Associate Professor Mark Baker

Mice have long been used as model organisms for investigating genetic features. The adaptation of clustered regularly interspaced palindromic repeats (CRISPR) and their associated nucleases (Cas) has increased our ability to utilise mice to further our knowledge of genetics as well as to create models of human disease conditions. Development of the CRISPR/Cas system is such that scientists can now create knockout/knockin mice in less than half the time previously required, as well as generate point mutations and insertion of small targeting sequences, allowing the finer detail of the genome to be examined. Current uses for the CRISPR/Cas system in mice also allows for deactivation of the cleavage domains of the Cas nuclease, permitting fusion of nickases or other activating/repressing effector proteins. This enables researchers to investigate timing of activation and localisation of a target locus and has even been used to correct genetic disease in the mouse. Mice are an essential part of the biological/biomedical research process and the CRISPR/Cas system has been and continues to be used in this organism with extremely promising results.

Posttranslational modifications represent a major mechanism by which cells control molecular events. Sample preparation is one of the biggest keys in terms of proteomic analysis, and especially when looking at phosphoproteomics. Here we describe two techniques that are used to identify phosphopeptides. These are Titanium Dioxide and Immobilized Metal Affinity Chromatography (IMAC) which enrich for phosphopeptides. These enrichment techniques typically often identify ~35 % of the same and ~65 % unique phosphopeptides. In this chapter, we describe the use of an ion-trap mass spectrometer for the identification of phosphopeptides. © 2013 Springer Science+Business Media, LLC.

Herein, we compare the different experimental regimes used to induce testicular heat stress and summarise their impact on sperm production and male fertility. Irrespective of the protocol used, scrotal heat stress causes loss of sperm production. This is first seen 1¿2 weeks post heat stress, peaking 4¿5 weeks thereafter. The higher the temperature, or the longer the duration of heat, the more pronounced germ cell loss becomes, within extreme cases this leads to azoospermia. The second, and often underappreciated impact of testicular hyperthermia is the production of poor-quality spermatozoa. Typically, those cells that survive hyperthermia develop into morphologically abnormal and poorly motile spermatozoa. While both apoptotic and non-apoptotic pathways are known to contribute to hyperthermic germ cell loss, the mechanisms leading to formation of poor-quality sperm remain unclear. Mechanistically, it is unlikely that testicular hyperthermia affects messenger RNA (mRNA) abundance, as a comparison of four different mammalian studies shows no consistent single gene changes. Using available evidence, we propose two novel models to explain how testicular hyperthermia impairs sperm formation. Our first model suggests aberrant alternative splicing, while the second model proposes a loss of RNA repression. Importantly, neither model requires consistent changes in RNA species.

Lipids are dynamic biological molecules that play key roles in metabolism, inflammation, cell signalling and structure. They are biologically significant in the physiology of conception and reproduction. Many of the mechanisms surrounding equine conception and the early feto-maternal dialogue are yet to be understood at a biochemical level. Recently, lipidomic technologies have advanced considerably and analytical strategies have been enhanced and diversified. Consequently, in-depth lipidomic exploration now has the potential to reveal new lipid biomarkers and biochemical relationships that improve our understanding of the processes leading to efficient and successful reproduction. This review considers the role of lipids in conception and establishment of pregnancy, providing new insights into the enigmatic pathways governing early reproductive physiology of the mare.

Evidence is presented for expression of the insulin receptor on the surface of mammalian spermatozoa as well as transcripts for the receptor substrate adaptor proteins (IRS1-4) needed to mediate insulin action. Exposure to this hormone resulted in insulin receptor phosphorylation (pTyr972), activation of AKT (pSer473) and the stimulation of sperm motility. Intriguingly, the male germ line is also shown to be capable of generating insulin, possessing the relevant mRNA transcript and expressing strong immunocytochemical signals for both insulin and C-peptide. Insulin could be released from the spermatozoa by sonication in a concentration-dependent manner but was not secreted in response to glucose, fructose or stimulation with progesterone. However, insulin release could be induced by factors present in human uterine lavages. Furthermore, the endometrium was also shown to possess the machinery for insulin production and action (mRNA, insulin, C-peptide, proprotein convertase and insulin receptor), releasing insulin into the uterine lumen prior to ovulation. These studies emphasize the fundamental importance of extra-pancreatic insulin in regulating the reproductive process, particularly in the support of spermatozoa on their perilous voyage to the site of fertilization.

