85 ergs/mm2, the kinetics of repair replication were linear to apparent saturation after about 0.5 generation and a replacement of 1.2% of the M. laidlawii B chromosome. A dose-response analysis showed that the amount of repair increased rapidly with dose at low u.v. doses (less than 5 ergs/mm2), but then more slowly to an apparent saturation at about 100 ergs/mm2. Both assay methods yielded essentially the same values, indicating no difference between the extent of repair replication ahead of and behind the normal replication growing point. Visible light illumination after ultraviolet light reduced the amount of repair replication, as consistent with the existence of an enzymic mechanism for photoreactivation in this organism. The existence of repair replication in the mycoplasmas, possibly the simplest of free-living organisms, demonstrates the importance of repair mechanisms for the maintenance of genetic continuity in living systems. © 1969.

Citations Scopus - 14
1968 Smith DW, Hanawalt PC, 'Macromolecular synthesis and thymineless death in Mycoplasma laidlawii B.', Journal of Bacteriology, 96 2066-2076 (1968)
Citations Scopus - 12
1968 Hanawalt PC, Pettijohn DE, Pauling EC, Brunk CF, Smith DW, Kanner LC, Couch JL, 'Repair replication of DNA in vivo.', Cold Spring Harbor Symposia on Quantitative Biology, 33 187-194 (1968)
Citations Scopus - 12
1967 Smith DW, Hanawalt PC, 'Properties of the growing point region in the bacterial chromosome', BBA Section Nucleic Acids And Protein Synthesis, 149 519-531 (1967)

1. A complex containing the newly replicated fragments of the bacterial chromosome in Escherichia coli strain TAU-bar can be separated from the bulk of the DNA fragments by zone s... [more]

1. A complex containing the newly replicated fragments of the bacterial chromosome in Escherichia coli strain TAU-bar can be separated from the bulk of the DNA fragments by zone sedimentation in sucrose gradients. 2. Pronase-treated lysozyme lysates of the bacteria are layered on a 5-20 % linear sucrose gradient over a 62 % sucrose shelf. After centrifugation for 1-5 h at 25 000 rev./min, the bulk of the DNA is still found in the upper half of the gradient, but the shelf contains DNA which is markedly enriched for the newly replicated (i.e., radioactively pulse-labeled) material; most of the cellular debris but very little DNA sediments through the shelf to the bottom of the tube. 3. Treatment with deoxycholate releases the pulse-labeled DNA from the complex and it then sediments with or somewhat more slowly than the bulk of the DNA. It is suggested that the growing point of the chromosome is bound to a lipid membrane complex in E. coli. 4. Partially replicated DNA fragments obtained from the sucrose shelf can be further characterized by subsequent equilibrium sedimentation in CsCl gradients. Such DNA fragments, if density-labeled with 5-bromouracil, form a band intermediate in buoyant density between that of normal and hybrid 5-bromouracil-containing DNA, as consistent with the interpretation that these fragments are Y-shaped partially replicated DNA units from the growing point region of the chromosome. © 1967.

Citations Scopus - 96
Show 118 more journal articles

Conference (15 outputs)

