Structural elucidation of the EPS of slime producing Brevundimonas vesicularis sp. isolated from a paper machine

The slime forming bacteria Brevundimonas vesicularis sp. was isolated from a paper mill and its EPS was produced on laboratory scale. After production, the exopolysaccharide (EPS) was purified and analysed for its purity and homogeneity, HPSEC revealed one distinct population with a molecular mass of more than 2,000 kDa. The protein content was around 9 w/w%. The sample was analysed to determine its chemical structure. The EPS was found to consist of rhamnose, glucose, galacturonic acid and glucuronic acid. Due to the presence of uronic acids the molar ratio between the four sugars found varies from 3:5:2:4 by sugar composition analyses after methanolysis to 1:1:1:1 found by NMR. A repeating unit with a molecular mass of 678 Da was confirmed by MALDI-TOF mass spectrometry after mild acid treatment. 13C and 1H hetero- and homonuclear 2D NMR spectroscopy of the native and partial hydrolysed EPS revealed a repeating unit, no non-sugar substituents were present.     

Expression of the xylulose 5-phosphate phosphoketolase gene, xpkA, from Lactobacillus pentosus MD363 is induced by sugars that are fermented via the phosphoketolase pathway and is repressed by glucose mediated by CcpA and the mannose phosphoenolpyruvate phosphotransferase system

Purification of xylulose 5-phosphate phosphoketolase (XpkA), the central enzyme of the phosphoketolase pathway (PKP) in lactic acid bacteria, and cloning and sequence analysis of the encoding gene, xpkA, from Lactobacillus pentosus MD363 are described. xpkA encodes a 788-amino-acid protein with a calculated mass of 88,705 Da. Expression of xpkA in Escherichia coli led to an increase in XpkA activity, while an xpkA knockout mutant of L. pentosus lost XpkA activity and was not able to grow on energy sources that are fermented via the PKP, indicating that xpkA encodes an enzyme with phosphoketolase activity. A database search revealed that there are high levels of similarity between XpkA and a phosphoketolase from Bifidobacterium lactis and between XpkA and a (putative) protein present in a number of evolutionarily distantly related organisms (up to 54% identical residues). Expression of xpkA in L. pentosus was induced by sugars that are fermented via the PKP and was repressed by glucose mediated by carbon catabolite protein A (CcpA) and by the mannose phosphoenolpyruvate phosphotransferase system. Most of the residues involved in correct binding of the cofactor thiamine pyrophosphate (TPP) that are conserved in transketolase, pyruvate decarboxylase, and pyruvate oxidase were also conserved at a similar position in XpkA, implying that there is a similar TPP-binding fold in XpkA.     

Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.     

Fusion of alphaviruses with liposomes is a non-leaky process

It has been reported that low-pH-induced fusion of influenza virus with liposomes results in rapid and extensive release of both low- and high-molecular-weight substances from the liposomes [Günther-Ausborn et al., J. Biol. Chem. 270 (1995) 29279-29285; Shangguan et al., Biochemistry 35 (1996) 4956-4965]. Here, we demonstrate retention of encapsulated water-soluble compounds during fusion of Semliki Forest virus (SFV) or Sindbis virus with liposomes at low pH. Under conditions allowing complete fusion of the liposomes, a limited fluorescence dequenching of liposome-encapsulated calcein was observed, particularly for SFV. Also, radioactively labeled inulin or sucrose were largely retained. Freezing and thawing of the viruses in the absence of sucrose resulted in an enhanced leakiness of fusion. These results support the notion that the alphavirus fusion event per se is non-leaky and may well involve a discrete hemifusion intermediate.     

Stochastic processes defining sensitivity and variability of internally calibrated quantitative NASBA-based viral load assays

For quantitative assessment of virus particles in patient plasma samples various assays are commercially available. Typical performance characteristics for such assays are sensitivity, precision and the range of linearity. In order to assess these properties it is common practice to divide the range of inputs into subranges in order to apply different statistical models to evaluate these properties separately. We developed a general statistical model for internally calibrated amplification based viral load assays that combines these statistical properties in one powerful analysis. Based on the model an unambiguous definition of the lower limit of the linear range can be given. The proposed method of analysis was illustrated by a successful application to data generated by the NucliSens EasyQ HIV-1 assay.     

