3H] norepinephrine binding by rat glial cells in culture. Lack of correlation between binding and adenylate cyclase activation.

Subcellular fractions prepared from rat glial cells in culture (clonal line c6) were used in an attempt to characterize the adrenergic receptor involved in adenylate cyclase activation. Both [3H] norepinephrine binding and enzyme activation were measured under identical experimental conditions. Binding sites for norepinephrine could be detected; their main characteristics were: apparant Km: 4 - 10-6 M, macimal capacity: 20 pmol/mg protein.Their stereospecificity towards structually related drugs was found to be different from the stereospecificity of the receptor involved in adenylate cyclase activation. Thus, 3-methoxydopamine (a competitive inhibitor of norepinephrine for adenylate cyclase activation), phenylephrine (a partial adrennergic agonist) and the blocking agent propranolol were unable to compete with [3H] norepinephrine for binding. On the other hand, several molecules like dopa bearing a catechol group and which are unable to interact with the adenylate cyclase as agonists or competitive inhibitors strongly inhibited [3H] norepinephrine binding. As in several other systems so far studied, the presence on the glial cell's membrane of a large number of "catechol-binding sites" makes it difficult to characterize the beta-adrenergic receptor.     

Muscarinic stimulation of inositol phosphate accumulation and acid secretion in gastric fundic mucosal cells

The muscarinic agonist, carbachol (CCh), was shown to stimulate the production of inositol phosphates (IP) in isolated cells from rabbit fundic mucosa. This stimulatory effect was time- and dose-dependent: EC50 values for IP1, IP2 and IP3 accumulation were not statistically different. The mean value was 30 +/- 8 microM (n = 6). The corresponding maximal stimulation (% of basal value) observed after 20 min incubation in the presence of 100 microM CCh was 160 +/- 15%. CCh-induced IP accumulation was abolished by atropine (Ki = 0.32 +/- 0.18 nM (n = 3)). The CCh concentrations leading to half-maximal inhibition of N-[3H]methylscopolamine binding and half-maximal IP accumulation were similar. The half-maximal value for CCh-induced aminopyrine accumulation was 8-times lower. These results indicate that IP3-mediated mobilization of intracellular Ca2+ might be involved in CCh-induced acid secretion by parietal cells.     

Participation of capsaicin-sensitive neurons in the cardiovascular effects of neurotensin in guinea pigs

Intravenous (IV) infusions of neurotensin (NT) in anesthetized guinea pigs elicited dose-dependent pressor effects and tachycardia. Both effects were significantly reduced or abolished in guinea pigs given a chronic treatment with the neurotoxin capsaicin. In guinea pig isolated atria NT evoked a positive inotropic and chronotropic effect. Both effects were completely abolished in atria derived from capsaicin-treated guinea pigs. The positive inotropic and chronotropic effects of NT in guinea pig atria were mimicked by capsaicin and calcitonin gene-related peptide (CGRP). These results were interpreted as an indication that NT produces its cardiovascular effects in guinea pigs by activating capsaicin-sensitive sensory neurons.     

Cellular Source and Mechanisms of High Transcriptome Complexity in the Mammalian Testis

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A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies

Camel single-domain antibody fragments or Nanobodies, are practical in a wide range of applications. Their unique biochemical and biophysical properties permit an intracellular expression and antigen targeting. The availability of an efficient intracellular selection step would immediately identify the best intracellularly performing functional antibody fragments. Therefore, we assessed a bacterial-two-hybrid system to retrieve such Nanobodies. With GFP as an antigen we demonstrate that antigen-specific Nanobodies of sub-micromolar affinity and stability above 30kJ/mol, at a titer of 10(-4) can be retrieved in a single-step selection. This was further proven practically by the successful recovery from an 'immune' library of multiple stable, antigen-specific Nanobodies of good affinity for HIV-1 integrase or nucleoside hydrolase. The sequence diversity, intrinsic domain stability, antigen-specificity and affinity of these binders compare favorably to those that were retrieved in parallel by phage display pannings.     

