Investigation of the species selectivity of a nonpeptide CGRP receptor antagonist using a novel pharmacodynamic assay

The recent discovery of several nonpeptide CGRP antagonists have led to significant advances in our understanding of CGRP receptor pharmacology. Specifically, these antagonists have demonstrated a clear species selectivity with >100-fold greater affinity for human CGRP receptor compared to receptors from other species, such as rat, rabbit and guinea pig. Therefore, nonhuman primate models are required to accurately assess the in vivo activity of these antagonists. The commonly used model in marmosets involves electrical stimulation of the trigeminal ganglia and is a technically difficult and terminal procedure. In this report, we describe a noninvasive pharmacodynamic model in which topical application of capsaicin is utilized to induce the release of endogenous CGRP and a vasodilatory response which can be measured using laser Doppler imaging. Using the potent and selective CGRP antagonist Compound 3, which is an analog of the well-characterized compound BIBN4096BS, we demonstrated 62% inhibition with 300 microg/kg, i.v., in the rat. When tested in the rhesus monkey, only 30 microg/kg of Compound 3 was needed to produce complete inhibition, suggesting that the rhesus CGRP receptor shares a pharmacological profile similar to marmoset and human receptors. Two separate measurements were obtained in this model to provide an indication of both the acute inhibitory effect as well as the prophylactic effect of the CGRP antagonist. At the doses studied, Compound 3 was equally effective on both the acute and prophylactic inhibition of CGRP-mediated vasodilation in rat and rhesus. In conclusion, this is the first report to describe and validate a noninvasive model in nonhuman primates that allows rapid evaluation of CGRP antagonist activity against endogenous CGRP.     

Saccharomyces cerevisiae Hsp104 enhances the chaperone capacity of human cells and inhibits heat stress-induced proapoptotic signaling

Hsp104, the most potent thermotolerance factor in Saccharomyces cerevisiae, is an unusual molecular chaperone that is associated with the dispersal of aggregated, non-native proteins in vivo and in vitro. The close cooperation between Hsp100 oligomeric disaggregases and specific Hsp70 chaperone/cochaperone systems to refold and reactivate heat-damaged proteins has been dubbed a "bichaperone network". Interestingly, animal genomes do not encode a Hsp104 ortholog. To investigate the biochemical and biological consequences of introducing into human cells a stress tolerance factor that has protein refolding capabilities distinct from those already present, Hsp104 was expressed as a transgene in a human leukemic T-cell line (PEER). Hsp104 inhibited heat-shock-induced loss of viability in PEER cells, and this action correlated with reduced procaspase-3 cleavage but not with reduced c-Jun N-terminal kinase phosphorylation. Hsp104 cooperated with endogenous human Hsp70 and Hsc70 molecular chaperones and their J-domain-containing cochaperones Hdj1 and Hdj2 to produce a functional hybrid bichaperone network capable of refolding aggregated luciferase. We also established that Hsp104 shuttles across the nuclear envelope and enhances the chaperoning capacity of both the cytoplasm and nucleoplasm of intact cells. Our results establish the fundamental properties of protein disaggregase function in human cells with implications for the use of Hsp104 or related proteins as therapeutic agents in diseases associated with protein aggregation.     

A 1200-Kilobase Transcription Map Encompassing theD6S105Locus at 6p21.3

The gene content of the MHC class I telomerically adjacent region, in linkage disequilibrium with hereditary hemochromatosis, has not been well characterized yet. In the present work, we established three bacterial clone contigs, including mainly P1-derived artificial chromosomes. These contigs cover 89% of the 1.2-Mb 6p-subtelomeric region encompassing locusD6S105.Terminal exon trapping was applied to selected clones from these contigs. Forty-six independent terminal exons were identified and mapped within the region, 2 of which matched perfectly to expressed sequence tags. These 3′ exons are all expressed in human fetal brain but differentially expressed in four tissues and two cell lines. The high number of exons identified indicates that the high gene density observed in the MHC class I region extends to this telomerically adjacent region.     

Systematic Sequencing of the Human HLA-A/HLA-F Region: Establishment of a Cosmid Contig and Identification of a New Gene Cluster within 37 kb of Sequence

The class I region of the human histocompatibility complex is characterized by a high density of genes and pseudogenes and a complex structural organization. To elucidate the complete structure of the HLA-A/HLA-F region with a view to defining its contents in genes and pseudogenes, we developed a strategy of systematic sequencing. This report describes the establishment of a cosmid contig spanning most of the region and the analysis of a 37-kb sequence from one of the cosmids. Four new genes, organized with the HCG-V gene in a clustered structure, have been identified. Two of these contain a zinc finger motif characteristic of DNA-binding proteins. The former, a member of the C3HC4 protein family, is highly expressed in prostate and contains a B30-2-like sequence identified in several genes mapped within the class I region. The latter, which is ubiquitously expressed, is the human equivalent of the yeast polymerase I A12.2 subunit and of the murine tctex6 gene. Of the two other genes, one remains an anonymous gene with no particular feature, while the fourth, specifically expressed in testis, is the human equivalent of the murine tctex4 gene. This cluster, located in a region corresponding to a syntenic unit between mouse and human, appears to be highly conserved.     

