Characterization of calf liver glucosidase I and its inhibition by basic sugar analogs

Glucosidase I, the enzyme catalyzing the first step of N-linked oligosaccharide processing, has been purified from calf liver crude membranes [H. Hettkamp, G. Legler, and E. Bause, (1984) Eur. J. Biochem. 142, 85-90]. Binding experiments with concanavalin A-Sepharose suggest that glucosidase I is a glycoprotein with high-mannose carbohydrate chain(s). The enzyme has a subunit molecular mass of approximately 83 kDa and specifically hydrolyzes the terminal alpha-1,2-linked glucose residue from the natural Glc3-Man9-GlcNAc2 oligosaccharide. Studies with a variety of substrates modified in the aglycon moiety suggest that the Glc2 branch rather than the more distant domains of the substrate molecule are important for binding and hydrolysis. Glucosidase I does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (dNM) and its N-alkyl derivatives. Ki values range from 0.07 microM for N-methyl-dNM to 1.0 microM for dNM, measured at the pH-optimum of enzyme activity. The pH dependence of inhibition indicates that the cationic form of the inhibitors is the active species. Comparison of the Ki for N-decanoyl-dNM (approximately 70 microM) with that of N-decyl-dNM (approximately 0.4 microM) suggests that electrostatic interactions at the catalytic site of the enzyme are important for inhibitor binding. 1-Deoxymannojirimycin, previously assumed to be a specific mannosidase inhibitor, as well as its N-methyl and N-5-carboxypentyl derivatives, inhibit glucosidase I with Ki values around 190, 17, and 100 microM, respectively. This apparent lack of specificity shows that in vivo experiments on N-glycoprotein processing as well as the interpretation of results with these mannosidase inhibitors may give misleading results when these compounds are used in the millimolar range.     

The complete nucleotide sequence of the colicinogenic plasmid ColA. High extent of homology with ColE1

Summary The complete nucleotide sequence of the colicinogenic plasmid ColA has been determined. The plasmid DNA consists of 6720 bp (molecular weight 4.48×10⁶). Fifteen putative biological functions have been identified using the functional map previously determined. These include 11 genes and 3 DNA sites. Nine genes encode proteins of which 3 have been fully characterized. The replication region of ColA coding for RNAI and RNAII is highly homologous to that of ColE1 andClo DF13. The same holds true for the site-specific recombination region containing palindromic symmetry and involved in stable maintenance of the plasmids. A high percentage of homology has been detected for putative mobility proteins encoded by ColA and ColE1. The exclusion proteins are also highly homologous.     

Human acid beta-glucosidase: affinity purification of the normal placental and Gaucher disease splenic enzymes on N-alkyl-deoxynojirimycin-sepharose

Two sepharose-bound 1-deoxynojirimycin N-alkyl derivatives, N-(9-carboxynonyl)- and N-(11-carboxyundecyl)-deoxynojirimycin, were used for the affinity purification of acid beta-glucosidase (beta-Glc) from normal and type-1 Ashkenazi Jewish Gaucher disease (AJGD) sources. The capacities of these nondegradable inhibitor supports were 0.5 and 0.75 mg of normal beta-Glc/ml of settled gel, respectively. The purified normal enzyme (14-18% yield) had a specific activity of 1.6 X 10(6) nmol/h/mg protein and was homogeneous as evidenced by a single protein species of Mr = 67,000 on sodium dodecylsulfate-polyacrylamide gel electrophoresis and reverse phase high-performance liquid chromatography (HPLC). Microsequencing demonstrated a single N terminus, and the sequence of the first 22 N-terminal amino acids was colinear with that predicted from the beta-Glc cDNA. Amino acid composition analyses of beta-Glc revealed a high content (35%) of hydrophobic amino acids. The N-decyl-deoxynojirimycin support facilitated the purification of the residual enzyme from type-1 AJGD spleen to about 7,500-fold in four steps with a yield of about 11%. These new affinity supports provided improved stability, capacity and/or specificity compared to other affinity or HPLC methods for purifying this lysosomal glycosidase.     

