Depth gradients of benthic standing stock and diversity on the continental margin at a high-latitude ice-free site (off Spitsbergen 79 degrees N)

High-latitude seas are mostly covered by multi-year ice, which impacts processes of primary production and sedimentation of organic matter. Because of the warming effect of West Spitsbergen Current (WSC), the waters off West Spitsbergen have only winter ice cover. That is uncommon for such a high latitude and enables to separate effects of multiyear-ice cover from the latitudinal patterns. Macrofauna was sampled off Kongsfjord (79°N) along the depth gradient from 300 to 3000 m. The density, biomass and diversity at shallow sites situated in a canyon were very variable. Biomass was negatively correlated with depth (R=-0.86R=-0.86, p<0.001), and ranged from 61 g ww m−2 (212 m) to 1 g ww m−2 (2025 m). The biomasses were much higher than in the multiyear-ice covered High Arctic at similar depths, while resembling those from temperate and tropical localities. Species richness (expressed by number of species per sample and species–area accumulation curves) decreased with depth. There was no clear depth-related pattern in diversity measures: Hurbert rarefaction, Shannon–Wiener or Pielou. The classic increase of species richness and diversity with depth was not observed. Species richness and diversity of deep-sea macrofauna were much lower in our study than in comparable studies of temperate North Atlantic localities. That is related to geographic isolation of Greenland–Icelandic–Norwegian (GIN) seas from the Atlantic pool of species.

Consequences of heat stress on reproduction of cattle

Heat stress represents one of the major environmental factors that adversely affect the reproductive performance of cattle. In this paper the behavioral adjustments, physical mechanisms and physiological responses to heat loss are described; bos indicus adaptive advantages with respect to bos Taurus, pathophysiology of heat stress and heat stress effects in animal reproduction, both the male and the female.

2,4-dinitrophenol as an activating reagent in a facile preparation of cyclic phosphate trimesters

Abstract 2,4-Dinitrophenol was employed with benzyloxy-bis-(diisopropylamino)phosphine to synthesise the cyclic phosphate derivatives of a series of alkane diols (HO–(CH2)n–OH, n=2–6) in good isolated yields. Tetrazole and DNP were compared by 31P NMR spectroscopy for their ability to catalyse the cyclisation at the P(III) stage. Investigation of the phosphate triester stability under various oxidation and chromatographic conditions resulted in the optimisation of the isolation procedures of the chemically unstable cyclic compounds. Conditions for debenzylation were developed to yield the corresponding cyclic phosphodiesters quantitatively. The methodology was further applied to the preparation and isolation of the cyclic phosphate derivative of a carbohydrate.

Multiple Enzymatic Activities Associated with Severe Acute Respiratory Syndrome Coronavirus Helicase

Severe acute respiratory syndrome coronavirus (SARS-CoV), a newly identified group 2 coronavirus, is the causative agent of severe acute respiratory syndrome, a life-threatening form of pneumonia in humans. Coronavirus replication and transcription are highly specialized processes of cytoplasmic RNA synthesis that localize to virus-induced membrane structures and were recently proposed to involve a complex enzymatic machinery that, besides RNA-dependent RNA polymerase, helicase, and protease activities, also involves a series of RNA-processing enzymes that are not found in most other RNA virus families. Here, we characterized the enzymatic activities of a recombinant form of the SARS-CoV helicase (nonstructural protein [nsp] 13), a superfamily 1 helicase with an N-terminal zinc-binding domain. We report that nsp13 has both RNA and DNA duplex-unwinding activities. SARS-CoV nsp13 unwinds its substrates in a 5'-to-3' direction and features a remarkable processivity, allowing efficient strand separation of extended regions of double-stranded RNA and DNA. Characterization of the nsp13-associated (deoxy)nucleoside triphosphatase ([dNTPase) activities revealed that all natural nucleotides and deoxynucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed slightly more efficiently than other nucleotides. Furthermore, we established an RNA 5'-triphosphatase activity for the SARS-CoV nsp13 helicase which may be involved in the formation of the 5' cap structure of viral RNAs. The data suggest that the (d)NTPase and RNA 5'-triphosphatase activities of nsp13 have a common active site. Finally, we established that, in SARS-CoV-infected Vero E6 cells, nsp13 localizes to membranes that appear to be derived from the endoplasmic reticulum and are the likely site of SARS-CoV RNA synthesis.

