Enzymatic production of β-D-glucose-1-phosphate from trehalose

β-D -Glucose-1-phosphate (βGlc1P) is an efficient glucosyl donor for both enzymatic and chemical glycosylation reactions but is currently very costly and not available in large amounts. This article provides an efficient production method of βGlc1P from trehalose and phosphate using the thermostable trehalose phosphorylase from Thermoanaerobacter brockii. At the process temperature of 60°C, Escherichia coli expression host cells are lysed and cell treatment prior to the reaction is, therefore, not required. In this way, the theoretical maximum yield of 26% could be easily achieved. Two different purification strategies have been compared, anion exchange chromatography or carbohydrate removal by treatment with trehalase and yeast, followed by chemical phosphate precipitation. In a next step, βGlc1P was precipitated with ethanol but this did not induce crystallization, in contrast to what is observed with other glycosylphosphates. After conversion of the product to its cyclohexylammonium salt, however, crystals could be readily obtained. Although both purification methods were quantitative (>99% recovery), a large amount of product (50%) was lost during crystallization. Nevertheless, a production process for crystalline βGlc1P is now available from the cheap substrates trehalose and inorganic phosphate.     

Modulation byn-alkanols of rat cardiac adenylate cyclase activity

Summary n-Alkanols (from methanol to decanol) have a biphasic effect on rat cardiac adenylate cyclase either basal or stimulated by GTP, GppNHp, NaF or hormones (isoproterenol, glucagon, secretin) in the presence of GTP. At high concentration, all the enzyme activities are inhibited. At low concentration, adenylate cyclase activity is either unchanged or potentiated depending on both the stimulus and the alkanols involved. Potentiation is due to an increase of maximum velocity with no change in the activation constant of the enzyme. Basal activity is unchanged as well as the isoproterenol-and glucagon-stimulated enzyme. The secretin-stimulated enzyme is potentiated. It is the guanyl nucleotide regulatory protein-mediated stimulation of adenylate cyclase which is mainly affected. An attempt was made to relate these effects on adenylate cyclase with physical parameters of the alkanols (partition coefficient). From the data obtained as a function of the alkanol chain-length and of temperature on the adenylate cyclase stimulated by GTP, GppNHp, NaF and permanently activated, it is concluded that the increase in efficacy observed in the presence of alkanol is due to an interaction with the protein moeity particularly with the guanyl nucleotide regulatory protein.     

M-MuLV-induced leukemogenesis: Integration and structure of recombinant proviruses in tumors

M-MuLV-specific DNA probes were used to establish the state of integration and amplification of recombinant proviral sequences in Moloney virus-induced tumors of Balb/Mo, Balb/c and 129 mice. The somatically acquired viral sequences contain both authentic M-MuLV genomes and recombinants of M-MuLV with endogenous viral sequences. All reintegrated genomes carry long terminal repeat (LTR) sequences at both termini of their genome. In the preleukemic stage a large population of cells exhibiting a random distribution of reintegrated M-MuLV genomes are seen, but during outgrowth of the tumor, selection of cells occurs leaving one or a few clonal descendants in the outgrown tumor. In this latter stage recombinant genomes can be detected. Although these recombinants constitute a heterogeneous group of proviruses, characteristic molecular markers are conserved among many individual proviral recombinants, lending credence to the notion that a certain recombinant structure is a prerequisite for the onset of neoplasia. The structure of these recombinants shows close structural similarities to the previously described mink cell focus-inducing (MCF)-type viruses.     

Sucrose phosphorylase as cross-linked enzyme aggregate: Improved thermal stability for industrial applications

Sucrose phosphorylase is an interesting biocatalyst that can glycosylate a variety of small molecules using sucrose as a cheap but efficient donor substrate. The low thermostability of the enzyme, however, limits its industrial applications, as these are preferably performed at 60°C to avoid microbial contamination. Cross-linked enzyme aggregates (CLEAs) of the sucrose phosphorylase from Bifidobacterium adolescentis were found to have a temperature optimum that is 17°C higher than that of the soluble enzyme. Furthermore, the immobilized enzyme displays an exceptional thermostability, retaining all of its activity after 1 week incubation at 60°C. Recycling of the biocatalyst allows its use in at least ten consecutive reactions, which should dramatically increase the commercial potential of its glycosylating activity.     

Exotic invaders of the meso-oligohaline zone of estuaries in the Netherlands: why are there so many?

The numbers of exotic species introduced into brackish waters (5–20 psu) and high-salinity waters (> 20 psu) in the Netherlands are hypothesized to reflect species richness in such waters elsewhere in the world. Notwithstanding the fact that species numbers in brackish waters all over the world are lower than in high-salinity waters, the numbers of introduced species in these waters in the Netherlands are about equal. Alternative hypotheses to explain this phenomenon are: (1) because most ports are situated in brackish regions, brackish-water species stand a better chance of being transported; (2) because brackish-water species are more tolerant of conditions in ballast water tanks, these species have a better chance of being transported alive than high-salinity species; and (3) because brackish waters have few species, it is easier for an introduced species to establish itself in brackish waters. None of the latter three hypotheses can be rejected and probably they all play a part in explaining the phenomenon. The third hypothesis, however, seems most likely.     

A strictly anaerobic betaproteobacterium Georgfuchsia toluolica gen. nov., sp. nov. degrades aromatic compounds with Fe(III), Mn(IV) or nitrate as an electron acceptor

A bacterium (strain G5G6) that grows anaerobically with toluene was isolated from a polluted aquifer (Banisveld, the Netherlands). The bacterium uses Fe(III), Mn(IV) and nitrate as terminal electron acceptors for growth on aromatic compounds. The bacterium does not grow on sugars, lactate or acetate. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain G5G6 belonged to the Betaproteobacteria . Its closest, but only distantly related, cultured relative is Sterolibacterium denitrificans Chol-1S^T (94.6% similarity of the 16S rRNA genes), a cholesterol-oxidizing, denitrifying bacterium. Strain G5G6 possesses the benzylsuccinate synthase A ( bssA ) gene encoding the α-subunit of Bss, which catalyzes the first step in anaerobic toluene degradation. The deduced BssA amino acid sequence is closely related to those of Azoarcus and Thauera species, which also belong to the Betaproteobacteria . Strain G5G6 is the first toluene-degrading, iron-reducing bacterium that does not belong to the Geobacteraceae within the Deltaproteobacteria . Based on phylogenetic and physiological comparison, strain G5G6 could not be assigned to a described species. Therefore, strain G5G6 (DSMZ 19032^T=JCM 14632^T) is a novel taxon of the Betaproteobacteria . We propose the name Georgfuchsia toluolica gen. nov., sp. nov.     

Plasma selenium levels in healthy blood bank donors in the central-eastern part of Belgium

Graphite furnace atomic absorption spectrometry, with Zeeman background correction and after improved matrix modification, was used to measure the plasma selenium content of healthy blood bank donors in the central part of Belgium.

The mean plasma selenium concentration of 80 men and 80 women was 79.7±4.4 ng/mL with a range of 55.0–117.4 ng/mL.

There was no gender difference observed. Plasma selenium level was significantly highest for the adult group, aged 45–64 years, compared to the others, except the young adults (18–24 years).

The mean plasma selenium concentration measured corresponded well with literature data for Belgium. The obtained values were found to be in the medium range, compared with recent literature values for the European countries.