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.     

Hypersensitivity of Bloom's syndrome fibroblasts to N-ethyl-N-nitrosourea

Fibroblast cells from two Japanese patients with Bloom's syndrome (BS) and normal donors were studied for the inactivation of colony-forming ability and the induction of sister-chromatid exchanges (SCEs) after N-ethyl-N-nitrosourea (ENU) treatment. The reduction of ENU-induced SCEs as a function of post-treatment incubation time was also compared between BS and normal fibroblasts. BS cells were approximately 4 times more sensitive than normal cells to the lethal effect of ENU and remarkably hypersensitive to the SCE induction by ENU. The post-treatment incubation of ENU-treated normal cells in the fresh medium resulted in a time-dependent decrease of the SCE level until 6 h after which time the SCE level remained the plateau of about 50% of the initial level. In contrast, the ENU-induced SCEs in BS cells decreased much more slowly with post-treatment incubation time and its half life was 24 h. These results collectively support the view that BS cells may be defective in the rapid repair of certain type(s) of DNA damages induced by ENU.     

Sister-chromatid exchanges induced by ultraviolet light in Bloom's syndrome fibroblasts

The relationships between the cytotoxic effect of ultraviolet light and the UV-induced sister-chromatid exchanges (SCEs) were compared among fibroblast cell strains from two unrelated Bloom's syndrome (BS) patients, one xeroderma pigmentosum (XP) patient belonging to complementation group A and two unrelated normal controls. The "net" induced SCEs as a function of UV fluence, obtained by subtracting spontaneous SCEs from observed SCEs, were much higher in both BS cells and XP group A cells than in normal cells. The relative efficiency of induced SCE, defined as the "net" induced SCEs as a function of surviving fraction after UV irradiation, was higher in BS cells than in normal and XP cells, and there was essentially no difference between XP and normal cells. These results imply that in addition to the extremely high frequency of spontaneous SCEs, the increased efficiency in UV induction of SCEs may reflect the intrinsic defect(s) in BS cells.     

Heterogeneity of intercellular adhesion in rat liver cells in culture

The intercellular homotypic adhesive properties of 14 clones derived from a nontumorigenic rat liver epithelial cell line (LEC), derived from neonatal Fischer rats, were examined and compared to those of the hepatoma H4-II-E cell line. Each clone was assayed also for the degree of chromosomal aneuploidy and the ability to grow in soft agar. Over 100-fold differences in adhesive properties were observed among the clones, but no correlation was observed between the degree of aneuploidy in the clones and intercellular adhesive properties. The parent LEC cell line and the clones derived from it were unable to grow in soft agar. The H4-II-E cells showed negligible capacity to reaggregate after dissociation into single cells and these cells readily formed colonies in soft agar. Many of the LEC clones were similar to the H4-II-E cells in their adhesive properties, which suggests that reduced cell-to-cell adhesiveness per se is not a necessary prerequisite of epithelial cells to be able to grow independent of anchorage. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of concanavalin A (Con A)-binding glycoproteins in the "most adhesive" clone 67 and the "least adhesive" clone 201 showed markedly elevated amounts of acidic 105 and 67-kDa glycoproteins in clone 67. Proteins with similar migration patterns in 2D-PAGE have previously been reported to participate in specific homotypic intercellular adhesion of liver cells. The Con A-binding glycoprotein pattern in H4-II-E cells was markedly different from that of LEC cells with a set of six proteins missing and nine proteins appearing new in the H4-II-E cells. It is suggested that, in addition to identifying known epithelial cell polypeptides, systematic screening of cell surface-associated glycoproteins in normal and transformed epithelial cells in vitro and in vivo may lead to identification of novel polypeptides intimately associated with the transformed phenotype.     

Biotransformation of 1-nitropyrene and 2-nitrofluorene to novel metabolites, the corresponding formylamino compounds, in animal bodies

The present study provides the first evidence that nitropolycyclic aromatic hydrocarbons such as 1-nitropyrene and 2-nitrofluorene are metabolized to the corresponding formylamino compounds in animal bodies. The study shows that the formation of such novel metabolites, 1-formylaminopyrene and 2-formylaminofluorene, is due to N-formylation of the nitroreduction products, 1-aminopyrene and 2-aminofluorene, by liver formamidase in the presence of N-formyl-L-kynurenine.     

Potentiation of harringtonine cytotoxicity by calcium antagonist diltiazem and biscoclaurine alkaloid cepharanthine in adriamycin-resistant P388 murine leukemia and K562 human leukemia cells

Harringtonine showed cross resistance in adriamycin-resistant murine leukemia P388 (P388/ADM) and human leukemia K562 (K562/ADM) cells. The relative resistance of the P388/ADM and K562/ADM cells to harringtonine was about 7 and 40, respectively. Calcium influx blockers, diltiazem and the biscoclaurine alkaloid cepharanthine enhanced the cytotoxicity of harringtonine in P388/ADM and K562/ADM cells. The extent of enhancement was different for the two drugs, and up to a 9- to 10-fold increase in harringtonine cytotoxicity occurred in P388/ADM cells, and 14- to 22-fold enhancement in K562/ADM cells with diltiazem or cepharanthine. Harringtonine resistance of P388/ADM was circumvented completely, and the resistance of K562/ADM was circumvented partially, by diltiazem or cepharanthine. The mechanism of enhanced cytotoxicity by diltiazem and cepharanthine is probably inhibition of active efflux of harringtonine in P388/ADM and K562/ADM cells.     

Instability of Mex- phenotype in human lymphoblastoid cell lines

Three lymphoblastoid cell lines (LCLs) had extremely low activities of O6-alkylguanine-DNA alkyltransferase (O6-AGT), and were classified as Mex-. They were highly sensitive to cell killing by 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitrosoure a hydrochloride (ACNU), whereas NMO2, a Mex+ LCL with a high O6-AGT activity, was resistant to the agent. Small fractions of these Mex- LCLs survived the treatment with 10 micrograms/ml of ACNU for 24 h, and the surviving cells were found to be resistant to subsequent treatments with the agent. In addition, they contained O6-AGT activities comparable to that of NMO2 and were therefore regarded as Mex+. These results suggest that the Mex- phenotype in LCLs is unstable.