Allosteric functioning of dimeric class C G-protein-coupled receptors

Whereas most membrane receptors are oligomeric entities, G-protein-coupled receptors have long been thought to function as monomers. Within the last 15 years, accumulating data have indicated that G-protein-coupled receptors can form dimers or even higher ordered oligomers, but the general functional significance of this phenomena is not yet clear. Among the large G-protein-coupled receptor family, class C receptors represent a well-recognized example of constitutive dimers, both subunits being linked, in most cases, by a disulfide bridge. In this review article, we show that class C G-protein-coupled receptors are multidomain proteins and highlight the importance of their dimerization for activation. We illustrate several consequences of this in terms of specific functional properties and drug development.     

Effects of Superior Cervical Ganglionectomy and Melatonin Replacement on Intra-hypothalamic LHRH Content and Pulsatile Luteinizing Hormone Release in the Mink

Long-term effects of subcutaneous melatonin implants on intrahypothalamic LHRH content and on pulsatile luteinizing hormone release have been investigated in ganglionectomized male mink. Animals were submitted to bilateral removal of the superior cervical ganglion in mid-April. A preliminary study revealed that plasma LH concentrations remain at a basal level throughout the year following ganglionectomy. In a second experiment, one month after ganglionectomy and transfer from the natural photoperiod environment to short daylengths (LD 4:20), melatonin pellets were subcutaneously implanted to overcome deafferentation of the pineal. Progressive effects of treatment were studied 7 days, 15 days, and one, two and three months after insertion of the melatonin implants. The intra-hypothalamic LHRH content in ganglionectomized mink was at a basal level similar to that observed during seasonally sexual quiescence, or after exposure to inhibitory long days (LD 20:4). A significant and transient elevation in LHRH content was observed already after fifteen days, and also one month after insertion of melatonin implants. This resulted in mean values similar to those observed during the breeding season, or after exposure to stimulatory short days (LD 4:20). A decrease in hypothalamic LHRH content started after two months. No pattern of pulsatile LH secretion was recorded in ganglionectomized untreated mink. A significant increase in all parameters of pulsatile LH secretion was observed fifteen days after the elevation of LHRH content induced by melatonin treatment, and maximum values were reached after two months. Pituitary activity tended to decrease after three months, characterized in particular by a significant decrease in the mean frequency of LH pulses. In addition, the increase in pulsatile characteristics of LH release occurred two months before the peripheral renewal of testicular activity. Apparently, the reproductive endocrine function in ganglionectomized mink treated with melatonin implants is restored more rapidly at the hypothalamic level than at the pituitary or testicular levels.     

Fluctuations naturelles et évolution artificielle des biocénoses macrozoobenthiques intertidales de trois estuaires des côtes françaises de la Manche

The study of the intertidal benthic population dynamics in three estuaries of the English Channel (Baie des Veys, Seine estuary, Baie de Somme:France) brings to light two types of species:

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key-species which directly respond to the local disturbance of the environmental conditions in their densities (Spionidae, Capitellidae) and in their growth rates (Cerastoderma edule);

target-species such as Macoma balthica which can endure brief changes in the environmental factors and shows no sign of long-lasting consequences on its population dynamics; yet, it fully integrates long-term changes through its numbers and productivity.

The parallel between such a regular study of the seasonal variations on selected sites and various base line surveys allows the authors to discuss the COST 647 sampling programm in order to selectrms, range of temperature) from human disturbances (embankments, chemical pollution, eutrophication). Diverse hypothesis are suggested which bring about several research topics to be developed within a european cooperation.     

ESR Spin Trapping Analysis of Gamma Induced Radicals in Sucrose: II

Radicals induced by gamma-irradiation of sucrose, in the solid state at different temperatures and in aqueous solution, have been investigated by the spin trapping method. Electron spin resonance (ESR) combined with high performance liquid chromatography (HPLC), followed by spectral analysis with a simulation program (Voyons) revealed seven main radical species. A comparative study of the ESR signals from spin trapped gamma-induced radicals in some glycosides, disaccharides, ¹³C specifically labelled carbohydrates, as well as in several deoxysucroses and fructans, led to the assignment of a chemical structure to five out of the seven sucrose-nitroxide adducts previously evidenced. Sucrose is shown to be a conceivable model for the study of fructans gamma-radiolysis mechanism in aqueous solution.     

