Characterization of heparan sulfate from the unossified antler of Cervus elaphus

The antler is the most rapidly growing tissue in the animal kingdom. According to previous reports, antler glycosaminoglycans (GAGs) consist of all kinds GAGs except for heparan sulfate (HS). Chondroitin sulfate is the major antler GAG component comprising 88% of the total uronic acid content. In the current study, we have isolated HS from antler for the first time and characterized it based on both NMR spectroscopy and disaccharide composition analysis. Antler GAGs were isolated by protease treatment and followed by cetylpyridinium chloride precipitation. The sensitivity of antler GAGs to heparin lyase III showed that this sample contained heparan sulfate. After incubation of antler GAGs with chondroitin lyase ABC, the HS-containing fraction was recovered by ethanol precipitation. The composition of HS disaccharides in this fraction was determined by its complete depolymerization with a mixture of heparin lyase I, II, and III and analysis of the resulting disaccharides by the reversed-phase (RP) ion pairing-HPLC, monitored by the fluorescence detection using 2-cyanoacetamide as a post-column labeling reagent. Eight unsaturated disaccharides (DeltaUA-GlcNAc, DeltaUA-GlcNS, DeltaUA-GlcNAc6S, DeltaUA2S-GlcNAc, DeltaUA-GlcNS6S, DeltaUA2S-GlcNS, DeltaUA2S-GlcNAc6S, DeltaUA2S-GlcNS6S) were produced from antler HS by digestion with the mixture of heparin lyases. The total content of 2-O-sulfo disaccharide units in antler HS was higher than that of heparan sulfate from most other animal sources.     

Changes to the rumen bacterial population of sheep with the addition of 2,4,6-trinitrotoluene to their diet

Previous work has shown that bacterial isolates from the sheep rumen are capable of detoxifying 2,4,6-trinitrotoluene (TNT) into polar constituents. In this study, the dietary effects of TNT on the sheep rumen microbial community were evaluated using molecular microbiology ecology tools. Rumen samples were collected from sheep fed with and without TNT added to their diet, genomic DNA was extracted, and the 16S rRNA-V3 gene marker was used to quantify changes in the microbial population in the rumen. Control and treatment samples yielded 533 sequences. Phylogenetic analyses were performed to determine the microbial changes between the two conditions. Results indicated the predominant bacterial populations present in the rumen were comprised of the phyla Firmicutes and Bacteroidetes, irrespective of presence/absence of TNT in the diet. Significant differences (P < 0.001) were found between the community structure of the bacteria under TNT (−) and TNT (+) diets. Examination of the TNT (+) diet showed an increase in the clones belonging to family Ruminococcaceae, which have previously been shown to degrade TNT in pure culture experiments.     

Biochemical synthesis of novel,self-assembling glycolipids from ricinoleic acid by a recombinant α-glucosidase from <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

Purpose of work  

To explore a novel glycolipid, we performed biochemical reactions using a recombinant α-glucosidase from Geobacillus sp. which shows excellent transglycosylation reaction to hydroxyl groups in a variety of compounds.     

Prediction of effective genome size in metagenomic samples

We introduce a novel computational approach to predict effective genome size (EGS; a measure that includes multiple plasmid copies, inserted sequences, and associated phages and viruses) from short sequencing reads of environmental genomics (or metagenomics) projects. We observe considerable EGS differences between environments and link this with ecologic complexity as well as species composition (for instance, the presence of eukaryotes). For example, we estimate EGS in a complex, organism-dense farm soil sample at about 6.3 megabases (Mb) whereas that of the bacteria therein is only 4.7 Mb; for bacteria in a nutrient-poor, organism-sparse ocean surface water sample, EGS is as low as 1.6 Mb. The method also permits evaluation of completion status and assembly bias in single-genome sequencing projects.     

Inventory and monitoring of wine microbial consortia

The evolution of the wine microbial ecosystem is generally restricted to Saccharomyces cerevisiae and Oenococcus oeni, which are the two main agents in the transformation of grape must into wine by acting during alcoholic and malolactic fermentation, respectively. But others species like the yeast Brettanomyces bruxellensis and certain ropy strains of Pediococcus parvulus can spoil the wine. The aim of this study was to address the composition of the system more precisely, identifying other components. The advantages of the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) approach to wine microbial ecology studies are illustrated by bacteria and yeast species identification and their monitoring at each stage of wine production. After direct DNA extraction, PCR-DGGE was used to make the most exhaustive possible inventory of bacteria and yeast species found in a wine environment. Phylogenetic neighbor-joining trees were built to illustrate microbial diversity. PCR-DGGE was also combined with population enumeration in selective media to monitor microbial changes at all stages of production. Moreover, enrichment media helped to detect the appearance of spoilage species. The genetic diversity of the wine microbial community and its dynamics during winemaking were also described. Most importantly, our study provides a better understanding of the complexity and diversity of the wine microbial consortium at all stages of the winemaking process: on grape berries, in must during fermentation, and in wine during aging. On grapes, 52 different yeast species and 40 bacteria could be identified. The diversity was dramatically reduced during winemaking then during aging. Yeast and lactic acid bacteria were also isolated from very old vintages. B. bruxellensis and O. oeni were the most frequent.     