STUDY QUESTION: What is the nuclear heterogeneity of high-density purified human spermatozoa typically used for IVF purposes. SUMMARY ANSWER: The data show that while density gradient separation has improved the overall sperm population, there is still a large degree of nuclear heterogeneity within these cells. WHAT IS KNOWN ALREADY: Chromomycin A3 (CMA3) is an important DNA binding fluorochrome for the assessment of malefactor fertility. It is typically used to predict IVF outcomes on entire sperm ejaculates with very high receiver operating characteristic. Here we used CMA3 to characterise typical populations of human spermatozoa that would be used for IVF purposes after density gradient separation. STUDY DESIGN, SIZE, DURATION: We compared the intensity of CMA3 binding within high-dense sperm populations obtained from men. Binding heterogeneity was confirmed through fluorescence microscopy and FACS analysis independently. We also looked at CMA3 staining directly with head morphology in this sperm population. Finally, we looked at electron micrographs of nuclear heterogeneity (vacuoles, chromatin compaction) of spermatozoa following density gradient sorting of CMA3-stained cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: We used sperm donors who had fathered one or more children. Semen was collected after 2 days abstinence and purified over Percoll gradients. Only the high-quality spermatozoa, the same used for assisted conception, were then used. Cells were stained with CMA3 and sorted using FACS. Following this, electron micrographs were used to assess nuclear heterogeneity of CMA3-dependent sorted spermatozoa. MAIN RESULTS AND THE ROLE OF CHANCE: CMA3 staining occurs within morphologically normal as well as abnormal spermatozoa. High-intensity CMA3-stained sperm possessed large vacuoles that were not seen in the low-CMA3 population. In addition, the high-CMA3 stained cells possess higher amounts of nuclear granulation. LIMITATIONS, REASONS FOR CAUTION: The present study only describes the issues within the chromatin of these cells and does not suggest an alternate selection technique. WIDER IMPLICATIONS OF THE FINDINGS: CMA3 is one of the better reported prognostic assays in predicting pregnancy outcomes, especially in cases where the male is at fault. However, it is clear that even in fractionated populations of human spermatozoa, there are sperm cells that are morphologically normal yet possess high levels of CMA3 staining and chromatin granulation. The implication of this is that the embryologist, whom selects on the basis of sperm morphology, may choose a cell with poor chromatin, which may lead to poor embryo outcomes. STUDY FUNDING/COMPETING INTEREST(S): The project was funded by the National Health and Medical Research council, APP1118943. The authors have no conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

It has recently been suggested that the human sperm genome is highly unstable, which may be a reasonable explanation as to why men, even fertile men, produce defective spermatozoa. Furthermore, an unstable genome may also explain why the semen profile of the same man changes from one ejaculate to the next. As such, we took multiple ejaculates (between 3 and 6) from 7 individuals over a 6-month period and isolated sperm through density gradients. We then compared the DNA of: (i) good and poor-quality spermatozoa within the same ejaculate; and (ii) from multiple ejaculates from the same individual. Our results suggest that on a global level, DNA present within spermatozoa is actually quite stable and similar between both good and poor sperm. This is important information for the assisted reproductive community when it comes to sperm selection.

