Anatomical evidence for glutamatergic transmission in primary sensory neurons and onto postganglionic neurons controlling penile erection in rats: an ultrastructural study with neuronal tracing and immunocytochemistry

In male rats, the dorsal penile nerve (DPN) conveys sensory information from the genitals to the lumbosacral spinal segments of the spinal cord. DPN is the afferent limb of a reflex loop that supports reflexive erections, and that includes a network of spinal interneurons and autonomic and somatic motoneurons to the penis and perineal striated muscles. Autonomic efferent pathways to the penis relay in the major pelvic ganglion (MPG). Glutamate (Glu) is a likely candidate as a neurotransmitter of reflexive erections. Both AMPA and NMDA glutamatergic receptor subunits are present in the lumbosacral spinal cord, and AMPA and NMDA receptor antagonists block reflexive erections. In the present study, we used tract-tracing experiments combined with immunohistochemical and immunocytochemical techniques to ascertain the presence of Glu at two different levels of the network controlling reflexive erections. DPN afferents were localized in the dorsal horn of the lumbosacral cord and displayed the characteristics of either C-fibers or Aδ fibers. DPN terminals (some of them glutamatergic) were mainly distributed in the medial edge of the dorsal horn in the L6 spinal segment. GluR1 subunits were present in some DPN afferents, suggesting that they could be autoreceptors. DPN fibers were also present in the MPG, as were Glu terminals and GluR4 subunits. The results reveal the presence of Glu in DPN fibers and terminals and suggest that both the spinal cord and the MPG use glutamatergic transmission to control reflexive erections. This work was supported by grant no. 5R01MH059811–03 from the NIH and by an institutional grant from the Institut National de la Recherche Agronomique.     

XRCC1 is phosphorylated by DNA-dependent protein kinase in response to DNA damage

The two BRCT domains (BRCT1 and BRCT2) of XRCC1 mediate a network of protein–protein interactions with several key factors of the DNA single-strand breaks (SSBs) and base damage repair pathways. BRCT1 is required for the immediate poly(ADP–ribose)-dependent recruitment of XRCC1 to DNA breaks and is essential for survival after DNA damage. To better understand the biological role of XRCC1 in the processing of DNA ends, a search for the BRCT1 domain-associated proteins was performed by mass spectrometry of GST-BRCT1 pulled-down proteins from HeLa cell extracts. Here, we report that the double-strand break (DSB) repair heterotrimeric complex DNA-PK interacts with the BRCT1 domain of XRCC1 and phosphorylates this domain at serine 371 after ionizing irradiation. This caused XRCC1 dimer dissociation. The XRCC1 R399Q variant allele did not affect this phosphorylation. We also show that XRCC1 strongly stimulates the phosphorylation of p53-Ser15 by DNA-PK. The pseudo phosphorylated S371D mutant was a much weaker stimulator of DNA-PK activity whereas the non-phosphorylable mutant S371L endowed with a DNA-PK stimulating capacity failed to fully rescue the DSB repair defect of XRCC1-deficient EM9 rodent cells. The functional association between XRCC1 and DNA-PK in response to IR provides the first evidence for their involvement in a common DSB repair pathway.     

Zinc supplementation does not alter plasma homocysteine,vitamin B12 and red blood cell folate concentrations in French elderly subjects

Background/aimsIn ageing, low folates and vitamin B12 status are frequent and can explain the increase of plasma homocysteine level. Zinc is involved in the folates and vitamin B12 metabolism with opposite actions. The aim of this study was to investigate the effects of zinc supplementation on homocysteine and vitamin B12 plasma levels as well as red blood cell folate level in French ageing subjects participating in the ZENITH study.MethodsApparently healthy middle-aged (55–70 years) and free-living older (70–85 years) subjects were enrolled. They were randomly allocated to three groups: 0, 15 or 30 mg Zn per day for 6 months as zinc gluconate in addition to their usual dietary intake.ResultsAt baseline, plasma homocysteine levels (15.2±3.5 μmol/L) in older people were higher than in the middle-aged subjects (12.7±2.7 μmol/L) and was negatively correlated with vitamin B12 values (p=0.0036, r=?0.215) and with RBC folate levels (p<0.0001, r=?0.30). These results are in agreement with previous data. However, we found no correlation between the biomarkers of zinc status and homocysteine, vitamin B12 or folate levels at baseline. Moreover, 6-month zinc supplementation did not modify homocysteine, vitamin B12 and RBC folate values in either of the groups.ConclusionsZinc supplementation at moderate doses do not lead to deleterious effect on folate or vitamin B12 status in ageing healthy free-living people, but does not have any beneficial effects on homocysteine metabolism either.     

Evolutionary Dynamics of Human Toll-Like Receptors and Their Different Contributions to Host Defense

Infectious diseases have been paramount among the threats to health and survival throughout human evolutionary history. Natural selection is therefore expected to act strongly on host defense genes, particularly on innate immunity genes whose products mediate the direct interaction between the host and the microbial environment. In insects and mammals, the Toll-like receptors (TLRs) appear to play a major role in initiating innate immune responses against microbes. In humans, however, it has been speculated that the set of TLRs could be redundant for protective immunity. We investigated how natural selection has acted upon human TLRs, as an approach to assess their level of biological redundancy. We sequenced the ten human TLRs in a panel of 158 individuals from various populations worldwide and found that the intracellular TLRs—activated by nucleic acids and particularly specialized in viral recognition—have evolved under strong purifying selection, indicating their essential non-redundant role in host survival. Conversely, the selective constraints on the TLRs expressed on the cell surface—activated by compounds other than nucleic acids—have been much more relaxed, with higher rates of damaging nonsynonymous and stop mutations tolerated, suggesting their higher redundancy. Finally, we tested whether TLRs have experienced spatially-varying selection in human populations and found that the region encompassing TLR10-TLR1-TLR6 has been the target of recent positive selection among non-Africans. Our findings indicate that the different TLRs differ in their immunological redundancy, reflecting their distinct contributions to host defense. The insights gained in this study foster new hypotheses to be tested in clinical and epidemiological genetics of infectious disease.     

