John V. Stokes

L-arginine enhances cell proliferation and reduces apoptosis in human endometrial RL95-2 cells

BackgroundL-arginine is considered to be one of the most versatile amino acids due to the fact that it serves as a precursor for many important molecules in cellular physiology. When supplemented in the diet, L-arginine can increase the number of implantation sites in mice and rats, suggesting an effect at the level of the endometrium. To this end, this study determined the effect that L-arginine has on apoptosis and cell proliferation in human endometrial RL95-2 cells.ResultsL-arginine at physiological (200 micromol/L) and supra-physiological (800 micromol/L) concentrations increased cell proliferation at days 2 and 4 post-treatment with a dose-dependent effect being observed on day 2. Additionally, inhibition of nitric oxide (NO) synthase and arginase, which are responsible for the conversion of L-arginine to NO and polyamines, respectively, reduced the proliferative effect of L-arginine. L-arginine also decreased the proportion of cells with TUNEL positive nuclei and increased the ratio of cells with healthy mitochondria compared to cells with a disrupted mitochondrial membrane potential, indicating that L-arginine prevents mitochondrial mediated apoptosis in endometrial RL95-2 cells. Furthermore, exposure to L-arginine did not affect total BAD protein expression; however, L-arginine increased the abundance of phosphorylated BAD protein.ConclusionsIn summary, L-arginine added to the culture media at physiological (200 micromol/L) and supraphysiological concentrations (800 micromol/L) enhanced endometrial RL95-2 cell proliferation through mechanisms mediated by NO and polyamine biosynthesis. In addition, L-arginine reduced endometrial RL95-2 mitochondrial mediated apoptosis through increased phosphorylation of BAD protein.

Bovine Viral Diarrhea Virus infection affects the expression of proteins related to professional antigen presentation in bovine monocytes.

The complete annotation of the cattle genome allows reliable protein identification by tandem mass spectrometry (MS(2)) and greatly facilitates proteomics. Previously, we reported that differential detergent fractionation (DDF) analysis of bovine monocytes reveals proteins related to antigen pattern recognition, uptake and presentation to immunocompetent lymphocytes. Here we have identified 47 bovine proteins, involved in immune function of professional antigen-presenting cells (APC) that have been significantly altered after cytopathic (cp) Bovine Viral Diarrhea Virus (BVDV) infection. In particular, proteins related to immune responses such as cell adhesion, apoptosis, antigen uptake, processing and presentation, acute phase response proteins, MHC class I- and II-related proteins and other molecules involved in immune function of professional antigen presentation have been significantly altered after BVDV infection. Our data suggest that cp BVDV, while promoting monocyte activation and differentiation, is inhibiting their antigen presentation to immunocompetent T cells, thus resulting in the uncontrolled inflammation mediated by activated macrophages, enhanced viral spread, and impaired anti-viral defense mechanisms in the host.

Interrelationships Between Apoptosis and Fertility in Bull Sperm

Abstract Male fertility, the ability of sperm to fertilize and activate the egg and support early embryogenesis, is vital for mammalian reproduction. Despite producing adequate numbers of sperm with normal motility and morphology, some males suffer from low fertility whose molecular mechanisms are not known. The objective was to determine apoptosis in sperm from high and low fertility bulls and its relationship with male fertility. DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) in the sperm were determined using TUNEL, Annexin V, and immunoblotting approaches, respectively. Amounts of apoptotic spermatozoa were 2.86 (± 1.31) and 3.00 (± 0.96) in high and low fertility bulls, respectively (P=0.548), and were not correlated with fertility. There was a negative correlation between early necrotic spermatozoa and viable spermatozoa (r = –0.99, P<0.0001). Fertility scores were correlated with live spermatozoa detected by eosin-nigrosin test and necrotic spermatozoa determined via flow cytometry (r = –0.49, P<0.006 and r = –0.266, P<0.0113, respectively). BAX level was higher in low fertile group than high fertile group; however, this difference was not statistically significant due to the variations of bull samples (Bull 1–3 vs. Bull 4–5) in low fertile group (P<0.283). BCL-2 was not detectable in any of the sperm samples. The results shed light onto molecular and cellular underpinnings of male fertility.

Exposure to an environmentally relevant mixture of organochlorine compounds and polychlorinated biphenyls Promotes hepatic steatosis in male Ob/Ob mice