Pachymedusa dacnicolor tryptophyllin-1 (PdT-1): structural characterization, pharmacological activity and cloning of precursor cDNA.

Tryptophyllins are a heterogenous group of amphibian skin peptides originally identified in skin extracts of Neotropical leaf frogs, Phyllomedusa sp., by chemical means. Until now, biosynthetic precursor structure and biological activity remain unreported. Here we describe the isolation of a novel, post-translationally modified tryptophyllin, Lys-Pro-Hyp-Ala-Trp-Val-Pro.amide (PdT-1), from the skin secretion of the Mexican leaf frog, Pachymedusa dacnicolor. Using a 3'- and 5'-RACE strategy and an in vitro skin cDNA library, the PdT-1-encoding precursor was cloned and found to consist of an open-reading frame of 62 amino acids with a single copy of PdT-1 located towards the C-terminus. A synthetic replicate of PdT-1 was found to be a potent myoactive agent, relaxing mammalian arterial smooth muscle and contracting small intestinal smooth muscle at nanomolar concentrations. PdT-1 is thus the first amphibian skin tryptophyllin to be pharmacologically characterized and the first whose precursor cDNA has been cloned.

The role of Y chromosome deletions in male infertility.

Male infertility affects approximately 2-7% of couples around the world. Over one in ten men who seek help at infertility clinics are diagnosed as severely oligospermic or azoospermic. Recent extensive molecular studies have revealed that deletions in the azoospermia factor region of the long arm of the Y chromosome are associated with severe spermatogenic impairment (absent or severely reduced germ cell development). Genetic research into male infertility, in the last 7 years, has resulted in the isolation of a great number of genes or gene families on the Y chromosome, some of which are believed to influence spermatogenesis.

Multivariate prediction of clarified butter composition using Raman spectroscopy

Raman spectroscopy has been used to predict the abundance of the FA in clarified butterfat that was obtained from dairy cows fed a range of levels of rapeseed oil in their diet. Partial least squares regression of the Raman spectra against FA compositions obtained by GC showed good prediction for the five major (abundance >5%) FA with R-2=0.74-0.92 and a root mean SE of prediction (RMSEP) that was 5-7% of the mean. In general, the prediction accuracy fell with decreasing abundance in the sample, but the RMSEP was 1.25%. The Raman method has the best prediction ability for unsaturated FA (R-2=0.85-0.92), and in particular trans unsaturated FA (best-predicted FA was 18:1 tDelta9). This enhancement was attributed to the isolation of the unsaturated modes from the saturated modes and the significantly higher spectral response of unsaturated bonds compared with saturated bonds. Raman spectra of the melted butter samples could also be used to predict bulk parameters calculated from standard analyzes, such as iodine value (R-2=0.80) and solid fat content at low temperature (R-2=0.87). For solid fat contents determined at higher temperatures, the prediction ability was significantly reduced (R-2=0.42), and this decrease in performance was attributed to the smaller range of values in solid fat content at the higher temperatures. Finally, although the prediction errors for the abundances of each of the FA in a given sample are much larger with Raman than with full GC analysis, the accuracy is acceptably high for quality control applications. This, combined with the fact that Raman spectra can be obtained with no sample preparation and with 60-s data collection times, means that high-throughput, on-line Raman analysis of butter samples should be possible.

