Substrate specificity and kinetic isotope effect analysis of the Eschericia coli ketopantoate reductase

Ketopantoate reductase (EC 1.1.1.169), an enzyme in the pantothenate biosynthetic pathway, catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate. The enzyme exhibits high specificity for ketopantoate, with V and V/K for ketopantoate being 5- and 365-fold higher than those values for alpha-ketoisovalerate and 20- and 648-fold higher than those values for alpha-keto-beta-methyl-n-valerate, respectively. For pyridine nucleotides, V/K for beta-NADPH is 3-500-fold higher than that for other nucleotide substrates. The magnitude of the primary deuterium kinetic isotope effects on V and V/K varied substantially when different ketoacid and pyridine nucleotide substrates were used. The small primary deuterium kinetic isotope effects observed using NADPH and NHDPH suggest that the chemical step is not rate-limiting, while larger primary deuterium isotope effects were observed for poor ketoacid and pyridine nucleotide substrates, indicating that the chemical reaction has become partially or completely rate-limiting. The pH dependence of (D)V using ketopantoate was observed to vary from a value of 1.1 at low pH to a value of 2.5 at high pH, while the magnitude of (D)V/K(NADPH) and (D)V/K(KP) were pH-independent. The value of (D)V is large and pH-independent when alpha-keto-beta-methyl-n-valerate was used as the ketoacid substrate. Solvent kinetic isotope effects of 2.2 and 1.2 on V and V/K, respectively, were observed with alpha-keto-beta-methyl-n-valerate. Rapid reaction analysis of NADPH oxidation using ketopantoate showed no "burst" phase, suggesting that product-release steps are not rate-limiting and the cause of the small observed kinetic isotope effects with this substrate pair. Large primary deuterium isotope effects on V and V/K using 3-APADPH in steady-state experiments, equivalent to the isotope effect observed in single turnover studies, suggests that chemistry is rate-limiting for this poorer reductant. These results are discussed in terms of a kinetic and chemical mechanism for the enzyme.     

HSV-2 infection of dendritic cells amplifies a highly susceptible HIV-1 cell target

Herpes simplex virus type 2 (HSV-2) increases the risk of HIV-1 infection and, although several reports describe the interaction between these two viruses, the exact mechanism for this increased susceptibility remains unclear. Dendritic cells (DCs) at the site of entry of HSV-2 and HIV-1 contribute to viral spread in the mucosa. Specialized DCs present in the gut-associated lymphoid tissues produce retinoic acid (RA), an important immunomodulator, able to influence HIV-1 replication and a key mediator of integrin α₄β₇ on lymphocytes. α₄β₇ can be engaged by HIV-1 on the cell-surface and CD4⁺ T cells expressing high levels of this integrin (α₄β₇ ^high) are particularly susceptible to HIV-1 infection. Herein we provide in-vivo data in macaques showing an increased percentage of α₄β₇ ^high CD4⁺ T cells in rectal mucosa, iliac lymph nodes and blood within 6 days of rectal exposure to live (n = 11), but not UV-treated (n = 8), HSV-2. We found that CD11c⁺ DCs are a major target of HSV-2 infection in in-vitro exposed PBMCs. We determined that immature monocyte-derived DCs (moDCs) express aldehyde dehydrogenase ALDH1A1, an enzyme essential for RA production, which increases upon HSV-2 infection. Moreover, HSV-2-infected moDCs significantly increase α₄β₇ expression on CD4⁺ T lymphocytes and HIV-1 infection in DC-T cell mixtures in a RA-dependent manner. Thus, we propose that HSV-2 modulates its microenviroment, influencing DC function, increasing RA production capability and amplifying a α₄β₇ ^highCD4⁺ T cells. These factors may play a role in increasing the susceptibility to HIV-1.     

Effects of semen fractionation and dilution ratio on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of semen fractionation and dilution ratio on motility parameters of stallion spermatozoa. In Experiment 1, three ejaculates from each of three stallions were divided into sperm-rich (SR) and sperm-poor (SP) fractions to determine the difference in sperm concentration. Mean sperm concentration in SR fractions (349.5 x 10(6)/ml) was greater (P < 0.001) than that of SP fractions (96.9 x 10(6)/ml). In Experiment 2, three ejaculates from each of two stallions were divided into SR and SP fractions. Fifty percent of the original volume of SR fractions was combined with 50% of the original volume of SP fractions for each ejaculate to represent total ejaculates. SR and total ejaculates were diluted with skim milk-glucose semen extender as follows: 1) no dilution, or dilution to 2) 100 x 10(6)sperm/ml, 3) 50 x 10(6)sperm/ml, or 4) 25 x 10(6)sperm/ml. Semen samples were evaluated at 0.5, 3, 6, 12, and 24 h postejaculation (25 degrees C storage temperature) for percentages of total spermatozoal motility (TSM) and progressive spermatozoal motility (PSM). Mean TSM was greater (P < 0.05) in SR ejaculates than total ejaculates at 12 and 24 h postejaculation. Mean TSM of undiluted semen was lower (P < 0.05) than other dilution ratios over all periods. Mean TSM was greater (P < 0.05) at a 25 x 10(6)sperm/ml dilution ratio than a 50 x 10(6)sperm/ml dilution ratio at 12 and 24 h postejaculation, and greater (P < 0.05) than a 100 x 10(6)sperm/ml dilution ratio from 3 to 24 h postejaculation. Similar patterns were found for PSM. Collection of SR ejaculates and dilution to 25 x 10(6)sperm/ml improved longevity of spermatozoal motility.     

