Systematics within Gyps vultures: a clade at risk

Background  

Populations of the Oriental White-backed Vulture (Gyps bengalensis) have declined by over 95% within the past decade. This decline is largely due to incidental consumption of the non-steroidal anti-inflammatory veterinary pharmaceutical diclofenac, commonly used to treat domestic livestock. The conservation status of other Gyps vultures in southern Asia is also of immediate concern, given the lack of knowledge regarding status of their populations and the continuing existence of taxonomic uncertainties. In this study, we assess phylogenetic relationships for all recognized species and the majority of subspecies within the genus Gyps. The continuing veterinary use of diclofenac is an unknown but potential risk to related species with similar feeding habits to Gyps bengalensis. Therefore, an accurate assessment of the phylogenetic relationships among Gyps vultures should aid in their conservation by clarifying taxonomic uncertainties, and enabling inference of their respective relatedness to susceptible G. bengalensis.     

Subtype-specific increase in G-protein alpha-subunit mRNA by interleukin 1 beta

The guanine nucleotide regulatory proteins (G-proteins) which are substrates for ADP-ribosylation by pertussis toxin (alpha i-1, alpha i-2, alpha i-3 and alpha o) transduce a variety of hormonal signals. Endothelial cells express mRNA for three alpha i subtypes although the level of alpha i-1 mRNA is very low. Interleukin 1 beta (IL 1 beta), a pleiotropic inflammatory mediator which stimulates a complex series of responses in human endothelial cells leading to increased coagulation and platelet adhesion, increases expression of one subtype of alpha i (alpha i-2) mRNA in human endothelial cells as determined by Northern blot analysis without affecting the level of mRNA for other alpha-subunits. These studies show that mRNA levels for alpha i subtypes are independently regulated, suggesting that there may be subtype specificity in the cell's requirements for the Gi class of signal-transducing proteins.     

13C NMR spectroscopy of Methanobacterium thermoautotrophicum. Carbon fluxes and primary metabolic pathways

The flux of 13C-labeled carbons from the soluble metabolite 2,3-cyclopyrophosphoglycerate (CPP), a novel compound found in high concentrations exclusively in methanobacteria and methanobrevibacter, into carbohydrate-containing material has been deduced by solid-state 13C NMR spectroscopy which strongly argues for a role in gluconeogenesis for this unique metabolite. The turnover rates, but not the steady-state levels, of CPP labeled by 13CO2 or [13C]acetate depend dramatically on cell growth conditions. When the demand for carbohydrate synthesis is reduced (i.e. in stationary phase), the rates of CPP biosynthesis and degradation decrease 10-fold, and the disaccharide alpha, alpha-trehalose accumulates. Valinomycin, a metabolic inhibitor of Methanobacterium thermoautotrophicum growth, does not affect steady-state levels of CPP, but does decrease 13C uptake into the CPP pool. The effects of these different conditions on CPP labeling suggest stringent regulation of CPP linked to cellular metabolism. Labeling of CPP by [6-(13)C]glucose, which does not serve as an energy or carbon source for this organism, provides strong evidence that glucose is cleaved by the reverse of the gluconeogenesis pathway. This metabolic pathway linking glucose with triose phosphate type precursors and an analysis of the 13C NMR spectrum of CPP labeled by incubating cells with [U-13C]glucose have established that in vivo phosphoenolpyruvate synthetase must be reversible.     

2'-Nor-cGMP: a seco-cyclic nucleotide with powerful anti-DNA-viral activity

As part of our study of antiherpetic acyclonucleosides, we synthesized a cyclic GMP analog, 9-[(2-hydroxy-1,3,2-dioxaphosphorinan-5-yl)oxymethyl]guanine P-oxide, sodium salt (2'-nor-cGMP), and discovered its potent and broad spectrum anti-DNA-viral activities. 2'-Nor-cGMP inhibits the replication of many DNA viruses, including herpes simplex virus, human cytomegalovirus, vaccinia, SV40, and adenovirus, but does not inhibit RNA viruses. In plaque reduction studies this potent antiviral agent is also approximately 10-fold more potent than 9-(1,3-dihydroxy-2-propoxymethyl)guanine (2'NDG) against varicella-zoster virus and inhibits cell transformation by bovine papilloma virus. Unlike 2'NDG, the potent activity of 2'-nor-cGMP against herpes virus is not dependent upon the action of virus-specified thymidine kinase. Intercellular metabolism of 2'-nor-cGMP produced small amounts of 2'NDG triphosphate which were insufficient to account for the antiviral activity observed, implying that this potent anti-DNA-viral agent operates by a mechanism different from that of known acyclonucleosides.     

