Chemical and physiological changes in filamentous fungi during autolysis. X. Changes in the concentration of polyol in autolysing cultures of several fungi

Botrytis cinerea, Colletotrichum gloeosporioides, Aspergillus niger andPenicillium griseofulvum were grown in flasks as stationary cultures in the ordinary Czapek-Dox medium at 24° C in the dark for long periods of time. At convenient intervals during autolysis samples of mycelium were harvested and its polyol content determined. The loss of mycelial mannitol during autolysis reached to a little more than 60 %, more than 70 %, to nearly 80 % and 90 % forC. gloeosporioides, P. griseofulvum, A. niger andB. cinerea, respectively. Seventy per cent of the arabitol contained inP. griseofulvum and 85 % of the arabitol inC. gloeosporioides disappeared during autolysis. Mannitol remained present in appreciable amounts in these four fungi throughout the whole period of autolysis described, whereas arabitol disappeared fromB. cinerea and fromA. niger during the process.

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Zusammenfassung Botrytis cinerea, Colletotrichum gloeosporioides, Aspergillus niger undPenicillium griseofulvum sind als stationäre Kulturen in Czapek-Dox Nährboden bei 24° C lange Zeit in der Dunkelheit gezüchtet worden. In geeigneten Zwischenzeiten sind während der Autolyse Myzeliumproben entnommen und ihr Polyol-Inhalt bestimmt. Der Verlust vom myzelialen Mannitol während der Autolyse erreichte mehr als 60 %, mehr als 70 %, beinahe 80 % und 90 % inC. gloeosporioides, P. griseofulvum, A. niger, undB. cinerea, beziehungsweise. Siebzig Prozent von Arabitol inP. griseofulvum und 85 % von Arabitol inC. gloeosporioides verschwanden während der Autolyse. Mannitol verblieb in beträchtlicher Menge in diesen vier Pilzen während der ganzen Periode der Autolyse, während Arabitol vonB. cinerea und vonA. niger in derselben Zeit verschwand.

     

Analysis and refinement of criteria for predicting the structure and relative orientations of transmembranal helical domains.

We are interested in modeling the membrane-spanning domain of the serotonin 5-HT1A G-protein coupled receptor. This superfamily of proteins is predicted to share the topology of the seven transmembrane helices of bacteriorhodopsin (BR), even though no significant sequence homology had been identified. We found significant homologies by allowing for helix shuffling corresponding to minimal exon shuffling during evolution. Consequently, our strategy for building the model for the 5-HT1A receptor has been to construct hypotheses concerning helix-helix interactions, their orientations, and arrangement in bundles surrounded by lipid, based on the 3.5 A resolution structure of BR. Inferences resulting from such models were tested against the 2.3 A resolution structure of the photosynthetic reaction center (PRC) from Rhodobacter Viridis. These comparisons led us to a reevaluation of current methods for the identification and topological orientation of membrane-embedded alpha-helices. We find that methods used currently in the construction of helical transmembrane domains could be misleading if used indiscriminately. These methods include the hydrophobicity profile, the hydrophobic moment, helix amphiphilicity, and charge neutralization. A refinement is proposed here, based on empirical observations, molecular modeling, and physicochemical considerations designed to overcome some of the shortcomings inherent in the use of the above mentioned methods. Here we present the analysis of two of the motifs identified in our study that led to the proposed refinements: the distribution of acidic and basic residues in the transmembranal domains, and the kink induced by a Pro residue in an alpha-helix.     

Marine Communities on Oil Platforms in Gabon,West Africa: High Biodiversity Oases in a Low Biodiversity Environment

The marine biodiversity of Gabon, West Africa has not been well studied and is largely unknown. Our examination of marine communities associated with oil platforms in Gabon is the first scientific investigation of these structures and highlights the unique ecosystems associated with them. A number of species previously unknown to Gabonese waters were recorded during our surveys on these platforms. Clear distinctions in benthic communities were observed between older, larger platforms in the north and newer platforms to the south or closer to shore. The former were dominated by a solitary cup coral, Tubastraea sp., whereas the latter were dominated by the barnacle Megabalanus tintinnabulum, but with more diverse benthic assemblages compared to the northerly platforms. Previous work documented the presence of limited zooxanthellated scleractinian corals on natural rocky substrate in Gabon but none were recorded on platforms. Total estimated fish biomass on these platforms exceeded one ton at some locations and was dominated by barracuda (Sphyraena spp.), jacks (Carangids), and rainbow runner (Elagatis bipinnulata). Thirty-four percent of fish species observed on these platforms are new records for Gabon and 6% are new to tropical West Africa. Fish assemblages closely associated with platforms had distinct amphi-Atlantic affinities and platforms likely extend the distribution of these species into coastal West Africa. At least one potential invasive species, the snowflake coral (Carijoa riisei), was observed on the platforms. Oil platforms may act as stepping stones, increasing regional biodiversity and production but they may also be vectors for invasive species. Gabon is a world leader in terrestrial conservation with a network of protected areas covering >10% of the country. Oil exploration and biodiversity conservation currently co-exist in terrestrial and freshwater ecosystems in Gabon. Efforts to increase marine protection in Gabon may benefit by including oil platforms in the marine protected area design process.     

Molecular Cloning and Expression of a Human Phosphodiesterase 4C

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5′ end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5′ splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the K_m for cAMP of the enzyme produced in COS cells was 0.6 μM compared to 2.6 μM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC₅₀ of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [³H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.     