Year Citation Altmetrics Link
2015 Dickinson S, Smith K, Bigland M, Smith D, Jobling P, Graham B, 'Morphological analysis of microglial and astrocyte populations in the superficial dorsal horn of spinal cord in aged mice', JOURNAL OF NEUROCHEMISTRY (2015) [E3]
Co-authors Brett Graham, Phillip Jobling
2014 Khan SI, Hübner PP, Smith DW, Brichta AM, Migliaccio AA, 'Ageing reduces vestibulo-ocular reflex adaptation in mice J Vestib Res', Journal of Vestibular Research: Equilibrium and Orientation: an international journal of experimental and clinical vestibular science (2014) [E3]
DOI 10.3233/VES-140517
Co-authors Alan Brichta
2013 Bigland M, Parkinson G, Brichta AM, Smith DW, 'Evidence for mitochondrial DNA deletions in vestibular hair cells of the aged rat', Proceedings of the Australian Neuroscience Society (2013) [E3]
Co-authors Alan Brichta
2012 Parkinson GM, Smith DW, 'Age-related increase in mitochondrial DNA copy number in midbrain dopamine neurons of the rat', Abstracts. Australian Neuroscience Society 32nd Annual Meeting (2012) [E3]
2012 James MH, Charnley JL, Levi EM, Dunkley PR, Smith DW, Dickson PW, Dayas CV, 'A role for the mTOR pathway in the development of addiction', Abstracts. Australian Neuroscience Society 32nd Annual Meeting (2012) [E3]
Co-authors Phil Dickson, Peter Dunkley, Christopher Dayas
2012 Thomas J, Smith DW, Garg ML, 'Early molecular signature of neuro-protection in hippocampus of resveratrol diet fed C57BL/6 mice', Resveratrol 2012. 2nd International Conference of Resveratrol and Health (2012) [E3]
Co-authors Manohar Garg
2012 Brown AL, Flynn JR, Dayas CV, Smith DW, 'Altered gene expression in cell signalling pathways of midbrain dopamine neurons from addiction and relapse vulnerable animals', Drug and Alcohol Review: Abstracts of the Australasian Professional Society on Alcohol and other Drugs Conference 2012 (2012) [E3]
Co-authors Christopher Dayas, Jamie Flynn
2012 Dayas CV, Quinn RK, Goldie BJ, Brown AM, Levi EM, Smith DW, Cairns MJ, 'Association of miRNAs with addiction-relevant synaptic plasticity genes', Abstract Book. Biological Psychiatry Australia Scientific Meeting (2012) [E3]
Co-authors Christopher Dayas, Murray Cairns
2011 Thomas J, Smith DW, Garg ML, 'Dietary supplementation with resveratrol normalizes histone deacetylase (HDAC4) in the hippocampus of streptozotocin-induced diabetic mice', Australasian Medical Journal (2011) [E3]
Co-authors Manohar Garg
2011 Barreto RDA, Walker FR, Dunkley PR, Day TA, Smith DW, 'Alteration of neurotrophic factor pathway gene expression in the rat infralimbic medial prefrontal cortex by subchronic restraint stress is reversed by fluoxetine', Posters. Australian Neuroscience Society 31st Annual Meeting (2011) [E3]
Co-authors Rohan Walker, Peter Dunkley
2010 Thomas J, Smith DW, Garg ML, 'Dietary supplementation with resveratrol reduces erythrocyte arachidonic and docosahexaenoic acids levels in diabetic mice', Proceedings of the Nutrition Society of Australia (2010) [E3]
Co-authors Manohar Garg
2008 Rao F, Zhang L, Wessel J, Zhang K, Wen G, Kennedy BP, et al., 'Adrenergic polymorphism and the human stress response', Annals of the New York Academy of Sciences (2008)

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Does common genetic variation at human TH alter autonomic activity and predispose to cardiovas... [more]