Hepatic steatosis is recognized as an independent risk factor for the development of cardiovascular disease. While obesity and type 2 diabetes are well‐established risk factors in the development of hepatic steatosis, recent studies have revealed exposure to mixtures of persistent organic pollutants (POPs), which are environmental contaminants in various fatty foods, can promote steatosis. Thus, the present study was designed to determine if exposure to a defined mixture of prevalent polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides or their metabolites promote hepatic steatosis in a genetically induced model of type 2 diabetes, the leptin‐deficient ob/ob mouse. Male C57BL/6J wild type (WT) or ob/ob mice were administered an environmentally relevant mixture of PCBs and OCs for 7 weeks via oral gavage. Exposure to POPs did not significantly alter fasting serum glucose or insulin levels. However, POPs exposure significantly increased hepatic triglyceride content in ob/ob animals, while decreasing serum triglyceride levels. This POPs‐mediated increase in hepatic triglyceride content did not appear to be associated with significantly increased inflammation in either the liver or adipose. Exposure to POPs significantly induced the expression of cytochrome P450 3a11 in WT animals, yet the expression of this cytochrome was significantly downregulated in ob/ob animals regardless of POPs exposure. Taken together, the present data indicate exposure to an environmentally relevant mixture of both PCBs and OC pesticides in ob/ob mice promotes hepatic steatosis while decreasing hypertriglyceridemia, which demonstrates exposure to a defined mixture of POPs alters systemic lipid metabolism in a genetically induced model of obesity and type 2 diabetes.

Bovine viral diarrhea viruses differentially alter the expression of the protein kinases and related proteins affecting the development of infection and anti-viral mechanisms in bovine monocytes

Using a proteomics approach, we evaluated the effect of cytopathic (cp), and non-cytopathic (ncp) bovine viral diarrhea viruses (BVDV) on the expression of protein kinases and related proteins in bovine monocytes. Proteins were isolated from membrane and cytosolic fractions with the differential detergent fractionation (DDF) method and identified with 2D-LC ESI MS2. Of approximately 10,000 proteins identified, 378 proteins had homology with known protein kinases or related proteins. Eighteen proteins involved in cell differentiation and activation, migration, anti-viral mechanisms (interferon/apoptosis), biosynthesis, sugar metabolism and oncogenic transformation were significantly altered in BVDV-infected monocytes compared to the uninfected controls. Six proteins, mostly related to cell migration, anti-viral mechanisms, sugar metabolism and possibly tumor resistance were differentially expressed between the ncp and cp BVDV-infected monocytes. Particularly, the expression of the receptor of activated C kinase (RACK), of pyridoxal kinase (PK), diacyglycerol kinase (DGK) and Brutons tyrosine kinase (BTK) was decreased in monocytes infected with cp BVDV compared to ncp BVDV, possibly contributing to the cytopathic effect of the virus. This and other findings are discussed in view of the possible role the identified proteins play in the development of viral infection and oncogenic transformation of cells.

Profiling of relaxin and its receptor proteins in boar reproductive tissues and spermatozoa

BackgroundRelaxin levels in seminal plasma have been associated with positive effects on sperm motility and quality, and thus having potential roles in male fertility. However, the origin of seminal relaxin, within the male reproductive tract, and the moment of its release in the vicinity of spermatozoa remain unclear. Here, we assessed the longitudinal distribution of relaxin and its receptors RXFP1 and RXFP2 in the reproductive tract, sex accessory glands, and spermatozoa of adult boars.MethodsSpermatozoa were harvested from three fertile boars and reproductive tract (testes and epididymis) and sex accessory gland (prostate and seminal vesicles) tissues were collected post-mortem from each boar. Epididymis ducts were sectioned into caput, corpus, and cauda regions, and spermatozoa were mechanically collected. All samples were subjected to immunofluorescence and/or western immunoblotting for relaxin, RXFP1, and RXFP2 detection. Immunolabeled-spermatozoa were submitted to flow cytometry analyses and data were statistically analyzed with ANOVA.ResultsBoth receptors were detected in all tissues, with a predominance of mature and immature isoforms of RXFP1 and RXFP2, respectively. Relaxin signals were found in the testes, with Leydig cells displaying the highest intensity compared to other testicular cells. The testicular immunofluorescence intensity of relaxin was greater than that of other tissues. Epithelial basal cells exhibited the highest relaxin immunofluorescence intensity within the epididymis and the vas deferens. The luminal immunoreactivity to relaxin was detected in the seminiferous tubule, epididymis, and vas deferens ducts. Epididymal and ejaculated spermatozoa were immunopositive to relaxin, RXFP1, and RXFP2, and epididymal corpus-derived spermatozoa had the highest immunoreactivities across epididymal sections. Both vas deferens-collected and ejaculated spermatozoa displayed comparable, but lowest immunofluorescence signals among groups. The entire sperm length was immunopositive to both relaxin and receptors, with relaxin signal being robust in the acrosome area and RXFP2, homogeneously distributed than RXFP1 on the head of ejaculated spermatozoa.ConclusionsImmunolocalization indicates that relaxin-receptor complexes may have important roles in boar reproduction and that spermatozoa are already exposed to relaxin upon their production. The findings suggest autocrine and/or paracrine actions of relaxin on spermatozoa, either before or after ejaculation, which have possible roles on the fertilizing potential of spermatozoa.

