Phosphorothioate analogues of 2',5'-oligoadenylate. Enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioates from adenosine 5'-O-(2-thiotriphosphate) and adenosine 5'-O-(3-thiotriphosphate)

The chiral and achiral phosphorothioate analogues of 2',5'-oligoadenylates (2-5A) have been enzymatically synthesized from the Sp and Rp isomers of adenosine 5'-O-(2-thiotriphosphate) [(Sp)-ATP beta S and (Rp)-ATP beta S, respectively] and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) by 2-5A synthetase from L929 cells and lysed rabbit reticulocytes. These 2',5'-phosphorothioate analogues were separated, purified, and structurally characterized. While ATP gamma S and (Sp)-ATP beta S were as efficient substrates for the 2-5A synthetase as was ATP, (Rp)-ATP beta S was more than 50-fold less efficient a substrate. The beta- and gamma-phosphorothioates were more resistant to enzymatic hydrolysis than was authentic 2-5A. Compared to 2-5A, there were marked differences in the biological activities of the 2',5'-phosphorothioates as determined by (i) binding to 2-5A-dependent endoribonuclease (RNase L), (ii) activation of RNase L to hydrolyze RNA, and (iii) inhibition of protein synthesis in intact L929 cells. These studies extend previous reports on the elucidation of the stereochemical requirements of 2-5A synthetase and RNase L [Karikó, K., Sobol, R. W., Jr., Suhadolnik, L., Li, S. W., Reichenbach, N. L., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (first of three papers in this issue); Karikó, K., Li, S. W., Sobol, R. W., Jr., Suhadolnik, R. J., Charubala, R., & Pfleiderer, W. (1987) Biochemistry (second of three papers in this issue)] with the phosphorothioate analogues of 2-5A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of the Danish (skive) variant of mouse liver alcohol dehydrogenase

The partially inbred Danish (Skive) strain of mice exhibits a form of liver alcohol dehydrogenase (ADH) which differs in electrophoretic mobility from that of all other inbred mouse strains thus far examined, e.g., C57BL/10, DBA/2J, and BALB/c. In order to compare the catalytic and molecular properties of the variant and normal enzyme forms, they were purified to homogeneity by ion-exchange and affinity chromatography. Tryptic peptides of reduced and carboxymethylated subunits of the normal and variant ADH forms were mapped by thin-layer two-dimensional electrophoresis and chromatography and by reversed-phase high-performance liquid chromatography. A unique nonapeptide in the Danish mouse liver ADH which did not appear in enzymes from C57BL/10, DBA/2J, or BALB/c mice was identified by both methods. Amino acid sequencing of this peptide revealed that the Arg residue at position 124, as predicted from the cDNA sequence of ADH in DBA/2J mice, has been replaced by Leu in the Danish variant. The Leu for Arg substitution in the variant form appears to account for its decreased cathodic mobility with electrophoresis in starch gels at pH 7.2. The K _m and V _max of ADH from the Danish strain for three primary alcohols and three aldehydes were similar in value to those of ADH from the C57BL/10, DBA/2J, and BALB/c strains. Based on the X-ray structure of horse liver ADH, position 124 is on the solvent-exposed surface of the catalytic domain. The finding that the kinetic constants are similar for the normal and variant forms is consistent with the observation that this residue is not in the active site and that there is no known role for it in the ADH catalytic mechanism.This work was supported by NIAAA Grant AA-04307.     

Nonadditive interactive effects of polychlorinated biphenyl congeners in rats: role of the 2,3,7,8-tetrachlorodibenzo-p-dioxin receptor

Administration of 3,3',4,4',5,5'-hexa-,3,3',4,4',5-penta-, and 2,3,3'4,4'5-hexa-chlorobiphenyl to immature male Wistar rats caused a thymic atrophy at high dose levels (1.25, 1.0, and 100 mumol/kg, respectively) and induced the hepatic cytochrome P-448 dependent monooxygenases (benzo[a]pyrene hydroxylase and ethoxyresorufin O-deethylase) at both high and low (0.25, 0.01, and 5 mumol/kg, respectively) doses. In contrast, 2,2',4,4',5,5'-hexachlorobiphenyl (HCBP) (300 mumol/kg) did not elicit any of these effects but elevated hepatic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) cytosolic receptor protein levels (threefold) as previously reported. The effects of hepatic receptor modulation by 2,2',4,4',5,5'-HCBP (300 mumol/kg) on the enzyme induction activities of 3,3'4,4',5-penta-, 3,3'4,4',5,5'-hexa-, and 2,3,3',4,4',5-hexa-chlorobiphenyl were dose-dependent; no interactive effects were observed at high (toxic) doses of these compounds, whereas apparent synergistically increased hepatic microsomal monooxygenase induction activities were noted at the lower submaximal induction doses. It was concluded that the increased responsiveness of the rats was due to elevated hepatic 2,3,7,8-TCDD receptor levels.     

Complete Nucleotide Sequence of the Mouse Lactate Dehydrogenase-a Functional Gene: Comparison of the Exon-Intron Organization of Dehydrogenase Genes

The complete sequence of 12,851 nucleotides of the mouse lactate dehydrogenase-A (LDH-A) gene has been determined. It includes eight exons, seven introns, promoter and regulatory regions. The B1 repetitive elements present in intron III and VI are oriented in opposite orientation, and they share 72% sequence homology. The exon-intron organization of mouse LDH-A gene is compared with the organizations of other dehydrogenase genes, and the molecular evolution of the nicotinamide adenine dinucleotide binding domains is discussed.     

