Purification and steady-state kinetic characterization of human liver beta 3 beta 3 alcohol dehydrogenase

Human liver alcohol dehydrogenase catalyzes the NAD+-dependent oxidation of alcohols. Isoenzymes are produced in liver by five different genes, two of which are polymorphic. We have studied the three beta beta isoenzymes produced at ADH2 because they exhibit very different kinetic properties and they appear with different frequencies in different racial populations. The beta 3 beta 3 isoenzyme which appears in 25% of black Americans was purified to homogeneity, and conditions were found to stabilize this labile isoenzyme. The comparison of substrate specificity among beta beta isoenzymes for primary straight-chain alcohols indicates that there is a positive correlation between Vmax/KM and the log octanol/water partition coefficient for alcohols with beta 2 beta 2 and beta 3 beta 3 but not with beta 1 beta 1. Methyl substitutions at C1 or C2 of these alcohols reduce the catalytic efficiency with all three isoenzymes. The KM and Ki values of beta 3 beta 3 for NAD+ and NADH are substantially higher than values for beta 1 beta 1 or beta 2 beta 2. The Vmax of beta 3 beta 3 ethanol oxidation is 90 times that of beta 1 beta 1. Sequencing of the beta 3 subunit and gene indicates that the polymorphism results from a single amino acid exchange of Cys-369 in beta 3 for Arg-369 in beta 1 and beta 2 [Burnell et al. (1987) Biochem. Biophys. Res. Commun. 146, 1227-1233]. In horse alcohol dehydrogenase and beta 1 beta 1, the guanidino group of Arg-369 is thought to stabilize the NAD(H)-enzyme complex by bonding to one of the pyrophosphate oxygens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progress toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer

Mammalian cloning by nuclear transfer from somatic cells has created new opportunities to generate animal models of genetic diseases in species other than mice. Although genetic mouse models play a critical role in basic and applied research for numerous diseases, often mouse models do not adequately reproduce the human disease phenotype. Cystic fibrosis (CF) is one such disease. Targeted ablation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in mice does not adequately replicate spontaneous bacterial infections observed in the human CF lung. Hence, several laboratories are pursuing alternative animal models of CF in larger species such as the pig, sheep, rabbits, and ferrets. Our laboratory has focused on developing the ferret as a CF animal model. Over the past few years, we have investigated several experimental parameters required for gene targeting and nuclear transfer (NT) cloning in the ferret using somatic cells. In this review, we will discuss our progress and the hurdles to NT cloning and gene-targeting that accompany efforts to generate animal models of genetic diseases in species such as the ferret.     

Enzymatic properties of human glucuronyltransferase and a sensitive method for its assay in a stable B lymphocyte cell line

Properties of lymphocyte glucuronyltransferase were studied in homogenates of SN1006 cells. A sensitive assay procedure for lymphocyte glucuronyltransferase was developed utilizing radioactive testosterone as the acceptor substrate and TLC for separation of the metabolite. The method is capable of detecting picomolar quantities of the product. The enzyme activity exhibited a broad pH optimum, and was subject to activation by the detergent Lubrol WX and Mn++ ions. The activity conformed to the Michaelis-Menten kinetics giving apparent Km values of 0.8 mM and 11 microM, for UDPGA and testosterone, respectively. 4-Methylumbelliferone, a-naphthol and p-nitrophenol behaved as competitive inhibitors of testosterone glucuronidation. The results indicate that the method could be used for genetic studies of human lymphocyte glucuronyltransferase, and that the enzyme is of consequence in detoxication of exogenous as well as endogenous substrates.     

A thin-film electrochemical study of the "blue" copper proteins,auracyanin A and auracyanin B,from the photosynthetic bacterium <Emphasis Type="Italic">Chloroflexus</Emphasis><Emphasis Type="Italic">aurantiacus</Emphasis>: the reduction potential as a function of pH

The reversible formal potentials of auracyanin A and auracyanin B, two closely related "blue" copper proteins from the photosynthetic bacterium Chloroflexus aurantiacus, have been determined by protein film voltammetry in the range 4相似文献   

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

Biological Data on <Emphasis Type="Italic">Anovia punica</Emphasis> Gordon (Coleoptera: Coccinellidae), a Predator of <Emphasis Type="Italic">Crypticerya multicicatrices</Emphasis> Kondo &amp; Unruh (Hemiptera: Monophlebidae)

The coccinellid beetle Anovia punica Gordon (Coleoptera: Coccinellidae: Noviini) is an important predator of the Colombian fluted scale, Crypticerya multicicatrices Kondo & Unruh (Hemiptera: Monophlebidae). In order to gather information on the biological traits of A. punica, we conducted a series of studies, including of the developmental time of each life history stage, estimation of life table parameters, and predation rates under laboratory conditions [25.1 ± 1.6°C, with 70.5 ± 7.3% RH, and natural light regime, approx. 12:12 (L:D) h]. Developmental stages of A. punica were categorized as follows: egg stage, four larval instars, prepupal instar, pupal instar, and adult. Developmental time from egg to adult emergence averaged 29.41 ± 1.85 days, and 47.6% of the eggs developed to adulthood. Female and male survival was 94.42 and 90 days, respectively. Life table parameters show that one female of A. punica is replaced by 86 females (R ₀), the intrinsic growth rate (r _m ) was 0.1115, the average generation time (T) was 40 days, and the doubling time (D _t ) was 6.2 days. The life table parameters suggest that A. punica can be used as a potential predator of C. multicicatrices and, more importantly, provided baseline information for a mass-rearing protocol. This is the first detailed study on the biology of A. punica that reports the potential of this predator as a biological control agent for scale insects of the tribe Iceryini.