Actin and spectrin-like (Mr= 260–240 000) proteins in gregarines

In Gregarina blaberae a M_r = 47 000 and a M_r = 260–240 000 doublet polypeptides reacted in immunoblotting: i) with a polyclonal monospecific rabbit antibody to frog muscular actin, a monoclonal anti-actin antibody against chicken gizzard; and ii) with polyclonal and monoclonal antibodies to human erythrocyte β-spectrin, respectively. The M_r = 47 000 actin-like protein is associated with the ghost and a contractille cytoplasmic extract. The presence of an actin-like protein in Gregarina and Lecudina and its cellular distribution in the cortex indicated that the gliding movement might involve an actin-myosin system in contrast to previous studies. Immunofluorescence showed clear differences between the anterior part of Gregarina and Lecudina which illustrated the high cell polarity of these protozoa. The M_r = 260–240 000 doublet was detected in SDS-PAGE from G. blaberae trophozoite ghosts but not in the cytoplasmic extracts or in extracts from sexual stages, indicating that the presence of these spectrin-like proteins is stage-dependent. Visualization of the M_r = 260–240 000 by immunofluorescence showed clear species differences, with rings arranged perpendicular to the longitudinal narrow folds of G. blaberae, with longitudinal lines underlying the folds of L. pellucida and with lines separating the large folds of Selenidium pendula. The cellular distribution is consistent with a stabilizer function of the spectrin-like proteins in the scaffolding of the cortex of gregarines according to the high diversity of the cell-shape and the cell motility systems in gregarines. The presence of spectrin-like proteins in protozoa and particularly in parasites from primitive arthropods indicated that ancestral spectrin genes could the M_r = 260–240 000 form.     

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.     

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.     

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.     

Astroglial Cells Express Large Amounts of GABAA Receptor Proteins in Mature Brain

Abstract: GABA_A receptors were characterized in cellular fractions isolated from adult bovine brain. The fraction enriched in cortical astrocytes is very rich in high-affinity binding sites for [³H]flunitrazepam and other "central-type" benzodiazepine ligands. The amount of specific [³H]flunitrazepam binding was more than five times higher in the glial fraction than in synaptosomal and perikaryal fractions. [³H]Flunitrazepam was displaced by low concentrations of clonazepam and other specific ligands for central GABA_A receptors. Specific binding sites for GABA, flunitrazepam, barbiturates, and picrotoxin-like convulsants were characterized. Allosteric interactions between the different sites were typical of central-type GABA_A receptors. The presence of α-subunit(s), as revealed by [³H]flunitrazepam photoaffinity labeling, was demonstrated in all brain fractions at molecular mass 51–53 kDa. Photoaffinity labeling was highest in the glial fraction. However, in primary cultured astrocytes from neonate rat cortex, no photoaffinity labeling was detected. Information obtained from astrocytes in culture should thus be taken with caution when extrapolated to differentiated astroglial cells. Our results actually show that, in mature brain, most of the fully pharmacologically active GABA_A receptors are extrasynaptic and expressed in astroglia.