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.     

Bioconversion of starch processing waste to Phellinus linteus mycelium in solid-state cultivation

The objective of the experiment was to use starch processing waste as an alternative growth medium for cultivation of mycelia of the mushroom Phellinus linteus and to find an optimum condition under solid-state cultivation. Response surface analysis along with a central composite design was successfully applied to approximate the simultaneous effects of the substrate concentration (16-36 g l(-1)), pH (4.5-6.5), and temperature (25-35 degrees C) on the mycelial growth rate. In the model, pH and temperature significantly affected the mycelial growth but substrate concentration did not. The optimal substrate concentration, pH, and temperature for maximizing growth rate of P. linteus mycelia were found to be 16.5 g l(-1), pH 6.0, and 29.7 degrees C, respectively. Subsequent verification of these levels agreed with model predictions and the maximum mycelial growth rate at these conditions was 6.1 +/- 0.8 mm day(-1). Therefore, the results of the experiments suggest that starch processing waste could be utilized as a growth substrate for the cultivation of the mushroom mycelia of P. linteus, enhancing the usefulness of this byproduct of the starch manufacturing industry. This approach is likely to be useful for establishing similar parameters for the cultivation of other fungi.     

Comparison of the substrate specificity of the two bacterial desulfurization systems

Using cell-free extracts of a desulfurizing mesophile, Rhodococcus erythropolis KA2-5-1 (the Dsz system) and Escherichia coli JM109, which possesses the desulfurizing genes of a thermophile Paenibacillus sp. A11-2 (the Tds system), the reactivity of desulfurizing enzymes toward 4,6-dialkyl dibenzothiophenes (4,6-dialkyl DBTs) and 7-alkyl benzothiophenes (7-alkyl BTs) was investigated. Both systems desulfurized all the 4,6-dialkyl DBTs, except 4,6-dibutyl DBT. Although some alkylated BTs were degraded by the Dsz system, no desulfurized compounds were detected. The reactivity of the Tds system toward alkylated BTs was higher than that of DBT. In contrast to the Dsz system, the Tds system yielded desulfurized compounds from all of the alkylated BTs examined.     

RELATIONSHIPS AMONG MORPHOLOGICAL STATUS, STEROID HORMONES, AND POST-THAWING VIABILITY OF FROZEN SPERMATOZOA OF MALE MINKE WHALES (BALAENOPTERA ACUTOROSTRATA)

Spermatozoa from 21 mature minke whales ( Balaenoptera acutorostrata ) taken in the Antarctic Ocean for Japanese research were recovered from vasa deferentia, diluted 1:9 in a Tris-based diluent, and frozen at - 80°C on board the vessel. After a period ranging from 45 to 125 d, the samples were transferred to liquid nitrogen and transported to the laboratory. After thawing at 37°C the motility (percentage of motile spermatozoa), vitality (proportion of live spermatozoa), and sperm concentration were determined for each sample. These values were tested for correlations with morphological measurements (body size, body weight, testis weight) and serum concentrations of progesterone (Pd), estradiol-17β (E2), and testosterone (T). Ten of 21 samples had motile spermatozoa (2%-40%). Although no motile spermatozoa were observed in 1.1 samples, all sperm samples were examined by eosinnigrosin staining and showed vitality levels of 3%44%. It was found that the motility (Y = 0.54) and vitality (r = 0.53) of the spermatozoa were significantly (P < 0.01) correlated with the E₂ levels (8.50 ± 1.80 pg/ml). Serum T levels (0.07 ± 0.02 ngml) were significantly correlated with the E₂ levels (r = 0.58, P < 0.01>, but sperm concentrations were not correlated with either Ea or T levels. The present study demonstrates that spermatozoa of minke whales can be successfully cryopreserved.     

Down-regulation of trypsinogen expression is associated with growth retardation in alpha1,6-fucosyltransferase-deficient mice: attenuation of proteinase-activated receptor 2 activity

Alpha1,6-fucosyltransferase (Fut8) plays important roles inphysiological and pathological conditions. Fut8-deficient (Fut8^–/–)mice exhibit growth retardation, earlier postnatal death, andemphysema-like phenotype. To investigate the underlying molecularmechanism by which growth retardation occurs, we examined themRNA expression levels of Fut8^–/– embryos (18.5days postcoitum [dpc]) using a cDNA microarray. The DNA microarrayand real-time polymerase chain reaction (PCR) analysis showedthat a group of genes, including trypsinogens 4, 7, 8, 11, 16,and 20, were down-regulated in Fut8^–/– embryos.Consistently, the expression of trypsinogen proteins was foundto be lower in Fut8^–/– mice in the duodenum, smallintestine, and pancreas. Trypsin, an active form of trypsinogen,regulates cell growth through a G-protein-coupled receptor,the proteinase-activated receptor 2 (PAR-2). In a cell culturesystem, a Fut8 knockdown mouse pancreatic acinar cell carcinoma,TGP49-Fut8-KDs, showed decreased growth rate, similar to thatseen in Fut8^–/– mice, and the decreased growth ratewas rescued by the application of the PAR-2-activating peptide(SLIGRL-NH₂). Moreover, epidermal growth factor (EGF)-inducedreceptor phosphorylation was attenuated in TGP49-Fut8-KDs, whichwas highly associated with a reduction of trypsinogens mRNAlevels. The addition of exogenous EGF recovered c-fos, c-jun,and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, theEGF-induced up-regulation of c-fos and c-jun mRNA expressionwas significantly blocked by the protein kinase C (PKC) inhibitor.Our findings clearly demonstrate a relationship between Fut8and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathwayin controlling cell growth and that the EGFR-trypsin-PAR-2 pathwayis suppressed in TGP49-Fut8-KDs as well as in Fut8^–/–mice.     

New records of polycentropodid caddisflies from Korea (Insecta: Trichoptera)

Four species of the family Polycentropodidae –Plectrocnemia baculifera, P. kusnezovi, Paranyctiophylax hjangsanchonus and Polyplectropus nocturnus– are recorded from Korea. Polyplectropus nocturnus is newly recorded from the Korean peninsula. The other three species are recorded from the Republic of Korea for the first time.     

Crankshaft motions of the polypeptide backbone in molecular dynamics simulations of human type-α transforming growth factor

Summary Order parameters for the backbone N–H and C–H bond vectors have been calculated from a 150 ps molecular dynamics (MD) simulation of human type- transforming growth factor in H₂O solvent. Two kinds of crankshaft motions of the polypeptide backbone are observed in this MD trajectory. The first involves small-amplitude rocking of the rigid peptide bond due to correlated changes in the backbone dihedral angles _i–1 and _i. These high-frequency librational crankshaft motions are correlated with systematically smaller values of motional order parameters for backbone N–H bond vectors compared to C–H bond vectors. In addition, infrequent crankshaft flips of the peptide bond from one local minimum to another are observed for several amino acid residues. These MD simulations demonstrate that comparisons of N–H and C–H order parameters provide a useful approach for identifying crank-shaft librational motions in proteins.     

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.