The status quo for combating uprising antibacterial resistance is to employ synergistic combinations of antibiotics. Nevertheless, the currently available combination therapies are fast becoming untenable. Combining antibiotics with various FDA-approved non-antibiotic drugs has emerged as a novel strategy against otherwise untreatable multi-drug resistant (MDR) pathogens. The apex of this study was to investigate the mechanisms of antibacterial synergy of the combination of polymyxin B with the phenothiazines against the MDR Gram-negative pathogens Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. The synergistic antibacterial effects were tested using checkerboard and static time-kill assays. Electron microscopy (EM) and untargeted metabolomics were used to ascertain the mechanism(s) of the antibacterial synergy. The combination of polymyxin B and the phenothiazines showed synergistic antibacterial activity in checkerboard and static time-kill assays at clinically relevant concentrations against both polymyxin-susceptible and polymyxin-resistant isolates. EM revealed that the polymyxin B-prochlorperazine combination resulted in greater damage to the bacterial cell compared to each drug monotherapy. In metabolomics, at 0.5 h, polymyxin B monotherapy and the combination (to a greatest extent) disorganised the bacterial cell envelope as manifested by a major perturbation in bacterial membrane lipids (glycerophospholipids and fatty acids), peptidoglycan and lipopolysaccharide (LPS) biosynthesis. At the late time exposure (4 h), the aforementioned effects (except LPS biosynthesis) perpetuated mainly with the combination therapy, indicating the disorganising bacterial membrane biogenesis is potentially behind the mechanisms of antibacterial synergy. In conclusion, the study highlights the potential usefulness of the combination of polymyxin B with phenothiazines for the treatment of polymyxin-resistant Gram-negative infections (e.g. CNS infections).

The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not individually predictive of fertilizing potential. The aim of this study was to harness mass spectrometry to compare the proteomic profiles of high- and low-quality stallion spermatozoa, with the ultimate goal of identifying fertility biomarker candidates. Extended stallion semen (n = 12) was fractionated using Percoll density gradients to isolate low-quality and high-quality sperm populations. Motility and morphological assessments were carried out, and proteomic analyses was conducted using UHPLC-MS/MS. High-quality spermatozoa recorded higher total (95.2 ± 0.52% vs 70.6 ± 4.20%; P = 0.001) and progressive motilities (43.4 ± 3.42% vs 27.3 ± 4.32%; P = 0.05), and a higher proportion of morphologically normal cells (50.2 ± 4.34% vs 38.8 ± 2.72%; P = 0.05). In total, 1069 proteins were quantified by UHPLC-MS/MS, of which 22 proteins were significantly more abundant in the high-quality sperm population (P = 0.05). A-kinase anchor protein 4 (AKAP4) and Hexokinase 1 (HK1) were considered possible biomarker candidates and their differential expression was confirmed by immunoblot. Protein expression was significantly correlated with total (AKAP4 R2 = 0.38, P = 0.01; HK1 R2 = 0.46, P = 0.001) and progressive motilities (AKAP4 R2 = 0.51, P = 0.001; HK1 R2 = 0.55, P = 0.01), percentage rapid (AKAP4 R2 = 0.29, P = 0.05; HK1 R2 = 0.58, P = 0.001), straight-line velocity (HK1 R2 = 0.50, P = 0.01) and straightness (HK1 R2 = 0.40, P = 0.01). Furthermore, AKAP4 was highly susceptible to adduction by 4-hydroxynonenal (4HNE), which resulted in a global reduction in the phosphorylation profiles following capacitation. In conclusion, the proteomic profiles of high- and low-quality stallion spermatozoa differ substantially, and proteins such as AKAP4 and HK1 could serve as biomarkers of ejaculate quality.

The central characteristic of uterine fibroids is excessive deposition of extracellular matrix (ECM), which contributes to fibroid growth and bulk-type symptoms. Despite this, very little is known about patterns of ECM protein expression in fibroids and whether these are influenced by the most common genetic anomalies, which relate to MED12. We performed extensive genetic and proteomic analyses of clinically annotated fibroids and adjacent normal myometrium to identify the composition and expression patterns of ECM proteins in MED12 mutation-positive and mutation-negative uterine fibroids. Genetic sequencing of tissue samples revealed MED12 alterations in 39 of 65 fibroids (60%) from 14 patients. Using isobaric tagged-based quantitative mass spectrometry on three selected patients (n = 9 fibroids), we observed a common set of upregulated (.1.5-fold) and downregulated (,0.66-fold) proteins in small, medium, and large fibroid samples of annotated MED12 status. These two sets of upregulated and downregulated proteins were the same in all patients, regardless of variations in fibroid size and MED12 status. We then focused on one of the significant upregulated ECM proteins and confirmed the differential expression of periostin using western blotting and immunohistochemical analysis. Our study defined the proteome of uterine fibroids and identified that increased ECM protein expression, in particular periostin, is a hallmark of uterine fibroids regardless of MED12 mutation status. This study sets the foundation for further investigations to analyze the mechanisms regulating ECM overexpression and the functional role of upregulated ECM proteins in leiomyogenesis.

