Genetic variation in the Bribri and Cabecar Amerindians from Talamanca, Costa Rica.

A screening of more than 40 loci was performed in the Bribri and Cabecar Amerindian populations of Talamanca in Southeastern Costa Rica. Some differences were found in the distribution (presence or absence) of the variants (some of them private) of the GOT, PEPA, RH, TF and TPI loci when the results reported here were compared with those of the Bribris and Cabecars that inhabit in the Pacific side of the country. Admixture with non-Indians was detected at the ABO, ADA, and G6PD loci. The proportion of polymorphic loci (P) found were 17.1 in the Bribris and 19.4 in the Cabecars; their average heterozygosites (H) estimates were 0.039 and 0.049 respectively. Fst values (0.019 in the Bribris and 0.056 in the Cabecars) seem to indicate that random events have played important roles in the divergence of the Cabecars. Low effective population sizes of the emigrant groups to the Pacific side and probable gene flow in the case of the Bribris, could be the cause of these differences.     

Effects of periodate oxidation and glycosidases on structural and functional properties of the acetylcholine receptor and the non-receptor,peripheral ν-polypeptide (Mr 43,000)

NaIO₄ oxidation, exo- and endo-glycosidase treatments and combinations thereof have been applied to acetylcholine receptor from Torpedo marmorata in its membrane-bound and detergent-solubilised forms. The effects of these chemical and enzymatic treatments are made apparent in the electrophoretic properties of the four receptor subunits (α, β, γ and δ) and of the non-receptor polypeptides, their thermal and proteolytic susceptibility, and the steady-state and kinetic parameters of receptor-toxin complex formation. The electrophoretic pattern of the membrane polypeptides is found to depend on the redox state of the membranes, presence or absence of the non-receptor peripheral ν-peptide (M_r 43,000), pH and temperature. Very low NaIO₄ concentrations (50 μM) suffice to prevent the penetration of the ν-peptide into NaDodSO₄ polyacrylamide gels. This effect could be abolished by N-ethylmaleimide alkylation of free sulphydryl groups, suggesting the involvement of easily oxidizable vicinal thiols in the aggregation of the peptide. Higher reagent concentrations resulted in the altered mobility and subsequent splitting of the receptor subunit carrying the ligand recognition site (α, M_r 40,000) into a doublet. In contrast, NaIO₄ treatment of the detergent-solubilized receptor aggregated the α-subunit, presumably via chemical groups hidden in the membrane but exposed in detergent. Only this subunit underwent such NaIO₄-dependent changes within the concentration range in which (a) an increase of the 13-S dimeric receptor species at the expense of the 9-S monomeric form was observed and (b) half-maximal quenching of the intrinsic fluorescence occurred (～2 mM NaIO₄).Neuraminidase digestion affected exclusively the γ- and δ-subunits of the receptor, suggesting the presence of substantial amounts of sialic acid residues in these subunits. β-Glucosidase and endoglycosidase D had no effect on the electrophoretic properties of receptor and non-receptor polypeptides. Neither NaIO₄ nor neuroaminidase treatments had any effect on the thermal sensitivity of the receptor. Similarly, the equilibrium and kinetic properties of receptor-α-neurotoxin complex formation were not modified by such treatment nor was the susceptibility to tryptic digestion. The thermal and proteolytic sensitivities were affected by acid pH (5.2) and β-glucosidase treatments. The latter enzymatic digestion reduced the α-toxin binding capacity of the receptor by 35% and increased the equilibrium dissociation constant by 2-fold.     

The role of the medium in long-range electron transfer

 The role of the polypeptide matrix in electron transfer processes in proteins has been studied in two distinct systems: first in a protein where the induced ET is artificial, and second as part of the catalytic cycle of an enzyme. Azurins are structurally well-characterized blue single-copper proteins consisting of a rigid β-sheet polypeptide matrix. We have determined rate constants and activation parameters for intramolecular long-range electron transfer between the disulfide radical anions (generated by pulse radiolysis) and the copper(II) centre as a function of driving force and nature of the intervening medium in a large number of wild-type and single-site-mutated proteins. In ascorbate oxidase, for which the three-dimensional structure is equally well characterized, the internal ET from the type-I Cu(I) to the trinuclear Cu(II) centre has been studied. We find that the results correlate well with distance through well-defined pathways using a through-bond electron tunnelling mechanism. Received: 2 January 1997 / Accepted: 6 February 1997     

Calcium-Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein

A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed.     

Characterization by cytofluorometry of the different types of adenohypophyseal cells in the rat

The different antehypophysical cell types which synthetize and release somatotroph (GH), corticothroph (ACTH), gonadotroph (LH-FSH) and lactotroph (PRL) hormones were analysed. The experiments were performed on hypophyses from five groups of animals: adult males, 14 days-old female, adult females, gestating adult females and lactating adult females. The cells were analysed by immunofluorescence using flow cytometry. For each of the hormones studied, there was a characteristic spectral distribution of cells. The evolution of cell size and granular content with respect to sex and physiological state of each group was studied by the analysis of diffused light. Small, slightly granular cells represented 50% of the cell population in males and 14 day-old females but only 8% in gestating or lactating females. The study of the cell cycle showed the presence of dividing cells in the population of large, granular cells from gestating and from lactating females. No features of cell division were observed in the population of small, slightly granular cells. This study indicates the potential value of multiparametric analysis in the separation of pure sub-populations of antehypophysial cells.     

Isoenzyme pattern and subcellular localisation of enzymes involved in fumaric acid accumulation by Rhizopus oryzae

Summary Electrophoretic studies of fumarase and nicotine adenine dinucleotide (NAD)-malate dehydrogenase were carried out in the fumaric acid-accumulating fungus Rhizopus oryzae. The analyses revealed two fumarase isoenzymes, one localised solely in the cytosol and the other found both in the cytosol and in the mitochondrial fraction. The activity of the cytosolic isoenzyme of fumarase was higher during the acid production stage than during growth. Addition of cycloheximide inhibited fumaric acid production and decreased the activity of the cytosolic isoenzyme of fumarase. These results suggested that de novo protein synthesis is required for increase in the activity of the cytosolic isoenzyme and that such an increase in activity is essential for fumaric acid accumulation. Three distinct isoenzymes of NAD-malate dehydrogenase could be detected in R. oryzae. No changes were observed in the isoenzyme pattern of malate dehydrogenase during fumaric acid production.