Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.     

Ultrastructure of Hendra virus and Nipah virus within cultured cells and host animals

The ultrastructure of Hendra and Nipah viruses is described in cultured cells, pigs, horses and humans. Differences in ultrastructure between the viruses are evident within infected cell cultures and lungs from infected amplifier hosts. These differences are important in viral identification and differentiation and understanding the pathogenesis of disease.     

Molecular and functional characterization of genes encoding horse MHC class I antigens

Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by alloreactive cytotoxic T lymphocytes. A total of 3815 bp of the genomic clone were sequenced, extending from 429 bp upstream (5') of the leader peptide through the 3' untranslated region. Promoter region motifs and an intron-exon structure characteristic of MHC class I genes of other species were found. A subclone containing 407 bp of the promoter region was inserted into a chloramphenicol acetyl transferase reporter plasmid, tested in transient transfection assays, and found to have promoter activity in heterologous cells. This genomic clone will enable detailed studies of MHC class I gene regulation in horse trophoblasts, and in horse retroviral infections.     

A comparative mitogenomic analysis of the potential adaptive value of Arctic charr mtDNA introgression in brook charr populations (Salvelinus fontinalis Mitchill)

Wild brook charr populations (Salvelinus fontinalis) completely introgressed with the mitochondrial genome (mtDNA) of arctic charr (Salvelinus alpinus) are found in several lakes of northeastern Québec, Canada. Mitochondrial respiratory enzymes of these populations are thus encoded by their own nuclear DNA and by arctic charr mtDNA. In the present study we performed a comparative sequence analysis of the whole mitochondrial genome of both brook and arctic charr to identify the distribution of mutational differences across these two genomes. This analysis revealed 47 amino acid replacements, 45 of which were confined to subunits of the NADH dehydrogenase complex (Complex I), one in the cox3 gene (Complex IV), and one in the atp8 gene (Complex V). A cladistic approach performed with brook charr, arctic charr, and two other salmonid fishes (rainbow trout [Oncorhynchus mykiss] and Atlantic salmon [Salmo salar]) revealed that only five amino acid replacements were specific to the charr comparison and not shared with the other two salmonids. In addition, five amino acid substitutions localized in the nad2 and nad5 genes denoted negative scores according to the functional properties of amino acids and, therefore, could possibly have an impact on the structure and functional properties of these mitochondrial peptides. The comparison of both brook and arctic charr mtDNA with that of rainbow trout also revealed a relatively constant mutation rate for each specific gene among species, whereas the rate was quite different among genes. This pattern held for both synonymous and nonsynonymous nucleotide positions. These results, therefore, support the hypothesis of selective constraints acting on synonymous codon usage.     

A neutral beta-D-glucan from dates of the date palm,Phoenix dactylifera L

Polysaccharides extracted from Libyan dates with hot water and 0.05 M NaOH were fractionated and purified by ion-exchange and gel-filtration chromatography. According to the methylation and hydrolysis analyses, the results indicate the D-glucan to be linear and to contain both (1-->3)- and (1-->4)-linkages. The anomeric NMR measurements confirm that the sugar residues are beta-glycosidically linked. This is the first report on the isolation of a neutral beta-D-glucan from dates.     

Pattern recognition and call preferences in treefrogs (Anura: Hylidae): a quantitative analysis using a no-choice paradigm

Studies of mate attraction have traditionally employed one of two experimental methods: choice tests in which female preferences from among two or more signals are tabulated, or single-stimulus tests in which the female response to one signal is quantified. Choice tests have long been preferred for examining mate attraction in anurans. Inspired by Wagner's (1998, Animal Behaviour,55, 1029-1042) discussion of the strengths and weaknesses of the two experimental approaches, we used a single-stimulus design to examine mate attraction in two sibling species of treefrogs (Hyla versicolor and H. chrysoscelis). We quantified female responses based upon the relative time required to approach signals varying in pulse rate, pulse rise time and pulse number. The data were used to generate response functions providing a quantitative measure of female attraction to the stimuli. Comparisons with data from choice experiments reveal broad similarities, as well as properties of female responses that had not been detected with choice tests. The results are discussed with regard to female selectivity for call parameters that are likely to mediate sexual selection and homospecific pairing.     

Vasopressin-dependent inhibition of the C-type natriuretic peptide receptor,NPR-B/GC-B,requires elevated intracellular calcium concentrations

Natriuretic peptides bind their cognate cell surface guanylyl cyclase receptors and elevate intracellular cGMP concentrations. In vascular smooth muscle cells, this results in the activation of the type I cGMP-dependent protein kinase and vasorelaxation. In contrast, pressor hormones like arginine-vasopressin, angiotensin II, and endothelin bind serpentine receptors that interact with G(q) and activate phospholipase Cbeta. The products of this enzyme, diacylglycerol and inositol trisphosphate, activate the conventional and novel forms of protein kinase C (PKC) and elevate intracellular calcium concentrations, respectively. The latter response results in vasoconstriction, which opposes the actions of natriuretic peptides. Previous reports have shown that pressor hormones inhibit natriuretic peptide receptors NPR-A or NPR-B in a variety of different cell types. Although the mechanism for this inhibition remains unknown, it has been universally accepted that PKC is an obligatory component of this pathway primarily because pharmacologic activators of PKC mimic the inhibitory effects of these hormones. Here, we show that in A10 vascular smooth muscle cells, neither chronic PKC down-regulation nor specific PKC inhibitors block the AVP-dependent desensitization of NPR-B even though both processes block PKC-dependent desensitization. In contrast, the cell-permeable calcium chelator, BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester), abrogates the AVP-dependent desensitization of NPR-B, and ionomycin, a calcium ionophore, mimics the AVP effect. These data show that the inositol trisphosphate/calcium arm of the phospholipase C pathway mediates the desensitization of a natriuretic peptide receptor in A10 cells. In addition, we report that CNP attenuates AVP-dependent elevations in intracellular calcium concentrations. Together, these data reveal a dominant role for intracellular calcium in the reciprocal regulation of these two important vasoactive signaling systems.