Lipase-Catalyzed Regioselective Acylation and Deacylation of Glucose Derivatives

In the development of an efficient synthesis of 1-O-decanoyl-2,3,4,6-tetra-O-acetyl-β-D-glucose (β-2) several lipase-based approaches have been explored. Among five immobilized Upases tested, the lipase from Candida antarctica proved particularly efficient for catalyzing selective hydrolysis in the 1-position of 1,2,3,4,6-penta-O-acetyl-β-D-glucose (β-1). Using triethylamine as catalyst, the hydrolysis product 2,3,4,6-tetra-O-acetyl-D-glucose (3) can be esterified with decanoyl chloride to form β-2 selectively, thereby providing an efficient chemo-enzymatic synthesis starting from readily available raw materials. Attempts to produce β-2 from β-1 by lipase-catalyzed interesterification or to esterify 3 with decanoic acid using a lipase as catalyst were unsuccessful. The latter finding was explained by the hemiacetal OH group of glucose being unable to act as nucleophile in the lysis of the lipase acyl-enzyme intermediate. Furthermore, β-2 was found to bee a too bulky substrate to fit into the active site of any of the lipases tested.     

The lcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium.

Virulent bacteria of the genus Yersinia secrete a number of virulence determinants called Yops. These proteins lack typical signal sequences and are not posttranslationally processed. Two gene loci have been identified as being involved in the specific Yop secretion system (G. Cornelis, p. 231-265, In C. E. Hormache, C. W. Penn, and C. J. Smythe, ed., Molecular Biology of Bacterial Infection, 1992; S. C. Straley, G. V. Plano, E. Skrzypek, P. L. Haddix, and K. A. Fields, Mol. Microbiol. 8:1005-1010, 1993). Here, we have shown that the lcrB/virB locus (yscN to yscU) encodes gene products essential for Yop secretion. As in previously described secretion apparatus mutants, expression of the Yop proteins was decreased in the yscN/U mutants. An lcrH yscR double mutant expressed the Yops at an increased level but did not secrete Yops into the culture supernatant. The block in Yop expression of the ysc mutants was also circumvented by overexpression of the activator LcrF in trans. Although the Yops were expressed in elevated amounts, the Yops were still not exported. This analysis showed that the ysc mutants were unable to secrete Yops and that they were also affected in the negative Ca(2+)-regulated loop. The yscN/U genes showed remarkably high homology to the spa genes of Shigella flexneri and Salmonella typhimurium with respect to both individual genes and gene organization. These findings indicate that the genes originated from a common ancestor.     

Genetic analysis of the minimal replicon of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid

Using a combination of mutagenesis with the transposon and polymerase chain reaction subcloning, the essential elements of the replication region of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid have been identified. An open reading frame, coding for a protein with homology to Rep proteins from other Lactococcus plasmids, is essential. This protein is trans-acting and could not be replaced by the Rep protein from another Lactococcus plasmid. A second open reading frame immediately downstream from the first could be removed or inactivated with no apparent effect on plasmid replication. A region containing two 10 by direct repeats and three tandem repeats of a 22 by sequence, immediately upstream of the essential open reading frame, is also essential and probably includes the origin of replication. A 181-bp DNA fragment containing this region was sufficient to allow replication in Lactococcus if the trans-acting protein was provided on another replicon. Single-stranded replication intermediates could not be detected, suggesting that the citrate plasmid uses theta replication rather than rolling-circle replication.     

Recolonization following past persecution questions the importance of persistent snow cover as a range limiting factor for wolverines

Globally, climate is changing rapidly, which causes shifts in many species' distributions, stressing the need to understand their response to changing environmental conditions to inform conservation and management. Northern latitudes are expected to experience strongest changes in climate, with milder winters and decreasing snow cover. The wolverine (Gulo gulo) is a circumpolar, threatened carnivore distributed in northern tundra, boreal, and subboreal habitats. Previous studies have suggested that wolverine distribution and reproduction are constrained by a strong association with persistent spring snow cover. We assess this hypothesis by relating spatial distribution of 1589 reproductive events, a fitness-related proxy for female reproduction and survival, to snow cover over two decades. Wolverine distribution has increased and number of reproductive events increased 20 times in areas lacking spring snow cover during our study period, despite low monitoring effort where snow is sparse. Thus, the relationship between reproductive events and persistent spring snow cover weakened during this period. These findings show that wolverine reproductive success and hence distribution are less dependent on spring snow cover than expected. This has important implications for projections of future habitat availability, and thus distribution, of this threatened species. Our study also illustrates how past persecution, or other factors, that have restricted species distribution to remote areas can mask actual effects of environmental parameters, whose importance reveals when populations expand beyond previously restricted ranges. Overwhelming evidence shows that climate change is affecting many species and ecological processes, but forecasting potential consequences on a given species requires longitudinal data to revisit hypotheses and reassess the direction and magnitude of climate effects with new data. This is especially important for conservation-oriented management of species inhabiting dynamic systems where environmental factors and human activities interact, a common scenario for many species in different ecosystems around the globe.     