Analysis of UDP-galactose 4'-epimerase mutations associated with the intermediate form of type III galactosaemia

Type III galactosaemia is a hereditary disease caused by reduced activity in the Leloir pathway enzyme, UDP-galactose 4'-epimerase (GALE). Traditionally, the condition has been divided into two forms-a mild, or peripheral, form and a severe, or generalized, form. Recently it has become apparent that there are disease states which are intermediate between these two extremes. Three mutations associated with this intermediate form (S81R, T150M and P293L) were analysed for their kinetic and structural properties in vitro and their effects on galactose-sensitivity of Saccharomyces cerevisiae cells that were deleted for the yeast GALE homologue Gal10p. All three mutations result in impairment of the kinetic parameters (principally the turnover number, k(cat)) compared with the wild-type enzyme. However, the degree of impairment was mild compared with that seen with the mutation (V94M) associated with the generalized form of epimerase deficiency galactosaemia. None of the three mutations tested affected the ability of the protein to dimerize in solution or its susceptibility to limited proteolysis in vitro. Finally, in the yeast model, each of the mutated patient alleles was able to complement the galactose-sensitivity of gal10 Delta cells as fully as was the wild-type human allele. Furthermore, there was no difference from control in metabolite profile following galactose exposure for any of these strains. Thus we conclude that the subtle biochemical and metabolic abnormalities detected in patients expressing these GALE alleles likely reflect, at least in part, the reduced enzymatic activity of the encoded GALE proteins.

Redescription of the deep-sea octopod Benthoctopus normani (Massy 1907) and a description of a new species from the Northeast Atlantic

A long-synonymized species Benthoctopus normani (Massy 1907) (Cephalopoda: Octopodidae) is redescribed from material collected over 30 years by the National Oceanography Centre, Southampton and the National Museums of Scotland. It can be distinguished from other octopodid specimens found in deep waters of the Northeast Atlantic by its biserial suckers, lack of ink sac, and simple ligula, which lacks transverse ridges. Examination of the collections led to the identification of a new species of Benthoctopus from the Northeast Atlantic, which is described herein.

Dioxygenase-catalysed mono-, di- and tri-oxygenation of dialkyl sulfides and thioacetals: chemoenzymatic synthesis of enantiopure cis-diol sulfoxides

Toluene dioxygenase (TDO)-catalysed monooxygenation of methylsulfanylmethyl phenyl sulfide 1 and methylsulfanylmethyl 2-pyridyl sulfide 4, using whole cells of Pseudomonas putida UV4, occurred exclusively at the alkyl aryl sulfur centre to yield the alkyl aryl sulfoxides 2 and 5 respectively. These sulfoxides, accompanied by the dialkyl sulfoxides 3 and 6, were also obtained from naphthalene dioxygenase (NDO)-catalysed sulfoxidation of thioacetals 1 and 4 using intact cells of P. putida NCIMB 8859. Enzymatic oxidation of methyl benzyl sulfide 7, 2-phenyl-1,3-dithiane 19, and 2-phenyl-1,3-dithiolane 23, using TDO, gave the corresponding dialkyl sulfoxides 8, 20 and 24 as minor bioproducts. TDO-catalysed dioxygenation of the alkyl benzyl sulfides 7, 15 and 17 and the thioacetals 19 and 23, with P. putida UV4, yielded the corresponding enantiopure cis-dihydrodiols 9, 16, 18, 21 and 25 as major metabolites and cis-dihydrodiol sulfoxides 14, 22 and 26 as minor metabolites, resulting from a tandem trioxygenation of substrates 7, 19 and 23 respectively. Chemical oxidation, of the enantiopure cis-dihydrodiol sulfides 9, 16, 18 and 21 with dimethyldioxirane (DMD), gave separable mixtures of the corresponding pairs of cis-dihydrodiol sulfoxide diastereoisomers 14 and 27, 28 and 29, 30 and 31, 22 and 32. While dialkyl sulfoxide bioproducts 3, 6, 20 and 24 were of variable enantiopurity (27-greater than or equal to 98% ee), alkyl aryl monosulfoxides 2 and 5, cis-dihydrodiols 9, 16, 18, 21 and 25 and cis-dihydrodiol sulfoxide bioproducts 14, 22 and 26 were all single enantiomers (greater than or equal to 98% ee). The absolute configurations of the products, obtained from enzyme-catalysed (TDO and NDO) and chemical (DMD) oxidation methods, were determined by stereochemical correlation, circular dichroism, and X-ray crystallographic methods.

