The enzymatic decarboxylation of hydroxypyruvate associated with purified pyruvate decarboxylase from wheat germ

Highly purified pyruvic decarboxylase (EC 4.1.1.1) from wheat germ catalyses the decarboxylation of hydroxypyruvate. A kinetic analysis of the activity of the enzyme with pyruvate and hydroxypyruvate as substrates suggests that a single enzyme is involved. The kinetics of decarboxylation are autocatalytic. The time lag before maximum activity is reached is affected by the concentration of hydroxypyruvate and the pH. The question whether or not hydroxypyruvate is a natural substrate for the enzyme remains unresolved, but it may be significant that at physiological pH (ca 7.5) the enzyme shows optimum activity with hydroxypyruvate, but negligible activity with pyruvate.     

Mesoflavibacter zeaxanthinifaciens gen. nov., sp. nov., a novel zeaxanthin-producing marine bacterium of the family Flavobacteriaceae

A novel strictly aerobic, gliding, Gram-negative, rod-shaped, halo- and mesophilic bacterium (TD-ZX30(T)) was isolated from a seawater sample collected on the Pacific coastline of Japan near Kamakura City (Fujisawa, Kanagawa). The temperature range for growth of TD-ZX30(T) was between 16 and 44 degrees C. The DNA G+C content was 32.0mol%. The predominant fatty acids were iso-C(15:1) G, iso-C(15:0), iso-C(16:0) 3-OH, iso-C(15:0) 3-OH, Summed feature (iso-C(15:0) 2-OH and/or C(16:1)omega7c), iso-C(17:0) 3-OH, and C(15:0). MK-6 was the only respiratory quinone. Zeaxanthin was the major carotenoid pigment produced but flexirubin-type pigments were not produced. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that TD-ZX30(T) belonged to a distinct lineage in the family Flavobacteriaceae, sharing 93.9% sequence similarity with the nearest species Olleya marilimosa. TD-ZX30(T) could be distinguished from the other members of the family Flavobacteriaceae by a number of chemotaxonomic and phenotypic characteristics. The results of polyphasic taxonomic analyses suggested that TD-ZX30(T) represents a novel genus and a novel species, for which the name Mesoflavibacter zeaxanthinifaciens gen. nov., sp. nov. is proposed. The type strain is TD-ZX30(T) (=NBRC 102119=CCUG 53614=DSM 18436).     

Oxidation of combined ingestion of glucose and fructose during exercise.

The purpose of the present study was to examine whether combined ingestion of a large amount of fructose and glucose during cycling exercise would lead to exogenous carbohydrate oxidation rates >1 g/min. Eight trained cyclists (maximal O(2) consumption: 62 +/- 3 ml x kg(-1) x min(-1)) performed four exercise trials in random order. Each trial consisted of 120 min of cycling at 50% maximum power output (63 +/- 2% maximal O(2) consumption), while subjects received a solution providing either 1.2 g/min of glucose (Med-Glu), 1.8 g/min of glucose (High-Glu), 0.6 g/min of fructose + 1.2 g/min of glucose (Fruc+Glu), or water. The ingested fructose was labeled with [U-(13)C]fructose, and the ingested glucose was labeled with [U-(14)C]glucose. Peak exogenous carbohydrate oxidation rates were approximately 55% higher (P < 0.001) in Fruc+Glu (1.26 +/- 0.07 g/min) compared with Med-Glu and High-Glu (0.80 +/- 0.04 and 0.83 +/- 0.05 g/min, respectively). Furthermore, the average exogenous carbohydrate oxidation rates over the 60- to 120-min exercise period were higher (P < 0.001) in Fruc+Glu compared with Med-Glu and High-Glu (1.16 +/- 0.06, 0.75 +/- 0.04, and 0.75 +/- 0.04 g/min, respectively). There was a trend toward a lower endogenous carbohydrate oxidation in Fruc+Glu compared with the other two carbohydrate trials, but this failed to reach statistical significance (P = 0.075). The present results demonstrate that, when fructose and glucose are ingested simultaneously at high rates during cycling exercise, exogenous carbohydrate oxidation rates can reach peak values of approximately 1.3 g/min.     

Oxidation of exogenous glucose, sucrose, and maltose during prolonged cycling exercise.

