Enantioselective hydrolysis of epoxides: the employment of the soluble fraction from Vicia sativa seedlings

Biocatalytic hydrolysis of meso and racemic aryl- and alkyl-oxiranes was accomplished by employing the epoxide hydrolase activity of the soluble fraction of Vicia sativa seedlings. Whereas meso epoxides were not hydrolyzed by this fraction, racemic compounds were transformed into the corresponding diols by formal anti-stereoselective water attack. Both substrate and product enantioselectivity were strongly influenced by the chains length and the presence of a hydroxyl group.     

Antheridiol, RNA polymerase II and sexual development in the aquatic fungus, Achlya

The fungus, Achlya, is one of the most primitive eukaryotes known to secrete and respond to diffusible steroid sex hormones (pheromones). Antheridiol, which is produced by female strains of Achlya induces male strains to differentiate male sex organs. Induction of male strains with antheridiol elicits several changes in macromolecular synthesis including a quantitative enhancement in the synthesis of poly adenylated messenger RNA. We have examined whether this quantitative change is due to the regulation of RNA polymerase II. The level of polymerase increases as a response to the pheromone. This was examined using two different approaches, one of which included an extremely sensitive enzyme-linked immunosorbant assay (ELISA). Furthermore, the specific activities of polymerase II preparations isolated from pheromone-stimulated cultures was significantly higher than the specific activities of enzyme preparations isolated from control. A comparison of the polypeptide subunit composition of polymerase II preparations isolated from both pheromone-treated and control cultures on SDS polyacrylamide gels indicated no qualitative differences. Apparent differences in the stoichiometry of two specific subunits were reproducibly observed. The subunits of 140,000 and of 69,000 stained much more intensely in RNA polymerase II preparations isolated from 4h antheridiol-treated cultures.     

Oceans and society: feedbacks between ocean and human health

The concentration of human population along coastlines has far-reaching effects on ocean and societal health. The oceans provide benefits to humans such as food, coastal protection and improved mental well-being, but can also impact negatively via natural disasters. At the same time, humans influence ocean health, for example, via coastal development or through environmental stewardship. Given the strong feedbacks between ocean and human health there is a need to promote desirable interactions, while minimising undesirable interactions. To this end, we articulate two scenarios for 2030. First, Business-as-Usual, named ‘Command and (out of) Control’, focuses on the anticipated future based on our current trajectory. Second, a more sustainable scenario called ‘Living and Connecting’, emphasises the development of interactions between oceans and society consistent with achieving the Sustainable Development Goals. We describe a potential pathway to achieving the ‘Living and Connecting’ scenario, centred on improving marine citizenship, achieving a more equitable distribution of power among stakeholders, and more equitable access to resources and opportunities. The constituent actions of this pathway can be categorised into four groups: (i) improved approaches to science and health communication that account for society’s diverse values, beliefs and worldviews, (ii) a shift towards more trusted relationships among stakeholders to enable two-way knowledge exchange, (iii) economic incentives that encourage behavioural changes necessary for achieving desired sustainability outcomes, and (iv) stronger regulations that simultaneously focus on ocean and human health. We contend that these changes will provide improved outcomes for both oceans and society over the United Nations Decade of Ocean Science.

Graphic abstract

     

Species identification of bivalve-inhabiting marine hydrozoans of the genus Eugymnanthea

Abstract. Species‐level identification is difficult in the symbiotic bivalve‐inhabiting hydrozoans of the genus Eugymnanthea (Cnidaria, Hydrozoa). Morphological differences are detected only in the adult medusoid stage. Eugymnanthea is known only from the Mediterranean and the western Pacific, and doubt persists over whether the two localities are inhabited by different species. Because the bivalve host, Mytilus galloprovincialis, is thought to have been introduced by humans from the Mediterranean to the western Pacific, there has been speculation that the Mediterranean Eugymnanthea was also introduced along with its host. Here, we evaluate the species status of the two hydrozoan forms with breeding experiments, morphology, and two recently developed tools for discrimination: a mesoglea cell adhesion and spreading test, and 16S rDNA comparison. Reciprocal crosses of the two forms failed to produce normal offspring, providing evidence that they are indeed different species according to the biological species concept, and suggesting that the Pacific form is not an invasion of the Mediterranean form. The tissue‐grafting test failed to distinguish between the two forms, while the morphological and genetic evidence corroborated the breeding results.     

