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11.
12.
In an attempt to identify the brain structures that synthesize melatonin and that probably mediate the photoperiodic response of the heteronemertean Myoisophagos lacteus, we utilized immunocytochemical techniques and employed immunoglobulins directed against hydroxyindole-O-methyltransferase (HIOMT, EC 2.1.1.4). This enzyme catalyzes the last step of melatonin biosynthesis. In immunocytochemically treated head sections of Myoisophagos lacteus, antibodies labelled a few cells in the dorsal region of the dorsal cerebral ganglia. Previous studies have shown that melatonin is present both in the brain and eyes of this nemertean species and that melatonin is involved in control of the worm reproduction. Other studies have demonstrated the presence of photoreceptor-like cells in the same region of the worm brain that showed HIOMT immunostaining. Therefore, anatomical findings of the present study, coupled with results of previous works, provide strong evidence that this region of the worm brain houses a photoperiodic receptor involved in melatonin biosynthesis. J. Exp. Zool. 290:156-162, 2001. 相似文献
13.
Vernet P Rock E Mazur A Rayssiguier Y Dufaure JP Drevet JR 《Molecular reproduction and development》1999,54(4):362-370
We have previously characterized and cloned a secreted sperm-bound selenium-independent glutathione peroxidase protein (GPX5), the expression of which was found to be restricted to the mouse caput epididymidis. Because of the lack of selenium (Se) in the active site of this enzyme, unlike the other animal GPXs characterized to date, it was suspected that GPX5 does not function in the epididymis as a true glutathione peroxidase in vivo. In the present report, following dietary selenium deprivation which is known to reduce antioxidant defenses and favor oxidative stress in relation with depressed Se-dependent GPX activities, we show that the epididymis is still efficiently protected against increasing peroxidative conditions. In this model, the caput epididymides of selenium-deficient animals showed a limited production of lipid peroxides, a total GPX activity which was not dramatically affected by the shortage in selenium availability and an increase in GPX5 mRNA and protein levels. Altogether, these data strongly suggest that the selenium-independent GPX5 could function as a back-up system for Se-dependent GPXs. 相似文献
14.
Role of vascular endothelial-cadherin in vascular morphogenesis 总被引:24,自引:0,他引:24
Gory-Fauré S Prandini MH Pointu H Roullot V Pignot-Paintrand I Vernet M Huber P 《Development (Cambridge, England)》1999,126(10):2093-2102
Vascular endothelial (VE)-cadherin is an adhesive transmembrane protein specifically expressed at interendothelial junctions. Its extracellular domain exhibits Ca2+-dependent homophilic reactivity, promoting cell-cell recognition. Mice deficient in VE-cadherin die at mid-gestation resulting from severe vascular defects. At the early phases of vascular development (E8.5) of VE-cadherin-deficient embryos, in situ differentiation of endothelial cells was delayed although their differentiation program appeared normal. Vascularization was defective in the anterior part of the embryo, while dorsal aortae and vitelline and umbilical arteries formed normally in the caudal part. At E9.25, organization of endothelial cells into large vessels was incomplete and angiogenesis was impaired in mutant embryos. Defects were more severe in extraembryonic vasculature. Blood islands of the yolk sac and clusters of angioblasts in allantois failed to establish a capillary plexus and remained isolated. This was not due to defective cell-cell recognition as endothelial cells formed intercellular junctions, as shown by electron microscopy. These data indicate that VE-cadherin is dispensable for endothelial homophilic adhesion but is required for vascular morphogenesis. 相似文献
15.