Effect of heat treatment on the antioxidant activity, color, and free phenolic acid profile of malt

Green malt was kilned at 95 degrees C following two regimens: a standard regimen (SKR) and a rapid regimen (RKR). Both resulting malts were treated further in a tray dryer heated to 120 degrees C, as was green malt previously dried to 65 degrees C (TDR). Each regimen was monitored by determining the color, antioxidant activity (by both ABTS(center dot+) and FRAP methods), and polyphenolic profile. SKR and RKR malts exhibited decreased L* and increased b* values above approximately 80 degrees C. TDR malts changed significantly less, and color did not develop until 110 degrees C, implying that different chemical reactions lead to color in those malts. Antioxidant activity increased progressively with each regimen, although with TDR malts this became significant only at 110-120 degrees C. The RKR malt ABTS(center dot+) values were higher than those of the SKR malt. The main phenolics, that is, ferulic, p-coumaric, and vanillic acids, were monitored throughout heating. Ferulic acid levels increased upon heating to 80 degrees C for SKR and to 70 degrees C for RKR, with subsequent decreases. However, the levels for TDR malts did not increase significantly. The increase in free phenolics early in kilning could be due to enzymatic release of bound phenolics and/or easier extractability due to changes in the matrix. The differences between the kilning regimens used suggest that further modification of the regimens could lead to greater release of bound phenolics with consequent beneficial effects on flavor stability in beer and, more generally, on human health.

Enzyme-catalysed synthesis and reactions of benzene oxide/oxepine derivatives of methyl benzoates

A series of twelve benzoate esters was metabolised, by species of the Phellinus genus of wood-rotting fungi, to yield the corresponding benzyl alcohol derivatives and eight salicylates. The isolation of a stable oxepine metabolite, from methyl benzoate, allied to evidence of the migration and retention of a carbomethoxy group ( the NIH Shift), during enzyme-catalysed ortho-hydroxylation of alkyl benzoates to form salicylates, is consistent with a mechanism involving an initial arene epoxidation step. This mechanism was confirmed by the isolation of a remarkably stable, optically active, substituted benzene oxide metabolite of methyl 2-( trifluoromethyl) benzoate, which slowly converted into the racemic form. The arene oxide was found to undergo a cycloaddition reaction with 4-phenyl-1,2,4-triazoline-3,5-dione to yield a crystalline cycloadduct whose structure and racemic nature was established by X-ray crystallography. The metabolite was also found to undergo some novel benzene oxide reactions, including epoxidation to give an anti-diepoxide, base-catalysed hydrolysis to form a trans-dihydrodiol and acid-catalysed aromatisation to yield a salicylate derivative via the NIH Shift of a carbomethoxy group.

Activity against Mycobacterium smegmatis and M. tuberculosis by extract of South African medicinal plants

Seven ethnobotanically selected medicinal plants were screened for their antimycobacterial activity. The mininium inhibitory concentration (MIC) of four plants namely Artemisia afra, Dodonea angustifolia, Drosera capensis and Galenia africana ranged from 0.781 to 6.25 mg/mL against Mycobacterium smegmatis. G. africana showed the best activity exhibiting an MIC of 0.78 mg/mL and a minimum bactericidal concentration (MBC) of 1.56 mg/mL. The MICs of ethanol extracts of A angustifolia and G. africana against M. tuberculosis were found to be 5.0 and 1.2 mg/mL respectively. The mammalian cytotoxicity IC50 value of the most active antimycobacterial extract, from G. africana, was found to be 101.3 mu g/mL against monkey kidney Vero cells. Since the ethanol G. africana displayed the best antimycobacterial activity, it was subjected to fractionation which led to the isolation of a flavone, 5,7,2'-trihydroxyflavone. The MIC of this compound was found to be 0.031 mg/mL against M. smegmatis and 0.10 mg/mL against M. tuberculosis. This study gives some scientific basis to the 14 traditional use of these plants for TB-related symptoms. Copyright (C) 2008 John Wiley & Sons, Ltd.

