Light-dependent GTP-binding proteins in squid photoreceptors.

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5'-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.     

A review of the in vitro and in vivo neurochemical characterization of the NMDA/PCP/Glycine/Ion channel receptor macrocomplex

Conclusions Current neurochemical studies of the NMDA receptor macromolecular complex are yielding new insights into the interactions of the subunits of this complex and the associated potential clinical benefits of selective modulation of these subnits. Such studies offer the great potential for a new generation of pharmacotherapies for a wide range of CNS disorders, including stroke, a condition for which there is currently no effective pharmacological treatment. However, it is essential to understand that the first generation products in this area may not be optimal pharmacotherapies, such that haracterization of possible receptor subtypes and understanding the molecular biology of the component proteins of the receptor complex will be crucial in the design of the optimal pharmacological modulators of the NMDA receptor complex.Special issue dedicated to Dr. Erminio Costa     

Codon usage in selected AT-rich bacteria

The relationship between DNA base composition and codon bias in very AT-rich bacteria was analyzed. Five clostridial genes, five mycoplasmal genes and three rickettsial genes constituted the data base. In the genes of these three organisms, the rule for codon bias was very simple: use U or A in the first and third positions of the codon when possible. This was contrasted with the bias found in Bacillus subtilis and Escherichia coli. The rule for Bacillus subtilis was equally straightforward: use all codons without bias. Only in E. coli, amongst the species examined, did the codon bias appear to be a complicated codon 'choice'.     

Amplification by glycine of the effect of the GABA transport inhibitor THPO on synaptosomal GABA level

Mice were injected intramuscularly (2 mmol/kg) with the glia-selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) 60 min prior to sacrifice, or with glycine (10 mmol/kg) 45 min before death, or with a combination of both. After decapitation of the animals, the brains were removed and synaptosomes prepared and analyzed for content of GABA, taurine, glutamine, serine, glutamate and aspartate. While no differences as compared with control animals were found for aspartate, serine and glutamine, synaptosomal GABA levels were increased significantly after injections with either THPO or glycine. The individual effects of THPO and glycine were found to be additive. Taurine levels were decreased to a similar extent in animals which had received either THPO alone or THPO in conjunction with glycine. Treatment with THPO and glycine in combination led to a decrease in the synaptosomal glutamate content. The findings are consistent with the previously observed synergistic anticonvulsant actions of THPO and glycine being mediated via the GABA neurotransmitter system.     

Characterization of immunoreactive epitopes of the HIV-1 p41 envelope protein using fusion proteins synthesized in Escherichia coli

We have identified several immunoreactive epitopes on the human immunodeficiency virus (HIV) type 1 transmembrane envelope protein by synthesizing various regions of the protein as fusions to the trpE gene in Escherichia coli. Ten fusion clones which expressed overlapping peptides were found to contain epitopes reactive with antibodies in sera of North American (NAm) and West African (WAf) patients with acquired immune deficiency syndrome (AIDS). An immunodominant epitope which reacted with all HIV-infected patients' sera was mapped to a 51-amino acid sequence in the N terminus of p41. A novel epitope was also identified in the C terminus of p41 which was reactive with 41% and 48% of the sera tested from NAm and WAf, respectively. In addition, several minor epitopes were identified. We observed that sera from WAf reacted more strongly to minor HIV-1 p41 epitopes than did sera from similarly infected individuals in NAm. We also report on the detection of antibodies from patients with HIV-2 infection in WAf which cross react with HIV-1 p41 recombinant envelope antigens.     

Breakdown of crystalline cellulose by synergistic action between cellulase components from Clostridium thermocellum and Trichoderma koningii

Abstract Certain isolated components of fungal cellulases, which cannot effect the breakdown of highly ordered cellulose individually, interact together synergistically to do so when recombined. Suprisingly, not all fungal cellulase components exhibit this property, and no such synergism has been observed so far between fungal and bacterial cellulases.
The cellulase complex of Clostridium thermocellum cannot effect the extensive breakdown of highly ordered cellulose unless Ca²⁺ and dithiothreitol (DTT) are present. However, we now report that isolated cellobiohydrolase from Trichoderma koningii can combine with C. thermocellum cellulase to effect the breakdown of cellulose in the absence of Ca²⁺ and DTT. enhanced activity is observed if Ca²⁺ and DTT are present.
This finding may have important applications in industry: it certainly has important implications for those interested in the basic mechanism of cellulase action in C. thermocellum .     

