Inhibition of in vitro SV40 DNA replication by ultraviolet light

Ultraviolet light-induced DNA damage was found to inhibit SV40 origin-dependent DNA synthesis carried out by soluble human cell extracts. Replication of SV40-based plasmids was reduced to approx. 35% of that in unirradiated controls after irradiation with 50-100 J/m2 germicidal ultraviolet light, where an average of 3-6 pyrimidine dimer photoproducts were formed per plasmid circle. Inhibition of the DNA helicase activity of T antigen (required for initiation of replication in the in vitro system) was also investigated, and was only significant after much higher fluences, 1000-5000 J/m2. The data indicate that DNA damage by ultraviolet light inhibits DNA synthesis in cell-free extracts principally by affecting components of the replication complex other than the DNA helicase activity of T antigen. The soluble system could be used to biochemically investigate the possible bypass or tolerance of DNA damage during replication.     

Effects of phorbol esters on endothelial cell microfilaments: Laser scanning confocal microscopy and quantitative morphometry of dose dependent changes

Phorbol esters are known to alter microfilaments but it is not clear if the changes correspond to modulation of the phosphoinositide turnover/protein kinase C system. The novel technique of laser scanning confocal epifluorescence was used to study fiber orientation in phorbol ester treated cells. We treated endothelial cells with control agents and agents known to stimulate protein kinase C: 4 alpha-phorbol, phorbol 12-myristate 13-acetate (PMA), phorbol dibutyrate (PDB), or lipopolysaccharide. After incubation with the test agents, the endothelial cell microfilaments were stained with rhodamine pholloidin and viewed by conventional epifluorescence and by laser scanning confocal epifluorescence microscopy. The images obtained by the confocal microscopy corresponded to a thin optical section through the cells, 300 nm or more in thickness. The microfilaments extended predominantly in the plane of focus. After exposure of the cells to phorbol esters, the stress fibers became more nearly parallel in arrangement or were shortened, but remained in the plane of focus. The modification of microfilaments in response to phorbol esters was quantitated by a single blind analysis. In order to compare the morphological changes with a biochemical action of the phorbol esters, we measured phosphoinositide turnover. The dose-dependence of morphological changes was compared and contrasted to the dose-dependent effect of phorbol esters on bradykinin-stimulated phosphoinositide turnover. PMA had about the same EC50 (1-5 nM) for both biochemical and morphological processes. PDB was less potent in inducing the disruption of microfilament structure than in inhibiting phosphoinositide turnover. Lipopolysaccharide was ineffective in inducing a morphological change under these conditions. A simple activation of protein kinase C is insufficient to explain the dose-dependent effects of phorbol esters. Thus a morphometric analysis can help distinguish the potency of cytoskeleton modulators.     

The mechanism of fungal cellulase action. Synergism between enzyme components of Penicillium pinophilum cellulase in solubilizing hydrogen bond-ordered cellulose.

Studies on reconstituted mixtures of extensively purified cellobiohydrolases I and II and the five major endoglucanases of the fungus Penicillium pinophilum have provided some new information on the mechanism by which crystalline cellulose in the form of the cotton fibre is rendered soluble. It was observed that there was little or no synergistic activity either between purified cellobiohydrolases I and II, or, contrary to previous findings, between the individual cellobiohydrolases and the endoglucanases. Cotton fibre was degraded to a significant degree only when three enzymes were present in the reconstituted enzyme mixture: these were cellobiohydrolases I and II and some specific endoglucanases. The optimum ratio of the cellobiohydrolases was 1:1. Only a trace of endoglucanase activity was required to make the mixture of cellobiohydrolases I and II effective. The addition of cellobiohydrolases I and II individually to endoglucanases from other cellulolytic fungi resulted in little synergistic activity; however, a mixture of endoglucanases and both cellobiohydrolases was effective. It is suggested that current concepts of the mechanism of cellulase action may be the result of incompletely resolved complexes between cellobiohydrolase and endoglucanase activities. It was found that such complexes in filtrates of P. pinophilium or Trichoderma reesei were easily resolved using affinity chromatography on a column of p-aminobenzyl-1-thio-beta-D-cellobioside.     

Refolding human serum albumin at relatively high protein concentration

The conditions for refolding reduced and denatured human serum albumin (HSA) were investigated with a view to maximising the yield of native monomeric albumin. Refolding by dialysis was found to be preferable to dilution as a means of chaotrope (urea) and reductant (2-mercaptoethanol) removal. Dialysis of denatured HSA solutions containing 4-8 M urea and 14 mM 2-mercaptoethanol at pH 10.0 was found to be optimal for HSA refolding. The yield of monomeric HSA was maximal (94%) for dialysis in the presence of EDTA (1 mM) and sodium palmitate (20 microM). Using this protocol it was possible to refold HSA at concentrations in excess of 5 mg.ml-1 whilst maintaining a high recovery of native monomer. These results represent a considerable improvement on established methods of HSA refolding.     

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.