The purpose of the present study was to investigate whether combined ingestion of two carbohydrates (CHO) that are absorbed by different intestinal transport mechanisms would lead to exogenous CHO oxidation rates of >1.0 g/min. Nine trained male cyclists (maximal O(2) consumption: 64 +/- 2 ml x kg body wt(-1) x min(-1)) performed four exercise trials, which were randomly assigned and separated by at least 1 wk. Each trial consisted of 150 min of cycling at 50% of maximal power output (60 +/- 1% maximal O(2) consumption), while subjects received a solution providing either 1.8 g/min of glucose (Glu), 1.2 g/min of glucose + 0.6 g/min of sucrose (Glu+Suc), 1.2 g/min of glucose + 0.6 g/min of maltose (Glu+Mal), or water. Peak exogenous CHO oxidation rates were significantly higher (P < 0.05) in the Glu+Suc trial (1.25 +/- 0.07 g/min) compared with the Glu and Glu+Mal trials (1.06 +/- 0.08 and 1.06 +/- 0.06 g/min, respectively). No difference was found in (peak) exogenous CHO oxidation rates between Glu and Glu+Mal. These results demonstrate that, when a mixture of glucose and sucrose is ingested at high rates (1.8 g/min) during cycling exercise, exogenous CHO oxidation rates reach peak values of approximately 1.25 g/min.     

Purification and characterization of two thermostable protease fractions from Bacillus megaterium

Protease enzyme from Bacillus megaterium was successively purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-200. The purification steps of protease resulted in the production of two protease fractions namely protease P1 and P2 with specific activities of 561.27 and 317.23 U mg^?1 of protein, respectively. The molecular weights of B. megaterium P1 and P2 were 28 and 25 KDa, respectively. The purified fractions P1 and P2 were rich in aspartic acid and serine. Relatively higher amounts of alanine, leucine, glycine, valine, thereonine valine and glutamic acid were also present. The maximum protease activities for both enzyme fractions were attained at 50 °C, pH 7.5, 1% of gelatine concentration and 0.5 enzyme concentrations. P1 and P2 fractions were more stable over pH 7.0–8.5 and able to prolong their thermal stability up to 80 °C. The effect of different inhibitors on the protease activity of both enzyme fractions was also studied. The enzyme was found to be serine active as it had been affected by lower concentrations of phenylmethylsulfonyl fluoride (PMSF). Complete dehairing of the enzyme-treated skin was achieved in 12 h, at room temperature.     

Physiology and mRNA expression in rainbow trout (Oncorhynchus mykiss) after long-term exposure to the new antifoulant medetomidine

Medetomidine is under evaluation for use as an antifouling agent, and its effects on non-target aquatic organisms are therefore of interest. In this study, rainbow trout was exposed to low (0.5 and 5.0nM) concentrations of medetomidine for up to 54 days. Recently we have reported on effects on paleness and melanophore aggregation of medetomidine in these fish. Here, specific growth rates were investigated together with a broad set of physiological parameters including plasma levels of growth hormone (GH), insulin-like growth factor-I (IGF-I) and leptin, glucose and haemoglobin (Hb), hematocrit (Ht), condition factor, liver and heart somatic indexes (LSI, HSI). Hepatic enzyme activities of CYP1A (EROD activity), glutathione S-transferases (GST) and glutathione reductase (GR) were also measured. Additionally, hepatic mRNA expression was analysed through microarray and quantitative PCR in fish sampled after 31 days of exposure. Medetomidine at both concentrations significantly lowered blood glucose levels and the higher concentration significantly reduced the LSI. The mRNA expression analysis revealed few differentially expressed genes in the liver and the false discovery rate was high. Taken together, the results suggest that medetomidine at investigated concentrations could interfere with carbohydrate metabolism of exposed fish but without any clear consequences for growth.     

Stability and resilience of the intestinal microbiota in children in daycare – a 12 month cohort study

Background

We performed a 12-month cohort study of the stability and resilience of the intestinal microbiota of healthy children in daycare in Denmark in relation to diarrheal events and exposure to known risk factors for gastrointestinal health such as travelling and antibiotic use. In addition, we analyzed how gut microbiota recover from such exposures.

Results

We monitored 32 children in daycare aged 1–6?years. Fecal samples were submitted every second month during a one-year observational period. Information regarding exposures and diarrheal episodes was obtained through questionnaires. Bacterial communities were identified using 16S rRNA gene sequencing. The core microbiota (mean abundance >?95%) dominated the intestinal microbiota, and none of the tested exposures (diarrheal events, travel, antibiotic use) were associated with decreases in the relative abundance of the core microbiota. Samples exhibited lower intra-individual variation than inter-individual variation. Half of all the variation between samples was explained by which child a sample originated from. Age explained 7.6–9.6% of the variation, while traveling, diarrheal events, and antibiotic use explained minor parts of the beta diversity. We found an age-dependent increase of alpha diversity in children aged 1–3?years, and while diarrheal events caused a decrease in alpha diversity, a recovery time of 40–45?days was observed.Among children having had a diarrheal event, we observed a 10x higher relative abundance of Prevotella. After travelling, a higher abundance of two Bacteroides species and 40% less Lachnospiraceae were seen. Antibiotic use did not correlate with changes in the abundance of any bacteria.

Conclusion

We present data showing that Danish children in daycare have stable intestinal microbiota, resilient to the exposures investigated. An early age-dependent increase in the diversity was demonstrated. Diarrheal episodes decreased alpha diversity with an estimated recovery time of 40–45?days.