Toxic shock syndrome toxin-1 induces inositol phospholipid turnover, protein kinase C translocation, and calcium mobilization in human T cells

Toxic shock syndrome toxin-1 (TSST-1) is a 22-kDa exotoxin produced by most Staphylococcus aureus strains responsible for toxic shock syndrome. TSST-1 is a mitogen for human T cells. The mechanism of T cell activation by TSST-1 was investigated. TSST-1 induced IL-2R expression, IL-2 synthesis, and proliferation in T cells in a monocyte-dependent fashion. Neither IL-1 nor IL-2, alone or in combination, substituted for monocytes in supporting TSST-1-induced mitogenesis. We investigated the mechanism by which TSST-1 induces initogenesis. TSST-1 failed to induce ADP-ribosylation of T cell membrane proteins. However, the toxin induced transient translocation of protein kinase C from cytosol to plasma membranes and also induced the mobilization of cellular Ca2+ stores in both PBMC and the Jurkat human tumor T cell line, suggesting that TSST-1 triggered inositol phospholipid turnover. This was directly demonstrated to be the case in both cellular preparations studied. TSST-1 induced the increased synthesis of the inositol phospholipid phosphatidyl inositol, phosphatidyl inositol-4 phosphate, and phosphoinositol inositol-4,5-bisphosphate, and induced the breakdown of inositol phospholipid as evidence by the accumulation of phosphatidic acid and inositol phosphates. We conclude that the action of TSST-1 involves the induction of inositol phospholipid turnover, protein kinase C activation, and mobilization of cellular Ca2+ stores. This effect is similar to that of mitogenic lectins and of anti-CD3 antibodies.     

A comparison of the lipid-lowering and intestinal morphological effects of cholestyramine, chitosan, and oat gum in rats

Cholestyramine, chitosan, and oat gum are lipid-lowering compounds. Cholestyramine use in humans may contribute to colonic adenocarcinoma; chitosan and oat gum are being studied in the rat to determine their potential for human use. To compare these compounds, we fed three groups of 10 male Sprague-Dawley rats one of the substances at 5% of diet with 1% cholesterol and 0.2% cholic acid; two other groups were fed cellulose with and without 1% cholesterol and 0.2% cholic acid. All groups had similar food intake and weight gains. Cholesterol feeding increased total liver lipids almost 3-fold and liver cholesterol concentration almost 10-fold. Cholestyramine, oat gum, and chitosan all significantly lowered liver cholesterol with cholestyramine feeding yielding levels identical to the noncholesterol-fed basal group. Chitosan and oat gum lowered liver cholesterol moderately. Cholestyramine and chitosan both significantly lowered serum cholesterol compared to the cellulose group. Oat gum was less effective. Hemoglobin and serum iron were similar in all groups except the oat gum group, which had decreased serum iron. Histological examination of small and large bowel with morphometry revealed statistically significant increases in both proximal and distal small bowel and distal large bowel mucosal thickness in the cholestyramine-fed group. No changes were noted in the proximal large bowel. Neither chitosan nor oat gum produced mucosal change other than an increase in the distal small bowel with the oat gum diet. Chitosan may have lipid-lowering effects similar to those of cholestyramine without the deleterious changes in intestinal mucosa.     

Effect of mutations at Met-88 and Met-90 on the biotination of Lys-89 of the apo 1.3S subunit of transcarboxylase

The apo 1.3S subunit of transcarboxylase contains the sequence Ala-87-Met-88-Lys-89-Met-90, and it is Lys-89 that is biotinated. This sequence is highly conserved in all the biotin enzymes that have been sequenced (with the exception of acetyl-CoA carboxylase from chicken liver, which has Val in place of Ala). The role of Met-88 and Met-90 in specifying Lys-89 for biotination by synthetase was examined by site-directed mutagenesis. Genes of the 1.3S subunit coding for Thr-88, Leu-88, or Leu-90 were generated by oligonucleotide-directed in vitro mutagenesis and expressed in Escherichia coli. The mutated apo 1.3S subunits were isolated and the biotination by homogeneous synthetase from Propionibacterium shermanii was compared with that of the apo wild-type subunit. The Vmax for the apo mutants was the same as that for the apo wild type, but when Leu was substituted for Met-88 or Met-90, the Km for the mutant was lower than that of the wild-type or mutant Thr-88. The activity of the synthetase of E. coli was determined by an in vivo assay. During the early log phase of growth, a smaller portion of mutants Thr-88 and Leu-90 was biotinated than with the wild-type or mutant Leu-88. When the cultures progressed to stationary phase, mutants and the wild type were biotinated to the same extent. The overall results show that Met-88 and Met-90 are not required for biotination of the apo 1.3S subunit by the synthetases.