Biochemistry and molecular biology international最新文献

The effect of various organic acids on hydroxyl radical (.OH) generation in the Fenton reaction were examined by the ESR spin trapping technique, where 5,5-dimethyl-1-pyroline-N-nitroxide (DMPO) and alpha-phenyl-tert-butyl nitrone (PBN) were used as the spin trapping reagents. alpha-Hydroxy acids such as lactic acid, glycolic acid and 2-hydroxy isobutyric acid were found to markedly enhance .OH generation in the reaction. In contrast, beta-hydroxy acid, alpha-keto acid, esters of alpha-hydroxy acids, aldehydes and other straight chain organic acids had no such enhancing activity. alpha-Amino acids had also no enhancing effect. The results suggest that the alpha-hydroxy acid moiety is prerequisite for the enhancement of .OH generation in the Fenton reaction. Superoxide dismutase did not inhibit the enhancing effect of alpha-hydroxy acids whereas catalase completely inhibited the .OH generation. Thus, alpha-hydroxy acids directly enhanced the .OH generation via the Fenton reaction but not the Haber-Weiss reaction. Possible role of lactic acid manipulating .OH generation is discussed in relation to the ischemia-reperfusion cell damage.

When cybrids with a point mutation, which locates in the tRNALeu(UUR) gene of mtDNA and causes a mitochondrial encephalomyopathy (MELAS syndrome), were exposed to a high concentration of oxygen (95%), the peroxide production markedly increased by 6 h of oxygen exposure, whereas the peroxide production was similar among the cybrids under a normal concentration of oxygen. The peroxide production by oxygen exposure was enhanced particularly in cybrids with high proportions of the mutant mtDNA and low respiratory capacities. The appearance of apoptotic cells by oxygen exposure was high in cybrids with the impaired respiratory function due to the mutation. An antioxidant NAC successfully suppressed both the peroxide production and apoptosis. These results imply that the peroxide production plays an important role in inducing apoptosis in cells carrying the mtDNA mutation causing encephalomyopathy.

Presence of homo- and hetero-tetrameric rice sucrose synthase (RSS) isoforms in etiolated rice seedlings was demonstrated by immunoprecipitation using monospecific antibodies. Three RSS isozymes with various pI values were purified by ammonium sulfate fractionation, Sepharose CL-6B gel filtration, DEAE-Sephacel and Mono-Q ion exchange chromatographies. They were characterized as heterotetramers composed of RSS1 and RSS2 subunits. All of them used UDP as the best substrate. The presence of divalent metal ions increased the activities of synthesis but inhibited the cleavage of sucrose.

Apoptosis induced by peroxynitrite in cultured cerebellar granule cells was confirmed morphologically by chromatin condensation and biochemically by DNA laddering. A 30 min exposure to peroxynitrite (10 microM) initiated oxidative stress, which caused the formation of thiobarbituric acid-reactive substances (TBARS) and the alteration of cell membrane fluidity. Peroxynitrite treatment also caused ATP decrease and thus activated the apoptotic program. Pre-treating cells with antioxidant EPC-K1 (L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H -1- benzopyran-6-yl-hydrogen phosphate] potasium salt), a new water-soluble derivative of vitamin C and vitamin E, attenuated oxidative injury and prevents cells from apoptosis. The results suggest that EPC-K1 might be used as a potential therapeutic agent for diseases associated with NO/ONOO(-)-mediated neuronal injury.

Cry proteins from Bacillus thuringiensis have insecticidal properties. The function of domains I and II has been described but domain III has so far eluded understanding. Domain III from Cry1Ab and Cry1Ac has been cloned, expressed in E. coli and injected to rabbits with the aid of characterizing them immunologically. Interestingly, polyclonal antibodies against Cry1Ab fragment did not recognize either the native Cry1Ab toxin or the Cry1Ac fragment while those against the latter did recognize either the native Cry1Ac toxin or the Cry1Ab protein fragment. A combination of information from sequence comparison and hydrophobicity profile indicates that these protein fragments possibly adopt different spatial dispositions within the respective toxins.

Using new time-resolved multichannel experimental and simulated data, analyzed by SVD, it is concluded that the second order binding rate constant for O2-binding to fully reduced mammalian cytochrome aa3 is approximately five times higher than the currently accepted value of approximately 1.5 x 10(8) M-1 s-1.

Effect of two angiotensin convertase inhibitors, enalapril and captopril, on blood plasma and erythrocyte lipid peroxidation and plasma peroxyl radical-trapping capacity was studied in rats with streptozotocin-induced diabetes. A progressive increase in blood erythrocyte malondialdehyde (MDA) level was observed in diabetic rats after 6 and 12 weeks. Blood plasma MDA level increased while plasma peroxyl radical-trapping capacity was decreased after 12 weeks. Captopril (2 mg/kg body weight) augmented the diabetes-induced changes in MDA content after 6 weeks and prevented them after 12 weeks increasing also the peroxyl radical-trapping capacity. Enalapril (1 mg/kg body weight) counteracted the diabetes-induced changes in MDA content after both 6 and 12 weeks but did not affect the plasma peroxyl radical-trapping capacity. These results suggest a possibility of a therapeutic use of angiotensin convertase inhibitors to attenuate the effects of oxidative stress in diabetes.

The conformational changes of the active site of creatine kinase (ATP: creatine N-phosphotransferase EC 2.7.3.2.) during thermal denaturation was followed by changes in fluorescence at the active site of the enzyme labeled by o-phthalaldehyde. Conformational changes of the active site occurred at the same time as inactivation of the enzyme. The active site changes occurred before the denaturation of the enzyme molecule as a whole was detected. The above results showed that the thermal inactivation of the creatine kinase was due to the conformational changes of its active sites.

We have generated a polyclonal antibody (CKG2) against native chicken gizzard ecto-ATPase for immunolocalization and immunoprecipitation. Active ecto-ATPase is immunoprecipitated from solubilized chicken and rat membranes and shown to be localized to the plasma membrane of the chicken smooth muscle cells. This antibody is specific for the ecto-ATPases, since the more abundant chicken stomach ecto-apyrase is not recognized in immunoprecipitation, western blot or immunolocalization analyses. The CKG2 antibody cross-reacts with mammalian (rat) ecto-ATPase in western blots, with testis being the most abundant source. Interestingly, when the same rat membranes are analyzed by western blot under non-reducing conditions, the 66 kDa ecto-ATPase is not recognized, instead a 200 kDa protein is detected, previously postulated to be an oligomer of ecto-ATPase. However, this 200 kDa cross-reacting protein is not related to the ecto-ATPases, but is instead an immunoglobulin binding protein, comprised of 50 kDa subunits.

Increasing evidence indicates that redox regulation is an important signaling mechanism. Protein tyrosine phosphatases (PTPases) are sensitive to oxidative inactivation and are potential targets of redox regulation. In this study, we analyzed the reversibility of oxidative inactivation of the PTPase SHP-1, which negatively regulates protein tyrosine kinase signaling. H2O2 inactivated SHP-1 in vitro. Incubation of the H2O2-inactivated SHP-1 with dithiothreitol recovered 44-99% of the PTPase activity, depending on the H2O2 concentrations used to inactivate SHP-1. Glutathione and N-acetylcysteine also reactivated H2O2-treated SHP-1. Stimulation of SHP-1-transfected HeLa cells with H2O2 rapidly decreased SHP-1 activity, which was completely reversed within 15 min. Thus, oxidative inactivation of SHP-1 is a reversible process.