Octapeptins are cyclic lipopeptides with a broader spectrum of activity against fungi and polymyxin-resistant Gram-negative and Gram-positive bacteria. In the present study, we investigated the interaction of octapeptin A3 with asymmetric outer membrane models of Gram-negative pathogen Pseudomonas aeruginosa using neutron reflectometry, together with fluorimetric and calorimetry methods. For the first time, our neutron reflectometry results reveal that the interaction of octapeptin A3 with the Gram-negative outer membrane involves an initial transient polar interaction with the phospholipid and lipid A headgroups, followed by the penetration of the entire octapeptin molecule into the fatty acyl core of the outer membrane. This mechanism contrasts with that of polymyxin B, which specifically targets lipid A, whereas octapeptins appear to target both lipid A and phospholipids. Furthermore, the mechanism of octapeptins does not appear to be highly dependent on an initial complementary electrostatic interaction with lipid A, which accounts for their ability to bind to lipid A of polymyxin-resistant Gram-negative bacteria that is modified with cationic moieties that act to electrostatically repel the cationic polymyxin molecule. The presented findings shed new light on the mechanism whereby octapeptins penetrate the outer membrane of polymyxin-resistant Gram-negative pathogens and highlight their potential as candidates for development as new antibiotics against problematic multi-drug-resistant pathogens.

This in vitro study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with 2 nm silver nanoparticles (NPs) against Gram-negative pathogens commonly isolated from the cystic fibrosis (CF) lung. The in vitro synergistic activity of polymyxin B with silver NPs was assessed using the checkerboard assay against polymyxinsusceptible and polymyxin-resistant Pseudomonas aeruginosa isolates from the lungs of CF patients. The combination was also examined against the Gram-negative species Haemophilus influenzae, Burkholderia cepacia, Burkholderia pseudomallei, Stenotrophomonas maltophilia, Klebsiella pneumoniae and Acinetobacter baumannii that are less common in the CF lung. The killing kinetics of the polymyxin B-silver NPs combinations was assessed against P. aeruginosa by static time-kill assays over 24 h. Polymyxin B and silver NPs alone were not active against polymyxin-resistant (MIC = 4 mg/L) P. aeruginosa. Whereas, the combination of a clinically-relevant concentration of polymyxin B (2 mg/L) with silver NPs (4 mg/L) successfully inhibited the growth of polymyxin-resistant P. aeruginosa isolates from CF patients as demonstrated by = 2 log10 decrease in bacterial count (CFU/mL) after 24 h. Treatment of P. aeruginosa cells with the combination induced cytosolic GFP release and an increase of cellular reactive oxygen species. In the nitrocefin assay, the combination displayed a membrane permeabilizing activity superior to each of the drugs alone. The combination of polymyxin B and silver NPs displays excellent synergistic activity against highly polymyxin-resistant P. aeruginosa and is potentially of considerable clinical utility for the treatment of problematic CF lung infections.

It is hard to fathom that one of the most highly differentiated cells in the body, the spermatozoon, spends over half of its developmental life without the capacity for nuclear protein biosynthesis. This is even more incredible when considering that protein synthesis is switched off long before the sperm is mature. As such, in order to obtain full functionality, spermatozoa rely on post-translational modifications (PTM) of existing proteins. Many PTM have been shown to play a role in the development of a sperm cell. These include phosphorylation and glycosylation events that occur both in the epididymis and during capacitation. In addition, several other PTM such as disulfide cross-linking, ubiquitination, acetylation and methylation all play a role to both develop and enable a spermatozoon to achieve its final destiny.