Flocculation of microalgae using cationic starch

Due to their small size and low concentration in the culture medium, cost-efficient harvesting of microalgae is a major challenge. We evaluated the potential of cationic starch as a flocculant for harvesting microalgae using jar test experiments. Cationic starch was an efficient flocculant for freshwater (Parachlorella, Scenedesmus) but not for marine microalgae (Phaeodactylum, Nannochloropsis). At high cationic starch doses, dispersion restabilization was observed. The required cationic starch dose to induce flocculation increased linearly with the initial algal biomass concentration. Of the two commercial cationic starch flocculants tested, Greenfloc 120 (used in wastewater treatment) was more efficient than Cargill C*Bond HR 35.849 (used in paper manufacturing). For flocculation of Parachlorella using Greenfloc 120, the cationic starch to algal biomass ratio required to flocculate 80% of algal biomass was 0.1. For Scenedesmus, a lower dose was required (ratio 0.03). Flocculation of Parachlorella using Greenfloc 120 was independent of pH in the pH range of 5 to 10. Measurements of the maximum quantum yield of PSII suggest that Greenfloc 120 cationic starch was not toxic to Parachlorella. Cationic starch may be used as an efficient, nontoxic, cost-effective, and widely available flocculant for harvesting microalgal biomass.     

Catalytic properties of β-cyclodextrin glucanotransferase from alkalophilic<Emphasis Type="Italic">Bacillus</Emphasis> sp. BL-12 and intermolecular transglycosylation of stevioside

The catalytic properties of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilicBacillus sp. BL-12 specific for the intermolecular transglycosylation of stevioside were investigated. The molecular mass of purified β-CGTase by ultra-filtration and β-cyclodextrin polymer affinity chromatography was estimated to be 90 kDa, which is high compared to other known bacterial CGTases. The optimal pH and temperature were 9.0 and 50°C, respectively, and thermal stability at 40°C was elevated 10-fold in the presence of 1% maltodextrin. The kinetic parameters of the new β-CGTase from alkalophilicBacillus sp. BL-12 indicate that it is more suitable for transglycosylation than the cyclization reaction. Maltodextrin was the most suitable glycosyl donor for transglycosylation of stevioside. The transglycosylation of stevioside was carried out using 60 units of CGTase per gram of maltodextrin, 20 g/L stevioside as the glycosyl acceptor, and 50 g/L maltodextrin as the gycosyl donor at 40°C for 6 h, and a conversion yield of stevioside as high as 76% was obtained.     

Molecular cloning,characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01

Aims

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.

Methods and Results

The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l^?1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni²⁺‐NTA) agarose column and their activities characterized. BSH A hydrolysed tauro‐conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco‐conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.

Conclusions

BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate‐binding sites, these remain functional through motif conservation.

Significance and Impact of the Study

This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco‐conjugated or tauro‐conjugated bile salts. Future structural homology studies and site‐directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.     

Novel adjuvant for immunization against tuberculosis: DNA vaccine expressing <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> antigen 85A and interleukin-15 fusion product elicits strong immune responses in mice

Objectives

To investigate the potential of interleukin (IL)-15 as a novel adjuvant for Mycobacterium tuberculosis (Mtb) antigen 85A (Ag85A) vaccine.

Results

C57BL/6 mice were intramuscularly immunized three times with a plasmid expressing the Ag85A-IL-15 fusion protein (pcDNA3.1-Ag85A-IL-15), with the empty pcDNA3.1 vector and the pcDNA3.1-Ag85A as control. Mice vaccinated with pcDNA3.1-Ag85A-IL-15 generated more secretory IgA (sIgA) into their lung (209 ± 21 μg/ml) and acquired an enhanced serum IgG response to Ag85A. IgG2a/IgG1 ratios were upregulated, natural killer cell activity was augmented and Ag85A-specific splenic T cell proliferation was enhanced in these mice as well. Vaccination with pcDNA3.1-Ag85A-IL-15 promoted the polarization of CD4⁺ T cells towards a Th1 type in the spleen, and significantly upregulated the serum level of interferon (IFN)-γ (458 ± 98 pg/ml), a typical Th1 cytokine. IFN-γ-expressing CD8⁺ cells were also increased in the spleen after pcDNA3.1-Ag85A-IL-15 immunization.

Conclusions

A superior immune type I response in mice vaccinated with plasmid Ag85A-IL-15 has been achieved.