The influence of exercise on the composition of developing equine joints

An overview is given of the direct and long-term effects of exercise on the biochemical characteristics of cartilage and subchondral bone, and on the metabolic activity of chondrocytes in the juvenile horse. In the experimental setup 43 foals were reared until weaning at 5 months of age under similar conditions, except for the type and amount of exercise. Fifteen foals remained at pasture (Pasture group and also control group), 14 foals were kept in box stalls (Box group), and 14 foals were kept in the same box stalls but were subjected daily to an increasing number of gallop sprints (Training group). After weaning 8 foals from each group were euthanised. All remaining 19 animals were housed together in a loose box with access to a small paddock to study a possible reversibility of exercise-induced effects. Post mortem subchondral bone and cartilage samples were collected and analysed for bone morphogenic enzymes, matrix composition, chondrocyte metabolic activity, and bone mineral density.It resulted that lack of exercise leads to a retardation of the normal development of the joint. This is largely compensated for when afterwards a more normal exercise regimen is followed. Most parameters in the Training group approximated those of the pastured foals at age 5 months. However, at age 11 months there were indications for a reduced performance of the investigated tissues in this group.It is concluded that regular, sub-maximal loading, as occurred in the Pasture group, seems best for an optimal development of the musculoskeletal tissues. The combination of short bouts of heavy exercise superimposed on a basic box rest regimen appears to have adverse effects on long-term viability of the tissues and may hence lead to an impaired resistance to injury.     

Structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution

Oxalate decarboxylase is a manganese-dependent enzyme that catalyzes the conversion of oxalate to formate and carbon dioxide. We have determined the structure of oxalate decarboxylase from Bacillus subtilis at 1.75 A resolution in the presence of formate. The structure reveals a hexamer with 32-point symmetry in which each monomer belongs to the cupin family of proteins. Oxalate decarboxylase is further classified as a bicupin because it contains two cupin folds, possibly resulting from gene duplication. Each oxalate decarboxylase cupin domain contains one manganese binding site. Each of the oxalate decarboxylase domains is structurally similar to oxalate oxidase, which catalyzes the manganese-dependent oxidative decarboxylation of oxalate to carbon dioxide and hydrogen peroxide. Amino acid side chains in the two metal binding sites of oxalate decarboxylase and the metal binding site of oxalate oxidase are very similar. Four manganese binding residues (three histidines and one glutamate) are conserved as well as a number of hydrophobic residues. The most notable difference is the presence of Glu333 in the metal binding site of the second cupin domain of oxalate decarboxylase. We postulate that this domain is responsible for the decarboxylase activity and that Glu333 serves as a proton donor in the production of formate. Mutation of Glu333 to alanine reduces the catalytic activity by a factor of 25. The function of the other domain in oxalate decarboxylase is not yet known.     

Triggered release of doxorubicin following mixing of cationic and anionic liposomes

In many applications, an ability of liposomes to retain drug and then rapidly release it at some later time would be of benefit. In this work, we investigate the ability of cationic large unilamellar vesicles (LUV) to promote rapid release of doxorubicin from anionic LUV. It is shown that the addition of cationic liposomes containing cholesterol, dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) and the cationic lipid N,N-dioleyl-N,N-dimethylammonium chloride (DODAC) to doxorubicin-containing LUV composed of cholesterol, DOPE, DSPC and the anionic lipid dioleoyphosphatidylglycerol (DOPG) can result in release of more than 90% of the drug in times of 30 s or less. Further, it is shown that these release characteristics are exquisitely dependent on the presence of DOPE and cholesterol. In the absence of DOPE, much slower release rates are observed, with maximum release levels of 50% after a 2-h incubation at 20 degrees C. Remarkably, threshold levels of more than 10 mol% cholesterol are required before any appreciable release is observed. [31P]NMR spectroscopy and freeze-fracture electron microscopy studies reveal that systems giving rise to rapid release of doxorubicin exhibit limited formation of inverted hexagonal (H(II)) phase, suggesting that these lipids facilitate drug release by formation of local regions of non-bilayer structure. It is concluded that drug release triggered by mixing anionic and cationic liposomes could be of utility in drug delivery applications.     

Tackling speciose genera: species composition and phylogenetic position of Senecio sect. Jacobaea (Asteraceae) based onplastid and nrDNA sequences

The molecular phylogeny of Senecio sect. Jacobaea (Asteraceae; Senecioneae) was studied to clarify species composition and interspecific relationships of Senecio sect. Jacobaea. This information is necessary for studies seeking explanations of the evolutionary success of Senecio, in terms of high species numbers and the evolution of chemical defense mechanisms. Parsimony analyses with 60 species of the tribe Senecioneae, representing 23 genera and 11 sections of Senecio, based on DNA sequence data of the plastid genome (the trnT-L intergenic spacer, the trnL intron, and two parts of the trnK intron, flanking both sides of the matK gene) and nuclear genome (ITS1, 5.8S, and ITS2 gene and spacers) show that sect. Jacobaea is a strongly supported monophyletic group. Fifteen species have been identified as members of section Jacobaea, including three species that have been consistently ascribed to this section in taxonomic literature and 12 species that were either placed in other sections of Senecio or not exclusively ascribed to sect. Jacobaea. This section was traditionally circumscribed as a group of European, biennial, or perennial herbs with pinnately incised leaves, but the results of this study show that one annual species, a species from northeastern Asia, and a species growing in the Himalayas are members of sect. Jacobaea as well. Furthermore, not all species in the section have pinnately incised leaves. The genera Emilia, Packera, and Pseudogynoxys form the sister clade of sect. Jacobaea, but this relationship lacks strong bootstrap support and thus remains provisional.