Cardiac mitochondrial NADP+-isocitrate dehydrogenase is inactivated through 4-hydroxynonenal adduct formation: an event that precedes hypertrophy development

Mitochondrial NADP+-isocitrate dehydrogenase activity is crucial for cardiomyocyte energy and redox status, but much remains to be learned about its role and regulation. We obtained data in spontaneously hypertensive rat hearts that indicated a partial inactivation of this enzyme before hypertrophy development. We tested the hypothesis that cardiac mitochondrial NADP+-isocitrate dehydrogenase is a target for modification by the lipid peroxidation product 4-hydroxynonenal, an aldehyde that reacts readily with protein sulfhydryl and amino groups. This hypothesis is supported by the following in vitro and in vivo evidence. In isolated rat heart mitochondria, enzyme inactivation occurred within a few minutes upon incubation with 4-hydroxynonenal and was paralleled by 4-hydroxynonenal/NADP+-isocitrate dehydrogenase adduct formation. Enzyme inactivation was prevented by the addition of its substrate isocitrate or a thiol, cysteine or glutathione, suggesting that 4-hydroxynonenal binds to a cysteine residue near the substrate's binding site. Using an immunoprecipitation approach, we demonstrated the formation of 4-hydroxynonenal/NADP+-isocitrate dehydrogenase adducts in the heart and their increased level (210%) in 7-week-old spontaneously hypertensive rats compared with control Wistar Kyoto rats. To the best of our knowledge, this is the first study to demonstrate that mitochondrial NADP+-isocitrate dehydrogenase is a target for inactivation by 4-hydroxynonenal binding. Furthermore, the pathophysiological significance of our finding is supported by in vivo evidence. Taken altogether, our results have implications that extend beyond mitochondrial NADP+-isocitrate dehydrogenase. Indeed, they emphasize the implication of post-translational modifications of mitochondrial metabolic enzymes by 4-hydroxynonenal in the early oxidative stress-related pathophysiological events linked to cardiac hypertrophy development.     

LIPOPHILIC PIGMENTS FROM CYANOBACTERIAL (BLUE-GREEN ALGAL) AND DIATOM MATS IN HAMELIN POOL,SHARK BAY,WESTERN AUSTRALIA1

Lipophilic pigments were examined in microbial mat communities dominated by cyanobacteria in the intertidal zone and by diatoms in the subtidal and sublittoral zones of Hamelin Pool, Shark Bay, Western Australia. These microbial mats have evolutionary significance because of their similarity to lithified stromatolites from the Proterozoic and Early Paleozoic eras. Fucoxanthin, diatoxanthin, diadinoxanthin, β-carotene, and chlorophylls a and c characterized the diatom mats, whereas cyanobacterial mats contained myxoxanthophyll zeaxanthin, echinenone, β-carotene, chlorophyll a and, in some cases, sheath pigment. The presence of bacteriochlorophyll a with in the mats suggest a close association of photosynthetic bacteria with diatoms and cyanobacteria. The high carotenoids: chlorophyll a ratios (0.84–2.44 wt/wt) in the diatom mats suggest that carotenoids served a photoprotective function in this high light environment. By contrast, cyanobacterial sheath pigment may have largely supplanted the photoprotective role of carotenoids in the intertidal mats.     

Heparin and its derivatives bind to HIV-1 recombinant envelope glycoproteins, rather than to recombinant HIV-1 receptor, CD4