A user''s guide for avoiding errors in absorbance image cytometry: a review with original experimental observations

Summary The sources of errors which may occur when cytophotometric analysis is performed with video microscopy using a charged-coupled device (CCD) camera and image analysis are reviewed. The importance of these errors in practice has been tested, and ways of minimizing or avoiding them are described. Many of these sources of error are known from scanning and integrating cytophotometry; they include the use of white instead of monochromatic light, the distribution error, glare, diffraction, shading distortion, and inadequate depth of field. Sources of errors specifically linked with video microscopy or image analysis are highlighted as well; these errors include blooming, limited dynamic range of grey levels, non-linear responses of the camera, contrast transfer, photon noise, dark current, read-out noise, fixed scene noise and spatial calibration. Glare, contrast transfer, fixed scene noise, depth of field and spatial calibration seem to be the most serious sources of errors when measurements are not carried out correctly. We include a table summarizing all the errors discussed in this review and procedures for avoiding them. It can be concluded that if accurate calibration steps are performed and proper guidelines followed, image cytometry can be applied safely for quantifying amounts of chromophore per cell or per unit volume of tissue in sections, even when relatively simple and inexpensive instrumentation is being used.     

HIV‐1 Nef induces p47phox phosphorylation leading to a rapid superoxide anion release from the U937 human monoblastic cell line

The Nef protein of the human immunodeficiency virus type 1 (HIV‐1) plays a crucial role in AIDS pathogenesis by modifying host cell signaling pathways. We investigated the effects of Nef on the NADPH oxidase complex, a key enzyme involved in the generation of reactive oxygen species during the respiratory burst in human monocyte/macrophages. We have recently shown that the inducible expression of HIV‐1 Nef in human macrophages cell line modulates in bi‐phasic mode the superoxide anion release by NADPH oxidase, inducing a fast increase of the superoxide production, followed by a delayed strong inhibition mediated by Nef‐induced soluble factor(s). Our study is focused on the molecular mechanisms involved in Nef‐mediated activation of NADPH oxidase and superoxide anion release. Using U937 cells stably transfected with different Nef alleles, we found that both Nef membrane localization and intact SH3‐binding domain are needed to induce superoxide release. The lack of effect during treatment with a specific MAPK pathway inhibitor, PD98059, demonstrated that Nef‐induced superoxide release is independent of Erk1/2 phosphorylation. Furthermore, Nef induced the phosphorylation and then the translocation of the cytosolic subunit of NADPH oxidase complex p47^phox to the plasma membrane. Adding the inhibitor PP2 prevented this process, evidencing the involvement of the Src family kinases on Nef‐mediated NADPH oxidase activation. In addition, LY294002, a specific inhibitor of phosphoinositide 3‐kinase (PI3K) inhibited both the Nef‐induced p47^phox phosphorylation and the superoxide anion release. These data indicate that Nef regulates the NADPH oxidase activity through the activation of the Src kinases and PI3K. J. Cell. Biochem. 106: 812–822, 2009. © 2009 Wiley‐Liss, Inc.     

A rapid and simple staining method, using Toluidine Blue, for analysing mitotic figures in tissue sections

Summary Mitotic index is a clinically important parameter in cancer pathology. We developed a staining method using Toluidine Blue to detect efficiently and rapidly mitotic figures in sections of formalin-fixed paraffin-embedded human and rat tisues. Sections were stained at acid pH with a 0.01% Toluidine Blue solution after removal of RNA with hydrochloric acid or ribonuclease. The optimal pH of the TB staining solution was found to be 4.5 for rat tissues and 3.5 for human tissues. This procedure stained mitotic figures much more intensely than other (extra)cellular structures. A quantitative estimate of the total number of nuclei in the field where mitotic figures were counted, was obtained in an adjacent section hydrolysed in 5 N hydrochloric acid and stained by the Feulgen reaction with a Schiff-type reagent containing 0.01% Toluidine Blue. This method specifically stained interphase and mitotic nuclei and the field cellularity could be quantified by image cytometry. When these procedures were performed on two consecutive serial sections, a mitotic index could be determined accurately by relating the count of mitotic figures to the number of tumour cells.     

