Molecular Diversity of Rumen Methanogens from Sheep in Western Australia

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order Methanobacteriales. Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet. Methanobrevibacter strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.     

Enzymatic hydrolysis and fermentation of palm kernel press cake for production of bioethanol

Palm kernel press cake (PKC) is a residue of palm oil extraction, which was found to contain 48.5% of total carbohydrates of which 35.2% was mannan. The present study examines enzymatic hydrolysis of polysaccharides from the cell-wall material present in PKC to obtain monosaccharides that can be substrate in various fermentation processes such as ethanol production. The requirements for pretreatment were investigated and it was found that mannan in PKC was readily hydrolysed without any pretreatment. Several enzyme preparations were tested and Mannaway 25L was found as the best for releasing mannose, and Gammanase 1.0L worked well in degrading cellulose and mannose. Binary mixtures of enzymes were tested to increase the conversion, and 1:1 mixture of Mannaway 25L and Gammanase 1.0L showed good synergistic effect releasing 30% more mannose than the sum obtained using these enzymes individually. Using an enzyme loading of 2.3 mg protein/g PKC resulted in 63% of mannan in PKC being hydrolysed to mannose in 24 h, and in 96 h a total of 365 g mannose and glucose could be produced per kg PKC. Finally, PKC was hydrolysed and fermented using Saccharomyces cerevisiae with an ethanol yield of 125 g/kg PKC.     

Sensitive Troponins – Which Suits Better for Hemodialysis Patients? Associated Factors and Prediction of Mortality

Background

In hemodialysis patients, elevated plasma troponin concentrations are a common finding that has even increased with the advent of newly developed sensitive assays. However, the interpretation and relevance of this is still under debate.

Methods

In this cross-sectional study, we analyzed plasma concentrations of sensitive troponin I (TnI) and troponin T (TnT) in stable ambulatory hemodialysis patients (n = 239) and investigated their associations with clinical factors and mortality.

Results

In all of the enrolled patients, plasma TnI or TnT was detectable at a median concentration of 14 pg/ml (interquartile range: 7–29) using the Siemens TnI ultra assay and 49 pg/ml (31–74) using the Roche Elecsys high sensitive TnT assay. Markedly more patients exceeded the 99th percentile for TnT than for TnI (95% vs. 14%, p<0.0001). In a multivariate linear regression model, TnT was independently associated with age, gender, systolic dysfunction, time on dialysis, residual diuresis and systolic blood pressure, whereas TnI was independently associated with age, systolic dysfunction, pulse pressure, time on dialysis and duration of a HD session. During a follow-up period of nearly two years, TnT concentration above 38 pg/mL was associated with a 5-fold risk of death, whereas elevation of TnI had a gradual association to mortality.

Conclusion

In hemodialysis patients, elevations of plasma troponin concentrations are explained by cardiac function and dialysis-related parameters, which contribute to cardiac strain. Both are highly predictive of increased risk of death.     

Lost in the hybridisation vortex: high-elevation <Emphasis Type="Italic">Senecio hercynicus</Emphasis> (Compositae,Senecioneae) is genetically swamped by its congener <Emphasis Type="Italic">S. ovatus</Emphasis> in the Bavarian Forest National Park (SE Germany)

Hybridisation is an important evolutionary process. The investigation of hybridisation along elevational gradients, with their steep abiotic and biotic clines, provides insight into the adaptation and maintenance of species in adjacent habitats. The rare Senecio hercynicus and its spreading congener S. ovatus are vertically vicariant species that show hybridisation in their range overlaps. In the present study, we used AFLP fingerprinting of 689 individuals from 38 populations to analyse population structure and introgression patterns along four elevational transects (650–1350 m) in the Bavarian Forest National Park, Gemany. Subsequently, we searched for loci putatively under divergent selection that may maintain ‘pure’ species despite hybrid formation by identifying taxon-specific alleles. A maximum-likelihood based hybrid index shows that the overall genetic differentiation among all populations was very low with a vanishingly small number of pure parental individuals. Almost 75% of the investigated individuals were classified as backcrosses towards S. ovatus. The highest S. hercynicus ancestry was found in the uppermost populations of two transects. Further, we found seven taxon-specific alleles being under divergent selection that correlated with climatic variables and deviating from neutral introgression. According to our results, hybridisation of S. ovatus and S. hercynicus has reached an advanced state of genetic swamping and there seems to be no driving force that is strong enough to keep both species as different lineages. Rather, S. ovatus appears to benefit through putatively adaptive introgression.     

