Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH2 of substance P using [3-prolinoleucine10]SP analogues.

Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue, cis- and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. [trans-3-prolinoleucine10]SP retained affinity and potency similar to SP whereas [cis-3-prolinoleucine10]SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results, the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucine-NHMe, Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the chi1 angle and a trans conformation around the chi2 angle in the bioactive conformation. Together with previously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor.     

On-line tools for sequence retrieval and multivariate statistics in molecular biology

We have developed a World-Wide Web server for browsing sequencecollections structured under the ACNUC format and for performingmultivariate analyses on sequences. General collections (likeGenBank or EMBL), as well as specialized data banks (like Hovergenand NRSub) can be accessed. This system allows complex queriesto be constructed, and the result of each query, representedby a list of sequences, is stored on the server. It is thenpossible to reuse this list to compute multivariate analyseson the sequences. Two examples of applications are shown. Thefirst one consists in a study of codon usage with correspondenceanalysis on all the protein genes of Haemophilus influenzaeRd. This study allows the highly expressed genes and the integralmembrane proteins of this organism to be identified. The secondone consists in an ordering of 70 aligned protein sequencesof growth hormone with principal coordinate analysis. With thismethod, we are able to re-establish the patterns of relationshipsbetween the sequences previously determined with tree buildingprograms.     

Tyrosine protein kinase assays

Protein kinases form a large family of enzymes that play a major role in a number of live processes. The study of their action is important for the understanding of the transformation mechanisms and of the normal and pathological growth events. The quality of an enzyme assay is often the key point of an enzymatic study. It must be flexible and compatible with various experimental conditions, such as those for the purification process, the screening of inhibitors and the substrate specificity studies. As will be shown in the present review, two categories of substrates, peptidic and proteic, should be distinguished. The use of peptide substrates facilitates the determination of the recognition requirements of the enzyme and of the kinetic effects of even minute variations in their sequence. These linear peptide structures are assumed to mimic a complex interaction between the enzyme and a proteic substrate in which distant amino acids in the sequence are vicinal in the folded substrate. Less amenable to a systematic study, but probably more adequate to investigate the natural substrate of a given kinase, are the proteic substrates. Obviously the tools to measure protein kinase activities are not the same in these two cases. The main difficulty in assaying protein kinases is the use of labelles γ-ATP, mostly at large excess concentration, since the final product of the reaction has to be separated from the non-reacted labelled ATP. In the case of peptide substrates, the difficulty is to separate them from ATP basing on differences of molecular mass. Despite the efforts of many investigators to rely upon differences in solubility, in charges or in “affinity”, this separation, which is crucial for the assay, is still an unsolved experimental problem. Chromatographic, as well as electrophoretic assays appeared relatively late in this domain, and more work in assessing new methodologies might bring new breakthroughs in the next few years. Specific, simple and reliable kinase assays are still a major challenge. Their improvement will help to conduct specificity studies, to elucidate complex growth mechanisms in which they are involved and to discover more selective potent inihibitors.     

Nitric oxide does not promote iron release from ferritin

It has been previously reported that iron release from ferritin could be promoted by nitric oxide (NO) generated from sodium nitroprusside. It was thus proposed that some of the toxic effects of NO could be related to its ability to increase intracellular free iron concentrations and generate an oxidative stress. On the contrary, the iron exchange experiments reported here show that NO from S-nitrosothiols is unable to promote iron release from ferritin. The discrepancy may be explained by the disregarded ability of ferrozine, the ferrous trap used in the previous report, to mobilize iron both from ferritin and from sodium nitroprusside spontaneously.     

Dominance competition through affiliation and support in Japanese macaques: An experimental study