Embryo retrieval and kin recognition in an amphipod (Crustacea)

Active maternal care directed towards embryos within the brood pouch has been identified in amphipod crustaceans from harsh aquatic environments. This involves 'curl' and 'stretch' components and brood flushing that alters in distinct ways in response to developmental and environmental cues. However, a cost of active brood care in crustaceans is the susceptibility to embryo loss, this being further predisposed by the structure of the amphipod brood pouch. We found embryo retrieval by females of the rock-pool amphipod Apherusa jurinei, whereby females inserted experimentally offered embryos into their brood pouches. Females early in brood development retrieved embryos to a greater degree than both nonovigerous and later stage females. In this experiment, all offered embryos were from other females, indicating a motivation to retrieve embryos that often overrides any kin recognition. In a second experiment, we found kin discrimination, with both early stage and late stage females retrieving more of their own embryos than those from other females. Recognition was not simply of embryos of similar developmental stages. There were high levels of embryo cannibalism in both experiments, but females were significantly less likely to consume their own compared to foreign embryos. We thus further show that 'lower' crustaceans such as amphipods engage in elaborate active maternal care including kin recognition and discrimination. Their maternal behaviour appears to balance the costs and benefits of embryo retrieval, minimizing fitness reductions due to embryo loss and adoption of foreign embryos. (C) 2008 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Establishing Clonal Cell Lines with Endothelial-Like Potential from CD9(hi), SSEA-1(-) Cells in Embryonic Stem Cell-Derived Embryoid Bodies

Background. Differentiation of embryonic stem cells (ESCs) into specific cell types with minimal risk of teratoma formation could be efficiently directed by first reducing the differentiation potential of ESCs through the generation of clonal, self-renewing lineage-restricted stem cell lines. Efforts to isolate these stem cells are, however, mired in an impasse where the lack of purified lineage-restricted stem cells has hindered the identification of defining markers for these rare stem cells and, in turn, their isolation. Methodology/Principal Findings. We describe here a method for the isolation of clonal lineage-restricted cell lines with endothelial potential from ESCs through a combination of empirical and rational evidence-based methods. Using an empirical protocol that we have previously developed to generate embryo-derived RoSH lines with endothelial potential, we first generated E-RoSH lines from mouse ESC-derived embryoid bodies (EBs). Despite originating from different mouse strains, RoSH and E-RoSH lines have similar gene expression profiles (r(2) = 0.93) while that between E-RoSH and ESCs was 0.83. In silico gene expression analysis predicted that like RoSH cells, E-RoSH cells have an increased propensity to differentiate into vasculature. Unlike their parental ESCs, E-RoSH cells did not form teratomas and differentiate efficiently into endothelial-like cells in vivo and in vitro. Gene expression and FACS analysis revealed that RoSH and E-RoSH cells are CD9(hi), SSEA-1(-) while ESCs are CD9(lo), SSEA-1(+). Isolation of CD9(hi), SSEA-1(-) cells that constituted 1%-10% of EB-derived cultures generated an E-RoSH-like culture with an identical E-RoSH-like gene expression profile (r(2) = 0.95) and a propensity to differentiate into endothelial-like cells. Conclusions. By combining empirical and rational evidence-based methods, we identified definitive selectable surface antigens for the isolation and propagation of lineage-restricted stem cells with endothelial-like potential from mouse ESCs.