Globally, ~1 in 15 men of reproductive age are infertile, yet the precise mechanisms underlying their gamete failure are unknown. Although a semen analysis is performed to determine fertilizing potential, the diagnostic suitability of this analysis has been questioned in several reports, as many men, classified as infertile according to their semen analysis, subsequently turn out to be fertile. Herein, we have used a quantitative (phospho)-proteomic analysis, using enrichment on titanium dioxide followed by ion-trap mass spectrometry (LC-MS/MS), to compare the semen of infertile versus fertile males. One protein, namely outer dense fiber 1 (ODF1), was dramatically reduced in infertile males. Using specific antibodies, we then screened the gametes of a cohort of suspected infertile men and demonstrated a reduction in the amount of ODF1 compared with fertile controls. Stress treatment of sperm deficient in ODF1 caused the head to decapitate, suggesting why these gametes fail to initiate fertilization. Interestingly, electron micrographs of ODF1-deficient spermatozoa revealed an abnormal connecting piece, indicating several developmental defects with both the implantation plate and the thin laminated fibers. In some cases, the implantation plate appeared to be reduced in size or was overburdened by granular material near the connecting piece. Hence, a strong reduction ODF1 is a marker of idiopathic male infertility and a potential driver of this condition.

Ertapenem is an important first-line carbapenem antibiotic used for the treatment of aerobic Gram-negative bacterial infections. It is the only marketed carbapenem that is highly bound to plasma proteins and displays a concentration-dependent and saturable plasma protein binding profile. To date, the plasma components responsible for sequestering ertapenems antibacterial activity remain uncharacterized. In the present study, we have employed an orthogonal, multiplatform approach, including novel compound-centric displacement proteomics and surface plasmon resonance to characterize the plasma protein binding proteome of ertapenem. In proof-of-concept, the capacity of physiological cocktails of the identified plasma proteins to inhibit the antibacterial activity of ertapenem was assessed with in vitro microbiological assays. We show that fibrinogen, complement C4, haptoglobulin, a-1-antitrypsin, fibronectin, transferrin, immunoglobulin G, hemopexin, and humans serum albumin are responsible for the majority of the sequestering activity in plasma. No binding was observed to a-1-acid-glycoprotein. The findings of this study have broad reaching implications for antibiotic drug design and for dose tailoring to suit the plasma protein levels of individual patients in order to maximize the clinical efficacy of important first-line antibiotics such as ertapenem.

The concurrent increases in global population and sexually transmitted infection (STI) demand a search for agents with dual spermicidal and microbicidal properties for topical vaginal application. Previous attempts to develop the surfactant spermicide, nonoxynol-9 (N-9), into a vaginal microbicide were unsuccessful largely due to its inefficiency to kill microbes. Furthermore, N-9 causes damage to the vaginal epithelium, thus accelerating microbes to enter the women¿s body. For this reason, antimicrobial peptides (AMPs), naturally secreted by all forms of life as part of innate immunity, deserve evaluation for their potential spermicidal effects. To date, twelve spermicidal AMPs have been described including LL-37, magainin 2 and nisin A. Human cathelicidin LL-37 is the most promising spermicidal AMP to be further developed for vaginal use for the following reasons. First, it is a human AMP naturally produced in the vagina after intercourse. Second, LL-37 exerts microbicidal effects to numerous microbes including those that cause STI. Third, its cytotoxicity is selective to sperm and not to the female reproductive tract. Furthermore, the spermicidal effects of LL-37 have been demonstrated in vivo in mice. Therefore, the availability of LL-37 as a vaginal spermicide/microbicide will empower women for self-protection against unwanted pregnancies and STI.

Spermatozoa are flagellated cells whose role in fertilization is dependent on their ability to move towards an oocyte. The structure of the sperm flagella is highly conserved across species, and much of what is known about this structure is derived from studies utilizing animal models. One group of proteins essential for the movement of the flagella are the dyneins. Using the advanced technology of CRISPR/Cas9 we have targeted three dynein group members; Dnaic1, Wdr63 and Ccdc63 in mice. All three of these genes are expressed strongly in the testis. We generated mice with amino acid substitutions in Dnaic1 to analyze two specific phosphorylation events at S124 and S127, and generated simple knockouts of Wdr63 and Ccdc63. We found that the targeted phosphorylation sites in Dnaic1 were not essential for male fertility. Similarly, Wdr63 was not essential for male fertility; however, Ccdc63 removal resulted in sterile male mice due to shortened flagella. This study demonstrates the versatility of the CRISPR/Cas9 system to generate animal models of a highly complex system by introducing point mutations and simple knockouts in a fast and efficient manner.