Within‐generation consequences of postsettlement mortality for trait composition in wild populations: An experimental test

There is a critical need to understand patterns and causes of intraspecific variation in physiological performance in order to predict the distribution and dynamics of wild populations under natural and human‐induced environmental change. However, the usual explanation for trait differences, local adaptation, fails to account for the small‐scale phenotypic and genetic divergence observed in fishes and other species with dispersive early life stages. We tested the hypothesis that local‐scale variation in the strength of selective mortality in early life mediates the trait composition in later life stages. Through in situ experiments, we manipulated exposure to predators in the coral reef damselfish Dascyllus aruanus and examined consequences for subsequent growth performance under common garden conditions. Groups of 20 recently settled D. aruanus were outplanted to experimental coral colonies in Moorea lagoon and either exposed to natural predation mortality (52% mortality in three days) or protected from predators with cages for three days. After postsettlement mortality, predator‐exposed groups were shorter than predator‐protected ones, while groups with lower survival were in better condition, suggesting that predators removed the longer, thinner individuals. Growth of both treatment groups was subsequently compared under common conditions. We did not detect consequences of predator exposure for subsequent growth performance: Growth over the following 37 days was not affected by the prior predator treatment or survival. Genotyping at 10 microsatellite loci did indicate, however, that predator exposure significantly influenced the genetic composition of groups. We conclude that postsettlement mortality did not have carryover effects on the subsequent growth performance of cohorts in this instance, despite evidence for directional selection during the initial mortality phase.     

Human metabolic phenotypes link directly to specific dietary preferences in healthy individuals

Individual human health is determined by a complex interplay between genes, environment, diet, lifestyle, and symbiotic gut microbial activity. Here, we demonstrate a new "nutrimetabonomic" approach in which spectroscopically generated metabolic phenotypes are correlated with behavioral/psychological dietary preference, namely, "chocolate desiring" or "chocolate indifferent". Urinary and plasma metabolic phenotypes are characterized by differential metabolic biomarkers, measured using 1H NMR spectroscopy, including the postprandial lipoprotein profile and gut microbial co-metabolism. These data suggest that specific dietary preferences can influence basal metabolic state and gut microbiome activity that in turn may have long-term health consequences to the host. Nutrimetabonomics appears as a promising approach for the classification of dietary responses in populations and personalized nutritional management.     

The Susceptibility of Pseudomonas aeruginosa Strains from Cystic Fibrosis Patients to Bacteriophages

Phage therapy may become a complement to antibiotics in the treatment of chronic Pseudomonas aeruginosa infection. To design efficient therapeutic cocktails, the genetic diversity of the species and the spectrum of susceptibility to bacteriophages must be investigated. Bacterial strains showing high levels of phage resistance need to be identified in order to decipher the underlying mechanisms. Here we have selected genetically diverse P. aeruginosa strains from cystic fibrosis patients and tested their susceptibility to a large collection of phages. Based on plaque morphology and restriction profiles, six different phages were purified from “pyophage”, a commercial cocktail directed against five different bacterial species, including P. aeruginosa. Characterization of these phages by electron microscopy and sequencing of genome fragments showed that they belong to 4 different genera. Among 47 P. aeruginosa strains, 13 were not lysed by any of the isolated phages individually or by pyophage. We isolated two new phages that could lyse some of these strains, and their genomes were sequenced. The presence/absence of a CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and Crisper associated genes) was investigated to evaluate the role of the system in phage resistance. Altogether, the results show that some P. aeruginosa strains cannot support the growth of any of the tested phages belonging to 5 different genera, and suggest that the CRISPR-Cas system is not a major defence mechanism against these lytic phages.     

Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA)

Genomic Representational Difference Analysis (gRDA) is a subtractive DNA method to clone the differences between two related genomes, called tester and driver. We have evaluated this method to obtain polymorphic DNA markers for pedigree dogs. Amplified size-selected genomic restriction fragments (amplicons) of two dog littermates were repeatedly hybridized to each other in order to remove (subtract) those restriction fragments common to both sibs. Already after two rounds of subtractive hybridization, a clear enrichment of presumably tester-specific restriction fragments was observed, which was even more pronounced after the third round of subtraction. A plasmid library of 3000 recombinant clones was constructed of the second round and of the third round difference product. DNA sequence determination of randomly chosen clones of each difference product showed that approximately 1000 unique clones were obtained in the second-round difference product and approximately 500 in the third-round difference product. About half of the clones identified in the second-round difference product were also present in the third-round difference product. Of the second-round difference product, 39 different gRDA fragments could be identified, of which 21 were tester specific. In the third-round difference product, 22 different gRDA fragments were identified, of which 18 were tester specific. There were 13 fragments in common, resulting in a total of 48 different fragments. In order to establish the localization of these markers, we performed mapping using the dog radiation hybrid panel RHDF5000. Of 39 mapped clones, 29 were mapped to 20 existing RH groups, and 10 remained unlinked. It is concluded that gRDA is suitable to generate DNA markers to track disease genes within lines of pedigree dogs. Received: 26 April 2000 / Accepted: 11 May 2000