Anterograde Axonal Transport of Peptidylglycine α-Amidating Monooxygenase in Rat Sciatic Nerves

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.     

Mitochondrial DNA haplogrouping of the brown bear,Ursus arctos (Carnivora: Ursidae) in Asia,based on a newly developed APLP analysis

Sequence analyses of the complete brown bear, Ursus arctos, mitochondrial DNA (mtDNA) genome have detected scattered single nucleotide polymorphisms (SNPs) that define distinct mtDNA haplogroups in phylogeographical studies. The degraded DNA in historical samples, such as stuffed or excavated specimens, however, is often not suitable for sequence analyses. To address this problem, we developed an amplified product length polymorphism (APLP) analysis for mtDNA‐haplogrouping U. arctos specimens by detecting haplogroup‐specific SNPs. We verified the validity and utility of this method by analysing up to 170‐year‐old skin samples from U. arctos specimens collected widely across continental Eurasia. We detected some of the same haplogroups as those occurring in eastern Hokkaido (Japan) and eastern Alaska in continental Eurasia (the Altai and the Caucasus). Our results show that U. arctos in eastern Hokkaido and eastern Alaska descended from a common ancestor in continental Eurasia, and suggest that U. arctos occupied several refugia in southern Asia during the Last Glacial Maximum. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111 , 627–635.     

Responses of the melanophores of the medaka, Oryzias latipes, to adrenergic drugs: evidence for involvement of alpha 2 adrenergic receptors mediating melanin aggregation

1. Melanin-aggregation response of the medaka melanophores to a series of adrenergic drugs were examined. 2. Concentration-response curves for the drugs indicated that the melanin-aggregating effects of alpha 2 adrenergic agonists, naphazoline, tramazoline and clonidine, were more than 100-fold greater than that of alpha 1 agonists, phenylpropanolamine, phenylephrine, oxymetazoline and methoxamine. 3. The inhibitory effect of alpha 2 antagonist, yohimbine, on the cell responses to the agonists were also about 100-fold greater than that of alpha 1 antagonists, corynanthine and prazosin. 4. These results indicate that adrenergic receptors which mediate melanin-aggregation response of the cells are alpha 2 in nature.     

Analysis of human terminal deoxynucleotidyl transferase cDNA expressible in mammalian cells

Human Molt3 cDNA library was constructed using pcD vector system which permits the expression of cDNA inserts in mammalian cells. Nearly full-length human terminal deoxynucleotidyltransferase (TdT) cDNA was cloned using a fragment of bovine TdT cDNA as a probe. The human TdT cDNA contains an open reading frame of 1,557 bp coding for 519 amino acids, including 31 bp and 341 bp from 5' and 3' untranslated regions, respectively. The TdT cDNA was transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 59,495. The cloned TdT cDNA hybridized with poly A+ RNAs of 2,100 b and 3,300 b from stable T-cell leukemia Molt3 and Molt4 cells.     

Antitumor effect of RBS (rice bran saccharide) on ENNG-induced carcinogenesis

We examined whether orally administered RBS (rice bran saccharide), prepared from rice bran by hot water extraction, increases immunocompetence, inhibits gastrointestinal carcinogenesis with N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) or shows an antitumor effect. After the administration of RBS, phytohemagglutinin (PHA)- and pokeweed mitogen (PWM)-stimulated blastogenesis of lymphocytes derived from the mesenteric lymph nodes and peripheral blood was enhanced, and the helper/ suppressor T-cell ratio was elevated, and migration activity of peritoneal macrophages was also increased in rats treated continuously with ENNG. ENNG-induced gastrointestinal carcinomas were observed in 43% of those administered RBS (ENNG-RBS) as compared with 88% in the control (ENNG) and 94% in the prednisolone (PRD) group (ENNG-PRD). The 12-month survival rate of rats bearing gastrointestinal cancer was 58% in the ENNG-RBS group as compared with 25% in the ENNG group and 15% in the ENNG-PRD group. RBS prevented the reduction in immunocompetence in the course of carcinogenesis, suppressed carcinogenesis, and prolonged the survival of rats with gastrointestinal cancer. Antitumor activities of RBS are thought to be a kind of host mediated action. The growth inhibition ratio of transplantable ENNG-induced cancer in Wistar rats was 42.1% in the RBS and 51.8% in the 5-FU group. Since little is known about the potent antitumor activity of -glucan, it would be interesting to consider the relationship between the structure and the biological activities of polysaccharides.     

Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

Combined mutagenicity of cobalt(II) salt and heteroaromatic compounds in Salmonella typhimurium

Mutagenic activities of 4-aminopyridine (4AP), 4-aminoquinoline (4AQ), 9-aminoacridine (9AA) and harman (HM) were examined by the Salmonella test system in the presence of cobalt(II) chloride (CoCl2), which itself is non-mutagenic in this system. Mutagenic activity of the mixture of 9AA and CoCl2 was found to be much higher than that of 9AA alone in strains TA1537 and TA2637. A similar enhancing phenomenon was observed in 4AQ-CoCl2 and HM-CoCl2 mixtures but not in that of 4AP-CoCl2. Judging from visible and nuclear magnetic resonance spectral data, this increased mutagenicity may be attributable to the formation of moderate to weak complexes between these chemicals and the Co(II) cation. A survey of the mutagenicity of several Co(II) complexes supported this interpretation.