Anoctamin 1 dysregulation alters bronchial epithelial repair in cystic fibrosis

Cystic fibrosis (CF) airway epithelium is constantly subjected to injury events due to chronic infection and inflammation. Moreover, abnormalities in CF airway epithelium repair have been described and contribute to the lung function decline seen in CF patients. In the last past years, it has been proposed that anoctamin 1 (ANO1), a Ca^2 +-activated Cl^? channel, might offset the CFTR deficiency but this protein has not been characterized in CF airways. Interestingly, recent evidence indicates a role for ANO1 in cell proliferation and tumor growth. Our aims were to study non-CF and CF bronchial epithelial repair and to determine whether ANO1 is involved in airway epithelial repair. Here, we showed, with human bronchial epithelial cell lines and primary cells, that both cell proliferation and migration during epithelial repair are delayed in CF compared to non-CF cells. We then demonstrated that ANO1 Cl^? channel activity was significantly decreased in CF versus non-CF cells. To explain this decreased Cl^? channel activity in CF context, we compared ANO1 expression in non-CF vs. CF bronchial epithelial cell lines and primary cells, in lung explants from wild-type vs. F508del mice and non-CF vs. CF patients. In all these models, ANO1 expression was markedly lower in CF compared to non-CF. Finally, we established that ANO1 inhibition or overexpression was associated respectively with decreases and increases in cell proliferation and migration. In summary, our study demonstrates involvement of ANO1 decreased activity and expression in abnormal CF airway epithelial repair and suggests that ANO1 correction may improve this process.     

Enzyme-catalyzed acylation of homoserine: mechanistic characterization of the Escherichia coli metA-encoded homoserine transsuccinylase

The first unique step in bacterial and plant methionine biosynthesis involves the activation of the gamma-hydroxyl of homoserine. In Escherichia coli, this activation is accomplished via a succinylation reaction catalyzed by homoserine transsuccinylase. The activity of this enzyme is closely regulated in vivo and therefore represents a critical control point for cell growth and viability. We have cloned homoserine transsuccinylase from E. coli and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the succinyl group of succinyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-succinylhomoserine. The maximal velocity, V/K(succinyl)(-)(CoA), and V/K(homoserine) all exhibited a bell-shaped pH dependence with apparent pK's of 6.6 and approximately 7.9. The enzyme was inhibited by iodoacetamide in a pH-dependent manner, with an apparent pK of the group being inactivated of 6.4. This suggests the presence of an active site cysteine which forms a succinyl-cysteine intermediate during enzymatic turnover. Solvent kinetic isotope effect studies yielded inverse effects of 0.7 on V and 0.61 on V/K in the reverse reaction only. On the basis of these observations, we propose a detailed chemical mechanism for this important member of the acyltransferase family.     

Multiple fruiting of the edible mushroomCoprinus cinereus from explants of the stipe

Summary Carpophores were developed on defined medium from explants of stipes produced in the dark using an expansionless mutant (no. 190) ofCoprinus cinereus. Explants taken from stipes within 24 h of formation developed multiple fruit within 3–12 days without formation of mycelia. Fruiting levels were affected by stipe age (24 h), medium composition, explant size, and polarity of the explant on fruiting medium. This method offers a new tool for developmental studies and may also be of use to commercial mushroom growers.     

Identification of a alpha-NeuAc-(2----3)-beta-D-galactopyranosyl N-acetyl-beta-D-galactosaminyltransferase in human kidney

Microsomal preparations from human kidney were found to contain enzymic activity capable to transfer N-acetylgalactosamine from UDP-N-acetylgalactosamine to native bovine fetuin. The acceptor structures on the fetuin molecules were identified as N- as well as O-linked glycans with a markedly higher incorporation into the N-linked carbohydrate chains. Analysis of the alkali-labile transferase products by thin-layer chromatography indicated that the enzyme is able to synthesize structures having mobilities identical with those found on glycophorin from Cad erythrocytes. Mild acid treatment and enzymic hydrolysis with N-acetylhexosaminidase from jack beans of the N-linked transferase products suggested that beta-D-GalpNAc-(1----4)-[alpha-NeuAc-(2----3)]-beta-D-Galp-(1----s tructures were formed by the enzymic reaction on both N- and O-linked acceptors. The enzyme might, therefore, be involved in the biosynthesis of Sda (and Cad) antigenic structures. By use of various oligosaccharides, glycopeptides, and glycolipids having well characterized carbohydrate sequences, the acceptor-substrate specificity of the N-acetylgalactosaminyltransferase was determined. The enzyme generally recognized alpha-NeuAc-(2----3)-beta-D-Gal groups as acceptors, but in a certain conformation. Thus, tri- and tetra-saccharide alditols, native human glycophorin A, and GM3 were not acceptor substrates although they carry the potential disaccharide acceptor unit. When these structures were presented as sialyl-(2----3)-lactose or as a tryptic peptide from glycophorin A, they were shown to be rather good acceptor substrates for the N-acetyl-beta-D-galactosaminyltransferase from human kidney.