Polymerase chain reaction assay specific for pathogenic Leptospira based on the gene hap1 encoding the hemolysis-associated protein-1

In this study, we used Southern hybridization of genomic DNA with the integral hap1 gene as a probe to show that this gene is only present in pathogenic Leptospira strains. We then selected PCR primers based on the hap1 gene, and tested them on several Leptospira strains and biological samples. Specific amplification was obtained for all pathogenic strains tested. Negative PCR results were observed with all saprophytic leptospire strains used as well as with other spirochetes and bacteria commonly found in biological samples. The results of direct PCR performed on biological samples, such as blood, urine or kidneys correlated with the results obtained with the classical Leptospira tests (culture and MAT). A PCR assay based on this gene would be a very useful tool for the rapid, sensitive and specific identification of pathogenic leptospires in samples for diagnosis or epidemiological survey.     

Aerobic and anaerobic biodegradability of 1-Anthraquinone sulphonate

 The present work investigates 1-anthraquinone sulphonate (1-AS) biodegradation under (i) aerobic conditions using domestic activated sludge as inoculum, (ii) anaerobic conditions using sludge from an anaerobic domestic wastewater treatment digestor in a sulphate-containing or methanogenic environment, (iii) a combination of anaerobic followed by aerobic conditions. The process was evaluated in terms of primary degradation, i.e. 1-AS elimination and ultimate degradation, as total dissolved organic carbon removal. It was shown that aerobic conditions lead to the complete primary and ultimate degradation, of 1-AS. By contrast, neither under sulphato-reductive nor methanogenic conditions does anaerobic digestion lead to the significant degradation of 1-AS. The use of anaerobic treatment followed by aerobic treatment did not improve degradation. Indeed aerobic post-treatment resulted in the re-appearance of pollutant in the medium even though this had been partly degraded under anaerobic conditions. Received: 12 October 1995/Received revision: 18 December 1995/Accepted: 8 January 1996     

Chemical and immunochemical characterization of caseins and the major whey proteins of rabbit milk.

Caseins were separated from whey proteins by acid precipitation of skimmed rabbit milk. Whole casein was resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis into three major bands with apparent relative molecular masses (Mr of 31 000, 29 000 and 25 000. On agarose/urea-gel electrophoresis whole casein gave three bands with electrophoretic mobilities alpha, beta and gamma. The three components were purified by DEAE-cellulose chromatography under denaturing and reducing conditions. Each was shown to have a different amino acid, hexose and phosphorus content, as well as non-identical peptide fragments after proteinase digestion. The 31 000 Da (dalton) protein, of alpha-electrophoretic mobility, had a high phosphorus content (4.38%, w/w); the 29 000 Da peptide, of gamma-mobility, had the highest hexose content (2.2%, w/w), contained 0.8 cysteine residue per 100 amino acid residues and was susceptible to chymosin digestion corresponding thus to kappa-casein; the 25 000 Da protein migrated to the beta-position. The rabbit casein complex is composed of at least three caseins, two of which (alpha- and kappa-caseins) are analogous to the caseins from ruminants. Although caseins are poor immunogens, specific antibodies were raised against total and purified polypeptides. The antiserum directed against whole casein recognized each polypeptide, each casein corresponding to a distinct precipitation line. The antisera directed against each casein polypeptide reacted exclusively with the corresponding casein and no antiserum cross-reaction occurred between the three polypeptides. From whey, several proteins were isolated, characterized and used as antigens to raise specific antibodies. An iron-binding protein with an apparent Mr of 80 000 was shown to be immunologically and structurally identical with serum transferrin.     

Role of IL‐15 in spinal cord and sciatic nerve after chronic constriction injury: regulation of macrophage and T‐cell infiltration

The release of inflammatory mediators from immune and glial cells either in the peripheral or CNS may have an important role in the development of physiopathological processes such as neuropathic pain. Microglial, then astrocytic activation in the spinal cord, lead to chronic inflammation, alteration of neuronal physiology and neuropathic pain. Standard experimental models of neuropathic pain include an important peripheral inflammatory component, which involves prominent immune cell activation and infiltration. Among potential immunomodulators, the T‐cell cytokine interleukin‐15 (IL‐15) has a key role in regulating immune cell activation and glial reactivity after CNS injury. Here we show, using the model of chronic constriction of the sciatic nerve (CCI), that IL‐15 is essential for the development of the early inflammatory events in the spinal cord after a peripheral lesion that generates neuropathic pain. IL‐15 expression in the spinal cord was identified in both astroglial and microglial cells and was present during the initial gliotic and inflammatory (NFκB) response to injury. The expression of IL‐15 was also identified as a cue for macrophage and T‐cell activation and infiltration in the sciatic nerve, as shown by intraneural injection of the cytokine and activity blockage approaches. We conclude that the regulation of IL‐15 and hence the initial events following its expression after peripheral nerve injury could have a future therapeutic potential in the reduction of neuroinflammation.     

Antiobesity designed multiple ligands: Synthesis of pyrazole fatty acid amides and evaluation as hypophagic agents

Searching for new antiobesity agents, a new series of fatty acid amide derivatives of 1,5-diarylpyrazole have been synthesized as dual peroxisome proliferator activated receptor alpha (PPARalpha)/cannabinoid receptor ligands. The compounds have been evaluated in vivo and in vitro as PPARalpha activators and as cannabinoids in two tests of the mouse tetrad. In vivo, food intake studies have been performed with all the compounds. No significant cannabinoid activity has been found but some compounds behaved as potent PPARalpha activators. Several compounds showed anorexigenic properties reducing food intake in rats.     

Dispersed mammary epithelial cells. Receptors of lactogenic hormones in virgin, pregnant, and lactating rabbits.