How translation termination factor eRF1 Euplotes does not recognise UGA stop codon

In universal-code eukaryotes, a single class-1 translation termination factor eRF1 decodes all three stop codons, UAA, UAG, and UGA. In some ciliates with variant genetic codes one or two stop codons are used to encode amino acid(s) and are not recognized by eRF1. In Stylonychia, UAG and UAA codons are reassigned as glutamine codons, and in Euplotes, UGA is reassigned as cysteine codon. In omnipotent eRF1s, stop codon recognition is associated with the N-terminal domain of eRF1. Because variant-code ciliates most likely evolved from universal code ancestor(s), structural features should exist in ciliate eRF1s that restrict their stop codon recognition. To find out amino acid residues which confer UAR-only specificity to Euplotes aediculatus eRF1, eRFI chimeras were constructed by swapping eRF1 E. aediculatus N-terminal domain sequences with the matching ones from the human protein. In these chimeras the MC-domain was from human eRF1. Functional analysis of these chimeric eRFI highlighted the crucial role of the two regions (positions 38-50 and 123-145) in the N-terminal domain of E. aediculatus eRF1 that restrict E. aediculatus eRF1 specificity toward UAR codons. Possibly, restriction of eRF1 specificity to UAR codons might have been an early event occurring in independent instances in ciliate evolutionary history, possibly facilitating the reassignment of UGA to sense codons.     

Genetic identity of clones and methods to explore DNA

Cloning by nuclear transfer has made it possible to produce genetically identical animals in terms of nuclear DNA content. Recent molecular biology tools are offering scientific ways to get an insight into the identity issues, by exploring and comparing genomes of cloned animals in order to test their genetic identity and methylation differences. We have initiated a study to compare genomic DNA of bovine adult clones, of normal phenotype. We have used, in parallel, the AFLP technique (amplification fragment length polymorphism) and one of its variant, MSAP (methylation-sensitive amplification polymorphism). We are also investigating other techniques leading to the detection of sequence polymorphisms between two genomes based on genomes hybridisation. We chose the representational difference analysis (RDA) methods that can be combined with mismatch-specific recognition or mismatch binding property of some proteins (CEL I, MutS). We plan to use these RDA methods for genome-wide detection of subtle mutations, then to focus on changes affecting the methylation status of promoting genomic regions in abnormal clones. This will be achieved using MSAP with NotI and applying, in parallel, the RLGS (restriction landmark genome scanning) technique. This study will hopefully improve the molecular and functional characterizations of these "new animals."     

Production of heterologous and chimeric scaffoldins by Clostridium acetobutylicum ATCC 824

Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents. This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes. To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C. acetobutylicum. A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum. Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.     

Disruption of protein kinase A interaction with A-kinase-anchoring proteins in the heart in vivo: effects on cardiac contractility, protein kinase A phosphorylation, and troponin I proteolysis

Protein kinase A (PKA)-dependent phosphorylation is regulated by targeting of PKA to its substrate as a result of binding of regulatory subunit, R, to A-kinase-anchoring proteins (AKAPs). We investigated the effects of disrupting PKA targeting to AKAPs in the heart by expressing the 24-amino acid regulatory subunit RII-binding peptide, Ht31, its inactive analog, Ht31P, or enhanced green fluorescent protein by adenoviral gene transfer into rat hearts in vivo. Ht31 expression resulted in loss of the striated staining pattern of type II PKA (RII), indicating loss of PKA from binding sites on endogenous AKAPs. In the absence of isoproterenol stimulation, Ht31-expressing hearts had decreased +dP/dtmax and -dP/dtmin but no change in left ventricular ejection fraction or stroke volume and decreased end diastolic pressure versus controls. This suggests that cardiac output is unchanged despite decreased +dP/dt and -dP/dt. There was also no difference in PKA phosphorylation of cardiac troponin I (cTnI), phospholamban, or ryanodine receptor (RyR2). Upon isoproterenol infusion, +dP/dtmax and -dP/dtmin did not differ between Ht31 hearts and controls. At higher doses of isoproterenol, left ventricular ejection fraction and stroke volume increased versus isoproterenol-stimulated controls. This occurred in the context of decreased PKA phosphorylation of cTnI, RyR2, and phospholamban versus controls. We previously showed that expression of N-terminal-cleaved cTnI (cTnI-ND) in transgenic mice improves cardiac function. Increased cTnI N-terminal truncation was also observed in Ht31-expressing hearts versus controls. Increased cTnI-ND may help compensate for reduced PKA phosphorylation as occurs in heart failure.