Production of polysaccharides by Arthrobacter globiformis associated with Anabaena azollae in Azolla leaf cavity

Abstract The composition of the capsular polysaccharides (CPS) and exopolysaccharides (EPS) of three strains of Arthrobacter globiformis , isolated from the leaf cavities of Azolla caroliniana (strain B1), A. filiculoides (strains A3 and L1) and A. globiformis ATCC 8010 have been analysed by HPLC and enzymatic assays. Glucose and galactose were detected in the EPS of all the strains, while rhamnose was present only in the EPS of the strain L1 and uronic acids in B1 and ATCC 8010. Traces of fructose were detected by enzymatic assays in all the strains. The CPS contained glucose, galactose and rhamnose, while uronic acids were present only in strain B1. In all the strains the amount of EPS was higher than CPS. The reactivity to different dyes and lectins of the mucilagineous matrix of the algal packets extracted from the fern and of the bacterial mucilage were similar.     

The rabbit liver microsomal biotransformation of 1,1-dialkylethylenes: Enantioface selection of epoxidation and enantioselectivity of epoxide hydrolysis

The rabbit liver microsomal biotransformation of α-methylstyrene ( 1a ), 2-methyl-1-hexene ( 1b ), 2,4,4-trimethyl-1-pentene ( 1c ), and 2,3,3-trimethyl-1-butene ( 1d ) has been investigated with the aim at establishing the enantioface selection of the cytochrome P-450-promoted epoxidation of the double bond and the enantioselectivity of microsomal epoxide hydrolase (mEH)-catalyzed hydrolysis of the resulting epoxides. GLC on a Chiraldex G-TA (ASTEC) column was used to determine the enantiomeric composition of the products. The epoxides 2 first produced in incubations carried out in the presence of an NADPH regenerating system were not detected, being rapidly hydrolyzed by mEH to diols 3 . The enantiomeric composition of the latter showed that no enantioface selection occurred in the epoxidation of 1c and 1d , and a very low (8%) ee of the (R)-epoxide was formed from 1b . Incubation of racemic epoxides 2b–d with the microsomal fraction showed that the mEH-catalyzed hydrolysis of 2c and 2d was practically nonenantioselective, while that of 2b exhibited a selectivity E = 4.9 favoring the hydrolysis of the (S)-enantiomer. A comparison of these results with those previously obtained for linear and branched chain alkyl monosubstituted oxiranes shows that the introduction of the second alkyl substituent suppresses the selectivity of the mEH reaction of the latter and reverses that of the former substrates. © 1994 Wiley-Liss, Inc.     

High Sensitivity of Glutamate Uptake to Extracellular Free Arachidonic Acid Levels in Rat Cortical Synaptosomes and Astrocytes

By using both synaptosomes and cultured astrocytes from rat cerebral cortex, we have investigated the inhibitory action of arachidonic acid on the high-affinity glutamate uptake systems, focusing on the possible physiological significance of this mechanism. Application of arachidonic acid (1-100 microM) to either preparation leads to fast (within 30 s) and largely reversible reduction in the uptake rate. When either melittin (0.2-1 microgram/ml), a phospholipase A2 activator, or thimerosal (50-200 microM), which inhibits fatty acid reacylation in phospholipids, is applied to astrocytes, both an enhancement in extracellular free arachidonate and a reduction in glutamate uptake are seen. The two effects display similar dose dependency and time course. In particular, 10% uptake inhibition correlates with 30% elevation in free arachidonate, whereas inhibition greater than or equal to 60% is paralleled by threefold stimulation of arachidonate release. In the presence of albumin (1-10 mg/ml), a free fatty acid-binding protein, inhibition by either melittin, thimerosal, or arachidonic acid is prevented and an enhancement of glutamate uptake above the control levels is observed. Our data show that neuronal and glial glutamate transport systems are highly sensitive to changes in extracellular free arachidonate levels and suggest that uptake inhibition may be a relevant mechanism in the action of arachidonic acid at glutamatergic synapses.