Multiresidue determination of quinolone antibacterials in eggs of laying hens by liquid chromatography with fluorescence detection

An analytical method for the simultaneous determination of seven quinolones (ciprofloxacin, enrofloxacin, danofloxacin, difloxacin, flumequine, oxolinic acid and sarafloxacin) in egg samples of laying hens was developed. Their use is totally prohibited in animals from which eggs are produced for human consumption. Protein precipitation was achieved by addition of acetonitrile and ammonia, removal of acetonitrile with dichloromethane, the quinolones remaining in the basic aqueous extract. The aqueous extract was analysed by liquid chromatography with fluorescence detection (LC-FD). The mobile phase was composed of acetonitrile and 10 mM citrate buffer solution of pH 4.5, with an initial composition of acetonitrile-water (12:88, v/v) and using linear gradient elution. Norfloxacin was used as an internal standard. The limits of detection found were 4-12 ng g(-1). These values were lower than the maximum residue limits (MRLs) established by the European Union for these compounds in different tissues of eggs-producing animals.     

Effects of Magnesium Sulfate Administration During Hypoxia on CaM Kinase IV and Protein Tyrosine Kinase Activities in the Cerebral Cortex of Newborn Piglets

The present study tested the hypothesis that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in Ca²⁺/Calmodulin-dependent-kinase (CaM Kinase) IV and Protein Tyrosine Kinase (PTK ) activities. Animals were randomly divided into normoxic (Nx), hypoxic (Hx) and magnesium-pretreated hypoxic (Mg²⁺-Hx) groups. Cerebral hypoxia was confirmed biochemically by measuring ATP and phosphocreatine (PCr) levels. CaM Kinase IV and PTK activities were determined in Nx, Hx and Mg²⁺-Hx newborn piglets. There was a significant difference between CaM kinase IV activity (pmoles/mg protein/min) in Nx (270 ± 49), Mg²⁺-Hx (317 ± 82) and Hx (574 ± 41, P < 0.05 vs. Nx and Mg²⁺-Hx) groups. Similarly, there was a significant difference between Protein Tyrosine Kinase activity (pmoles/mg protein/h) in normoxic (378 ± 68), Mg²⁺-Hx (455 ± 67) and Hx (922 ± 66, P < 0.05 vs. Nx and Mg²⁺-Hx ) groups. We conclude that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in CaM Kinase IV and Protein Tyrosine Kinase activities. We propose that by blocking the NMDA receptor ion-channel mediated Ca²⁺-flux, magnesium sulfate administration inhibits the Ca²⁺/calmodulin-dependent activation of CaMKIV and prevents the generation of nitric oxide free radicals and the subsequent increase in PTK activity. As a result, phosphorylation of CREB and Bcl-2 family of proteins is prevented leading to prevention of programmed cell death.     

Linkage at 12q24 with systemic lupus erythematosus (SLE) is established and confirmed in Hispanic and European American families

Systemic lupus erythematosus (SLE) is a chronic, complex, and systemic human autoimmune disease, with both an environmental component and a heritable predisposition. Clinical studies, reinforced by epidemiology and genetics, show impressive variation in disease severity, expression, prevalence, and incidence by ethnicity and sex. To identify the novel SLE susceptibility loci, we performed a genomewide scan with 318 markers on 37 multiplex Hispanic families, using a nonparametric penetrance-independent affected-only allele-sharing method. Three chromosomal regions (12q24, 16p13, and 16q12-21) exceeded our predetermined threshold (Zlr>2.32; nominal P<.01) for further evaluation. Suspected linkages at 12q24, 16p13, and 16q12-21 were tested in an independent data set consisting of 92 European American (EA-1) and 55 African American (AA) families. The linkage at 12q24 was replicated in EA-1 (Zlr=3.06; P=.001) but not in AA (Zlr=0.37; P=.35). Although neither the 16p13 nor the 16q12-21 was confirmed in EA-1 or AA, the suggestive linkage (Zlr=3.06; P=.001) at 16q12-21 is sufficient to confirm the significant linkage, reported elsewhere, at this location. The evidence for linkage at 12q24 in the 129 combined (Hispanic and EA-1) families exceeded the threshold for genomewide significance (Zlr=4.39; P=5.7x10-6; nonparametric LOD=4.19). Parametric linkage analyses suggested a low-penetrance, dominant model (LOD=3.72). To confirm the linkage effect at 12q24, we performed linkage analysis in another set of 82 independent European American families (EA-2). The evidence for linkage was confirmed (Zlr=2.11; P=.017). Therefore, our results have detected, established, and confirmed the existence of a novel SLE susceptibility locus at 12q24 (designated "SLEB4") that may cause lupus, especially in Hispanic and European American families.     

Introgression of 1Dx5+1Dy10 into Tritordeum

The uses of hexaploid tritordeum as a crop for human consumption require improvement of its bread-making quality. For this purpose chromosome 1D of bread wheat with the Glu-D1 allele encoding for high-molecular-weight glutenin subunits Dx5+Dy10 was introgressed into tritordeum. Different primary tritordeums were crossed with wheats carrying subunits Dx5+Dy10. The hybrids were backcrossed to tritordeum and seeds for the next backcross (or selfing) were selected for the presence of chromosome 1D using SDS-PAGE. Forty two chromosome plants carrying subunits Dx5+Dy10 were obtained after two backcrosses and selfing. Chromosome characterization of these plants using fluorescence in situ hybridisation (FISH) proved that either chromosome substitution 1H(ch)/1D or 1A/1D had been obtained. A homozygous plant with a translocation of the entire 1DL arm to 1H(ch)S was also obtained. The complete chromosome substitution lines have better agronomic characteristics than the lines with translocations.