Biochemistry and molecular biology international最新文献

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.

The action of flavonoids on bovine leukemia virus-transformed lamb fibroblasts (line FLK) and HL-60 cells was accompanied by lipid peroxidation, their toxicity was partly prevented by iron chelator desferrioxamine and antioxidant N,N'-diphenyl-p-phenylene diamine. This pointed out to the involvement of oxidative stress in flavonoid cytotoxicity. The concentration of compound for 50% survival of FLK cells (cL50) did not show correlation with polarographic oxidation half-peak potential (Ep/2) and/or partition coefficient (log P) of flavonoids; however, their toxicity to HL-60 cells was described by equation log cL50 (microM) = 3.0161 + 1.1099 Ep/2 (V) - 0.3369 log P. The toxicity of quercetin was partly prevented by nontoxic concentrations of other flavonoids examined, thus pointing out to potential neutralization of quercetin cytotoxicity by intake of flavonoid mixtures.

A D-galactose-binding lectin which does not require Ca2+ or reducing reagents to induce activity was purified from skin of sea hare, Aplysia kurodai, by affinity chromatography. Skin lectin was confirmed to be a disulfide-bonded heteromultimer with molecular mass of 200 kDa, consisting of 28 kDa and 26 kDa subunits by SDS-PAGE and two-dimensional SDS-PAGE. Human rhabdomyosarcoma cells attached to and spread on plastic plates coated with lectin. Cell adhesion induced by lectin was completely inhibited by the addition of lactose.

BB rat sublines, all developing an insulin-dependent diabetes, differ in several phenotypic traits and in the genotype. To study the genetic basis of phenotypic differences diabetic BB/OK and BB/Mol rats characterized by significantly different frequency and age at onset of diabetes were reciprocally crossed. F1 females of both crosses were backcrossed onto diabetic BB/Mol rats resulting 94 BC1 hybrids which were analyzed for 30 polymorphic microsatellite markers on 14 chromosomes. For the first time it is shown that a diabetes protective locus on chromosome X and a gene around the D10Mit9 locus on chromosome 10 can explain the low frequency (ca. 50%) and the late age at onset of diabetes (ca. 130 days) in the BB/OK rat subline, respectively.

Recombinant TIS21 protein was overexpressed in Escherichia coli harboring the expression vector plasmid pQE-30 carrying the TIS21 cDNA coding sequence containing an extra 120 nucleotides upstream. Employing this protein consisting of 158 amino acid residues of the main chain plus 40 residues of the fusion peptide. It was found that one of the protein methylase I group [S-adenosylmethionine:nuclear protein/histone-arginine N-methyltransferase; BC 2.1.1.23; J. Biol. Chem., 269, 1075 (1994)] methylated this protein. The methylation products were identified as guanidino-N-methylated arginines. Some of the kinetics of the reaction are described.

We have carried out a comparison of KM and Vmax values for various primers in the polymerization reaction catalyzed by the HIV-1 RT. The affinity of RT for complementary d(pT)6 containing two different 5'-end pyranone derivatives was 2-3 orders of magnitude higher (KM = 3-15 nM) than that of d(pT)6 (KM = 12.6 mM). Oligodeoxynucleotides (ODNs) noncomplementary to poly(A) template were not elongated by RT. However, derivatives of d(CAGGTG) containing the 5'-terminal chromone and coumarin related groups were efficient primers showing KM (30-300 nM) and Vmax (75-93%) values comparable with that for d(pT)10 (800 nM; 100%). The [d(CAGGTG)]ddT ODN derivatives were effective inhibitors of RT. The primer function of derivatives of noncomplementary ODNs appears to be due to the additional interactions of their 5'-terminal groups with the enzyme tRNA-binding site.

In the present investigation, the inactivation by N-bromosuccinimide of acid phosphatase from penaeus penicillatus has been studied using the kinetic method of the substrate reaction during modification of enzyme activity as previously described by Tsou [(1988, Adv. Enzymemol. Related Areas Mol. Biol. 61, 381-436]. The results show that inactivation of the enzyme by N-bromosuccinimide is a slow, reversible reaction. The results also clearly show that the modification of the tryptophan residues of penaeus penicillatus acid phosphatase by high concentrations of N-bromosuccinimide led to the complete inactivation of the enzyme. The microscopic rate constants were determined for the reaction of the inactivator with the free enzyme and with the enzyme-substrate complex. Comparison of the obtained microscopic rate constants indicates that the presence of the substrate offers marked protection of the enzyme against inactivation by N-bromosuccinimide. The above results suggest that the tryptophan residue is essential for activity and may be situated at the active site of the enzyme.

The nucleotide sequences of the S-RNA of Akabane viruses JaGAr-39, OBE-1, Iriki and the newly isolated PT-17 strains and the Aino virus were determined and compared. The results reveal that the S-RNAs of the four Akabane strains share 96.9% homology in nucleotide sequences. Only one amino acid difference out of the 233 amino acids of the nucleocapsid protein (N) and three amino acid differences in the 91 amino acids of the nonstructural protein (NSs) were found among the Akabane viruses. Amino acid sequences of N and NSs proteins of the Aino virus have approximately 80% identity as compared with the Akabane viruses. The results also demonstrate that the four Akabane viruses and the Aino virus can be clearly differentiated by RFLP (restriction fragments length polymorphism) analysis using RT-PCR generated nucleocapsid protein genes and digested with HaeIII and HindIII. The phylogenetic tree based on the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) analysis of the sequences of nucleocapsid protein genes and the S-DNAs revealed that the newly isolated PT-17 strain is most closely related to Iriki strain, than the JaGAr-39 or OBE-1 strains.

Translational regulation of HPV16 E7 mRNA is of particular interest since the viral E7 protein has oncogenic activity. Here we report that additional supplementation of cell-free reticulocyte/wheat-germ translation systems with rat liver tRNA pool favors the expression of the viral oncoprotein E7 which otherwise is scarcely translatable. The translational activation is explained by the positive correlation between the frequency of the glutamic and aspartic codons in E7 mRNA, and the increased concentration of the corresponding tRNAs following tRNA supplementation. The present in vitro data suggest a link between E7 expression and cell conditions conductive to tRNA changes such as development, cell proliferation and aging.

The changes of Na,K-ATPase activity and its regulation have been investigated in the renal cortex and its basolateral membrane of aldosterone-induced hypertensive rat. Ouabain-sensitive Na,K-ATPase activity and [3H]ouabain-binding site (Bmax) in the hypertensive rat were significantly increased than those in the control. The levels of Na,K-ATPase alpha 1- and beta 1-subunit mRNA of the renal cortex in hypertensive rat were more increased than those in the control, and their increases were repressed by actinomycin-D. These results suggest that the increase of Na,K-ATPase activities and ouabain binding sites in aldosterone-induced hypertensive rat may be correlated with transcriptional regulation of Na,K-ATPase gene expression.