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Does common genetic variation at human TH alter autonomic activity and predispose to cardiovascular disease? We undertook systematic polymorphism discovery at the TH locus, and then tested variants for contributions to sympathetic function and blood pressure. We resequenced 80 ethnically diverse individuals across the TH locus. One hundred seventy-two twin pairs were evaluated for sympathetic traits, including catecholamine production and environmental (cold) stress responses. To evaluate hypertension, we genotyped subjects selected from the most extreme diastolic blood pressure percentiles in the population. Human TH promoter haplotype/reporter plasmids were transfected into chromaffin cells. Forty-nine single nucleotide polymorphisms (SNPs) and one tetranucleotide repeat were discovered, but coding region polymorphism did not account for common phenotypic variation. A block of linkage disequilibrium spanned four common variants in the proximal promoter. Catecholamine secretory traits were significantly heritable, as were stress-induced blood pressure changes. In the TH promoter, significant associations were found for urinary catecholamine excretion, as well as blood pressure response to stress. TH promoter haplotype #2 (TGGG) showed pleiotropy, increasing both norepinephrine excretion and blood pressure during stress. In hypertension, a case-control study (1266 subjects, 53% women) established the effect of C-824T in determination of blood pressure. We conclude that human catecholamine secretory traits are heritable, displaying joint genetic determination (pleiotropy) with autonomic activity and finally with blood pressure in the population. Catecholamine secretion is influenced by genetic variation in the adrenergic pathway encoding catecholamine synthesis, especially at the classically rate-limiting step, TH. The results suggest novel pathophysiological links between a key adrenergic locus, catecholamine metabolism, and blood pressure, and suggest new strategies to approach the mechanism, diagnosis, and treatment of systemic hypertension. © 2008 New York Academy of Sciences.

DOI 10.1196/annals.1410.085
Citations Scopus - 9
2007 McInerny SC, Brown AL, Smith DW, 'Brain region-specific decrease in mitochondrial D-loop in aged rats (Poster)', 7th IBRO 2007 World Congress of Neuroscience Program (2007) [E3]
1995 Day TA, Smith DW, Buller KM, 'Regulation of neurosecretory cell activity by A1 noradrenergic neurons', 1st Joint World Congress of Neurohypophysis and Vasopressin (1995)
1995 Day TA, Smith DW, Buller KM, 'Activation of corticotrophin releasing factor cells by ventrolateral medulla catecholamine neurons', Journal of UOEH (1995)
Show 12 more conferences
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Grants and Funding

Summary

Number of grants 19
Total funding $5,160,249

Click on a grant title below to expand the full details for that specific grant.


20172 grants / $370,324

Cholesterol metabolism in the ageing brain – implications for dementia$25,000

Funding body: Hunter Medical Research Institute

Funding body Hunter Medical Research Institute
Project Team Associate Professor Doug Smith
Scheme Project Grant
Role Lead
Funding Start 2017
Funding Finish 2017
GNo G1700349
Type Of Funding Grant - Aust Non Government
Category 3AFG
UON Y

20152 grants / $45,000

Maintaining our cognitive abilities as we grow old: preventing the effects of ageing on the brain$25,000

Funding body: Hunter Medical Research Institute

Funding body Hunter Medical Research Institute
Project Team Associate Professor Doug Smith, Associate Professor Rohan Walker
Scheme Project Grant
Role Lead
Funding Start 2015
Funding Finish 2015
GNo G1500078
Type Of Funding Grant - Aust Non Government
Category 3AFG
UON Y

Dizzy and Deaf - restoring signals from the inner ear$20,000

Funding body: Hunter Medical Research Institute

Funding body Hunter Medical Research Institute
Project Team Doctor Rebecca Lim, Professor Alan Brichta, Professor Robert Callister, Associate Professor Doug Smith
Scheme Project Grant
Role Investigator
Funding Start 2015
Funding Finish 2015
GNo G1501395
Type Of Funding Grant - Aust Non Government
Category 3AFG
UON Y

20131 grants / $35,000

20122 grants / $313,375

The effects of ageing on the peripheral vestibular system. Can ageing-related functional decline be reduced or prevented?$298,375

Funding body: Garnett Passe and Rodney Williams Memorial Foundation

Funding body Garnett Passe and Rodney Williams Memorial Foundation
Project Team Associate Professor Doug Smith
Scheme Project Grant
Role Lead
Funding Start 2012
Funding Finish 2014
GNo G1100935
Type Of Funding Aust Competitive - Non Commonwealth
Category 1NS
UON Y

