Hypoxia Increases the Susceptibility to Oxidant Stress and the Permeability of the Blood-Brain Barrier Endothelial Cell Monolayer

Abstract: Using a cell culture model of the blood-brain barrier (BBB), we investigated the brain capillary endothelial cell (EC) response to hypoxia. The activities of antioxidant enzymes such as glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase and the GSH level of brain capillary ECs alone or in coculture with astrocytes, as well as those of pericytes, were compared with those obtained with freshly isolated microvessels. These results demonstrated that brain capillary ECs cocultured with astrocytes and used in the presence of a coculture-conditioned medium provided a relevant in vitro model for studying the effect of hypoxia-reoxygenation at the BBB level. The effect of hypoxia on antioxidant enzymes, GSH, and ATP levels was studied, as well as the modification of the permeability to small weight molecules. A decrease in all enzymes and the GSH level could explain an increase in the susceptibility of the brain capillary ECs to further oxidant injury. Second, profound rearrangements of F-actin filaments of the ECs and a decrease in the ATP level could be associated with an increase in the permeability of the monolayer. Furthermore, an apoptotic process was detected by in situ end labeling of DNA. These results indicate that hypoxia distorts the function of ECs and that these cells in culture provide a valuable tool for exploring mechanisms after hypoxia-reoxygenation.     

Precise physical mapping of theEscherichia coli rnb gene, encoding ribonuclease II

Ribonuclease II (encoded byrnb) is one of the two main exonucleases involved in mRNA degradation inEscherichia coli. We report the precise physical mapping ofrnb to 29 min on the chromosomal map in the vicinity ofpyrF, and clarify the genetic and physical maps of thisE. coli chromosomal region. The results were confirmed by the construction of a strain partially deleted forrnb.     

Further farnesyl-homogentisic acid derivatives from Otoba parvifolia

The seeds of Otoba parvifolia contain three novel compounds apparently derived from homogentisic acid, rel-(1′R,5′R)-2-(1′-farnesyl-5′-hydroxy-2′-oxocyclohex-3′-en-1′-yl)-acetic acid and its acetate as well as rel-(1′R,4′S,5′R)-2-(1′-farnesyl-4′,5′-dihydroxy-2′-oxocyclohexan-1′-yl)-acetic acid δ-lactone. The structure of an additional isolate, previously described as 2-(1′-farnesyl-2′-hydroxy-5′-oxocyclohex-3′-en-1′-yl)-acetic acid γ-lactone was revised to rel-(1′R,5′R)-2-(1′-farnesyl-5′-hydroxy-2′-oxocyclohex-3′-en-1′-yl)-acetic acid δ-lactone.     

Dihydrochalcones, coumarins and alkaloids from Metrodorea nigra

Two novel dihydrochalcones, 2′,3,4′,6′-tetrahydroxy-4-methoxy-3′,5-di-(3,3-dimethylallyl)-dihydrochalcone and 2′,.3,6′-trihydroxy-4-methoxy-5-(3,3-dimethylallyl)-3′,4′-(2″,2″-dimethyldihydropyran)-dihydrochalcone, have been isolated from fresh fruits of Metrodorea nigra. Stems and leaves showed a similar composition and we have isolated common steroids, simple coumarins, several furocoumarins, furoquinoline alkaloids and a furofuran lignan. From stems, we have also isolated the pentacyclic 6-C-monoterpenyl-5,7-dioxycoumarin, deoxybruceol. Structures of the isolated compounds were elucidated on the basis of spectral data.     

Polymorphism in the Ostertagiinae Lopez-Neyra, 1947 and comments on the systematics of these nematodes

A list of 19 polymorphic species of the generaOstertagia, Orloffia, Teladorsagia, Marshallagia andSpiculopteragia is presented. The morphs of the polymorphic species in question have identical females and show similarities in some male features, e.g. the structure of the oesophagus, the synlophe and the shape of the rays of the copulatory bursa. However, they differ by characters which are recognised as generic features, e.g. the structure of spicules, gulbernaculum and genital cone. The presence or lack of minor morphs is a generic feature in the Ostertagiinae. Each polymorphic genus has a defined type of minor morph.     

Ionic iridium-gold and rhodium-gold perfluorobenzenethiolato binuclear complexes: syntheses and solution behavior

By reacting [(C₅Me₅)M(SR_F)₂] (forM = Ir, Rf = C₆F₅ (1a) or C₆F₄H-p (1b); for M = Rh, Rf = C₆F₅ (2a) or C₆F₄H-p (2a)) in toluene with Na[AuCl₄], ionic binuclear compounds with the general formula [(C₅Me₅)M(μ-SR_F)₂AuCl₂]Cl for M = Ir, R = C₆F₅ (3a) or C₆F₄H-p (3a); for M = Rh, R_F = C₆F₅ (4a) or C₆F₄H-p (4b) can be obtained, together with small amounts of [(C₅Me₅)₂Rh₂(μ-SR_F)(μ-Cl)₂]Cl (R_F = C₆F₅ (5a) or C₆F₄H-p (5b)) as by-products when 2a and 2b were used.     

Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. (c) 1995 John Wiley & Sons, Inc.     