Cloning of the 3'-phosphoadenylyl sulfate reductase and sulfite reductase genes from Escherichia coli K-12

The structural genes for 3'-phosphoadenylyl sulfate (PAPS) reductase (cysH) and sulfite reductase (alpha and beta subunits; EC 1.8.1.2)(cysI and cysJ) of Escherichia coli K-12 have been cloned by complementation. pCYSI contains two PstI fragments (18.3 and 2.9 kb) which complement cysH-, cysI-, and cysJ- mutants. Subcloning showed that the cysH gene is located on a 1.6-kb ClaI subfragment (pCYSI-3) whereas cysI and most of cysJ are carried on a 3.7-kb ClaI subfragment (pCYSI-5). The PAPS reductase gene is closely linked to the sulfite reductase genes, but its expression is regulated by a unique promoter. The cysI and cysJ genes, on the other hand, are transcribed as an operon and the promoter precedes the cysI gene. Maxicell analysis demonstrated that pCYSI encodes three polypeptides of Mr 27,000, 57,000, and 60,000, in addition to the tetracycline-resistance determinant. The 60- and 57-kDa proteins are most likely the alpha and beta subunits, respectively, of E. coli sulfite reductase while the 27-kDa protein is putatively identified as PAPS reductase. Preliminary data suggest that the alpha and beta subunits of sulfite reductase are encoded by cysI and cysJ, respectively.     

Cyclosporin treatment of IgA nephropathy: a short term controlled trial.

Cyclosporin''s known regulatory effects on the immune system suggest that it may be useful in treating patients with IgA nephropathy. A randomised prospective single blind study of 19 patients with IgA nephropathy and proteinuria (greater than 1.5 g/day) was conducted to determine the therapeutic value of cyclosporin. The patients were divided into two groups: nine patients were given oral cyclosporin (5 mg/kg/day) for 12 weeks and 10 patients a placebo. The two groups were comparable in age of presentation, ratio of men to women, plasma creatinine and serum IgA concentrations, creatinine clearance, daily urinary protein excretion, severity of renal histopathological changes, and prevalence of hypertension. A significant reduction of proteinuria and an increase of plasma albumin concentration was observed with treatment with cyclosporin. Nevertheless, a significant rise of plasma creatinine concentration and a fall in creatinine clearance was found in patients after six weeks'' treatment with cyclosporin, although the plasma cyclosporin concentrations were maintained within a narrow therapeutic range. Serum IgA concentrations were reduced in seven patients. Renal function improved within eight weeks after treatment was stopped. Three months after treatment was stopped proteinuria remained less than half of the pretreatment values in three patients. No similar biochemical changes were observed in the controls. Short term cyclosporin therapy may be beneficial in reducing proteinuria in some patients with IgA nephropathy. As transient renal impairment was seen, despite cyclosporin concentrations being maintained within a narrow therapeutic range, indiscriminate use of cyclosporin in glomerulonephritis should be discouraged.     

长滩果引种驯化研究

长滩果系罗汉果最佳品种。原产区分布于永福和临桂县交界400—600米山区,但不适应于低丘陵和平原地区,因而不能在低丘陵和平原地区推广。为了解决这个问题。采用了(1)选择湿润和半阴的生长环境;(2)采用适应性强的砧木;(3)促成栽培;(4)加强秋旱期的科学管理等措施并取得成功,试验结果表明长滩果产量和品质达到或略超过原产区水平。     

水翁花蕾和水翁叶精油的化学成分研究

水翁的花营和鲜叶经水蒸汽蒸馏得到一种淡黄色的精油,前者出油率为0.18％。后者为0.08％。我们应用毛细管气相色谱,气相色谱/质谱联用,红外光谱和紫外光谱等方法,对两种精油进行化学分成分析,分别鉴定出35个和27个已知化学成分。两者相同的化学成分有:β-罗勒烯(Z)、β-罗勒烯(E)、α-蒎烯、β-蒎烯、月桂烯、小茴香烯、香叶醇、顺式-丁香烯、橙花叔醇等23个化学成分,分别占花营精油全油的90％和叶精油95％以上。     

Some physicochemical properties of rice mitochondrial DNA

Summary Certain physicochemical properties of rice mitochondrial DNA (mtDNA) were determined. Certain low-molecular-weight mtDNA bands were found in addition to the major mtDNA band. Rice mtDNA appeared in the electron microscope as a collection of linear molecules with heterogeneous length in the range of 1–156 kb. The major distribution area was 60–105 kb. A small fraction (less than 5%) of rice mtDNA was found in the form of a circular molecule. Some molecules had the appearance of being supercoiled. Replication fork structures were found in both circular and linear mtDNA molecules. In one rice species, Jin Nante, 15 different circular molecules were found. Rice mtDNA was digested with different restriction enzymes. The total molecular weight of rice mtDNA was calculated to be about 300 kb according to the data of restriction enzyme digestion and electron microscopy.