Cycle of the vasoactive intestinal peptide and its binding site in a human adenocarcinoma cell line (HT 29)

The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60-80% loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37 degrees C with a VIP concentration of 100 nM. The t1/2 of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in 125I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was specific since, with the same conditions of preincubation, the specific binding of 125I-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that 125I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37 degrees C. All the data strongly suggest that: internalization of VIP is receptor-mediated; upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.     

Anti-cell surface monoclonal antibodies which antagonize the action of VIP in a human adenocarcinoma cell line (HT 29 cells)

Hybridoma cells have been obtained by fusing RCY 3 Ag 1-2-3 rat myeloma cells with spleen cells from a rat hyperimmunized with human adenocarcinoma cells (HT 29 cell line) grown in serum-free medium. Immunoglobulins secreted by hybridomas were screened for: (i) specific binding to HT 29 cells; (ii) their ability to inhibit the binding of [125I]-vasoactive intestinal peptide (VIP) to HT 29 cells; (iii) their capacity to modulate the cAMP production induced by VIP. The monoclonal antibodies we have obtained from clones 109-10-16 and 109-10-19 compete for the binding of radiolabelled VIP to HT 29 cells and partially inhibit the production of cAMP induced by VIP while they are ineffective in reducing the intracellular level of cAMP attained after stimulation of HT 29 cells by isoproterenol. We never found antibodies which increase the cAMP level in HT 29 cells. The binding of the purified monoclonal antibody 109-10-16 Ig gamma 2c to HT 29 cells was visualized by indirect immunofluorescence and was not present at the surface of all cells. These observations strongly support the hypothesis that the monoclonal antibodies we have characterized interact with an antigenic determinant which belongs to the VIP receptor or at least to a cell surface component closely associated with the receptor.     

Is cytoplasmic pH involved in the regulation of cell cycle in plants?

Modifications of cytoplasmic pH has biological significance in animal and plant cell development. Many observations suggest an important function of cytopiasmic pH in mitotic signalling in animal ceils. In Bidens pilosa cultivated under white light, acidification of cytoplasm, observed after mechanical trauma, is associated with an inhibition of DNA synthesis and a decrease in mitotic frequency. In contrast, in Bidens pilosa cultivated under blue light, mechanical stimulation induces an increase of cytoplasmic pH and stimulation of DNA duplication and mitotic activity. A correlation has been established between transient variations of cytoplasmic pH and rapid modification in cell development. The critical role of cytoplasmic pH in the regulation of the cell cycle in plants is discussed.     

Brain stem NO modulates ventilatory acclimatization to hypoxia in mice.

The objective of our study was to assess the role of neuronal nitric oxide synthase (nNOS) in the ventilatory acclimatization to hypoxia. We measured the ventilation in acclimatized Bl6/CBA mice breathing 21% and 8% oxygen, used a nNOS inhibitor, and assessed the expression of N-methyl-d-aspartate (NMDA) glutamate receptor and nNOS (mRNA and protein). Two groups of Bl6/CBA mice (n = 60) were exposed during 2 wk either to hypoxia [barometric pressure (PB) = 420 mmHg] or normoxia (PB = 760 mmHg). At the end of exposure the medulla was removed to measure the concentration of nitric oxide (NO) metabolites, the expression of NMDA-NR1 receptor, and nNOS by real-time RT-PCR and Western blot. We also measured the ventilatory response [fraction of inspired O(2) (Fi(O(2))) = 0.21 and 0.08] before and after S-methyl-l-thiocitrulline treatment (SMTC, nNOS inhibitor, 10 mg/kg ip). Chronic hypoxia caused an increase in ventilation that was reduced after SMTC treatment mainly through a decrease in tidal volume (Vt) in normoxia and in acute hypoxia. However, the difference observed in the magnitude of acute hypoxic ventilatory response [minute ventilation (Ve) 8% - Ve 21%] in acclimatized mice was not different. Acclimatization to hypoxia induced a rise in NMDA receptor as well as in nNOS and NO production. In conclusion, our study provides evidence that activation of nNOS is involved in the ventilatory acclimatization to hypoxia in mice but not in the hypoxic ventilatory response (HVR) while the increased expression of NMDA receptor expression in the medulla of chronically hypoxic mice plays a role in acute HVR. These results are therefore consistent with central nervous system plasticity, partially involved in ventilatory acclimatization to hypoxia through nNOS.     

Mendelian randomization supports causality between maternal hyperglycemia and epigenetic regulation of leptin gene in newborns

Leptin is an adipokine that acts in the central nervous system and regulates energy balance. Animal models and human observational studies have suggested that leptin surge in the perinatal period has a critical role in programming long-term risk of obesity. In utero exposure to maternal hyperglycemia has been associated with increased risk of obesity later in life. Epigenetic mechanisms are suspected to be involved in fetal programming of long term metabolic diseases. We investigated whether DNA methylation levels near LEP locus mediate the relation between maternal glycemia and neonatal leptin levels using the 2-step epigenetic Mendelian randomization approach. We used data and samples from up to 485 mother-child dyads from Gen3G, a large prospective population-based cohort. First, we built a genetic risk score to capture maternal glycemia based on 10 known glycemic genetic variants (GRS₁₀) and showed it was an adequate instrumental variable (β = 0.046 mmol/L of maternal fasting glucose per additional risk allele; SE = 0.007; P = 7.8 × 10⁻¹¹; N = 467). A higher GRS₁₀ was associated with lower methylation levels at cg12083122 located near LEP (β = −0.072 unit per additional risk allele; SE = 0.04; P = 0.05; N = 166). Direction and effect size of association between the instrumental variable GRS₁₀ and methylation at cg12083122 were consistent with the negative association we observed using measured maternal glycemia. Lower DNA methylation levels at cg12083122 were associated with higher cord blood leptin levels (β = −0.17 log of cord blood leptin per unit; SE = 0.07; P = 0.01; N = 170). Our study supports that maternal glycemia is part of causal pathways influencing offspring leptin epigenetic regulation.     

Multiplex degenerate PCR coupled with an oligo sorbent array for human endogenous retrovirus expression profiling

Human endogenous retroviruses (HERVs) can be divided into distinct families of tens to thousands of paralogous loci. The expression of HERV elements has been detected in all tissues tested to date, particularly germ cells, embryonic tissues and neoplastic tissues. Hence, the study of HERV expression could represent added value in cancer diagnosis. We developed a quantitative assay combining a multiplex degenerate PCR (MD-PCR) amplification, based on the relative conservation of the pol genes, and a colorimetric Oligo Sorbent Array (OLISA^®). Nine HERV families were selected and amplification primers and capture probes were designed for each family. The features required to achieve efficient amplification of most of the elements of each HERV family and balanced co-amplification of all HERV families were analyzed. We found that MD-PCR reliability, i.e. equivalence of amplification and dose-effect relationship, relied on the adjustment of three critical parameters: the primer degeneracy, the relative concentration of each primer and the total amount of primers in the amplification mixture. The analysis of tumoral versus normal tissues suggests that this assay could prove useful in tumor phenotyping.