p-cresol methylhydroxylase from a denitrifying bacterium involved in anaerobic degradation of p-cresol

A bacterium, strain PC-07, previously isolated as part of a coculture capable of growing on p-cresol under anaerobic conditions with nitrate as the acceptor was identified as an Achromobacter sp. The first enzyme of the pathway, p-cresol methylhydroxylase, which converts its substrate into p-hydroxybenzyl alcohol, was purified. The enzyme had an Mr of 130,000 and the spectrum of a flavocytochrome. It was composed of flavoprotein subunits of Mr 54,000 and cytochrome subunits of Mr 12,500. The midpoint redox potential of the cytochrome was 232 mV. The Km and kcat for p-cresol were 21 microM and 112 s-1 respectively, and the Km for phenazine methosulfate, the artificial acceptor used in the assays, was determined to be 1.7 mM. These properties place the enzyme in the same class as the p-cresol methylhydroxylases from aerobically isolated Pseudomonas spp.     

Impact of Nutrition and Salinity Changes on Biological Performances of Green and White Sturgeon

Green and white sturgeon are species of high conservational and economic interest, particularly in the San Francisco Bay Delta (SFBD) for which significant climate change-derived alterations in salinity and nutritional patterns are forecasted. Although there is paucity of information, it is critical to test the network of biological responses underlying the capacity of animals to tolerate current environmental changes. Through nutrition and salinity challenges, climate change will likely have more physiological effect on young sturgeon stages, which in turn may affect growth performance. In this study, the two species were challenged in a multiple-factor experimental setting, first to levels of feeding rate, and then to salinity levels for different time periods. Data analysis included generalized additive models to select predictors of growth performance (measured by condition factor) among the environmental stressors considered and a suite of physiological variables. Using structural equation modeling, a path diagram is proposed to quantify the main linkages among nutrition status, salinity, osmoregulation variables, and growth performances. Three major trends were anticipated for the growth performance of green and white sturgeon in the juvenile stage in the SFBD: (i) a decrease in prey abundance will be highly detrimental for the growth of both species; (ii) an acute increase in salinity within the limits studied can be tolerated by both species but possibly the energy spent in osmoregulation may affect green sturgeon growth within the time window assessed; (iii) the mechanism of synergistic effects of nutrition and salinity changes will be more complex in green sturgeon, with condition factor responding nonlinearly to interactions of salinity and nutrition status or time of salinity exposure. Green sturgeon merits special scientific attention and conservation effort to offset the effects of feed restriction and salinity as key environmental stressors in the SFBD.     

Purification and expression of glutathione S-transferase from a Malaysian population of Aedes albopictus (Diptera: Culicidae)

Glutathione S-transferase (GST) from the 4^th instar larvae of the dengue vector Aedes albopictus was purified by glutathione-agarose affinity chromatography and characterised using SDS-PAGE. The expression of the purified enzyme in the life stages and insecticide treated populations of Ae. albopictus as well as its cross-reactivity with larval GST of two dipteran species Aedes aegypti and Batrocera papayae were observed using western blotting. The purified GST had a specific activity of 196.0 ± 11 μmol/min/mg with a purification fold and yield of 28 and 69%, respectively. The SDS-PAGE analysis of the purified GST depicted a single band size of 23 kDa. The GST was expressed in all the larval and adult stages of Ae. albopictus with the exception of the pupal stage. However, the expression level in the adult stage was visibly reduced as compared to the larval stages. Western blotting analysis showed no cross-reactivity with the GST of Ae. aegypti (4^th instar) and B. papayae (3^rd instar) larvae. The expression of this enzyme was not inducible by exposure to the insecticides dichlorodiphenyltrichloroethane (1.25 mg/L) and malathion (0.3125 mg/L).     

Gene expression profiles of human inner cell mass cells and embryonic stem cells

Human embryonic stem cell (hESC) lines are derived from the inner cell mass (ICM) of preimplantation human blastocysts obtained on days 5–6 following fertilization. Based on their derivation, they were once thought to be the equivalent of the ICM. Recently, however, studies in mice reported the derivation of mouse embryonic stem cell lines from the epiblast; these epiblast lines bear significant resemblance to human embryonic stem cell lines in terms of culture, differentiation potential and gene expression. In this study, we compared gene expression in human ICM cells isolated from the blastocyst and embryonic stem cells. We demonstrate that expression profiles of ICM clusters from single embryos and hESC populations were highly reproducible. Moreover, comparison of global gene expression between individual ICM clusters and human embryonic stem cells indicated that these two cell types are significantly different in regards to gene expression, with fewer than one half of all genes expressed in both cell types. Genes of the isolated human inner cell mass that are upregulated and downregulated are involved in numerous cellular pathways and processes; a subset of these genes may impart unique characteristics to hESCs such as proliferative and self-renewal properties.     

