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.     

Studies on peptides CLVIII. Model experiments for the synthesis of open-chain unsymmetrical cystine-peptides

Treatment of a mixture of Cys(R)(O) and Cys(R) with an acid was found to generate cystine in fairly good yields, when suitable R, R, and an acid were selected. An unsymmetrical cystine peptide was prepared by treatment of a mixture of Z(OMe)-Cys(R) (0)-Ala-NH₂ (R=Acm or MBzl) and Z(OMe)-Cys(MBzl)-Gly-OBzl with TFA or 1 M TFMSA/TFA.³ Oxytocin was obtained in an excellent yield by TFA treatment of the protected peptide containing Cys(Acm)(0) and Cys(MBzl). Thus, formation of the disulfide bond was found feasible at the position of Cys(R) (0).The following abbreviations are used Boc t-butyloxycarbonyl - Z(OMe) p-methoxybenzyloxycarbonyl - MBzl p-methoxybenzyl - Acm acetamidomethyl - Bzl benzyl - Ad l-adamantyl - tBu t-butyl - TFA trifluoroacetic acid - TFMSA trifluoromethanesulfonic acid - TMSOTf trimethylsilyl trifluoromethane sulfonate     

Progression of the phenotype of transformed cells after growth stimulation of cells by a human papillomavirus type 16 gene function.

Alteration of the growth properties of the established murine fibroblast cell lines NIH 3T3 and 3Y1 was studied in monolayer cultures and in cells suspended in semisolid medium after introduction of a cloned human papillomavirus type 16 (HPV16) DNA. HPV 16 DNA stimulated both cell lines to grow beyond their saturation densities in monolayer cultures without any apparent morphological changes or tendency to pile up. These cells were also stimulated to grow in soft agar. Since essentially all the cells that received the viral gene were stimulated to grow, the growth-stimulatory activity of HPV16 appeared to be due to the direct effect of a viral gene function. The NIH 3T3 cells showed an additional change in growth properties upon prolonged incubation of dense monolayers of cells containing the HPV16 DNA; morphologically recognizable dense foci appeared at a frequency of about 10(-3). These cells, when cloned from the foci, grew more rapidly in soft agar than the parental cells and were morphologically transformed. In other words, there were two sequential steps in cell transformation induced by HPV16. Practically all the viral DNAs were present in the cells as large rearranged multimers and were integrated into host chromosomal DNA. There was no obvious difference in the state of viral DNA in the cells of the original clone or the three subclones derived from it as dense foci. There was no difference in the amount or the number of viral RNA species expressed in the cells at these two stages. The secondary changes in the growth properties of NIH 3T3 cells appear to be due to some cellular alterations.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Continuous production of galacto-oligosaccharides from lactose using immobilized β-galactosidase from Bacillus circulans

Summary -Galactosidase-2 (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was purified using hydroxyapatite gel chromatography and immobilized onto Duolite ES-762 (phenolformaldehyde resin) and Merckogel (controlled pore silica gel) for continuous production of galacto-oligosaccharides using lactose as the substrate. The maximum amount of ologosaccharides produced by the immobilized enzyme was 35–40% of the total sugar during hydrolysis of 4.56% lactose. Partially purified -galactosidase from B. circulans was also immobilized onto various supports for the same purpose. The stability of the immobilized -galactosidase-2 or partially purified enzyme during a continuous reaction depended on their supports and specific activity. Of the supports tested, Merckogel was best for operational stability. With this support, the enzyme was quite stable with specific activity up to 15 units/g of wet gel; it was reversibly inactivated with more.     

Accuracy of endometrial aspiration in the diagnosis of endometrial cancer

A study was performed to evaluate the cytologic criteria for recognizing endometrial cancer and to determine the accuracy of endometrial aspiration in its detection. In addition to the conventional cytologic criteria for the diagnosis of endometrial cancer, the irregular chromatin distribution around the macronucleoli proved to be a useful criterion. Using all criteria, cytology was reported as positive in 18 of 19 patients (94.7%) with endometrial cancer. In 856 of 12,563 high-risk outpatients at Kinki University Hospital, aspiration using the Masubuchi apparatus was carried out in screening for endometrial cancer. Cancer was detected in 18 patients (2.1%), with 94.7% of the cancers detected by the cytologic screening. This result indicates that endometrial aspiration using the Masubuchi apparatus is a reliable and safe method of screening for endometrial cancer.     

A role of the B-oligomer moiety of islet-activating protein, pertussis toxin, in development of the biological effects on intact cells

Islet-activating protein (IAP), pertussis toxin, is an oligomeric protein (Tamura, M., Nogimori, K., Murai, S., Yajima, M., Ito, K., Katada, T., Ui, M., and Ishii, S. (1982) Biochemistry 21, 5516-5522), the biggest subunit (Mr = 28,000, referred to as the A-protomer) of which catalyzes transfer of the ADP-ribose moiety of NAD to the membrane Mr = 41,000 protein. The pentamer, termed the B-oligomer, consisting of the residual subunits was the moiety of IAP that was responsible for binding to the cell surface, as revealed by competitive inhibition of the development of the IAP actions on intact rat C6 glioma cells and rat adipocytes. The binding of the B-oligomer to its receptor proteins was divalent via the constituent two dimers; it stimulated mitosis of lymphocytes and caused an insulin-like action to enhance glucose oxidation in adipocytes, just as did concanavalin A, presumably as a result of cross-linking or aggregation of the membrane proteins. The A-promoter displayed its biological action on adipocytes only when the B-oligomer had been bound to the cells. Thus, IAP is a typical A-B toxin in which the B-oligomer is first bound to the cell surface proteins to enable the A-protomer to reach to the site of its action within the cell. Diverse biological actions of pertussis toxin may be accounted for by the mitogenic action of the B-oligomer as well as ADP-ribosyltransferase activity of the A-promoter.     

Effects of W-7 on catecholamine release and 45Ca2+ uptake in cultured adrenal chromaffin cells.

Effects of N-(6-aminohexyl)-5-chloro-1-naph-thalenesulfonamide (W-7), a calmodulin antagonist, on catecholamine (CA) release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. W-7 inhibited the carbamylcholine (CCh)-evoked CA release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner. The inhibitory effect of W-7 on CCh-evoked CA release was not overcome either by an increase in extracellular calcium or CCh concentration. Although W-7 inhibited the high K⁺-evoked CA release and ⁴⁵Ca²⁺ uptake, potency of the drug was approximately 50–100 fold less than when inhibiting the CCh-evoked CA release and ⁴⁵Ca²⁺ uptake. The inhibitory effects of W-7 were observed both in norepinephrine release and epinephrine release. Moreover, W-7 inhibited the CCh-evoked ⁴⁵Ca²⁺ efflux. These results suggest that the inhibition of CA release by W-7 in adrenal chromaffin cells is mainly due to its inhibition of calcium uptake. W-7 may influence the linkage between acetylcholine-receptor and calcium uptake with higher potency than depolarization-dependent calcium entry.