Choulier L Rauffer-Bruyère N Ben Khalifa M Martin F Vernet T Altschuh D 《Biochemistry》1999,38(12):3530-3537
Monoclonal antibody 57P, which was raised against tobacco mosaic virus protein, cross-reacts with a peptide corresponding to residues 134-146 of this protein. Previous studies using peptide variants suggested that the peptide in the antibody combining site adopts a helical configuration that mimics the structure in the protein. In this study, we carried out a detailed comparison of Fab-peptide and Fab-protein interactions. The same five amino acid substitutions were introduced in the peptide (residues 134-151) and the parent protein, and the effect of these substitutions on antibody binding parameters have been measured with a Biacore instrument. Fabs that recognize epitopes located away from the site of mutations were used as indirect probes for the conformational integrity of protein antigens. Their interaction kinetics with all proteins were similar, suggesting that the substitutions had no drastic effect on their conformation. The five substitutions introduced in the peptide and the protein had minor effects on association rate constants (ka) and significant effects on dissociation rate constants (kd) of the antigen-Fab 57P interactions. In four out of five cases, the effect on binding affinity of the substitutions was identical when the epitope was presented in the form of a peptide or a protein antigen, indicating that antibody binding specifity was not affected by epitope presentation. However, ka values were about 10 times larger and kd values about 5 times larger for the peptide-Fab compared to the protein-Fab interaction, suggesting a different binding mechanism. Circular dichroism measurements performed for three of the peptides showed that they were mainly lacking structure in solution. Differences in conformational properties of the peptide and protein antigens in solution and/or in the paratope could explain differences in binding kinetics. Our results demonstrate that the peptides were able to mimic correctly some but not all properties of the protein-Fab 57P interaction and highlight the importance of quantitative analysis of both equilibrium and kinetic binding parameters in the design of synthetic vaccines and drugs. 相似文献
16.
Gene and protein expression in the epididymis of infertile c-ros receptor tyrosine kinase-deficient mice 总被引:2,自引:0,他引:2
Cooper TG Wagenfeld A Cornwall GA Hsia N Chu ST Orgebin-Crist MC Drevet J Vernet P Avram C Nieschlag E Yeung CH 《Biology of reproduction》2003,69(5):1750-1762
Transgenic male mice bearing inactive mutations of the receptor tyrosine kinase c-ros lack the initial segment of the epididymis and are infertile. Several techniques were applied to determine differences in gene expression in the epididymal caput of heterozygous fertile (HET) and infertile homozygous knockout (KO) males that may explain the infertility. Complementary DNA arrays, gene chips, Northern and Western blots, and immunohistochemistry indicated that some proteins were downregulated, including the initial segment/proximal caput-specific genes c-ros, cystatin-related epididymal-spermatogenic (CRES), and lipocalin mouse epididymal protein 17 (MEP17), whereas other caput-enriched genes (glutathione peroxidase 5, a disintegrin and metalloproteinase [ADAM7], bone morphogenetic proteins 7 and 8a, A-raf, CCAAT/enhancer binding protein beta, PEA3) were unchanged. Genes normally absent from the initial segment (gamma-glutamyltranspeptidase, prostaglandin D2 synthetase, alkaline phosphatase) were expressed in the undifferentiated proximal caput of the KO. More distally, lipocalin 2 (24p3), CRISP1 (formerly MEP7), PEBP (MEP9), and mE-RABP (MEP10) were unchanged in expression. Immunohistochemistry and Western blots confirmed the absence of CRES in epididymal tissue and fluid and the continued presence of CRES in spermatozoa of the KO mouse. The glutamate transporters EAAC1 (EAAT3) and EAAT5 were downregulated and upregulated, respectively. The genes of over 70 transporters, channels, and pores were detected in the caput epididymidis, but in the KO, only three were downregulated and six upregulated. The changes in these genes could affect sperm function by modifying the composition of epididymal fluid and explain the infertility of the KO males. These genes may be targets for a posttesticular contraceptive. 相似文献
17.
Gene therapy of erectile dysfunction in the rat with penile neuronal nitric oxide synthase 总被引:3,自引:0,他引:3
Magee TR Ferrini M Garban HJ Vernet D Mitani K Rajfer J Gonzalez-Cadavid NF 《Biology of reproduction》2002,67(3):1033-1041
Gene transfer to the penile corpora cavernosa of constructs of the inducible and endothelial nitric oxide synthase (NOS) cDNAs ameliorates erectile dysfunction in aged rats. In this study, we investigated whether the neuronal NOS (nNOS) variant responsible for erection, penile nNOS (PnNOS), can exert a similar effect, and whether the combination of electroporation with a helper-dependent adenovirus (AdV) improves gene transfer. PnNOS and beta-galactosidase cDNAs were cloned in plasmid (pCMV-PnNOS; pCMV-beta-gal) and "gutless" AdV (AdV-CMV-PnNOS; AdV-CMV-beta-gal) vectors, and injected into the penis of adult (beta-gal) or aged (PnNOS) rats, with or without electroporation. Penile erection was measured at different times after PnNOS cDNA injection, by electrical field stimulation of the cavernosal nerve. The expression of beta-galactosidase or PnNOS was estimated in penile tissue by either histochemistry and luminometry or Western blot, and the effects of AdV-CMV-PnNOS on mRNA expression were examined by a DNA microarray. We found that electroporation increased pCMV-beta-gal uptake, and its expression was detectable at 56 days. In the aged rats treated with pCMV-PnNOS and electroporation, the maximal intracavernosal:mean arterial pressure ratios were elevated for 11 and 18 days when compared with those in controls. Electroporation intensified penile uptake of as few as 10(6) viral particles (vp) of AdV-CMV-beta-gal, and with 10(7) vp beta-galactosidase was still detectable at 60 days. Electroporated AdV-CMV-PnNOS (10(7) vp) was effective at 18 days in stimulating the erection of aged rats, without inducing the expression of cytotoxic genes. In conclusion, intracavernosal gene therapy with PnNOS cDNA corrected the aging-related erectile dysfunction for at least 18 days when given by electroporation in a helper-dependent AdV at low viral loads. 相似文献
18.