Analytical platform for metabolome analysis of microbial cells using methyl chloroformate derivatization followed by gas chromatography-mass spectrometry

This protocol describes an analytical platform for the analysis of intra- and extracellular metabolites of microbial cells (yeast, filamentous fungi and bacteria) using gas chromatography-mass spectrometry (GC-MS). The protocol is subdivided into sampling, sample preparation, chemical derivatization of metabolites, GC-MS analysis and data processing and analysis. This protocol uses two robust quenching methods for microbial cultures, the first of which, cold glycerol-saline quenching, causes reduced leakage of intracellular metabolites, thus allowing a more reliable separation of intra- and extracellular metabolites with simultaneous stopping of cell metabolism. The second, fast filtration, is specifically designed for quenching filamentous micro-organisms. These sampling techniques are combined with an easy sample-preparation procedure and a fast chemical derivatization reaction using methyl chloroformate. This reaction takes place at room temperature, in aqueous medium, and is less prone to matrix effect compared with other derivatizations. This protocol takes an average of 10 d to complete and enables the simultaneous analysis of hundreds of metabolites from the central carbon metabolism (amino and nonamino organic acids, phosphorylated organic acids and fatty acid intermediates) using an in-house MS library and a data analysis pipeline consisting of two free software programs (Automated Mass Deconvolution and Identification System (AMDIS) and R).     

Functional morphology of the fin rays of teleost fishes

Ray‐finned fishes are notable for having flexible fins that allow for the control of fluid forces. A number of studies have addressed the muscular control, kinematics, and hydrodynamics of flexible fins, but little work has investigated just how flexible ray‐finned fish fin rays are, and how flexibility affects their response to environmental perturbations. Analysis of pectoral fin rays of bluegill sunfish showed that the more proximal portion of the fin ray is unsegmented while the distal 60% of the fin ray is segmented. We examined the range of motion and curvatures of the pectoral fin rays of bluegill sunfish during steady swimming, turning maneuvers, and hovering behaviors and during a vortex perturbation impacting the fin during the fin beat. Under normal swimming conditions, curvatures did not exceed 0.029 mm^?1 in the proximal, unsegmented portion of the fin ray and 0.065 mm^?1 in the distal, segmented portion of the fin ray. When perturbed by a vortex jet traveling at approximately 1 ms^?1 (67 ± 2.3 mN s.e. of force at impact), the fin ray underwent a maximum curvature of 9.38 mm^?1. Buckling of the fin ray was constrained to the area of impact and did not disrupt the motion of the pectoral fin during swimming. Flexural stiffness of the fin ray was calculated to be 565 × 10^?6 Nm². In computational fluid dynamic simulations of the fin‐vortex interaction, very flexible fin rays showed a combination of attraction and repulsion to impacting vortex dipoles. Due to their small bending rigidity (or flexural stiffness), impacting vortices transferred little force to the fin ray. Conversely, stiffer fin rays experienced rapid small‐amplitude oscillations from vortex impacts, with large impact forces all along the length of the fin ray. Segmentation is a key design feature of ray‐finned fish fin rays, and may serve as a means of making a flexible fin ray out of a rigid material (bone). This flexibility may offer intrinsic damping of environmental fluid perturbations encountered by swimming fish. J. Morphol. 274:1044–1059, 2013. © 2013 Wiley Periodicals, Inc.     

Metabolite secretion in microorganisms: the theory of metabolic overflow put to the test

Introduction

Microbial cells secrete many metabolites during growth, including important intermediates of the central carbon metabolism. This has not been taken into account by researchers when modeling microbial metabolism for metabolic engineering and systems biology studies.

Materials and Methods

The uptake of metabolites by microorganisms is well studied, but our knowledge of how and why they secrete different intracellular compounds is poor. The secretion of metabolites by microbial cells has traditionally been regarded as a consequence of intracellular metabolic overflow.

Conclusions

Here, we provide evidence based on time-series metabolomics data that microbial cells eliminate some metabolites in response to environmental cues, independent of metabolic overflow. Moreover, we review the different mechanisms of metabolite secretion and explore how this knowledge can benefit metabolic modeling and engineering.