Non-linear loss of suitable wine regions over Europe in response to increasing global warming

Evaluating the potential climatic suitability for premium wine production is crucial for adaptation planning in Europe. While new wine regions may emerge out of the traditional boundaries, most of the present-day renowned winemaking regions may be threatened by climate change. Here, we analyse the future evolution of the geography of wine production over Europe, through the definition of a novel climatic suitability indicator, which is calculated over the projected grapevine phenological phases to account for their possible contractions under global warming. Our approach consists in coupling six different de-biased downscaled climate projections under two different scenarios of global warming with four phenological models for different grapevine varieties. The resulting suitability indicator is based on fuzzy logic and is calculated over three main components measuring (i) the timing of the fruit physiological maturity, (ii) the risk of water stress and (iii) the risk of pests and diseases. The results demonstrate that the level of global warming largely determines the distribution of future wine regions. For a global temperature increase limited to 2°C above the pre-industrial level, the suitable areas over the traditional regions are reduced by about 4%/°C rise, while for higher levels of global warming, the rate of this loss increases up to 17%/°C. This is compensated by a gradual emergence of new wine regions out of the traditional boundaries. Moreover, we show that reallocating better-suited grapevine varieties to warmer conditions may be a viable adaptation measure to cope with the projected suitability loss over the traditional regions. However, the effectiveness of this strategy appears to decrease as the level of global warming increases. Overall, these findings suggest the existence of a safe limit below 2°C of global warming for the European winemaking sector, while adaptation might become far more challenging beyond this threshold.     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

Stress-induced assortative mating and the evolution of stress resistance

We show with a model that variation in environmental stress between generations facilitates the evolution of stress resistance through assortative mating. Stress induces delayed maturation of susceptible phenotypes, segregating their fertile period from resistant phenotypes. Assortment of mates enhances the responsiveness of populations to natural selection by inflating genetic variance. Thus, positive selection and inflated genetic variance in stressful environments can cause a strong evolutionary increase in resistance. By contrast, benign environments do not segregate phenotypes, and the random mating among phenotypes deflates genetic variance, leading to a weaker response to selection against resistance, assuming that resistance is costly. When environments vary randomly from benign to stressful, populations respond asymmetrically to negative and positive selection. This asymmetry (1) accelerates fixation of a resistance allele if resistance is generally favoured (stressful generations more frequent) but delays the loss of the allele if it is generally disfavoured (benign generations more frequent), and (2) it can push a resistance allele to fixation even when long‐term costs modestly exceed benefits. When resistance alleles pleiotropically delay mating, stress‐induced random mating has complementary effects. Serial autocorrelation in the stressor amplifies these effects. These results suggest a novel mechanism for the persistence of resistance polymorphisms.     

Roles of bovine serum albumin and copper in the assay and stability of ammonia monooxygenase activity in vitro.

We investigated the effects of bovine serum albumin (BSA) on both the assay and the stability of ammonia-oxidizing activity in cell extracts of Nitrosomonas europaea. Ammonia-dependent O2 uptake activity of freshly prepared extracts did not require BSA. However, a dependence on BSA developed in extracts within a short time. The role of BSA in the assay of ammonia-oxidizing activity apparently is to absorb endogenous free fatty acids which are present in the extracts, because (i) only proteins which bind fatty acids, e.g., BSA or beta-lactoglobulin, supported ammonia-oxidizing activity; (ii) exogenous palmitoleic acid completely inhibited ammonia-dependent O2 uptake activity; (iii) the inhibition caused by palmitoleic acid was reversed only by proteins which bind fatty acids; and (iv) the concentration of endogenous free palmitoleic acid increased during aging of cell extracts. Additionally, the presence of BSA (10 mg/ml) or CuCl2 (500 microM) stabilized ammonia-dependent O2 uptake activity for 2 to 3 days at 4 degrees C. The stabilizing effect of BSA or CuCl2 was apparently due to an inhibition of lipolysis, because both additives inhibited the increase in concentrations of free palmitoleic acid in aging extracts. Other additives which are known to modify lipase activity were also found to stabilize ammonia-oxidizing activity. These additives included HgCl2, lecithin, and phenylmethylsulfonyl fluoride.     

Immunochemical and biological studies of antigens isolated from a strain of Bacteroides fragilis

Phenol/water-extracted lipopolysaccharide and a fraction HM, extracted with acetate buffer pH 2.0, from Bacteroides fragilis strain 62/73 are antigenically different as shown by immunodiffusion, passive haemagglutination, haemagglutination inhibition and preliminary chemical investigations. Biological activity, assessed with the local Shwartzmann reaction, was demonstrated for the lipopolysaccharide whereas antigen HM was almost inactive in this test. HM is immunogenic in rabbits. Antibodies against HM were detected in seven out of ten sera of healthy humans.