Effects of rubratoxin B on the kinetics of cationic and substrate activation of (Na+-K+)-ATPase and p-nitrophenyl phosphatase.

Rubratoxin B, a lactone-containing bisanhydride metabolite of certain toxigenic molds, inhibited (Na+-K+)-stimulated ATPase activity of mouse brain microsomes in a dose-dependent manner with an estimated IC50 of 6 x 10(-6) M. Hydrolysis of ATP was linear with time and enzyme concentration, with or without rubratoxin in reaction mixtures. Altered pH and activity curves for (Na+-K+)-ATPase demonstrated comparable inhibition by rubratoxin in buffered acidic, neutral, and alkaline pH ranges. Kinetic studies of cationic-substrate activation of (Na+-K+)-ATPase indicated classical competitive inhibition for Na+ and K+. Results also showed competitive inhibition for K+ activated p-nitrophenyl phosphatase as demonstrated by altered binding site parameters without change in the catalytic velocity of dephosphorylation of the enzyme . phosphoryl complex. Noncompetitive inhibition with regards to activation by ATP and p-nitrophenyl phosphate was indicated by altered Vmax values with no change in Km values. Inhibition was partially restored by repeated washings. Preincubation with sulfhydryl agents protected the enzyme from inhibition. Cumulative inhibition studies with rubratoxin and ouabain indicated possible interaction between the two inhibitors of (Na+-K+)-ATPase. Rubratoxin appeared to exert its effects on (Na+-K+)-ATPase by interacting at Na+ and K+ sites.     

Kinetics of carbon monoxide binding to singly reduced human methemoglobin

The kinetics of reaction of singly reduced methemoglobin (HbFe3(3+)Fe2+) with carbon monoxide have been investigated by the pulse radiolysis method. The rate constant for carbon monoxide binding to this form of hemoglobin is 4.1 X 10(6) M-1 S-1 at 24 degrees in our solutions. This value compares with existing values for various forms of hemoglobin ranging from 4 X 10(6) to 6.5 X 10(6) M-1 S-1. Addition of inositol hexaphosphate to the solutions results in a lower rate constant for carbon monoxide binding amounting to 1.1 X 10(5) M-1 S-1.     

A proton nuclear magnetic resonance investigation of proximal histidyl residues in human normal and abnormal hemoglobins. A probe for the heme pocket.

Proton nuclear magnetic resonance spectroscopy at 250 MHz has been used to investigate the conformations of proximal histidyl residues of human normal adult hemoglobin, hemoglobin Kempsey [beta 99(G1) Asp leads to Asn], hemoglobin Osler [beta 145(HC2) Tyr leads to Asp], and hemoglobin McKees Rocks [beta 145(HC2) Tyr leads to Term] around neutral pH in H2O at 27 degrees C, all in the deoxy form. Two resonances that occur between 58 and 76 ppm downfield from the water proton signal have been assigned to the hyperfine shifted proximal histidyl NH-exchangeable protons of the alpha- and beta-chains of deoxyhemoglobin. These two resonances are sensitive to the quaternary state of hemoglobin, amino acid substitutions in the alpha 1 beta 2-subunit interface and in the carboxy-terminal region of the beta-chain, and the addition of organic phosphates. The experimental results show that there are differences in the heme pockets among these four hemoglobins studied. The structural and dynamic information derived from the hyperfine shifted proximal histidyl NH-exchangeable proton resonances complement that obtained from the ferrous hyperfine shifted and exchangeable proton resonances of deoxyhemoglobin over the spectral region from 5 to 20 ppm downfield from H2O. The relationship between these findings and Perutz's stereochemical mechanism for the cooperative oxygenation of hemoglobin is discussed.     

Forskolin as a novel lipolytic agent

Forskolin is a novel lipolytic agent which elevates cAMP and FFA release in rat adipocytes in a manner different from existing lipolytic factors. This effect of Forskolin is potentiated by all lipolytic hormones tested, i.e. epinephrine, ACTH, and glucagon and is also reversible. The same batch of adipocytes can be repeatedly stimulated after washing. The effective concentration of Forskolin is in the micromolar range. Its action is due to an activation of cAMP synthesis by adenylate cyclase. There is no effect on cAMP hydrolysis. In contrast to stimulation by lipolytic hormones, Forskolin-activated membrane adenylate cyclase was not further stimulated by GPP(NH)P. These results suggest that Forskolin may be a useful analytical agent in the study of adenylate cyclase mediated function in intact adipocytes.     