We have employed a direct radiolabel binding assay to investigate the interaction between3H-heparin and recombinant envelope glycoproteins, rgp120s, derived from several different isolates of HIV-1. Comparable dose-dependent binding is exhibited by rgp120s from isolates IIIB, GB8, MN and SF-2. Under identical experimental conditions the binding of3H- heparin to a recombinant soluble form of the cellular receptor for gp120, CD4, is negligible. The binding of3H-heparin to rgp120 is competed for by excess unlabeled heparin and certain other, but not all, glycosaminoglycan and chemically modified heparins. Of a range of such polysaccharides tested, ability to compete with3H-heparin for binding was strictly correlated with inhibition of HIV-1 replication in vitro. Those possessing potent anti-HIV-1 activity were effective competitors, whereas those having no or little anti-HIV-1 activity were poor competitors. Scatchard analysis indicates that the K d of the interaction between heparin and rgp120 is 10 nM. Binding studies conducted in increasing salt concentrations confirm that the interaction is ionic in nature. Synthetic 33-35 amino acid peptides based on the sequence of the V3 loop of gp120 also bind to heparin with high affinity. V3 loop peptides that are cyclized due to terminal cysteine residues show more selective binding than their uncyclized counterparts. Overall, these data demonstrate further that heparin exerts its anti-HIV-1 activity by binding to the envelope glycoprotein of HIV-1, rather than its cellular receptor, CD4. This study confirms that the V3 loop of gp120 is the site at which heparin exerts its anti- HIV-1 activity. Moreover, it reveals that high affinity binding to heparin is shared by all four rgp120s examined, despite amino acid substitutions within the V3 loop.      

Quantitation of 1,3-butanediol and its acidic metabolites by gas chromatography-mass spectrometry

A number of problems present themselves during the gas chromatographic-mass spectrometric assay of R,S-1,3-butanediol as its bis-tert-butyldimethylsilyl ether. To circumvent these problems, three labeled internal standards were synthesized: (i) R,S-1,3-[3,4-13C2]-butanediol, (ii) R,S-1,3-[1,1,3-2H3]butanediol, and (iii) R,S-1,3-[1,1,3-2H3,3,4-13C2]butanediol. The availability of internal standards with different degrees of labeling allows (i) assaying of either unlabeled or 13C-labeled R,S-1,3-butanediol and (ii) analysis of 1,3-butanediol in either blood or urine samples. Reproducible standard curves were obtained using both electron impact and ammonia chemical ionization modes. The latter provides greater sensitivity and a lower limit of detection (5 microM). We have also designed an indirect assay of S-3-hydroxybutyrate, a catabolite of R,S-1,3-butanediol, which is difficult to analyze by conventional methods. This assay relies on the difference between (i) the concentration of R,S-3-hydroxybutyrate assayed by gas chromatography-mass spectrometry and (ii) the concentration of R-3-hydroxybutyrate assayed enzymatically.     

CFTR transgene expression in primary DeltaF508 epithelial cell cultures from human nasal polyps following gene transfer with cationic phosphonolipids

Cystic fibrosis (CF) is the most common autosomal lethal recessive disorder in the Caucasian population. The major cause of mortality is lung disease, owing to the failure of a functional protein from the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Today, even though the knowledge about the CFTR genomic is extensive, no efficient treatment has been developed yet. In this context, gene therapy represents a potential important advance on condition that it could develop efficient and safe transfection agents. Even though viral vectors have been used in most clinical trials owing to their high transfection efficiency, random integration and immunogenicity are still critical side effects. Consequently, all of these drawbacks brought forth the development of nonviral transfection systems. Although they engender few toxicity and immunogenicity problems, their low transfection efficiency is a hurdle that must be overcome. Over the past decade, we have developed an original family of monocationic lipids, cationic phosphonolipids, whose efficiency has been previously demonstrated both in vitro and in vivo. In this report, we observe that a new cationic phosphonolipid (KLN 30) can lead to the restoration of the CFTR protein following the ex vivo transfection of epithelial cells issuing from a F508 homozygous patient. The transgene expression and the cytotoxicity correlate with the charge ratio of the lipoplex. A kinetic study was performed, and a luminescent signal was detected until 35 d after transfection.     