2011 Emerging Research Leaders Program$15,000

Funding body: University of Newcastle

Funding body University of Newcastle
Project Team Associate Professor Doug Smith
Scheme Emerging Research Leaders Program
Role Lead
Funding Start 2012
Funding Finish 2012
GNo G1200307
Type Of Funding Internal
Category INTE
UON Y

20111 grants / $25,000

Brain Mechanisms Conferring Psychostimulant Addiction$25,000

Funding body: University of Newcastle

Funding body University of Newcastle
Project Team Associate Professor Chris Dayas, Emeritus Professor Peter Dunkley, Associate Professor Doug Smith
Scheme Near Miss Grant
Role Investigator
Funding Start 2011
Funding Finish 2011
GNo G1001052
Type Of Funding Internal
Category INTE
UON Y

20104 grants / $649,000

Laser microdissection microscopy system for cell and development biology$350,000

Funding body: ARC (Australian Research Council)

Funding body ARC (Australian Research Council)
Project Team Professor Eileen McLaughlin, Conjoint Professor Keith Jones, Laureate Professor John Aitken, Professor Brett Nixon, Doctor Shaun Roman, Professor Alan Brichta, Doctor Rick Thorne, Associate Professor Doug Smith, Associate Professor David McCurdy, Emeritus Professor Ray Rose, Professor Christopher Grof, Emeritus Professor Leonie Ashman, Professor Gordon Burns, Associate Professor Brett Graham, Associate Professor Paul Tooney, Laureate Professor Roger Smith, Laureate Professor Paul Foster, Professor Trevor Day, Professor Robert Callister
Scheme Linkage Infrastructure Equipment & Facilities (LIEF)
Role Investigator
Funding Start 2010
Funding Finish 2010
GNo G0190369
Type Of Funding Scheme excluded from IGS
Category EXCL
UON Y

ABI 7500 Real Time PCR System $34,000

Funding body: NHMRC (National Health & Medical Research Council)

20081 grants / $538,500

Dopamine mechanisms conferring resilience to depression: A new antidepressant target$538,500

Funding body: NHMRC (National Health & Medical Research Council)

Funding body NHMRC (National Health & Medical Research Council)
Project Team Professor Trevor Day, Emeritus Professor Peter Dunkley, Professor David Pow, Associate Professor Doug Smith
Scheme Project Grant
Role Investigator
Funding Start 2008
Funding Finish 2010
GNo G0187604
Type Of Funding Aust Competitive - Commonwealth
Category 1CS
UON Y

20072 grants / $40,000

Vulnerability to depression: the role of dopamine pathways$20,000

Funding body: University of Newcastle

Funding body University of Newcastle
Project Team Professor Trevor Day, Emeritus Professor Peter Dunkley, Associate Professor Doug Smith, Professor David Pow
Scheme Near Miss Grant
Role Investigator
Funding Start 2007
Funding Finish 2007
GNo G0187196
Type Of Funding Internal
Category INTE
UON Y

Characterisation of the brain mechanisms linking vulnerability to stress and vulnerability to drug addiction$20,000

Funding body: Hunter Medical Research Institute

Funding body Hunter Medical Research Institute
Project Team Professor Trevor Day, Associate Professor Chris Dayas, Associate Professor Doug Smith
Scheme Project Grant
Role Investigator
Funding Start 2007
Funding Finish 2007
GNo G0187255
Type Of Funding Contract - Aust Non Government
Category 3AFC
UON Y

20042 grants / $13,679

Characterisation of age-related gene expression changes in midbrain dopamine neurons$12,000

Funding body: University of Newcastle

Funding body University of Newcastle
Project Team Associate Professor Doug Smith
Scheme New Staff Grant
Role Lead
Funding Start 2004
Funding Finish 2004
GNo G0184998
Type Of Funding Internal
Category INTE
UON Y

Society for Neuroscience Annual Meeting, 23-27 October 2004$1,679

Funding body: University of Newcastle

Funding body University of Newcastle
Project Team Associate Professor Doug Smith
Scheme Travel Grant
Role Lead
Funding Start 2004
Funding Finish 2004
GNo G0184814
Type Of Funding Internal
Category INTE
UON Y

