Biochemistry and molecular biology international最新文献

The rae28 gene, a mouse homologue of the Drosophila polyhomeotic gene, is involved in the maintenance of the transcriptional repression states of Hox genes. In this study we synthesized the glutathione S transferase-RAE28 (GST-RAE28) fusion protein and examined sequence-specific DNA binding activity in the RAE28 protein by using the selected and amplified binding site method. After five rounds of enrichment, the eluted DNAs were amplified, cloned and sequenced. The sequences of individual oligonucleotides included the following consensus sequences; 5'-ACCA-3', 5'-ACCCA-3', 5'-CTATCA-3' and 5'-TGCC-3'. The oligonucleotides including these consensus sequences were show to have significant affinity with the GST-RAE28 fusion protein. The RAE28 protein was recently shown to form multimeric protein complexes with other members of mouse Pc-G proteins in the nucleus. These findings strongly suggest that the RAE28 protein constitutes a sequence-specific DNA binding domain in multimeric Pc-G protein complexes.

Macrophages/foam cells have a pivotal role in atherogenesis although little is known about the way lipid imbalance, a hallmark of atherosclerosis, leads to lipid accumulation in these cells. Modified low-density lipoproteins are associated with macrophage lipid dysfunction in atherosclerosis, but a possible role for altered lipogenesis leading to lipid accumulation remains to be elucidated. Since endothelium-derived nitric oxide (NO) and prostaglandins (PGs) are physiological autacoids whose production may be impaired in atherosclerosis, the effects of these mediators on de novo lipid synthesis in 24-h cultured rat peritoneal macrophages is investigated. In resident (unstimulated) cells, 1 microM PGE2 and the stable analog of PGI2 carbaprostacyclin (cPGI2, 1 microM) deviated the overall [1-14C]acetate from incorporation into cholesterol, free fatty acids and triacylglycerols favoring the formation of phospholipids. In inflammatory (thioglycollate-elicited) macrophages, these eicosanoids likewise reduced 14C-incorporations into all the lipid fractions tested. Also, cPGI2 and PGE2 reduced [4-14C]cholesterol uptake from inflammatory cells but did not interfere in 14C-cholesterol export. The PGE2-derivative PGA2 (10-20 microM) reduced 14C-incorporations into all the lipids in resident cells while it enhanced phospholipid synthesis by up to 129% at the expense of reduced incorporations into the other test lipids. The NO donor S-nitroso-N-acetylpenicillamine (SNAP, 1-10 microM), when added to macrophages in the presence of superoxide dismutase (SOD, to avoid the reaction of superoxide with NO), significantly reduced lipogenesis especially in inflammatory cells. These findings suggest that endothelium-derived NO and PGs may be associated with macrophage lipid accumulation by modulating lipogenesis and cholesterol uptake within these cells.

Changes of glycosylation of serum proteins of patients with psoriatic arthritis were detected by lectin blotting and a new enzyme-linked lectin assay (ELLA) using concanavalin A (Con A). A good linear correlation was found between the total Con A-reactivity of serum and the serum levels of C-reactive protein and interleukin-6, which is known to regulate the glycosylation pattern of proteins upon inflammation. A good linear correlation was also observed between the immunoreactivity of alpha 1-antitrypsin, measured by ELISA, using a monoclonal antibody sensitive to glycosylation changes, and the erythrocyte sedimentation rate and the serum concentrations of soluble interleukin-2 receptor, an index of lymphocyte activation which correlated with some inflammatory parameters of disease activity. These protein changes, which are described here for the first time, deserve to be studied in further detail in view of their possible clinical applications.

We examined effects of pravastatin on age-related changes in mitochondrial function in rats. Decline in the activity of complex I of the mitochondrial electron transport chain was observed in diaphragm and psoai major in rats aged 35 and 55 weeks, and that of complex IV in rats aged 55 weeks. Pravastatin accelerated significantly age-related decline in the activity of complex I of diaphragm mitochondria, though pravastatin did not show significant effect on normally observed age-associated decline in the activities of complex IV of psoai major and diaphragm mitochondria. Aging effect on mitochondrial respiratory function was not observed on heart muscle and liver in rats up to 55 weeks old, and pravastatin did not effect significantly heart and liver mitochondrial respiratory function. From these results, careful clinical examination on respiratory muscle function should be necessary in patients treated with pravastatin particularly in elderly patients.