Spatio-temporal distribution of the microphytobenthic biomass in intertidal flats of Tagus Estuary (Portugal)

A study on spatio-temporal distribution of microphytobhethos in intertidal zones of Tagus Estuary was carried out from 1990 to 1992. Near Lisbon, Portugal, Tagus Estuary is a shallow mesotidal estuary, covering an area of 320 km². The intertidal area ranges from 20 to 40% off the total area and it is constituted mainly by mudflats. Intertidal flats are richly populated by microalgae, diatoms being the most important and ubiquitos group.Spatial variation of microphytobethos was studied in spring 1990, 21 different sites were sampled. Microphytobenthos biomass was evaluated as chlorophyll a content of the surface centimeter, ranging from 10 to 240 mg m^–2. A Principal Component Analysis showed that 62% of the total variability found in intertidal flats of Tagus estuary could be attributed to two major factors: sediment type and tidal height. A hierarchical grouping defined 3 major groups of similar stations, each one representing a different strata of the ecosystem.One station from each group was chosen for the study of the temporal variation. A sampling, rogram took place from April 1991 to April 1992, with fortnightly sampling, the Chl a ranged from 20–300 mg m^–2. No clear seasonal variation was found, and our results indicated that tidal height of sampledsite played an essential role in temporal biomass evolution, thus upper littoral sites were influenced by climatic parameters, whereas in lower sites action of tides mainly controlled microphytic biomass.

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Résumé Une étude sur l'hétérogénéité spatio-temporelle du microphytobenthos dans les sédiments intertidaux de l'Estuaire du Tage a été accompli de 1990 á 1992.L'Estuaire du Tage, prés de Lisbonne (Portugal) est un estuaire peu profond, mesotidal, avec une aire total de 320 km². L'aire intertidale est comprise entre 20 et 40% du total, et constituéé surtout par des vasiéres. Ces slikkes sont peuplées par une communauté assez riche de microalgues, ou les diatomées sont les plus abundantes.La variation spatialle du microphytobenthos était évalué au Printemps 1990, ou 21 différentes stations étaient échantillonnées. La biomasse était évalué par la concentration enchlorophylle a du premier centimétre de sédiment, qui a varié de 10 á 240 mg Chl a m^–2. Une Analyse en Composants Principales a montré que 62% de la variabilité de la biomasse était lié á deux facteurs: le sédiment et l'hauteur vis-á-vis la marée. Une classification hiérarchique des stations par similitude a établi 3 groupes principaux, représantantles différents strates de écecosytéme.Une station de chaque groupement a été choisie pour l'étude de la variation temporelle, qui s'est deroulé d'avril 1991 á avril 1992, avec des prélévements deux fois par mois. Les valeurs de Chl a obtenus vont de 20 á 300 mg m^–2. Les variations saisonniéres observées ne sont pas claires: nos résultats indiquent que l'hauteur de la station (m) joue un rôle essentiel dans l'évolution temporel de la biomasse, c'est á dire, la biomasse microalgal des sites du supra-littoral est influencié par les paramétres climatiques, tandis que dans l'infra-littoral c'est l'action des marées le facteur principal.

     

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

S-2-(3 aminopropylamino) ethylphosphorothioic acid (WR-2721) shown to surpass radical scavenging thiols in their radioprotective efficacy in cancer-type diseases has been tested for its protective potential in the reperfused heart. We investigated the radical scavenger properties of the compound in a radical generating systemin vitro as well as in isolated rat hearts subjected to 30 min ischaemia and 30 min reperfusion with the electron-paramagnetic resonance spin trap technique. The action on high-energy phosphates is observed by means of phosphorus-31 nuclear magnetic resonance (NMR) spectroscopy while its influence on left ventricular systolic segmental length change (SSLC) during 60 min reperfusion following 60 min regional ischaemia was assessed with a fibreoptic system in anaesthetized open-chest rats. WR-2721 (0.1 mM) reduced the vascular concentration of radical adduct in isolated hearts by up to 78% (275±84% of pre-ischaemic baseline values vs 1260±413%, p<0.05) between 5 and 12.5 min reperfusion. This was accompanied by a reduction of the left ventricular end diastolic pressure to pre-ischaemic values at 30 min of reperfusion (9±6 mmHg vs 42±8 mmHg in the absence of WR-2721, p<0.02). An accelerated recovery of creatine phosphate levels (78±5% of pre-ischaemia values vs 41±5% within 60 min reperfusion; p<0.05) was observed under similar conditions with NMR-spectroscopy, although the post-ischaemic tissue content of adenosine triphosphate was not affected. The administration of WR-2721 (0.5 mmol/kg body weight) ledin situ to an accelerated restoration of contractile activity in the post-ligated left ventricular area reflected by the post-ischaemic recovery of SSLC (64±10% of pre-ischaemic values compared with 27±6% in control animals 60 min following reperfusion; p<0.02). The present data confirm an effective reduction in the exposure of the reperfused heart to endogenously released free radicals by WR-2721, a partial preservation of high-energy phosphates and an improvement in post-ischaemic contractility and encourage further investigation of such favourable action in injured myocardium.