Induction and decay of thermotolerance in rainbow trout fibroblasts

Thermotolerance was studied in the rainbow trout fibroblast cell line RTG-2. RTG-2 cultures that had been incubated at 28 degrees C for 24 h were better able to withstand ultimately lethal temperatures above 28 degrees C than RTG-2 cultures that had been maintained at the routine growth temperature of 22 degrees C. This thermotolerance developed rapidly between 3 and 6 h and was fully developed by 24 h at 28 degrees C. After development for 24 hr at 28 degrees C, thermotolerance showed little change over 72 h at 0 and 5 degrees C but approximately a 40 and 60% reduction at 10 and 22 degrees C, respectively. This is the first demonstration of heat-induced thermal resistance in the cells of a poikilothermic vertebrate.     

Polyisoprenylation of Ras in vitro by a farnesyl-protein transferase

Farnesylation of Ras occurs in vivo on a Cys residue in the C-terminal sequence -Cys-Val-Leu-Ser (termed a CAAX box). This modification is required for Ras membrane localization and cell transforming activity. Using [3H]farnesyl-PPi as precursor and Escherichia coli-expressed Ras, forms of Ras having the CAAX sequence were radiolabeled upon incubation with the cytosolic fraction of bovine brain. Forms of Ras having a deletion of the CAAX sequence or a Cys to Ser substitution in this sequence were not substrates. Radioactivity incorporated into Ras by bovine brain cytosol was released by treatment with iodomethane but not with methanolic KOH indicating a thioether linkage. High pressure liquid chromatography analysis of the cleavage products on a C-18 column showed a major peak of radioactivity that co-eluted with a farnesol standard. The enzyme responsible for Ras farnesylation in bovine brain was approximately 190 kDa as estimated by gel filtration and required a divalent cation for activity. Nonradioactive farnesyl-PPi, geranylgeranyl-PPi, and Ras peptides having the C-terminal sequence -Cys-Val-Leu-Ser competed in the assay with IC50 values of 0.7, 1.4, and 1-3 microM, respectively. Farnesol and Ras peptides having the sequence -Ser-Val-Leu-Ser were not inhibitory. These results identify a farnesyl-protein transferase activity that may be responsible for the polyisoprenylation of Ras in intact cells.     

Persistence and local extinction of lion prides in the Ngorongoro Crater, Tanzania

African lions (Panthera leo) live in social groups (prides) that exhibit group territorial behavior. Pride persistence is expected to depend on its ability to compete against neighboring prides as well as on average rates of reproduction and survival, thus providing a meaningful measure of intergenerational reproductive success. We used Akaike’s Information Criterion (AIC) to select the best approximating models explaining how demographic variables influenced pride persistence during a 30-year period in the Ngorongoro Crater, Tanzania, and identified landscape factors affecting those demographic variables. Pride persistence to 10 years depended on adult female density (pride size) and cub productivity (the ultimate source of new females). Average age of adult females had a weakly positive influence on pride persistence, while the effect of female mortality was weakly negative. Adult female mortality increased with disease epidemics and in territories with high human disturbance. Cub productivity was highest in territories closest to rivers and only slightly higher near swamps, and also high in areas of higher vegetative cover and high human use. No landscape variable significantly affected female density. The growth and population size of the Crater lions was closely linked to demographic performance of individual prides, while territorial behavior played a key role in mediating the interactive effects of landscape and demography.     

Benzodiazepine calcitonin gene-related peptide (CGRP) receptor antagonists: optimization of the 4-substituted piperidine

In our continuing effort to identify CGRP receptor antagonists for the acute treatment of migraine, we have undertaken a study to evaluate alternative 4-substituted piperidines to the lead dihydroquinazolinone 1. In this regard, we have identified the piperidinyl-azabenzimidazolone and phenylimidazolinone structures which, when incorporated into the benzodiazepine core, afford potent CGRP receptor antagonists (e.g., 18 and 29). These studies produced a potent analog (18) which overcomes the instability issues associated with the lead structure 1. A general pharmacophore for the 4-substituted piperidine component of these CGRP receptor antagonists is also presented.     

Kinetic and biophysical analysis of the m2 muscarinic receptor

The recombinant Pm2 muscarinic receptor expressed in Chinese hamster ovary (CHO) cells was used as a model system to examine receptor-effector coupling and ligand binding. In CHO cells, equilibrium binding studies and the dependence on receptor number per cell of the maximum response and EC50 values for agonist stimulation of phosphatidylinositol metabolism and inhibition of cAMP formation were consistent with a modified ternary complex model of signal transduction that included a physiologically noncompetent receptor state. Detailed kinetic studies of oxotremorine M (Oxo-M) binding to CHO cell membranes suggested that agonist interactions at the high affinity class of binding sites are complicated and depend on receptor expression levels. At low levels of expression, kinetic data were consistent with a special case of a mechanism in which Oxo-M shifts the equilibrium between two receptor conformations while at high levels of expression, it was necessary to evoke receptor-receptor interactions to explain the kinetic data. Far ultraviolet circular dichroism studies of the purified recombinant receptor showed a high content of alpha-helical secondary structure and small changes in secondary structure upon antagonist, but not agonist, binding.     

Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia

Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions—exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.