Three wild-type strains of Saccharomyces cerevisiae, viz. K, Y55 and sigma 1278b, two mutants lacking one or both of the putative K+ transporters, trk1 delta and trk1 delta trk2 delta, and a mutant in the plasma membrane H(+)-ATPase, viz. pma1-105, were compared in their extracellular acidification following addition of glucose and subsequent addition of KCl; in ATPase activity in purified plasma membranes; and in respiration on glucose. The glucose-induced acidification was the greater the higher the respiratory quotient, i.e. the higher the anaerobic metabolism. A markedly lower acidification was found in the ATPase-deficient pma1-105 strain but also in the TRK-deficient double mutant. The acidification pattern after addition of KCl corresponds to expectations in the TRK mutants; however, a similarly decreased acid production was found in the ATPase-deficient mutant pma1-105. The highest rate of ATP hydrolysis in vitro was found with the trk1 delta trk2 delta mutant where glucose-, as well as KCl-induced acidification were lowest. Likewise, the pma1-105 mutant with extremely low acidification showed only a minutely lower ATP hydrolysis than did its parent Y55 strain. Apparently, several different sources of acidity are involved in the glucose-induced acidification (including extrusion of organic acids); in fact, contrary to the general belief, the H(+)-ATPase may play a minor role in this process in some strains.

Human interleukin-17 (hIL-17) gene without the signal sequence was isolated from activated peripheral blood lymphocytes by RT-PCR, then highly expressed in the yeast Pichia pastoris in the form of the glycosylated monomer. The monomer of rhIL-17 stimulated mouse fibroblast 3T3 cells to secrete IL-6 and was specifically bound to its receptors on 3T3 cells.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a rare disease in North China. In the present investigation, DNA samples from 17 patients with G6PD deficiency from Tianjin area in North China were studied for the two G6PD common mutations (R459L and R463H) and two single nucleotide polymorphisms (1311C/T and 1365-13T/C) using a dideoxy fingerprinting method. Five patients were positive for mutation R459L, and six patients were positive for mutation R463H. Further haplotype analyses using three flanking dinucleotide repeat polymorphism loci, DXS1123, DXS1113, and F8C(IVS13), were performed on 14 patient families and 16 control Chinese females. The results indicated that the two common mutations were from different haplotypes. Also, the data suggested a possible allelic association between the two G6PD common mutations and the F8C(IVS13) locus and a different allelic distribution for loci DXS1113 and F8C(IVS13) between Chinese and Caucasian populations.

Experimentally induced diabetic rats were treated separately with insulin and vanadate. The activities of hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6PDH) were increased in reticulocyte hemolysate isolated from the diabetic rats and were restored to normal levels by insulin. The restoration was not detected in vanadate treated diabetic animals. The enzymes of glutathione metabolism namely glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-s-transferase (GST) exhibited increases in their activities with diabetes and were restored to almost control values by insulin treatment. Vanadate given to diabetic animals further increased GPx, and GST. The level of superoxide dismutase(SOD) decreased in the reticulocytes of diabetic rats and catalase (CAT) was unchanged. Both CAT and SOD had normal values when the diabetic rats were treated with insulin and vanadate. It is proposed that vanadate may cause an increase in the activity of GR which may stimulate glucose transporters and glucose metabolism.

A cDNA library of Rinderpest vaccine virus was prepared in Zap Express vector (Stratagene). The Rinderpest 'N' gene specific clones were selected, characterized and thereafter expressed in E. coli XLOLR strain. The expressed protein was found to be immunogenic in western blot with hyperimmune sera. It reacted with rinderpest and 'N' protein specific monoclonal antibodies in Enzyme Linked Immunosorbent Assay (ELISA). Prokaryotically expressed 'N' protein also gave precipitin band in counter immunoelectrophoresis test (CIE). The expression of N protein was sufficient for its utility as positive antigen in CIE and ELISA used for rinderpest diagnosis.

Three constitutive gelatinases in human plasma were identified and characterized relative to known matrix metalloproteinase (MMP) gelatinases: MMP-2 (fibroblast 72-kDa) and MMP-9 (neutrophil 92-, 130-, and 225-kDa). Substrate gel electrophoresis (gelatin zymography) revealed an apparent Mw of 78-, 82-, and 89-kDa for these gelatinases. Densitometry revealed that MMP-9 and MMP-2 were highly calcium sensitive requiring 50-150 microM and 500 microM calcium for half-maximal activity, respectively. Of the new gelatinases, only the 89-kDa form demonstrated slight calcium activation. The three gelatinases were unaffected by known MMP inhibitors: EDTA (5 mM), 1,10-phenanthroline (2 mM), and pepstatin (18 microM). Serine and thiol protease inhibitors (leupeptin, aprotinin, PMSF, TLCK, TPCK, antichymostatin, antipain) were also ineffective. Solution-phase IEF revealed that the 78- and 82-kDa forms focused at neutral pI 6.72-7.95 whereas the 89-kDa focused at an acidic pI 4.89-5.18 (similar to neutrophil and fibroblast forms). The data indicate that these gelatinases are not MMPs or partially activated MMPs. Their role in normal and pathological conditions is not known.