The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (~750-1300kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

One of the major causes of defective sperm function is oxidative stress, which not only disrupts the integrity of sperm DNA but also limits the fertilizing potential of these cells as a result of collateral damage to proteins and lipids in the sperm plasma membrane. The origins of such oxidative stress appear to involve the sperm mitochondria, which have a tendency to generate high levels of superoxide anion as a prelude to entering the intrinsic apoptotic cascade. Unfortunately, these cells have very little capacity to respond to such an attack because they only possess the first enzyme in the base excision repair (BER) pathway, 8-oxoguanine glycosylase 1 (OGG1). The latter successfully creates an abasic site, but the spermatozoa cannot process the oxidative lesion further because they lack the downstream proteins (APE1, XRCC1) needed to complete the repair process. It is the responsibility of the oocyte to continue the BER pathway prior to initiation of S-phase of the first mitotic division. If a mistake is made by the oocyte at this stage of development, a mutation will be created that will be represented in every cell in the body. Such mechanisms may explain the increase in childhood cancers and other diseases observed in the offspring of males who have suffered oxidative stress in their germ line as a consequence of age, environmental or lifestyle factors. The high prevalence of oxidative DNA damage in the spermatozoa of male infertility patients may have implications for the health of children conceivedin vitro and serves as a driver for current research into the origins of free radical generation in the germ line. © 2014 AJA, SIMM & SJTU. All rights reserved.

Spermatozoa are quite unique amongst cell types. Although produced in the testis, both nuclear gene transcription and translation are switched off once the pre-cursor round cell begins to elongate and differentiate into what is morphologically recognized as a spermatozoon. However, the spermatozoon is very immature, having no ability for motility or egg recognition. Both of these events occur once the spermatozoa transit a secondary organ known as the epididymis. During the ~12 day passage that it takes for a sperm cell to pass through the epididymis, post-translational modifications of existing proteins play a pivotal role in the maturation of the cell. One major facet of such is protein phosphorylation. In order to characterize phosphorylation events taking place during sperm maturation, both pure sperm cell populations and pre-fractionation of phosphopeptides must be established. Using back flushing techniques, a method for the isolation of pure spermatozoa of high quality and yield from the distal or caudal epididymides is outlined. The steps for solubilization, digestion, and pre-fractionation of sperm phosphopeptides through TiO2 affinity chromatography are explained. Once isolated, phosphopeptides can be injected into MS to identify both protein phosphorylation events on specific amino acid residues and quantify the levels of phosphorylation taking place during the sperm maturation processes.

During early development, apoptosis plays a major role in the ontogeny of the germ line as a means of regulating the germ cell:Sertoli cell ratio. In the adult, apoptosis fulfils another function in removing damaged germ cells from the seminiferous epithelium in response to a wide range of physiological and environmental triggers. These include various forms of electromagnetic radiation, chemotherapeutic agents and commonly encountered toxicants such as phthalate es-ters, bisphenol A and cadmium. This form of apoptosis can lead to spermatogenic arrest and is predominantly mediated by the Fas/FasL system. In addition, senescent mature spermatozoa can undergo a truncated form of apoptosis in order to ensure their efficient phagocytosis within the male and female reproductive tracts. This apoptotic cascade appears to be triggered by oxidative stress and lipid peroxidation, which leads to activation of mitochondrial reactive oxygen species (ROS) generation in a self-perpetuating redox cycle. The electrophilic aldehydes generated as a result of lipid peroxidation also lead to a rapid loss of sperm motility followed some hours later by caspase activation and phosphatidylserine exposure on the sperm surface. The nuclear DNA suffers oxidative damage during this process but there is no immediate DNA cleavage by endonucleases as there is in somatic cells. The reasons for this deviation from the normal pattern of apoptosis involve the unusual physical architecture of spermatozoa and the limited capacity these cells possess for base-excision repair. These findings have practical implications for the approaches that might be used to detect and prevent DNA damage in spermatozoa. © 2013 UBC Press.

