Synthesis of oxidative metabolites of CRA13 and their analogs: Identification of CRA13 active metabolites and analogs thereof with selective CB2R affinity

CRA13; a peripheral dual CB₁R/CB₂R agonist with clinically proven analgesic properties, infiltrates into CNS producing adverse effects due to central CB₁R agonism. Such adverse effects might be circumvented by less lipophilic compounds with attenuated CB₁R affinity. Metabolism produces less lipophilic metabolites that might be active metabolites. Some CRA13 oxidative metabolites and their analogues were synthesized as less lipophilic CRA13 analogues. Probing their CB₁R and CB₂R activity revealed the alcohol metabolite 8c as a more potent and more effective CB₂R ligand with attenuated CB₁R affinity relative to CRA13. Also, the alcohol analogue 8b and methyl ester 12a possessed enhanced CB₂R affinity and reduced CB₁R affinity. The CB₂R binding affinity of alcohol analogue 8b was similar to CRA13 while that of methyl ester 12a was more potent. In silico study provided insights into the possible molecular interactions that might explain the difference in the elicited biological activity of these compounds.     

Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the Acipenseriformes

The shortnose sturgeon Acipenser brevirostrum was revealed to have a larger number of chromosomes than previously reported for other sturgeon species. Its chromosome number ranged from 362 to 372 (of ten specimens examined), showing intraindividual variation. The karyotype of metaphase with the highest chromosome number (372) consisted of 89 pairs of macrochromosomes and 97 pairs of microchromosomes (fundamental number; NF=550). Although the microchromosomes were relatively shorter than the macrochromosomes, most of them had discernible arms and centromeres. Silver-stained nucleolar organizer regions (Ag-NORs) were localized on the telomeric regions of 5 pairs of chromosomes (Ag-NORs=10): 4 were made up of small meta/submetacentrics and 1 of acrocentrics. Polyploidy of A. brevirostrum should be hexaploid based on the karyotype, numerous chromosomes, Ag-NORs, and previously reported large genome size (ca. 13pg DNA/cell).Supplementary material to this paper is available in electronic format at http://dx.doi.org/10.1007/s10228-004-0257-z     

Helicobacter muricola sp. nov., a novel Helicobacter species isolated from the ceca and feces of Korean wild mouse (Mus musculus molossinus)

A slowly growing microaerophilic Helicobacter strain was isolated from the ceca and fecal pellets of Korean wild mice (Mus musculus molossinus). This bacterial strain possessed a pair of nonsheathed bipolar flagella, was positive for urease, catalase and oxidase, and reduced nitrate to nitrite. It proved susceptible to nalidixic acid and resistant to cephalodine, and did not hydrolyze hippurate. On the basis of phenotypic characteristics and 16S rRNA gene sequence analysis, the isolate represents a new species of the genus Helicobacter, for which the name Helicobacter muricola sp. nov. is proposed; the type strain of the new species is w-06T.     

Tissue expression and cellular localization of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA in male mice

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is an ubiquitous antioxidant enzyme, but the exact expression pattern in mammalian tissues is still unknown. The expression and cellular localization of PHGPx mRNA were examined in male mice using real time-polymerase chain reaction and in situ hybridization techniques. The rank order of PHGPx mRNA expression across tissues exhibiting substantial levels of expression was:testes ≫ heart > cerebrum ≥ ileum > stomach = liver = jejunum ≥ epididymis. In testes, PHGPx mRNA was highly expressed in spermiogenic cells and Leydig cells. The signal was also expressed in the molecular layer, Purkinje cell layer, and white matter of cerebellum, the pituicytes of neurohypophysis, the parafollicular cells and follicular basement membrane of thyroid, the exocrine portion of pancreas, the tubular epithelium of kidney, the smooth muscle cells of arteries, and the red pulp of spleen. In the gastrointestinal tract, PHGPx mRNA expression was mainly observed in the keratinized surface epithelium of forestomach, the submucosal glands and serosa layers, and further the Paneth cells of intestines. PHGPx mRNA appeared to be ubiquitously expressed in the parenchyma of heart, liver, and lung. These results indicate that PHGPx exhibits a cell- and tissue-specific expression pattern in mice.     

