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.     

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.      

Express Your LOV: An Engineered Flavoprotein as a Reporter for Protein Expression and Purification

In this work, we describe the utility of Light, Oxygen, or Voltage-sensing (LOV) flavoprotein domains from plant phototropins as a reporter for protein expression and function. Specifically, we used iLOV, an enhanced and more photostable variant of LOV. A pET-based plasmid for protein expression was constructed, encoding a C terminal iLOV-octahistidine (His8)-tag and a HRV 3C protease cleavage recognition site. Ten different proteins, with various sub-cellular locations, were cloned into the plasmid, creating iLOV-His8 tag fusions. To test protein expression and how iLOV could be used as a reporter, the proteins were expressed in three different cell lines, in four different culture media, at two different temperatures. To establish whether the presence of the iLOV tag could have an impact on the functionality, one of the proteins, EspG, was over-expressed and purified. EspG is an “effector” protein normally produced by enterohemorrhagic E. coli strains and “injected” into host cells via the T3SS. We tested functionality of EspG-iLOV fusion by performing functional studies of EspG in mammalian host cells. When EspG-iLOV was microinjected into the host cell, the Golgi apparatus was completely disrupted as had previously been observed for EspG.     

Mechanistic studies on thiamin phosphate synthase: evidence for a dissociative mechanism

Thiamin phosphate synthase catalyzes the coupling of 4-methyl-5-(beta-hydroxyethyl)thiazole phosphate (Thz-P) and 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate (HMP-PP) to give thiamin phosphate. In this paper, we demonstrate that 4-amino-5-(hydroxymethyl)-2-(trifluoromethyl)pyrimidine pyrophosphate (CF(3)-HMP-PP) is a very poor substrate [k(cat)(CH(3)) > 7800k(cat)(CF(3))] and that 4-amino-5-(hydroxymethyl)-2-methoxypyrimidine pyrophosphate (CH(3)O-HMP-PP) is a good substrate [k(cat)(OCH(3)) > 2.8k(cat)(CH(3))] for the enzyme. We also demonstrate that the enzyme catalyzes positional isotope exchange. These observations are consistent with a dissociative mechanism (S(N)1 like) for thiamin phosphate synthase in which the pyrimidine pyrophosphate dissociates to give a reactive pyrimidine intermediate which is then trapped by the thiazole moiety.     

Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase

Thiamin phosphate synthase catalyzes the formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxyethyl)-4-methylthiazole phosphate. Several lines of evidence suggest that the reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We report here seven new crystal structures primarily involving crystals of S130A thiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130A thiamin phosphate synthase with the intent of characterizing the enzyme-substrate complex. Surprisingly, in three thiamin phosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the electron density is provided by a model that consists of independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbenium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe is the species that is observed in the crystal structures.     

Spontaneous mutagenesis is enhanced in Apex heterozygous mice

Germ line DNA directs the development of the next generation and, as such, is profoundly different from somatic cell DNA. Spermatogenic cells obtained from young adult lacI transgenic mice display a lower spontaneous mutant frequency and greater in vitro base excision repair activity than somatic cells and tissues obtained from the same mice. However, spermatogenic cells from old lacI mice display a 10-fold higher mutant frequency. This increased spontaneous mutant frequency occurs coincidentally with decreased in vitro base excision repair activity for germ cell and testicular extracts that in turn corresponds to a decreased abundance of AP endonuclease. To directly test whether a genetic diminution of AP endonuclease results in increased spontaneous mutant frequencies in spermatogenic cell types, AP endonuclease heterozygous (Apex(+/-)) knockout mice were crossed with lacI transgenic mice. Spontaneous mutant frequencies were significantly elevated (approximately twofold) for liver and spleen obtained from 3-month-old Apex(+/-) lacI(+) mice compared to frequencies from Apex(+/+) lacI(+) littermates and were additionally elevated for somatic tissues from 9-month-old mice. Spermatogenic cells from 9-month-old Apex(+/-) lacI(+) mice were significantly elevated twofold compared to levels for 9-month-old Apex(+/+) lacI(+) control mice. These data indicate that diminution of AP endonuclease has a significant effect on spontaneous mutagenesis in somatic and germ line cells.     

CC chemokine receptor 2 expression in donor cells serves an essential role in graft-versus-host-disease

The complete repertoire of cellular and molecular determinants that influence graft-vs-host disease (GVHD) is not known. Using a well-established murine model of GVHD (B6-->bm12 mice), we sought to elucidate the role of the donor non-T cell compartment and molecular determinants therein in the pathogenesis of GVHD. In this model the acute GVHD-inducing effects of purified B6 wild-type (wt) CD4(+) T cells was inhibited by wt non-T cells in a dose-dependent manner. Paradoxically, unlike the chronic GVHD phenotype observed in bm12 mice transplanted with B6wt unfractionated splenocytes, bm12 recipients of B6ccr2-null unfractionated splenocytes developed acute GVHD and died of IFN-gamma-mediated bone marrow aplasia. This switch from chronic to acute GVHD was associated with increased target organ infiltration of activated CD4(+) T cells as well as enhanced expression of Th1/Th2 cytokines, chemokines, and the antiapoptotic factor bfl1. In vitro, ccr2(-/-) CD4(+) T cells in unfractionated splenocytes underwent significantly less activation-induced cell death than B6wt CD4(+) T cells, providing another potential mechanistic basis along with enhanced expression of bfl1 for the increased numbers of activated T cells in target organs of B6ccr2(-/-) splenocyte-->bm12 mice. Collectively, these findings have important clinical implications, as they implicate the donor non-T cell compartment as a critical regulator of GVHD and suggest that ccr2 expression in this cellular compartment may be an important molecular determinant of activation-induced cell death and GVHD pathogenesis.