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.     

Membrane glycoproteins involved in neurite fasciculation

Lectin affinity chromatography combined with mAb production was used to identify chick neural cell surface molecules related to L1 antigen, a mouse neural glycoprotein implicated in cell-cell adhesion (Rathjen, F. G., and M. Schachner, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1-10). A glycoprotein, G4 antigen, isolated by mAb G4 from adult chick brain is described which comprises a major 135-kD component, a minor doublet at 190 kD, and diffusely migrating bands at 80 and 65 kD in SDS PAGE. This molecule is structurally related to mouse L1 antigen according to NH2-terminal amino acid sequence (50% identity) as well as the behavior of its components in two-dimensional IEF/SDS PAGE gels. A second chicken glycoprotein, F11 antigen, was isolated from adult chick brain using mAb F11. This protein has also a major 135-kD component and minor components at 170 kD and 120 kD. Both immunotransfer analysis with polyclonal antibodies to mAb G4 and to mAb F11 isolate and the behavior on IEF/SDS PAGE gels indicates that the major 135-kD component of F11 antigen is distinct from G4 antigen components. However, the 135-kD component of F11 antigen shares with G4 antigen and the neural cell adhesion molecule (NCAM) the HNK-1/L2 carbohydrate epitope. In immunofluorescence studies, G4 and F11 antigenic sites were found to be associated mainly with the surface of process-bearing cells, particularly in fiber-rich regions of embryonic brain. Although Fab fragments of polyclonal antibodies to mAbs G4 or F11 immunoaffinity isolate only weakly inhibit the Ca2+-independent aggregation of neural cells, they strongly inhibit fasciculation of retinal axons. Together these studies extend the evidence that bundling of axons reflects the combined effects of a group of distinct cell surface glycoproteins.     

Binding properties of detergent-solubilized NCAM

An assay has been designed for the identification of NCAM-binding proteins present in an NP-40 detergent extract of brain membranes. This method, which is capable of analyzing both heterophilic and homophilic interactions, uses species-specific antibodies against NCAM in combination with radioiodination, so that after unlabeled chicken and iodinated frog brain membrane proteins were allowed to interact, the chicken NCAM could be specifically isolated by immunoaffinity adsorption. The radiolabeled frog proteins coisolated with chicken NCAM were then characterized by one- and two-dimensional gel electrophoresis in combination with immunoblotting. The only detectable NCAM-binding proteins were identified as the 140- and 180-kD forms of NCAM. The presence and absence of polysialic acid on NCAM did not change the amount or nature of the frog proteins immunopurified under these conditions. As an alternative for detecting heterophilic ligands, a simplified immunoprecipitation method was employed using either iodine or sulfate radiolabels. Again under these conditions only NCAM was detected. These results are consistent with the hypothesis that the major binding protein for NCAM is NCAM itself, and suggest that differences in polysialic acid content do not directly alter the properties of NCAM's homophilic binding site.     

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.      

NCAM polysialic acid can regulate both cell-cell and cell-substrate interactions

We have proposed previously that the polysialic acid (PSA) moiety of NCAM can influence membrane-membrane apposition, and thereby serve as a selective regulator of a variety of contact-dependent cell interactions. In this study, cell and tissue culture models are used to obtain direct evidence that the presence of PSA on the surface membrane can affect both cell-cell and cell-substrate interactions. Using a neuroblastoma/sensory neuron cell hybrid, it was found that removal of PSA with a specific neuraminidase (endo-N) augments cell-cell aggregation mediated by the L1 cell adhesion molecule as well as cell attachment to a variety of tissue culture substrates. In studies of embryonic spinal cord axon bundling, which involves both cell-cell and cell-substrate interactions, the pronounced defasciculation produced by removal of PSA is most easily explained by an increase in cell-substrate interaction. The fact that in both studies NCAM's intrinsic adhesion function was found not to be an important variable further illustrates that regulation of the cell surface by PSA can extend beyond binding mediated by the NCAM polypeptide.     

Intact but not truncated insulin-like growth factor binding protein-3 (IGFBP-3) blocks IGF I-induced stimulation of osteoblasts: control of IGF signalling to bone cells by IGFBP-3-specific proteolysis?

IGFBP-3 is the predominant IGFBP in serum and the major IGFBP secreted by osteoblasts. Native and recombinant IGFBP-3 and a truncated form lacking the carboxyterminal third were tested for their effects on 2 osteoblastic cell lines. Intact but not truncated IGFBP-3 blocked IGF I-stimulated DNA and glycogen synthesis. Inhibition was dose-dependent and found whenever the concentration of intact IGFBP-3 exceeded the concentration of IGF I. Truncated IGFBP-3 appears to result from proteolytic cleavage and does occur in vivo. The loss of inhibition by IGFBP-3 may be regulated at the site of IGF target cells and thus be essential for IGF I-induced osteoblast growth.     

Molecules involved in cell–cell adhesion during development

A peculiarity of nitrosamines is the high degree of cell and organ specificity in inducing tumors. There is substantial evidence that the initiation of the carcinogenesis process by carcinogens of this group is linked to the metabolic competence of the target tissue or cell to convert these carcinogens into mutagenic metabolites and to the binding of those metabolites to cellular DNA. Alkylation occurs in the DNA at the N-1, N-3, and N-7 positions of adenine; the N-3, N-7, and O⁶ of guanine; the N-3, and O² of cytosine; and the N-3, O⁴, and O² of thymine; and the phosphate groups. The initial proportion of each DNA adduct depends upon the alkylating agent used. The various DNA adducts are lost to a variable extent from DNA in vivo by spontaneous release of bases and Or by specific DNA repair processes. Studies conducted in vitro and in vivo indicate that alkylation at the oxygen atoms of DNA bases is more critical than alkylation at other positions in the mutagenesis and carcinogenesis induced by N-nitroso compounds. In particular, tissues in which tumors occur more frequently after a pulse dose of nitrosamine are those in which O⁶-alkylguanine persists longest in DNA, presumably resulting in an increased probability that a miscoding event (mutation) will take place during DNA synthesis. The more rapid removal of O⁶-methylguanine from the DNA of liver (as compared with cxtrahepatic tissues) of rats has been associated with the absence of tumor production in this organ by a single dose of dimethylnitrosamine; however, a significant incidence of liver tumors is observed if the same dose is given 24 hr after partial hepatectomy, and tumors arc induced by such a dose of dimethyl-nitrosamine in the liver of hamsters, which has a low capacity to remove O⁶-methylguanine from its DNA. These data also indicate that the rate of disappearance of 7-methylguanine from the liver or cxtrahepatic tissues is independent of the dose of dimethylnitrosamine; whereas O⁶-methylguanine is lost from DNA more rapidly after a low dose of this nitrosamine. It has been shown that in liver the removal of O⁶-methylguanine, but not of other DNA adducts, from DNA can be affected by pretreating the animals with N-nitroso compounds. The modulation of DNA repair processes observed after a single dose and after chronic treatment with nitrosamines is discussed in relation to the tissue-specific carcinogenic effect of this group of carcinogens.