The number and affinity of binding sites for lactogenic hormones have been determined in dispersed mammary cells from virgin, pregnant, and lactating rabbits. Dispersed epithelial cells, prepared from mammary glands by enzyme digestion, calcium chelation, and gentle shearing, were separated from nonepithelial cells by density centrifugation. 125I-labeled ovine prolactin (oPRL) and 125I-labeled human growth hormone (/GH) were used as tracers. Association and dissociation of 125I-oPRL or 125I-hGH were time- and temperature-dependent. The rate of association followed a second order reversible reaction with a rate constant of approximately 0.5 at 4 degrees C, approximately 2.0 at 23 degrees C, and approximately 9 x 10(7) M-1 min-1 at 37 degrees C. Maximum binding was achieved after 120 h at 4 degrees C, 48 h at 23 degrees C, and 2 to 4 h at 37 degrees C. Dissociation of 125I-oPRL or hGH from cells by unlabeled oPRL was complete at 4 degrees C after 160 h, following a first order reaction (5-1 = 9.9 x 10(-5) min) and incomplete at 23 degrees C and 37 degrees C even after prolonged time. Internalization of receptor-bound 125I-oPRL was studied by quantitative electron microscope autoradiography. Grain distribution over- and volume densities of cellular organelles was analyzed as a function of time and temperature. At 37 degrees C, there was a rapid and specific translocation of lactogenic hormones to intracellular organelles. Autoradiographic grains were found associated with vesicles, Golgi elements, lysosome-like structures, and the nucleus. One class of high affinity binding sites was estimated from Scatchard plot and direct kinetic analyses at 4 degrees C. Whereas the apparent affinity constant (approximately 10(10) M-1) did not change significantly throughout pregnancy and early lactation, the number of receptors extrapolated from Scatchard plots at 4 degrees C varied in an inverse relation to serum progesterone concentration. Thus, approximately 1900 sites were detected in virgin rabbits (progesterone, approximately 200 pg/ml), and midpregnancy (progesterone, approximately 15,000 pg/ml), and approximately 1800 during early lactation (progesterone, approximately 500 pg/ml). The binding properties of lactogenic hormones to dispersed cells was compared with those to Triton X-100 solubilized microsomal membrane preparations. Good correlation between the two systems was found indicating that cell dispersion did not alter binding properties. Our results indicate that dispersed mammary cells bind lactogenic hormones in a saturable and reversible process, that the number of exposed receptors varies throughout gestation and lactation, and finally that lactogenic hormones are internalized following interaction with their membrane receptors.     

One‐locus‐several‐primers: A strategy to improve the taxonomic and haplotypic coverage in diet metabarcoding studies

In diet metabarcoding analyses, insufficient taxonomic coverage of PCR primer sets generates false negatives that may dramatically distort biodiversity estimates. In this paper, we investigated the taxonomic coverage and complementarity of three cytochrome c oxidase subunit I gene (COI) primer sets based on in silico analyses and we conducted an in vivo evaluation using fecal and spider web samples from different invertivores, environments, and geographic locations. Our results underline the lack of predictability of both the coverage and complementarity of individual primer sets: (a) sharp discrepancies exist observed between in silico and in vivo analyses (to the detriment of in silico analyses); (b) both coverage and complementarity depend greatly on the predator and on the taxonomic level at which preys are considered; (c) primer sets’ complementarity is the greatest at fine taxonomic levels (molecular operational taxonomic units [MOTUs] and variants). We then formalized the “one‐locus‐several‐primer‐sets” (OLSP) strategy, that is, the use of several primer sets that target the same locus (here the first part of the COI gene) and the same group of taxa (here invertebrates). The proximal aim of the OLSP strategy is to minimize false negatives by increasing total coverage through multiple primer sets. We illustrate that the OLSP strategy is especially relevant from this perspective since distinct variants within the same MOTUs were not equally detected across all primer sets. Furthermore, the OLSP strategy produces largely overlapping and comparable sequences, which cannot be achieved when targeting different loci. This facilitates the use of haplotypic diversity information contained within metabarcoding datasets, for example, for phylogeography and finer analyses of prey–predator interactions.     

TRAIL/TRAIL receptor system and susceptibility to multiple sclerosis

The TNF-related apoptosis inducing ligand (TRAIL)/TRAIL receptor system participates in crucial steps in immune cell activation or differentiation. It is able to inhibit proliferation and activation of T cells and to induce apoptosis of neurons and oligodendrocytes, and seems to be implicated in autoimmune diseases. Thus, TRAIL and TRAIL receptor genes are potential candidates for involvement in susceptibility to multiple sclerosis (MS). To test whether single-nucleotide polymorphisms (SNPs) in the human genes encoding TRAIL, TRAILR-1, TRAILR-2, TRAILR-3 and TRAILR-4 are associated with MS susceptibility, we performed a candidate gene case-control study in the Spanish population. 59 SNPs in the TRAIL and TRAIL receptor genes were analysed in 628 MS patients and 660 controls, and validated in an additional cohort of 295 MS patients and 233 controls. Despite none of the SNPs withstood the highly conservative Bonferroni correction, three SNPs showing uncorrected p values<0.05 were successfully replicated: rs4894559 in TRAIL gene, p = 9.8×10⁻⁴, OR = 1.34; rs4872077, in TRAILR-1 gene, p = 0.005, OR = 1.72; and rs1001793 in TRAILR-2 gene, p = 0.012, OR = 0.84. The combination of the alleles G/T/A in these SNPs appears to be associated with a reduced risk of developing MS (p = 2.12×10⁻⁵, OR = 0.59). These results suggest that genes of the TRAIL/TRAIL receptor system exerts a genetic influence on MS.