20031 grants / $2,489,371

Genetic aberrations in HPRT deficiency$2,489,371

Funding body: National Institute of Neurological Disorders (NIH)

Funding body National Institute of Neurological Disorders (NIH)
Project Team

Theodore Friedmann

Scheme Project
Role Investigator
Funding Start 2003
Funding Finish 2007
GNo
Type Of Funding External
Category EXTE
UON N

20011 grants / $641,000

Array screening for Lesch-Nyhan disese$641,000

Funding body: NICH (NIH)

Funding body NICH (NIH)
Project Team

Theodore Friedmann

Scheme R21
Role Investigator
Funding Start 2001
Funding Finish 2003
GNo
Type Of Funding External
Category EXTE
UON N
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Research Supervision

Number of supervisions

Completed7
Current5

Total current UON EFTSL

PhD2.05

Current Supervision

Commenced Level of Study Research Title Program Supervisor Type
2017 PhD Investigation of Age-Related Neuro-Inflammatory and Neuro-Vascular Changes in the Central Nervous System PhD (Anatomy), Faculty of Health and Medicine, The University of Newcastle Principal Supervisor
2015 PhD Spinal Cord Glial Changes in Pain PhD (Anatomy), Faculty of Health and Medicine, The University of Newcastle Co-Supervisor
2012 PhD Ageing of the Inner Ear Balance System PhD (Anatomy), Faculty of Health and Medicine, The University of Newcastle Principal Supervisor
2012 PhD The Role of Mitochondrial DNA in the Post-Injury "Inflammatory" Response Following Major Trauma PhD (Medicine), Faculty of Health and Medicine, The University of Newcastle Co-Supervisor
2007 PhD Role of dopamine in depression Medical Science, University of Newcastle Principal Supervisor

Past Supervision

Year Level of Study Research Title Program Supervisor Type
2016 PhD Ageing of the Somatic Motor Nervous System: A Nuclear and Mitochondrial Genome Perspective PhD (Medical Biochemistry), Faculty of Health and Medicine, The University of Newcastle Principal Supervisor
2015 Masters Nucleotide Excision Repair of UVA-Induced DNA Damage: Regulation in Sunlight-Induced Melanoma M Philosophy (Medical Genetic), Faculty of Health and Medicine, The University of Newcastle Co-Supervisor
2014 PhD The Role of Cocaine- and Amphetamine-Regulated Transcript (CART) and Orexin in Drug-Seeking and Addiction-Related Behaviours PhD (Anatomy), Faculty of Health and Medicine, The University of Newcastle Co-Supervisor
2013 PhD Effects of Anti-Inflammatory Bioactives on Diabetes-Induced Changes in Cognition-Related Gene Expression in the Hippocampus PhD (Medical Biochemistry), Faculty of Health and Medicine, The University of Newcastle Co-Supervisor
2012 PhD Molecular Correlates of Dopamine Signalling in Addiction Vulnerability PhD (Anatomy), Faculty of Health and Medicine, The University of Newcastle Principal Supervisor
2012 PhD Dopaminergic Pathway Imbalance in the Neurobiology of Depression PhD (Anatomy), Faculty of Health and Medicine, The University of Newcastle Principal Supervisor
2006 Honours Role of mitochondrial D-loop in aging brain Medical Science, University of Newcastle Principal Supervisor
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Associate Professor Doug Smith

Position

Associate Professor
School of Biomedical Sciences and Pharmacy
Faculty of Health and Medicine

Focus area

Anatomy

Contact Details

Email douglas.smith@newcastle.edu.au
Phone (02) 4921 7108
Fax (02) 4921 8667

Office

Room MS306B
Building Medical Sciences
Location Callaghan
University Drive
Callaghan, NSW 2308
Australia
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