Hepatic clearance prediction, using scaled data obtained from hepatocytes and microsomes, often under-predicts the eventual observed clearance. This occurs commonly where the compound is highly bound and/or a sinusoidal transporter substrate. The authors' own laboratory observations and those reported in the literature indicate that consideration of transporter effects in vitro is not sufficient to provide a direct, quantitative estimate of hepatic clearance in vivo. The physiology of contributing processes has been reviewed and the processes were compiled into a kinetic model of compound disposition for a hepatocyte compartment. The model has variables describing the kinetic effects of plasma protein binding, sinusoidal uptake, passive permeability, and cellular disposition. Parameters were determined experimentally requiring, in some instances, assays less familiar or routine to Drug metabolism and pharmacokinetics (DMPK) laboratories. The model accurately fitted data for the hepatic disposition of a UCB-proprietary compound that did not undergo metabolism but was a substrate for protein binding and sinusoidal uptake. On addition of bovine serum albumin to the assay, the uptake kinetics approximated neither those for the free fraction nor the total concentration. However, the model accurately predicted the intermediate observed kinetics and illustrated the kinetic effects of plasma protein binding and the complex interplay between various competing and collaborating processes. Through the use of simulations the model illustrated the influence of different combinations and influences of hepatic kinetic processes. Possible causes behind the extraction of highly bound compounds were identified as (1) low compound permeability facilitating active uptake and (2) high permeability facilitating the role of intracellular metabolism. In almost all circumstances koff is not low enough (<1 s-1) to limit the extraction of plasma protein bound compounds; however, kon still exerts an effect through competition with the uptake process. Crucially the model indicated the complex nature of the combined processes, whose broad range of effects on hepatic disposition could only be accurately determined with a kinetic model. © 2007 Informa UK Ltd.

Although displacement from plasma protein binding (dPB) is usually of little clinical significance, it should be taken into account when interpreting changes in total plasma concentrations of drugs subject to metabolically based drug-drug interactions (mDDI). The aim of this study was to develop an approach to predict changes in the free fractions (fu) of pairs of drugs that compete for plasma binding, knowing their binding affinity constants, and to consider the implications of associated concentration- and time-dependence of such changes with respect to drug exposure. Experimental fu values of valproic acid and phenytoin in the presence of ibuprofen, diflunisal, or naproxen were predicted successfully (within 0.99- to 1.36-fold) by the model. In addition, the simulation of time-dependent changes in fu of valproic acid following administration of ibuprofen indicated different extents of dPB during 'first-pass' through the liver after oral absorption and on systemic recirculation. To understand the impact of the time-dependent change in fu, a full physiologically based pharmacokinetic model, that accounts for concentration-time profile of displacee and displacer and their mutual effect on each other, is required. The approach developed in this study is a first step towards the development of such a model. © 2006 Wiley-Liss, Inc.

Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1. GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis.

Mitochondrial porin or VDAC (Voltage Dependent Anion selective Channels) was identified for the first time in 1976, on the basis of the evolutionary similarity between the gram negative and mitochondrial outer membranes. Since this achievement VDAC has been extensively investigated: its functional features have been sharply defined upon reconstitution in artificial membranes and its sequence has been determined in many genomes. Unfortunately the tertiary structure has not yet been solved, mainly because it proved to be very difficult to get suitable crystals. Despite this established knowledge, in the last few years this protein has attracted renewed interest. There are two main reasons for this interest: the discovery, in most eukaryotes, of a family of genes encoding VDAC isoforms and the claims of VDAC involvement in the intrinsic pathway of apoptosis and in particular in the mechanism of cytochrome c release from mitochondria. We can affirm that nowadays the eukaryotic porin (or VDAC) is studied in a more general cellular contest, looking at the interactions and integration with other molecules, since VDAC is in a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. In this minireview we will briefly focus our attention onto the following topics: 1) recent advances about the structure of VDAC; 2) the VDAC-related multigene families; 3) the presence, targeting and function of VDAC in various cell membranes.