Gut microbiota modulate the metabolism of brown adipose tissue in mice

A two by two experimental study has been designed to determine the effect of gut microbiota on energy metabolism in mouse models. The metabolic phenotype of germ-free (GF, n = 20) and conventional (n = 20) mice was characterized using a NMR spectroscopy-based metabolic profiling approach, with a focus on sexual dimorphism (20 males, 20 females) and energy metabolism in urine, plasma, liver, and brown adipose tissue (BAT). Physiological data of age-matched GF and conventional mice showed that male animals had a higher weight than females in both groups. In addition, conventional males had a significantly higher total body fat content (TBFC) compared to conventional females, whereas this sexual dimorphism disappeared in GF animals (i.e., male GF mice had a TBFC similar to those of conventional and GF females). Profiling of BAT hydrophilic extracts revealed that sexual dimorphism in normal mice was absent in GF animals, which also displayed lower BAT lactate levels and higher levels of (D)-3-hydroxybutyrate in liver, plasma, and BAT, together with lower circulating levels of VLDL. These data indicate that the gut microbiota modulate the lipid metabolism in BAT, as the absence of gut microbiota stimulated both hepatic and BAT lipolysis while inhibiting lipogenesis. We also demonstrated that (1)H NMR metabolic profiles of BAT were excellent predictors of BW and TBFC, indicating the potential of BAT to fight against obesity.     

Jasmonate-induced lipid peroxidation in barley leaves initiated by distinct 13-LOX forms of chloroplasts

In addition to a previously characterized 13-lipoxygenase of 100 kDa encoded by LOX2:Hv:1 [V?r?s et al., Eur. J. Biochem. 251 (1998), 36-44], two full-length cDNAs (LOX2:Hv:2, LOX2:Hv:3) were isolated from barley leaves (Hordeum vulgare cv. Salome) and characterized. Both of them encode 13-lipoxygenases with putative target sequences for chloroplast import. Immunogold labeling revealed preferential, if not exclusive, localization of lipoxygenase proteins in the stroma. The ultrastructure of the chloroplast was dramatically altered following methyl jasmonate treatment, indicated by a loss of thylakoid membranes, decreased number of stacks and appearance of numerous osmiophilic globuli. The three 13-lipoxygenases are differentially expressed during treatment with jasmonate, salicylate, glucose or sorbitol. Metabolite profiling of free linolenic acid and free linoleic acid, the substrates of lipoxygenases, in water floated or jasmonate-treated leaves revealed preferential accumulation of linolenic acid. Remarkable amounts of free 9- as well as 13-hydroperoxy linolenic acid were found. In addition, metabolites of these hydroperoxides, such as the hydroxy derivatives and the respective aldehydes, appeared following methyl jasmonate treatment. These findings were substantiated by metabolite profiling of isolated chloroplasts, and subfractions including the envelope, the stroma and the thylakoids, indicating a preferential occurrence of lipoxygenase-derived products in the stroma and in the envelope. These data revealed jasmonate-induced activation of the hydroperoxide lyase and reductase branch within the lipoxygenase pathway and suggest differential activity of the three 13-lipoxygenases under different stress conditions.     

S100 and S100 fused-type protein families in epidermal maturation with special focus on S100A3 in mammalian hair cuticles

Epithelial Ca²⁺-regulation, which governs cornified envelope formation in the skin epidermis and hair follicles, closely coincides with the expression of S100A3, filaggrin and trichohyalin, and the post-translational modification of these proteins by Ca²⁺-dependent peptidylarginine deiminases. This review summarizes the current nomenclature and evolutional aspects of S100 Ca²⁺-binding proteins and S100 fused-type proteins (SFTPs) classified as a separate protein family with special reference to the molecular structure and function of S100A3 dominantly expressed in hair cuticular cells. Both S100 and SFTP family members are identified by two distinct types of Ca²⁺-binding loops in an N-terminal pseudo EF-hand motif followed by a canonical EF-hand motif. Seventeen members of the S100 protein family including S100A3 are clustered with seven related genes encoding SFTPs on human chromosome 1q21, implicating their association with epidermal maturation and diseases. Human S100A3 is characterized by two disulphide bridges and a preformed Zn²⁺-pocket, and may transfer Ca²⁺ ions to peptidylarginine deiminases after its citrullination-mediated tetramerization. Phylogenetic analysis utilizing current genome databases suggests that divergence of the S100A3 gene coincided with the emergence of hair, a defining feature of mammals, and that the involvement of S100A3 in epithelial Ca²⁺-cycling occurred as a result of a skin adaptation in terrestrial mammals.     