Effects of ammonium sulphate application on the chemistry of bulk soil,rhizosphere, fine roots and fine-root distribution in a Picea abies (L.) karst. stand

The effect of ammonium sulphate application on the bulk and rhizosphere soil chemistry, elemental concentration of living fine roots (<2 mm in diameter), amounts of living and dead fine roots, root length density and specific root length density were investigated in a 28 year old Norway spruce stand in SW Sweden. The treatments started in 1988. Core samples of the LFH layer and mineral soil layers were sampled in control (C) and ammonium sulphate (NS) treatment plots in 1988, 1989 and 1990. Soil pH and NO₃-S and SO₄-S, Al, Ca, Mg, Mn and K concentrations were measured for both the bulk soil and rhizosphere soil.The pH-values of the bulk and rhizosphere soil decreased in 1989 and 1990 in NS plots compared to control plots, while the SO₄-S concentration increased. The Ca, Mg and K concentration increased in the NS treatment in almost all layers in the bulk and the rhizosphere soil. Ammonium ions may have replaced these elements in the soil organic matter. The NS treatment reduced Mg concentration in fine roots in all layers in 1990. The Al concentrations in the rhizosphere and bulk soil were higher in NS plots in all layers, except at 0–10 cm depth, both in 1989 and 1990. The Al content of living fine roots was higher in NS plots than C plots but the differences were not significant. The NS addition did not affect the P and K contents of fine roots in any soil layer, but the S concentrations of fine roots were significantly higher in NS plots in 1989 and 1990. The fine root necromass was higher in NS than in C in 1990, in the LFH layer, indicating a gradual decrease in the vitality of the fine roots. It was suggested that the NS treatment resulted in displacement of Mg and K from exchange sites in the LFH layer leading to leaching of these cations to the mineral soil. Further application of ammonium sulphate may damage the fine roots and consequently adversely affect the water and nutrient uptake of root systems.     

An attempt to predict long-term effects of atmospheric nitrogen deposition on the yield of Norway spruce stands (Picea abies (L.) Karst.) in southwestern Sweden

Long-term field experiments in Norway spruce stands on fertile sites (site indices 27–35 m) in southwestern Sweden were analysed with respect to volume increment. Three treatments were included (0=No fertilization, N = Fertilization with N, NP = Fertilization with N and P).Volume growth was monitored for 18 years in 10 blocks. No significant differences in annual volume increment between the treatments were detected. Volume increments in the N treatment were 97%, 99% and 107% as high as those in the 0 treatment for the periods 1–5, 6–10 and 11–15 years after the first fertilization. Corresponding values for the NP treatment were 104%, 108% and 110%, indicating that P has a small positive effect.The amount of N-fertilization would correspond to an annual N deposition of 20 kg ha^-1 during the next 30 years in southwestern Sweden. For this period, the results imply that this N deposition would not affect the growth of Norway spruce stands on fertile sites.     

A reagentless amperometric electrode based on carbon paste, chemically modified with D-lactate dehydrogenase, NAD(+), and mediator containing polymer for D-lactic acid analysis. II. On-line monitoring of fermentation process

The production of D-lactic acid by Lactobacillus delbrueckii (ATCC 9649) during fermentation was monitored on-line with a reagentless D-lactate dehydrogenase modified carbon paste electrode in a flow injection system integrated with a filtration sampling device. The time delay between sampling and detection was approximately 6 min. The use of an electropolymerized ortho-phenylenediamine membrane on the elctrode resulted in a very selective sensor response with acceptable stability and sensitivity. The D-lactate concentrations determined on-line agreed well with those determined by a standard method, suggesting that this sensor system is suitable for on-line monitoring of fermentation processes. (c) 1995 John Wiley & Sons, Inc.     

Glycan modification of a thermostable recombinant (1-3,1-4)-β-glucanase secreted fromSaccharomyces cerevisiae is determined by strain and culture conditions

High level biosynthesis and secretion of the thermostable hybrid (1-3,1-4)--glucanase H(A16-M) has been achieved inSaccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1_p) and theBacillus macerans (1-3,1-4)--glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man_8-13GlcNAc₂, although only traces of Man₉GlcNAc₂ were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3-phosphoglycerate kinase promoter, each in two differentSaccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y. Abbreviations: AMY,B. amyloliquefaciens (1-3,1-4)--glucanse; MAC,B. macerans (1-3,1-4)--glucanase H(A16-M), H(A36-M), H(A78-M),H(A107-M) and H(A152-M), hybrid (1-3,1-4)--glucanases containing 16, 36, 78, 107 and 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC; similar enzyme abbreviations followed by Y, e.g. H(A16-M)-Y, denote the enzymes secreted from yeast cells; PCR, polymerase chain reaction; PGK_p, yeast 3-phosphoglycerate kinase promoter; PRB1_p, yeast protease B promoter; LB, Luria-Bertani medium; SC, minimal medium; CNBr, cyanogen bromide; Endo H_f, endoglycosidase H fusion protein; PNGase F, peptide:N-glycosidase F; HPAEC; high pH anion exchange chromatography; HVE, high voltage paper electrophoresis; CPY, yeast carboxypeptidase Y.