Patterns of divergence during evolution of alpha 1-proteinase inhibitors in mammals

alpha 1-Proteinase inhibitor (alpha 1-PI), a member of the serine proteinase inhibitor superfamily, has a primary role in controlling neutrophil elastase activity within the mammalian circulation. Several studies have indicated that the reactive center region of alpha 1-PI, the amino acid sequence of which is critical to recognition of and binding to target proteinases, is highly divergent within and among species. This appears to be a consequence of accelerated rates of evolution that may have been driven by positive Darwinian selection. In order to examine this and other features of alpha 1-PI evolution in more detail, we have isolated and sequenced cDNAs representing alpha 1- PI mRNAs of the mouse species Mus saxicola and Mus minutoides and have compared these with a number of other mammalian alpha 1-PI mRNAs. Relative to other mammalian mRNAs, the extent of nonsynonymous substitution is generally high throughout the alpha 1-PI mRNA molecule, indicating greater overall rates of amino acid substitution. Within and among mouse species, the 5'-half of the mRNA, but not the 3'-half, has been homogenized by concerted evolution. Finally, the reactive center is under diversifying or positive Darwinian selection in murid rodents (rats, mice) and guinea pigs yet is under purifying selection in primates and artiodactyls. The significance of these findings to alpha 1-PI function and the possible selective forces driving evolution of serpins in general are discussed.      

Mitochondrial DNA polymorphisms in subterranean mole-rats of the Spalax ehrenbergi superspecies in Israel, and its peripheral isolates

Patterns of mitochondrial DNA (mtDNA) variation were examined in 133 mole-rats constituting all four chromosomal species (2n = 52, 2n = 54, 2n = 58, and 2n = 60) of the Spalax ehrenbergi superspecies in Israel, as well as the peripheral isolates of 2n = 60. In the main range of the complex, a total of 28 mtDNA haplotypes were found in 64 mole-rats, with most haplotypes being unique to either a single chromosomal species or population. mtDNA divergence increased from low to high diploid number in a north-to-south direction in Israel. Overall levels of mtDNA diversity were unexpectedly the highest in the 2n = 60, the youngest species of the complex. The mtDNA haplotypes can be separated into two major groups, 2n = 52-54 and 2n = 58-60, and a phylogenetic analysis for each group revealed evidence of a few haplotypes not sorted by diploid number. The overall patterns of mtDNA divergence seen within and among the four chromosomal species are consistent with the parapatric mode of speciation as suggested from previous studies of allozyme and DNA hybridization. In a separate data set the patterns of mtDNA variation were examined across the main geographic range and across peripheral semi-isolates and isolates of the 2n = 60 chromosomal species. Fifteen haplotypes were found in 69 mole-rats. High levels of mtDNA diversity characterized the main range, semi-isolated, and even some desert isolated populations. The peripheral isolates contain much mtDNA diversity, including novel haplotypes.      

Growth hormone stimulation of amino acid transport and utilization by the perfused rat liver.

The effects of growth hormone, administered in vivo or added in vitro, on amino acid transport and utilization have been studied in perfused livers of normal and hypophysectomized rats. A perfusion system employing a nonrecirculating medium was used in all of the studies. Two nonmetalbolizable amino acid analogues, alpha-aminoisobutyric acid (AIB) and 1-aminocyclopentane carboxylic acid (cycloleucine) were used to study transport. Accumulation of AIB increased linearly over a 60-min perfusion period, reaching distribution ratios of between 1 and 2 for both groups of animals. Treatment of both normal and hypophysectomized rats with growth hormone 60 min prior to the start of perfusion increased AIB distribution ratios by up to 84 and 108%, respectively. Accumulation of cycloleucine was linear for only about 20 min of perfusion and then plateaued. Steady state distribution ratios of this analogue ranged between 1 and 2 for both groups of animals. Growth hormone treatment had no apparent effect on the time necessary to reach these steady state levels, but significantly increased them in livers of both normal and hypophysectomized rats by 16 and 42%, respectively. Studies designed to analyze the kinetic properties of these hormone effects revealed that growth hormone treatment caused 2-fold i-crease in the maximum velocities of both the AIB and cycloleucine transport systems. The substrate concentration for half-maximal transport velocity was increased slightly for both systems by growth hormone. Direct effects of growth hormone were demonstrated in studies where livers of hypophysectomized rats were perfused under conditions simulationg those of experiments in which the hormone was administered in vivo. Following an initial 45-min period of perfusion the medium during the 20 min. Growth hormone added to the medium during the entire 65-min perfusion at a concentration of 1 mug per ml caused a 30% increase in the cycloleucine distribution ratio. Under similar experimental conditions growth hormone directly stimulated three hepatic pathways of amino acid utilization: (a) incorporation of [14C]valine into protein, (b) urea formation and (c) conversion of 14-C-amino-acids to labeled glucose. Intracellular concentrations of seven amino acids, including threonine, serine, proline, glycine, alanine, lysine, and arginine, were increased significantly in livers perfused with medium containing growth hormone...