It has been proposed that monkeys direct grooming to high-ranking individuals in an attempt to obtain agonistic support in return. But whether these two categories of interactions are causally related has proven difficult to establish. Part of the problem stems from the fact that in stable groups social relationships reflect an equilibrium state and that behaviors need only be performed at low rates and long intervals to maintain the current social structure. In theory, however, if affiliative and supportive interactions are indeed causally related, it should be possible to accentuate their temporal relation, hence their causal dynamics. For example, destabilizing dominance relations can be expected to induce competition for status and force individuals to deploy behavioral tactics for settling new rank relations. We experimentally induced rank reversals in a captive group of Japanese macaques (Macaca fuscata) composed of three matrilines (A-B-C rank order). A reversed C-A-B order composed of three individuals per matriline was maintained for 2 weeks. The results show the close temporal relation among (i) asserting one’s rank, (ii) competing for access to dominants through affiliation and interferences in affiliation, (iii) receiving support from dominants against lower-ranking individuals, and (iv) supporting dominants against subordinates. These findings are compatible with one version of the affiliation-for-support hypothesis, namely that monkeys affiliate with dominants as a way to assert their position in the hierarchy. In a functional perspective, mutual selfishness provides a better explanation than reciprocal altruism because the possibility that both groomers and supporters derive immediate net benefits cannot be excluded.     

Effects of dark- and light-induced proton gradients in thylakoids on the Q and B thermoluminescence bands

Thermoluminescence experiments have been carried out to study the effect of a transmembrane proton gradient on the recombination properties of the S₂ and S₃ states of the oxygen evolving complex with Q_A ^- and Q_B ^-, the reduced electron acceptors of Photosystem II. We first determined the properties of the S₂Q_A ^- (Q band), S₂Q_B ^- and S₃Q_B ^- (B bands) recombinations in the pH range 5.5 to 9.0, using uncoupled thylakoids. The, a proton gradient was created in the dark, using the ATP-hydrolase function of ATPases, in coupled unfrozen thylakoids. A shift towards low temperature of both Q and B bands was observed to increase with the magnitude of the proton gradient measured by the fluorescence quenching of 9-aminoacridine. This downshift was larger for S₃Q_B ^- than for S₂Q_B ^- and it was suppressed by nigericin, but not by valinomycin. Similar results were obtained when a proton gradient was formed by photosystem I photochemistry. When Photosystem II electron transfer was induced by a flash sequence, the reduction of the plastoquinone pool also contributed to the downshift in the absence of an electron acceptor. In leaves submitted to a flash sequence above 0°C, a downshift was also observed, which was supressed by nigericin infiltration. Thus, thermoluminescence provides direct evidence on the enhancing effect of lumen acidification on the S₃S₂ and S₂S₁ reverse-transitions. Both reduction of the plastoquinone pool and lumen acidification induce a shift of the Q and B bands to lower temperature, with a predominance of lumen acidification in non-freezing, moderate light conditions.Abbreviations 9-AA 9-aminoacridine - E_A activation energy - F₀ constant fluorescence level - F_M maximum fluorescence, when all PS-II centers are closed - F_V variable fluorescence (F_M–F₀) - PS I, PS II Photosystem I, photosystem II - PQ plastoquinone - TL thermoluminescence     

Transformation of chicory and expression of the bacterial uidA and nptll genes in the transgenic regenerants

Transgenic chicory plants were obtained from different explantsco-cultured with Agrobacterium tumefaciens. Among tap-root,leaf and cotyledonary tissues, etiolated cotyledons showed thegreatest competence for transformation. The Agrobacterium strainsused contained either pGSGLUC1 or pTDE4 as a vector which carryboth the neomycin phosphotransferase II gene (nptll) for kanamycinresistance and ß-glucuronidase gene (uidA) under thecontrol of different promoters. Transformation was confirmedby NPTII enzymatic assay, histochemical analysis of GUS activityand DNA hybridization. Transgenic plants expressed both markergenes in root and shoot tissues. In leaves, GUS activity wasexpressed in all tissue types, whatever the nature of the promoter.Nevertheless, variable heterogeneous patterns of expressionwere observed in the different root tissues. Differential expression of the GUS fusions controlled by thedual TR or the CaMV 35S promoters are discussed. Key words: Chicory, genetic transformation, GUS activity, kanamycin resistance     

5'-(N-Ethylcarboxamido)adenosine Inhibits Ca2+ Influx and Activates a Protein Phosphatase in Bovine Adrenal Chromaffin Cells

Abstract: We investigated the effect of the adenosine receptor agonist 5'-( N -ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A_2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30–40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 ± 3.3%), slowly developing inhibition of [Ca²⁺]_i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca²⁺ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca²⁺]_i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca²⁺/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca²⁺ influx pathway (e.g., L-type Ca²⁺ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.