Biochemistry international最新文献

Carbamylation of haemoglobin by methyl isocyanate (MIC) was detected by high-performance liquid chromatography (HPLC) using a photodiode array detector following cyclisation of the N-terminal valine into methyl isopropyl hydantoin (MIH). MIH was also synthesised by reaction of MIC with valine, the chromatographic conditions standardised and the spectrum derived by a photodiode array detector recorded for confirmation of the identity of MIH. This HPLC method is specific, sensitive and suitable for the detection of exposure of blood samples to methyl isocyanate.

When basic proteins I and II from Trimeresurus flavoviridis (Habu snake) venom, which are Lys-49-phospholipase A2 (PLA2) with low activity, were injected intramuscularly into mice, myonecrosis was induced accompanied by a rapid increase of plasma creatine kinase level. This increase was about 2 times greater than that raised by highly active T. flavoviridis Asp-49-PLA2 which has been regarded as a myotoxic factor. Calcium-dependent depolarization of frog muscle fibers was observed with basic proteins I and II but not with Asp-49-PLA2. Indirect hemolysis of rat erythrocytes was induced by Asp-49-PLA2 but not by basic proteins I and II. Myotoxicity and depolarization effects appear to be not necessarily related to lipolytic activity of proteins although hemolytic effects is in parallel to lipolytic potency. Light microscopic observations of muscle preparations treated with three PLA2s showed similar histological changes, i.e., myolytic necrosis without hemorrhage and infiltration of polymorphonuclear cells. The structural and functional elements of PLA2s for eliciting myotoxicity are discussed.

The interaction of 12 antisense nucleosides with phenylalanine (Phe), and the effect of pH and salts on the strength of interaction was studied by charge-transfer reversed-phase thin-layer chromatography. Phe significantly decreased the lipophilicity of nucleosides. This effect may be due to the interaction between the more hydrophilic Phe and the more lipophilic nucleosides, resulting in charge-transfer complexes of moderate lipophilicity. The relative strength of interaction was the weakest in acidic and the strongest in alkaline environment. This finding indicates the partially or entirely hydrophilic character of the interaction. Salts influenced to a lesser extent the interaction, their effect depended both on the concentration and on the type of cation. The relatively low impact of salts on the strength of interaction suggests that other than hydrophilic forces are involved in the Phe - antisense nucleoside interaction.

The cytotoxicity of human beta-interferon (HuIFN-beta) produced in human glioma cells was examined by use of our liposomes entrapping two plasmids, pSV2neo and pSVMTV-IFN-beta. After the cells had been transfected with these genes by means of the liposomes, neomycin-resistant cells were selected. When the selected cells were subjected to a single exposure to dexamethasone, all of the cells were found to produce HuIFN-beta and were eliminated by 8 days. Accordingly, the effect of HuIFN-beta produced in human glioma cells is considered to be cytocidal.

Transfer RNAs were separated into a ribosome bound fraction and a supernatant (cytoplasmic) fraction. The nucleoside composition, 2-dimensional PAGE pattern and in vivo labeling were compared. 12 minor nucleosides were identified by HPLC. In general, the minor nucleosides, especially N2-methyl-guanidine and ribothymidine, were higher in the ribosome-bound fraction. The PAGE patterns were similar but there were quantitative and qualitative differences among the smaller spots. In vivo labeling by 32P showed that new tRNA goes preferentially to the ribosome but mixing does occur. The results suggest the existence of two compartments of tRNA.

Human placental trophoblast plasma membranes were prepared by differential centrifugation and solubilized in nonionic detergent. Transferrin receptors were isolated from the solubilized membranes by affinity chromatography on diferric transferrin-coupled Sepharose 4B. The trophoblast plasma membrane vesicles demonstrated NADH-ferricyanide oxidoreductive activity. However, NADH-Fe(III) oxidoreductive activity was very weak when Fe(III)-ammonium citrate or diferric transferrin was used as electron acceptor in the presence of bathophenanthroline disulfonate as an indicator of the reaction. After solubilization, only NADH-ferricyanide oxidoreduction was recovered. Affinity chromatography-purified transferrin receptors did not exhibit any measurable oxidoreductase activity. Thus, when these receptors are present in plasma membranes they mediate redox reactions, but biochemically isolated receptors do not mediate such reactions. These observation suggest that transferrin receptors in plasma membranes bind diferric transferrin, and, in an undetermined way, facilitate Fe(III) release so that iron reduction can occur.

A protease from house dust mite(Dermatophagoides farinae) having high specificity towards a substrate of blood coagulation factor XIIa catalyzes the activation of kallikrein-kinin system in plasma (Takahashi et al., 1990). To prevent the formation of kinin by the mite-protease, inhibition of the protease with its inhibitors was tested in vitro and in vivo. Its kinetic studies revealed that Ki values are 3.9 x 10(-10) M for aprotinin, 3.0 x 10(-9) M for soybean trypsin inhibitor (Kunitz) and 2.5 x 10(-8) M for gabexate mesylate. Enhancement of blood permeability in guinea pigs caused by the protease was markedly suppressed by these inhibitors.

Two main forms of protein kinase C (PKC 1 and PKC 2) are detected in homogenates of rat hepatocytes using DEAE-cellulose column chromatography. The activity of these forms paradoxically, is rapidly decreased by treatment in vivo with phorbol myristate acetate (PMA). The dose-response curves to PMA for decreasing the activities of PKC 1 and 2 were shifted to the right by the potent and selective PKC inhibitors, staurosporine and calphostin-C. The decreases induced by 100 nM PMA were dose-dependently blocked by these inhibitors. It is concluded that activation of PKC is required and precedes such decreases in activity induced by the active phorbol ester.

It was found that the detyrosination of tyrosinated tubulin by tubulin carboxypeptidase can occur when both the enzyme and the substrate are adsorbed on nitrocellulose. This, and the use of a specific antibody that recognizes detyrosinated tubulin allowed us to localize tubulin carboxypeptidase on a nitrocellulose membrane after agarose gel electrophoresis and blotting. The method was also extended to detect pancreatic carboxypeptidase A.

The susceptibility to peroxidative stress of low density lipoprotein (LDL), induced by incubation with CuSO4, has been studied in eleven diabetic patients and eleven control subjects. Our results suggest that oxidized LDL (OX-LDL) of diabetic patients have a significant higher reactivity to 2,4,6-trinitrobenzene sulfonic acid (TNBS) than controls, that indicates a lower susceptibility of LDL to oxidative stress. Furthermore using the fluorescence polarization (Pf) of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its phosphatidylcholine derivative (DPH-PC) we have shown that peroxidation induces a decrease of fluidity in OX-LDL of controls and diabetic patients, both at the lipoprotein surface, where is localized DPH-PC and at the interface between lipoprotein surface and core which is probed by DPH.