A set of epitope-tagging integration vectors for functional analysis in Saccharomyces cerevisiae

Functional analysis of genes from Saccharomyces cerevisiae has been the major goal after determination of genome sequences. Even though several tools for molecular-genetic analyses have been developed, only a limited number of reliable genetic tools are available to support functional assay at protein level. Epitope tagging is a powerful tool for detecting, purifying, and functional studying of proteins. But systematic tagging systems developed with integration vectors are not available. Here, we have constructed a set of integration vectors allowing a translational fusion of interested proteins to the four different epitope tags (HA, Myc, Flag, and GFP). To confirm function and expression of C-terminal-tagged proteins, we used Cdc11, a component of the septin filament that encircles the mother bud neck and consists of five major proteins: Cdc3, Cdc10, Cdc11, Cdc12, and Sep7. The tagged version of Cdc11 expressed under its endogenous promoter was found to be physiologically functional, as evidenced by localization at the neck and suppression of the growth defect associated with the temperature-sensitive mutation of cdc11-6. The expressed proteins were efficiently detected with antibodies against Cdc11 or the epitopes. When immunoprecipitated with anti-Myc antibody, each septin protein tagged with Myc was effectively copurified with other septin components, indicating formation of a stable septin complex. Because the modules of the tags were located under the same array of eighteen restriction sites on integration vectors containing four different markers (HIS3, TRP1, LEU2, or URA3), this tagging system provides efficient multiple tagging and stable expression of a gene of interest.     

Radiolytic transformation of rotenone with potential anti-adipogenic activity

Radiolytic transformation of the isoflavonoid rotenone (1) with γ-irradiation afforded two new degraded products, rotenoisins A (2) and (3). The structures of the two new rotenone derivatives were elucidated on the basis of spectroscopic methods. The new products 2 and 3 exhibited significantly enhanced inhibitory activities against pancreatic lipase and adipocyte differentiation in 3T3-L1 cells when compared to parent rotenone.     

Cystatin B homolog from rock bream Oplegnathus fasciatus: genomic characterization, transcriptional profiling and protease-inhibitory activity of recombinant protein

Cysteine proteases are present in all living organisms and, in animals, function in a vast array of physiological and pathological processes. Cysteine protease inhibitors act upon the cysteine proteases to regulate their activity. The cystatin superfamily of cysteine protease inhibitors has members represented in all living organisms studied to date. Here, we report the identification of a new member of the family 1 cystatin in Oplegnathus fasciatus rock bream (denoted as RbCyt B) and the characterization at the molecular level. The complete genomic sequence of RbCyt B consists of three exons and a promoter region. The open reading frame (ORF) encodes for a 100 amino acids length polypeptide with a single cystatin-like domain and a cysteine protease inhibitor signature motif. The conserved N-terminal glycine, glutamine-valine-glycine motif, QxVxG, and a variant of the proline-tryptophan, PW, motif were identified. RbCyt B showed closest phylogenetic distance to Dicentrarchus labrax cystatin B, and shared up to 73% amino acid identity and 90% amino acid similarity with known cystatin B genes. RbCyt B mRNA expression was detected in nine different tissues and was highly expressed in liver, spleen, gill, brain, intestine, kidney, head kidney, and blood, as compared with muscle. In vivo immune stimulation with Edwardsiella tarda bacteria caused significant up-regulation of RbCyt B mRNA in head kidney and spleen at 24h post-infection (P<0.05). Recombinant RbCyt B was expressed in Escherichia coli, and the purified protein demonstrated 82% papain inhibitory activity at 500 × 10(-3) μg μL(-1) in a concentration-dependent manner. These results suggest that RbCyt B is a member of family 1 cystatin with high homology to cystatin B, and is a biologically active protein possessing papain inhibitory activity and potentially involved in immune responses against invading Gram-negative bacteria in rock bream.