Endothelial cells within embryonic skeletal muscles: a potential source of myogenic progenitors

We investigated whether the vessel-associated or endothelial cells within mouse embryo muscles can be a source of myogenic progenitors. Immunodetection of the stem cell surface markers, CD34 and Flk1, which are known to characterize the endothelial lineage, was done throughout the course of embryo muscle development. Both markers appeared to be restricted to the vessel-associated cells. On the basis of CD34 labeling, the reactive cells were purified by magnetic-bead selection from the limb muscles of 17-dpc desmin+/-LacZ mouse embryos and characterized by fluorescence-activated cell sorting. The cells in the selected CD34(+) population appeared to be approximately 95% positive for Flk1, but usually negative for CD45. We demonstrated that in vitro the CD34(+)/Flk1(+) population differentiated into endothelial cells and skeletal myofibers. When transplanted into mdx mouse muscle, this population displayed a high propensity to disperse within the recipient muscle, fuse with the host myofibers, and restore dystrophin expression. The marked ability of the embryonic muscle endothelial cells to activate myogenic program could be related to their somitic origin.     

Characterization of pancreatic lipase-related protein 2 isolated from human pancreatic juice

Human pancreatic lipase-related protein 2 (HPLRP2) was identified for the first time in pancreatic juice using specific anti-peptide antibodies and purified to homogeneity. Antibodies were raised in the rabbit using a synthetic peptide from the HPLRP2 protein sequence deduced from cDNA. Western blotting analysis showed that these antibodies did not react with classical human pancreatic lipase (HPL) or human pancreatic lipase-related protein 1 (HPLRP1) but cross-reacted with native rat PLRP2 (RPLRP2), as well as with recombinant rat and guinea-pig PLRP2 (GPLRP2). Immunoaffinity chromatography was performed on immobilized anti-recombinant HPLRP2 polyclonal antibodies to purify native HPLRP2 after conventional chromatographic steps including gel filtration and chromatrography on an anion-exchanger. The substrate specificity of HPLRP2 was investigated using various triglycerides, phospholipids and galactolipids as substrates. The lipase activity on triglycerides was inhibited by bile salts and weakly restored by colipase. The phospholipase activity of HPLRP2 on phospholipid micelles was very low. A significant level of galactolipase activity was measured using monogalactosyldiglyceride monomolecular films. These data suggest that the main physiological function of HPLRP2 is the hydrolysis of galactolipids, which are the main lipids present in vegetable food.     

An experimental test of the dose-dependent effect of carotenoids and immune activation on sexual signals and antioxidant activity

Carotenoid-based sexual traits are thought to be reliable indicators of male quality because they might be scarce and therefore might indicate the ability of males to gather high-quality food and because they are involved in important physiological functions (as immune enhancers and antioxidants). We performed an experiment where male and female zebra finches (Taeniopygia guttata) were provided with increasing carotenoid doses in the drinking water during 4 weeks (bill color of this species is a carotenoid-based sexual signal). Simultaneously, birds were split into two groups: one receiving weekly injections of Escherichia coli lipopolysaccharide in order to activate the immune system, the other being injected with the same volume of phosphate buffered saline. We assessed how carotenoid availability and immune activation affected the amount of circulating plasma carotenoids, the beak color, and the antioxidant defenses (assessed as the resistance of red blood cells to a controlled free radical attack). Carotenoid availability affected the amount of circulating carotenoids and beak color; both variables reached a plateau at the highest carotenoid doses. Immune activation diverted carotenoids from plasma, and this in turn affected the expression of the sexual trait. Finally, we found a positive correlation between the change in circulating carotenoids and antioxidant defenses. These results support the idea that carotenoids have important physiological properties that ensure the honesty of carotenoid-based sexual traits.     

Glutathione depletion increases nitric oxide-induced oxidative stress in primary rat hepatocyte cultures: involvement of low-molecular-weight iron

Various drugs and chemicals can cause a glutathione (GSH) depletion in the liver. Moreover, nitric oxide (NO) can be generated in response to physiological and pathological situations such as inflammation. The aim of this study was to estimate oxidative stress when primary rat hepatocytes were exposed to GSH depletion after NO production. For this purpose, cells were preincubated with lipopolysaccharide (LPS) and gamma-interferon (IFN) for 18 h in order to induce NO production by NO synthase and then L-buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, was added for 5 h. In hepatocyte cultures preincubated with LPS and IFN before BSO addition, an increase in lipid peroxidation was noted. In those cells, an elevation of iron-bound NO and a decrease in free NO led us to suggest the involvement of low-molecular-weight iron (LMW iron) in the enhancement of oxidative stress. Indeed, addition of deferiprone, a chelator of LMW iron, reduced iron-bound NO levels and the extent of oxidative stress. Moreover, an important elevation of LMW iron levels was also observed. As both, N-acetylcysteine, a GSH precursor, and N(G)-monomethyl-L-arginine, a NO synthase inhibitor, totally inhibited the elevation of LMW iron and oxidative stress, a cooperative role could be attributed to NO production and GSH depletion.