The sugar phosphate specificity of rat hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The sugar phosphate specificity of the active site of 6-phosphofructo-2-kinase and of the inhibitory site of fructose-2,6-bisphosphatase was investigated. The Michaelis constants and relative Vmax values of the sugar phosphates for the 6-phosphofructo-2-kinase were: D-fructose 6-phosphate, Km = 0.035 mM, Vmax = 1; L-sorbose 6-phosphate, Km = 0.175 mM, Vmax = 1.1; D-tagatose 6-phosphate, Km = 15 mM, Vmax = 0.15; and D-psicose 6-phosphate, Km = 7.4 mM, Vmax = 0.42. The enzyme did not catalyze the phosphorylation of 1-O-methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, 2,5-anhydro-D-mannitol 6-phosphate, D-ribose 5-phosphate, or D-arabinose 5-phosphate. These results indicate that the hydroxyl group at C-3 of the tetrahydrofuran ring must be cis to the beta-anomeric hydroxyl group and that the hydroxyl group at C-4 must be trans. The presence of a hydroxymethyl group at C-2 is required; however, the orientation of the phosphonoxymethyl group at C-5 has little effect on activity. Of all the sugar monophosphates tested, only 2,5-anhydro-D-mannitol 6-phosphate was an effective inhibitor of the kinase with a Ki = 95 microM. The sugar phosphate specificity for the inhibition of the fructose-2,6-bisphosphatase was similar to the substrate specificity for the kinase. The apparent I0.5 values for inhibition were: D-fructose 6-phosphate, 0.01 mM; L-sorbose 6-phosphate, 0.05 mM; D-psicose 6-phosphate, 1 mM; D-tagatose 6-phosphate, greater than 2 mM; 2,5-anhydro-D-mannitol 6-phosphate, 0.5 mM. 1-O-Methyl-D-fructose 6-phosphate, alpha- and beta-methyl-D-fructofuranoside 6-phosphate, and D-arabinose 5-phosphate did not inhibit. Treatment of the enzyme with iodoacetamide decreased sugar phosphate affinity in the kinase reaction but had no effect on the sensitivity of fructose-2,6-bisphosphatase to sugar phosphate inhibition. The results suggest a high degree of homology between two separate sugar phosphate binding sites for the bifunctional enzyme.     

A dual role of extracellular DNA during biofilm formation of Neisseria meningitidis

Major pathogenic clonal complexes (cc) of Neisseria meningitidis differ substantially in their point prevalence among healthy carriers. We show that frequently carried pathogenic cc (e.g. sequence type ST‐41/44 cc and ST‐32 cc) depend on extracellular DNA (eDNA) to initiate in vitro biofilm formation, whereas biofilm formation of cc with low point prevalence (ST‐8 cc and ST‐11 cc) was eDNA‐independent. For initial biofilm formation, a ST‐32 cc type strain, but not a ST‐11 type strain, utilized eDNA. The release of eDNA was mediated by lytic transglycosylase and cytoplasmic N‐acetylmuramyl‐l ‐alanine amidase genes. In late biofilms, outer membrane phospholipase A‐dependent autolysis, which was observed in most cc, but not in ST‐8 and ST‐11 strains, was required for shear force resistance of microcolonies. Taken together, N. meningitidis evolved two different biofilm formation strategies, an eDNA‐dependent one yielding shear force resistant microcolonies, and an eDNA‐independent one. Based on the experimental findings and previous epidemiological observations, we hypothesize that most meningococcal cc display a settler phenotype, which is eDNA‐dependent and results in a stable interaction with the host. On the contrary, spreaders (ST‐11 and ST‐8 cc) are unable to use eDNA for biofilm formation and might compensate for poor colonization properties by high transmission rates.     

The Association between Plasma Adiponectin and Insulin Sensitivity in Humans Depends on Obesity

Objective: In humans, low plasma adiponectin concentrations precede a decrease in insulin sensitivity and predict type 2 diabetes independently of obesity. However, it is possible that the contribution of adiponectin to insulin sensitivity is not equally strong over the whole range of obesity. Research Methods and Procedures: We investigated the cross‐sectional association between plasma adiponectin levels and insulin sensitivity in different ranges of body fat content [expressed as percentage of body fat (PFAT)] in a large cohort of normal glucose‐tolerant subjects (n = 900). All individuals underwent an oral glucose tolerance test (OGTT), and 299 subjects additionally a euglycemic hyperinsulinemic clamp. In longitudinal analyses, the association of adiponectin at baseline with change in insulin sensitivity was investigated in a subgroup of 108 subjects. Results: In cross‐sectional analyses, the association between plasma adiponectin and insulin sensitivity, adjusted for age, gender, and PFAT, depended on whether subjects were lean or obese [p for interaction adiponectin × PFAT = <0.001 (OGTT) and 0.002 (clamp)]. Stratified by quartiles of PFAT, adiponectin did not correlate significantly with insulin sensitivity in subjects in the lowest PFAT quartile (R² = 0.10, p = 0.13, OGTT; and R² = 0.10, p = 0.57, clamp), whereas the association in the upper PFAT quartile was rather strong (R² = 0.36, p < 0.0001, OGTT; and R² = 0.48, p = 0.003, clamp). In longitudinal analyses, plasma adiponectin at baseline preceded change in insulin sensitivity in obese (n = 54, p = 0.03) but not in lean (n = 54, p = 0.68) individuals. Discussion: These data suggest that adiponectin is especially critical in sustaining insulin sensitivity in obese subjects. Thus, interventions to reduce insulin resistance by increasing adiponectin concentrations may be effective particularly in obese, insulin‐resistant individuals.