Covalent modification of guanine bases in double-stranded DNA. The 1.2-A Z-DNA structure of d(CGCGCG) in the presence of CuCl2

We have solved the single crystal structure to 1.2-A resolution of the Z-DNA sequence d(CGCGCG) soaked with copper(II) chloride. This structure allows us to elucidate the structural properties of copper in a model that mimics a physiologically relevant environment. A copper(II) cation was observed to form a covalent coordinate bond to N-7 of each guanine base along the hexamer duplex. The occurrence of copper bound at each site was dependent on the exposure of the bases and the packing of the hexamers in the crystal. The copper at the highest occupied site was observed to form a regular octahedral complex, with four water ligands in the equatorial plane and a fifth water along with N-7 of the purine base at the axial positions. All other copper complexes appear to be variations of this structure. By using the octahedral complex as the prototype for copper(II) binding to guanine bases in the Z-DNA crystal, model structures were built showing that duplex B-DNA can accommodate octahedral copper(II) complexes at the guanine bases as well as copper complexes bridged at adjacent guanine residues by a reactive dioxygen species. The increased susceptibility to oxidative DNA cleavage induced by copper(II) ions in solution of the bases located 5' to one or more adjacent guanine residues can thus be explained in terms of the cation and DNA structures described by these models.     

39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.     

Mutagenic DNA repair in enterobacteria.

Sixteen species of enterobacteria have been screened for mutagenic DNA repair activity. In Escherichia coli, mutagenic DNA repair is encoded by the umuDC operon. Synthesis of UmuD and UmuC proteins is induced as part of the SOS response to DNA damage, and after induction, the UmuD protein undergoes an autocatalytic cleavage to produce the carboxy-terminal UmuD' fragment needed for induced mutagenesis. The presence of a similar system in other species was examined by using a combined approach of inducible-mutagenesis assays, cross-reactivity to E. coli UmuD and UmuD' antibodies to test for induction and cleavage of UmuD-like proteins, and hybridization with E. coli and Salmonella typhimurium umu DNA probes to map umu-like genes. The results indicate a more widespread distribution of mutagenic DNA repair in other species than was previously thought. They also show that umu loci can be more complex in other species than in E. coli. Differences in UV-induced mutability of more than 200-fold were seen between different species of enteric bacteria and even between multiple natural isolates of E. coli, and yet some of the species which display a poorly mutable phenotype still have umu-like genes and proteins. It is suggested that umDC genes can be curtailed in their mutagenic activities but that they may still participate in some other, unknown process which provides the continued stimulus for their retention.     

鳅鮀鱼类鳔囊结构及系统发育研究(鲤形目:鲤科)

本文应用分支系统学原理对鳅(鱼它)鱼类的鳔囊、鳔及相关结构进行了特征分圻,并以(鱼句)亚科作为外类群。解剖结果表明,鳅(鱼它)鱼类的鳔分四种类型;分析结果进一步证实鳅(鱼它)鱼类是一个单源群,作为一亚科可分为异鳔鳅(鱼它)属和鳅(鱼它)属,后者又分为原鳅(鱼它)和鳅(鱼它)二个亚属。     

人源性流行性出血热病毒的形态特征

本文报道流行性出血热病毒(汉坦病毒)H-114株的电镜形态。发现形态发生以内质网膜和胞浆膜芽生为主。病毒颗粒为圆形或卵圆形。具有双层膜结构,大小为90～120nm。提出了汉坦病毒形态发生的理论观点。     

Determination of buprenorphine by high-performance liquid chromatography with fluorescence detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with fluorescence detection was developed for the determination of buprenorphine in human, rabbit, pig and dog plasma. It is comprised of only a one-step extraction procedure with hexane—isoamyl alcohol at pH 9.25 and reversed-phase chromatography on a μPorasil column. The recoveries of buprenorphine and nalbuphine (internal standard) were greater than 90%. Calibration graphs were linear over the concentration range 3–300 ng/ml with a coefficient of variation, both within-day and between-day, of less than 9% at any level. The limit of detection was 1.0 ng/ml of plasma based on a signal-to-noise ratio of 3. Eight other clinically used narcotics were investigated to check for potential interferences and their analytical conditions. The possible decomposed compounds of buprenorphine were also checked for the specificity of this assay. The method has been succesfully applied to the stability and pharmacokinetic studies of buprenorphine. Buprenorphine in plasma did not decompose significantly at −20°C for four weeks. Pharmacokinetic application in six rabbits and a surgical patient revealed that buprenorphine followed a linear three-compartment model with two distribution phases. The two distribution and elimination half-lives and the clearance of buprenorphine were 1.32, 24.8 and 230 min and 224 ml/min in human plasma, and 0.94, 12.5 and 232 min and 30 ml/min in rabbit plasma.