The narrow sheath duplicate genes: sectors of dual aneuploidy reveal ancestrally conserved gene functions during maize leaf development

The narrow sheath mutant of maize displays a leaf and plant stature phenotype controlled by the duplicate factor mutations narrow sheath1 and narrow sheath2. Mutant leaves fail to develop a lateral domain that includes the leaf margins. Genetic data are presented to show that the narrow sheath mutations map to duplicated chromosomal regions, reflecting an ancestral duplication of the maize genome. Genetic and cytogenetic evidence indicates that the original mutation at narrow sheath2 is associated with a chromosomal inversion on the long arm of chromosome 4. Meristematic sectors of dual aneuploidy were generated, producing plants genetically mosaic for NARROW SHEATH function. These mosaic plants exhibited characteristic half-plant phenotypes, in which leaves from one side of the plant were of nonmutant morphology and leaves from the opposite side were of narrow sheath mutant phenotype. The data suggest that the narrow sheath duplicate genes may perform ancestrally conserved, redundant functions in the development of a lateral domain in the maize leaf.     

The biology and ecology of lotic microcrustaceans

• 1 Copepoda, Ostracoda and ‘Cladocera’ are important meiobenthic Crustacea which can be both numerically abundant and species rich in running waters. Harpacticoids and ostracods are well adapted to benthic life because they are typical crawlers, walkers, and burrowers. Many cladocerans are substratum dwellers, but most benthic species among these can also swim. Cyclopoids which are generally good swimmers are nevertheless often bottom frequenters and actively colonise sediment interstices (the hyporheic zone).
  • 2 The subclass Copepoda includes 10 orders. With 53 families, the order Harpacticoida dominates the benthos. Only five of these families are represented in fresh waters (ca. 1 000 species and subspecies). The order Cyclopoida includes 12 families of which the Cyclopidae is well represented in freshwater habitats with 900 species and subspecies. Freshwater Ostracods belong to the order Podocopida (5 000 species) with three superfamilies occurring in running fresh waters. The group ‘Cladocera’ contains four orders, 12 families, more than 80 genera, and 450–600 freshwater species. Most of the benthic species are found in the families Chydoridae (39 genera), Macrothricidae, Ilyocryptidae and Sididae.
• 3 For each of the three major taxa, morphological characteristics are presented, specimen collection and preparation are described and references to available taxonomical keys are provided.
• 4 Biological characteristics are extremely diverse among and within the three taxa, resulting in a great variety of strategies in meiobenthic crustaceans. Characteristics of reproduction, sexual dimorphism, cyclomorphosis and population parameters (i.e. clutch size, lifespan, growth, moulting) are provided for some of the most common species.
• 5 Important differences between the three main taxa were found at the species level. Ecological requirements such as hydraulic microhabitats and geomorphologic features of the streambed are the major determinants of species diversity and abundance for benthic microcrustacea of lotic habitats. Many studies on the ecology of these communities are limited by a lack of knowledge of the life history characterisitics of lotic (especially interstitial) crustacean populations.

     

Mathematical simulation of the diel O, S, and C biogeochemistry of a hypersaline microbial mat

The creation of a mathematical simulation model of photosynthetic microbial mats is important to our understanding of key biogeochemical cycles that may have altered the atmospheres and lithospheres of early Earth. A model is presented here as a tool to integrate empirical results from research on hypersaline mats from Baja California Sur (BCS), Mexico into a computational system that can be used to simulate biospheric inputs of trace gases to the atmosphere. The first version of our model, presented here, calculates fluxes and cycling of O(2), sulfide, and dissolved inorganic carbon (DIC) via abiotic components and via four major microbial guilds: cyanobacteria (CYA), sulfate reducing bacteria (SRB), purple sulfur bacteria (PSB) and colorless sulfur bacteria (CSB). We used generalized Monod-type equations that incorporate substrate and energy limits upon maximum rates of metabolic processes such as photosynthesis and sulfate reduction. We ran a simulation using temperature and irradiance inputs from data collected from a microbial mat in Guerrero Negro in BCS (Mexico). Model O(2), sulfide, and DIC concentration profiles and fluxes compared well with data collected in the field mats. There were some model-predicted features of biogeochemical cycling not observed in our actual measurements. For instance, large influxes and effluxes of DIC across the MBGC mat boundary may reveal previously unrecognized, but real, in situ limits on rates of biogeochemical processes. Some of the short-term variation in field-collected mat O(2) was not predicted by MBGC. This suggests a need both for more model sensitivity to small environmental fluctuations for the incorporation of a photorespiration function into the model.