Immunomodulation By Subchronic Low Dose 2,3,7,8-Tetrachlorodibenzo-p-Dioxin in Experimental Autoimmune Encephalomyelitis in the Absence of Pertussis Toxin

Multiple sclerosis (MS) is an autoimmune neurodegenerative disorder, characterized by demyelination of neurons in the central nervous system. To investigate the pathogenicity of various T cell types in MS, especially IFN-γ- or IL-17-producing CD4(+ )cells (TH1 or TH17 cells, respectively), the mouse model, experimental autoimmune encephalomyelitis (EAE), is commonly used. One method by which EAE is induced is immunization with myelin oligodendrocyte glycoprotein (MOG) peptide (MOG35-55) followed by subsequent injections of pertussis toxin (PTX) as an adjuvant. We have an interest in the mechanisms by which EAE occurs in the absence of PTX because it induces a milder disease state more consistent with autoimmune disease onset and PTX inactivates Gi/o protein-coupled receptors, many of which contribute to immune homeostasis. Another receptor that plays a role in immune homeostasis is the aryl hydrocarbon receptor (AHR). In fact, the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to attenuate EAE pathogenesis by affecting CD4(+ )T and regulatory T (Treg) cells in an AHR-dependent manner. However, many of these studies have been conducted with an acute high dose TCDD. Thus, the goal of this work was to investigate the modulation of MOG-specific immune responses with subchronic low dose TCDD (0.1-1.0 μg/kg/d for 12 days) in EAE without PTX. The results demonstrate that subchronic, low dose exposure of TCDD attenuates the immune responses in EAE development in the absence of PTX, which is due in part to suppression of MOG-specific IL-17A and IFN-γ responses.

Polyamine transporter in Streptococcus pneumoniae is essential for evading early innate immune responses in pneumococcal pneumonia

Streptococcus pneumoniae is the most common bacterial etiology of pneumococcal pneumonia in adults worldwide. Genomic plasticity, antibiotic resistance and extreme capsular antigenic variation complicates the design of effective therapeutic strategies. Polyamines are ubiquitous small cationic molecules necessary for full expression of pneumococcal virulence. Polyamine transport system is an attractive therapeutic target as it is highly conserved across pneumococcal serotypes. In this study, we compared an isogenic deletion strain of S. pneumoniae TIGR4 in polyamine transport operon (ΔpotABCD) with the wild type in a mouse model of pneumococcal pneumonia. Our results show that the wild type persists in mouse lung 24 h post infection while the mutant strain is cleared by host defense mechanisms. We show that intact potABCD is required for survival in the host by providing resistance to neutrophil killing. Comparative proteomics analysis of murine lungs infected with wild type and ΔpotABCD pneumococci identified expression of proteins that could confer protection to wild type strain and help establish infection. We identified ERM complex, PGLYRP1, PTPRC/CD45 and POSTN as new players in the pathogenesis of pneumococcal pneumonia. Additionally, we found that deficiency of polyamine transport leads to up regulation of the polyamine synthesis genes speE and cad in vitro.

Sperm superoxide dismutase is associated with bull fertility

Decreasing mammalian fertility and sperm quality have created an urgent need to find effective methods to distinguish non-viable from viable fertilising spermatozoa. The aims of the present study were to evaluate expression levels of ?-tubulin 2C (TUBB2C), heat shock protein 10 (HSP10), hexokinase 1 (HXK1) and superoxide dismutase 1 (SOD1) in spermatozoa from Holstein bulls with varying fertility using western blotting and to analyse the biological networks of these key sperm proteins using a bioinformatics software (Metacore; Thomson-Reuters, Philadelphia, PA, USA). The rationales behind this study were that the sperm proteins play crucial roles in fertilisation and early embryonic development in mammals and ascertaining the biological networks of the proteins helps us better understand sperm physiology and early mammalian development. The results showed that expression of SOD1 was higher in spermatozoa from high fertility bulls (PPin vivo bull fertility. The findings are important because they illuminate molecular and cellular determinants of sperm viability and the identified protein markers can be used to determine bull fertility.

Cannabidiol (CBD) induces functional Tregs in response to low-level T cell activation.

Many effects of the non-psychoactive cannabinoid, cannabidiol (CBD), have been described in immune responses induced by strong immunological stimuli. It has also been shown that CBD enhances IL-2 production in response to low-level T cell stimulation. Since IL-2, in combination with TGF-β1, are critical for Treg induction, we hypothesized that CBD would induce CD4+CD25+FOXP3+ Tregs in response to low-level stimulation. Low-level T cell stimulation conditions were established based on minimal CD25 expression in CD4+ cells using suboptimal PMA/Io (4nM/0.05μM, S/o), ultrasuboptimal PMA/Io (1nM/0.0125μM, Us/o) or soluble anti-CD3/28 (400-800ng each, s3/28). CBD increased CD25+FOXP3+ cells from CD4+, CD4+CD25+, and CD4+CD25- T cells, as well as in CD4+ T cells derived from FOXP3-GFP mice. Most importantly, the Us/o+CBD-induced CD4+CD25+ Tregs robustly suppressed responder T cell proliferation, demonstrating that the mechanism by which CBD is immunosuppressive under low-level T cell stimulation involves induction of functional Tregs.