Hugo N Weidenhaupt M Beukes M Xu B Janson JC Vernet T Altschuh D 《Protein engineering》2003,16(5):381-386
Predictive engineering of antibodies exhibiting fast kinetic properties could provide reagents for biotechnological applications such as continuous monitoring of compounds or affinity chromatography. Based on covariance analysis of murine germline antibody variable domains, we selected position L34 (Kabat numbering) for mutational studies. This position is located at the VL/VH interface, at the base of the paratope but with limited antigen contacts, thus making it an attractive position for mild alterations of antigen binding properties. We introduced a serine at position L34 in two different antibodies: Fab (fragment antigen binding) 57P (Asn34Ser) and scFv (single chain fragment variable) 1F4 (Gln34Ser), that recognize peptides derived from the coat protein of tobacco mosaic virus and the oncoprotein E6, respectively. Both mutated antibodies exhibited similar properties: (i) expression levels of active fragments in Escherichia coli were markedly improved; (ii) thermostability was enhanced; and (iii) dissociation rate parameters (k(off)) were increased by 2- and at least 57-fold for scFv1F4 and Fab57P, respectively, while their association rate parameters (k(on)) remained unchanged. The L34 Ala and Thr mutants of both antibody fragments did not possess these properties. This first demontration of similar effects observed in two antibodies with different specificities may open the way to the predictive design of molecules with enhanced stability and fast dissociation rates. 相似文献
19.
The objectives of the present paper were to review and quantitatively determine the influence of the nutritional factors on whole body glucose turnover in growing and adult non-productive ruminants. A meta-analysis approach was used. The dietary grain: forage ratio significantly increased the slope of the relationship between glucose turnover and metabolisable energy intake. This effect was probably associated with the inclusion of maize rather than any other grain source in the diet. The analysis pointed out the possible differences in response between growing and adult non-productive animals, and suggested that the performance level of the animals (and their glucose requirements) could contribute to regulating whole body glucose turnover. This aspect would warrant further investigation. 相似文献
20.
Khalifa MB Weidenhaupt M Choulier L Chatellier J Rauffer-Bruyère N Altschuh D Vernet T 《Journal of molecular recognition : JMR》2000,13(3):127-139
The influence of framework residues belonging to VH and VL modules of antibody molecules on antigen binding remains poorly understood. To investigate the functional role of such residues, we have performed semi-conservative amino acid replacements at the VH-VL interface. This work was carried out with (i) variants of the same antibody and (ii) with antibodies of different specificities (Fab fragments 145P and 1F1h), in order to check if functional effects are additive and/or similar for the two antibodies. Interaction kinetics of Fab mutants with peptide and protein antigens were measured using a BIACORE instrument. The substitutions introduced at the VH-VL interface had no significant effects on k(a) but showed small, significant effects on k(d). Mutations in the VH module affected k(d) not only for the two different antibodies but also for variants of the same antibody. These effects varied both in direction and in magnitude. In the VL module, the double mutation F(L37)L-Q(L38)L, alone or in combination with other mutations, consistently decreased k(d) about two-fold in Fab 145P. Other mutations in the VL module had no effect on k(d) in 145P, but always decreased k(d) in 1F1h. Moreover, in both systems, small-magnitude non-additive effects on k(d) were observed, but affinity variations seemed to be limited by a threshold. When comparing functional effects in antibodies of different specificity, no general rules could be established. In addition, no clear relationship could be pointed out between the nature of the amino acid change and the observed functional effect. Our results show that binding kinetics are affected by alteration of framework residues remote from the binding site, although these effects are unpredictable for most of the studied changes. 相似文献