Interactive effects of food quality,temperature and rearing time on condition of juvenile black bream Acanthopagrus butcheri

A laboratory experiment was conducted to determine the interactive effects of temperature and diet on condition indices of juvenile black bream Acanthopagrus butcheri, reared for time periods ranging from 2 to 42 days. After fish were reared for varying periods, growth, morphometric (Fulton's K) and biochemical [RNA:DNA (R:D) ratios] indices were measured. Fulton's K responded primarily to temperature, with progressive decrease in condition over time for fish reared at high temperatures. In contrast, R:D ratios were primarily affected by diet composition, with the highest values observed for fish reared on fish‐based diets as opposed to vegetable‐based diets. Significant effects of rearing time were also observed for Fulton's K and R:D ratios, as were some interactive treatment effects. In addition, Fulton's K and R:D ratios were not significantly correlated, perhaps due to the different periods of time integrated by each index or their relative sensitivity to lipid and protein deposition. These results highlight the complex responses of these condition indices to environmental variables and nutritional status.     

Pentoxifylline reduces fibrin deposition and prolongs survival in neonatal hyperoxic lung injury.

Bronchopulmonary dysplasia is a leading cause of mortality and morbidity in preterm infants despite improved treatment modalities. Pentoxifylline, a phosphodiesterase inhibitor, inhibits multiple processes that lead to neonatal hyperoxic lung injury, including inflammation, coagulation, and edema. Using a preterm rat model, we investigated the effects of pentoxifylline on hyperoxia-induced lung injury and survival. Preterm rat pups were exposed to 100% oxygen and injected subcutaneously with 0.9% saline or 75 mg/kg pentoxifylline twice a day. On day 10, lung tissue was harvested for histology, fibrin deposition, and mRNA expression, and bronchoalveolar lavage fluid was collected for total protein concentration. Pentoxifylline treatment increased mean survival by 3 days (P = 0.0018) and reduced fibrin deposition by 66% (P < 0.001) in lung homogenates compared with untreated hyperoxia-exposed controls. Monocyte chemoattractant protein-1 expression in lung homogenates was decreased, but the expressions of TNF-alpha, IL-6, matrix metalloproteinase-12, tissue factor, and plasminogen activator inhibitor-1 were similar in both groups. Total protein concentration in bronchoalveolar lavage fluid was decreased by 33% (P = 0.029) in the pentoxifylline group. Pentoxifylline treatment attenuates alveolar fibrin deposition and prolongs survival in preterm rat pups with neonatal hyperoxic lung injury, probably by reducing capillary-alveolar protein leakage.     

Structural basis for lipoxygenase specificity. Conversion of the human leukocyte 5-lipoxygenase to a 15-lipoxygenating enzyme species by site-directed mutagenesis

Mammalian lipoxygenases constitute a heterogeneous family of lipid-peroxidizing enzymes, and the various isoforms are categorized with respect to their positional specificity of arachidonic acid oxygenation into 5-, 8-, 12-, and 15-lipoxygenases. Structural modeling suggested that the substrate binding pocket of the human 5-lipoxygenase is 20% bigger than that of the reticulocyte-type 15-lipoxygenase; thus, reduction of the active-site volume was suggested to convert a 5-lipoxygenase to a 15-lipoxygenating enzyme species. To test this "space-based" hypothesis of the positional specificity, the volume of the 5-lipoxygenase substrate binding pocket was reduced by introducing space-filling amino acids at critical positions, which have previously been identified as sequence determinants for the positional specificity of other lipoxygenase isoforms. We found that single point mutants of the recombinant human 5-lipoxygenase exhibited a similar specificity as the wild-type enzyme but double, triple, and quadruple mutations led to a gradual alteration of the positional specificity from 5S- via 8S- toward 15S-lipoxygenation. The quadruple mutant F359W/A424I/N425M/A603I exhibited a major 15S-lipoxygenase activity (85-95%), with (8S,5Z,9E,11Z,14Z)-8-hydroperoxyeicosa-5,9 ,11, 14-tetraenoic acid being a minor side product. These data indicate the principle possibility of interconverting 5- and 15-lipoxygenases by site-directed mutagenesis and appear to support the space-based hypothesis of positional specificity.     

Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value

The diagnosis of rheumatoid arthritis (RA) is primarily based on clinical symptoms, so it is often difficult to diagnose RA in very early stages of the disease. A disease-specific autoantibody that could be used as a serological marker would therefore be very useful. Most autoimmune diseases are characterized by a polyclonal B-cell response targeting multiple autoantigens. These immune responses are often not specific for a single disease. In this review, the most important autoantibody/autoantigen systems associated with RA are described and their utility as a diagnostic and prognostic tool, including their specificity, sensitivity and practical application, is discussed. We conclude that, at present, the antibody response directed to citrullinated antigens has the most valuable diagnostic and prognostic potential for RA.     

Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9

A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC-ESI-MS a potent tool for the routine characterization of SNPs in forensic science.     

Taurine in the marine hydrozoan Hydractinia echinata: stabilizer of the larval state?

Taurine (beta-aminoethane sulfonic acid) is present in high concentrations in tissue of planula larvae of the marine hydrozoan Hydractinia echinata. It has been proposed to function as a stabilizer of the larval state mainly because of the previous findings that larvae induced to undergo metamorphosis appeared to lose most of their taurine, and taurine added to the medium antagonizes metamorphosis. Release of taurine was assumed to be a necessary prerequisite for the onset of metamorphosis. The primary aim of the present study was to confirm this by determination of taurine release accompanying metamorphosis induction by inducers other than CsCl. However, a decrease of the larval tissue taurine content was not found, irrespective of schedule of treatment and the inducer applied. The cause for this difference from the preceding study could not be clarified. Taurine in the medium, even at low concentration, causes elevated tissue concentrations high enough to cause general adverse effects on cell physiology. In order to ascribe an alternative function to taurine in H. echinata variations of the free amino acid pool under osmotic stress were examined. The tissue concentration of beta-alanine strongly correlates with the salinity of the medium. Large amounts of gamma-aminobutyric acid (GABA) are present in animals adapted to high salinity. Taurine content appears not to depend on osmolarity of the medium. Nevertheless, taurine may constitute the foundation of the cellular organic osmolyte system of the H. echinata larva.     

The development of a transformation system for the dimorphic plant pathogen Holleya sinecauda based on Ashbya gossypii DNA elements

We have developed a transformation system for the dimorphic plant pathogenic fungus Holleya sinecauda based on an electroporation protocol used for the closely related filamentous fungus Ashbya gossypii. DNA-mediated transformation of the dominant selection marker kanMX generated H. sinecauda transformants that were resistant to the antibiotic drug G418/geneticin. Freely replicating plasmids could be established in H. sinecauda using an A. gossypii autonomously replicating sequence (ARS) element, whereas Saccharomyces cerevisiae ARS elements, which are functional in A. gossypii, were not functional in H. sinecauda. In addition, centromeric DNA of A. gossypii stabilized the maintenance of plasmids in H. sinecauda under non-selective conditions. We isolated a fragment of the HsLEU2 gene and used this locus for targeted integration of kanMX3, consisting of the kanMX gene flanked by direct repeats. This allowed the construction of a Hsleu2 strain which became G418 sensitive after direct repeat-induced marker excision. The Hsleu2 strain can be complemented by the ScLEU2 gene. Finally, we constructed high- and low-copy shuttle vectors for H. sinecauda.     

Zinc toxicity in various lung cell lines is mediated by glutathione and GSSG reductase activity

In a previous work, it was shown that in cells after a decrease of cellular glutathione content, toxic zinc effects, such as protein synthesis inhibition or GSSG (glutathione, oxidized form) increases, were enhanced. In this study, zinc toxicity was determined by detection of methionine incorporation as a parameter of protein synthesis and GSSG increase in various lung cell lines (A549, L2, 11Lu, 16Lu), dependent on enhanced GSSG reductase activities and changed glutathione contents. After pretreatment of cells with dl-buthionine-[R,S]-sulfoximine (BSO) for 72 h, cellular glutathione contents were decreased to 15–40% and GSSG reductase activity was increased to 120–135% in a concentration-dependent manner. In BSO pretreated cells, the IC₅₀ values of zinc for methionine incorporation inhibition were unchanged as compared to cells not pretreated. The GSSG increase in BSO pretreated cells by zinc was enhanced in L2, 11Lu, and 16Lu cells, whereas in A549 cells, the GSSG increase by zinc was enhanced only after pretreatment with the highest BSO concentration. Inhibition of GSSG reductase in alveolar epithelial cells was observed at lower zinc concentrations than needed for methionine incorporation inhibition, whereas in fibroblastlike cells, inhibition of GSSG reductase occurred at markedly higher zinc concentrations as compared to methionine incorporation inhibition. These results demonstrate that GSSG reductase is an important factor in cellular zinc susceptibility. We conclude that reduction of GSSG is reduced in zinc-exposed cells. Therefore, protection of GSH oxidation by various antioxidants as well as enhancement of GSH content are expected to be mechanisms of diminishing toxic cellular effects after exposure to zinc.     

Comparison of purple membrane from Halobacterium cutirubrum and Halobacterium halabium.

Direct comparison of purple membrane preparations from Halobacterium cutirubrum and Halobacterium halobium was carried out. Both preparations were found to be essentially identical with respect to their molecular weight, retinal content, lipid composition, fingerprinting of peptides from peptide digestion, electron micrographs and X-ray diffraction patterns, and behaviour as a light-activated proton pump. Thus, there would appear to be no species differences in the purple membranes from these two bacteria.