Genetic Divergence among Southeast and East Asian Populations of Rana limnocharis (Amphibia: Anura), with Special Reference to Sympatric Cryptic Species in Java

An electrophoretic survey was conducted for the Southeast and East Asian populations of Rana limnocharis. Results indicated that specimens from a single locality in Java derived from two genodemes showing complete allelic displacement at eight out of 25 presumptive loci examined. Within each of these genodemes, genotype frequencies at all polymorphic loci were well concordant with the Hardy-Weinberg expectation. These results strongly suggest that those specimens actually represent two biological species. Comprehensive distance analyses suggested that one of the two species from Java is genetically most divergent among all samples examined, whereas the other is most similar to the Laos sample and then to a group consisting of samples from Hongkong, western China, and the southern Ryukyus. Large values of genetic distances obtained for each combination of allopatric samples imply the presence of additional cryptic species in this nominal species.     

CO binding studies of nitric oxide synthase: effects of the substrate, inhibitors and tetrahydrobiopterin

The dissociation constant (K_d) for CO from neuronal nitric oxide synthase heme in the absence of the substrate and cofactor was less than 10⁻³ μM. In the presence of

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-Arg, it dramatically increased up to 1 μM. In the presence of inhibitors such as N^G-nitro-

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-arginine methyl ester and 7-nitroindazole (NI), the K_d value further increased up to more than 100 μM. Addition of the cofactor, 5,6,7,8-tetrahydrobiopterin (H₄B), increased the K_d value by 10-fold in the presence of

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-Arg, whereas it decreased the value to less than one 250th in the presence of NI. Addition of H₄B increased the recombination rate constant (k_on) for CO by more than two-fold in the presence of

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-Arg or N⁶-(1-iminoethyl)-

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-lysine, whereas it decreased the k_on value by three-fold in the presence of

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-thiocitrulline. Thus, the binding fashion of some of inhibitors, such as NI, may be different from that of

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-Arg with respect to the H₄B effect.     

Newly developed selective immunoinactivation assay revealed reduction in adipose triglyceride lipase activity in peripheral leucocytes from patients with idiopathic triglyceride deposit cardiomyovasculopathy

Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare and newly identified disease among patients requiring cardiac transplantation. TGCV is characterized by cardiomyocyte steatosis and triglyceride (TG)-deposit atherosclerosis, resulting from the abnormal intracellular metabolism of TG. TGCV is classified into primary and idiopathic types. Primary TGCV carries ultra-rare genetic mutations in the adipose triglyceride lipase (ATGL), a rate-liming enzyme that hydrolyzes intracellular TG in adipose and non-adipose tissues. Idiopathic TGCV, first identified among autopsied individuals with diabetes mellitus (DM) with severe heart diseases, shows no ATGL mutations and its causes and underlying mechanisms are still unknown. TGCV is difficult to diagnose in daily clinics, thereby demanding feasible diagnostic procedures. We aimed to develop an assay to measure ATGL activity using peripheral leucocytes. Human his6-ATGL was expressed in COS1 cells, purified to homogeneity, and used to raise a polyclonal antibody neutralizing TG-hydrolyzing activity of ATGL. We developed a selective immunoinactivation assay (SIIA) for the quantitation of ATGL activity in cell lysates of leucocytes by the antibody neutralizing ATGL activities. ATGL activity was measured in 13 idiopathic TGCV patients, with two patients with primary TGCV as the negative control. Healthy (non-DM) and DM controls without heart diseases were also subjected. The developed SIIA assay revealed significant reduction in ATGL activity in leucocytes from patients with idiopathic TGCV who did not carry ATGL mutations as compared with non-DM and DM controls. Thus, ATGL in leucocytes may be an important biomarker for the diagnosis of TGCV and our assay may provide insights into pathophysiology and elucidate the underlying mechanism of TGCV and related disorders.     

Prolonged effect of fluid flow stress on the proliferative activity of mesothelial cells after abrupt discontinuation of fluid streaming

Encapsulating peritoneal sclerosis (EPS) often develops after transfer to hemodialysis and transplantation. Both termination of peritoneal dialysis (PD) and transplantation-related factors are risks implicated in post-PD development of EPS, but the precise mechanism of this late-onset peritoneal fibrosis remains to be elucidated. We previously demonstrated that fluid flow stress induced mesothelial proliferation and epithelial-mesenchymal transition via mitogen-activated protein kinase (MAPK) signaling. Therefore, we speculated that the prolonged bioactive effect of fluid flow stress may affect mesothelial cell kinetics after cessation of fluid streaming. To investigate how long mesothelial cells stay under the bioactive effect brought on by fluid flow stress after removal of the stress, we initially cultured mesothelial cells under fluid flow stress and then cultured the cells under static conditions. Mesothelial cells exposed to fluid flow stress for a certain time showed significantly high proliferative activity compared with static conditions after stoppage of fluid streaming. The expression levels of protein phosphatase 2A, which dephosphorylates MAPK, in mesothelial cells changed with time and showed a biphasic pattern that was dependent on the duration of exposure to fluid flow stress. There were no differences in the fluid flow stress-related bioactive effects on mesothelial cells once a certain time had passed. The present findings show that fluid flow stress exerts a prolonged bioactive effect on mesothelial cells after termination of fluid streaming. These findings support the hypothesis that a history of PD for a certain period could serve as a trigger of EPS after stoppage of PD.     

Endoplasmic reticulum stress induces the phosphorylation of small heat shock protein, Hsp27

There are several reports describing participation of small heat shock proteins (sHsps) in cellular protein quality control. In this study, we estimated the endoplasmic reticulum (ER) stress-induced response of Hsp27 and alphaB-crystallin in mammalian cells. Treatment targeting the ER with tunicamycin or thapsigargin induced the phosphorylation of Hsp27 but not of alphaB-crystallin in U373 MG cells, increase being observed after 2-10 h and decline at 24 h. Similar phosphorylation of Hsp27 by ER stress was also observed with U251 MG and HeLa but not in COS cells and could be blocked using SB203580, an inhibitor of p38 MAP kinase. Other protein kinase inhibitors, like G?6983, PD98059, and SP600125, inhibitors of protein kinase C (PKC), p44/42 MAP kinase, and JNK, respectively, were without major influence. Prolonged treatment with tunicamycin but not thapsigargin for 48 h caused the second induction of the phosphorylation of Hsp27 in U251 MG cells. Under these conditions, the intense perinuclear staining of Hsp27, with some features of aggresomes, was observed in 10%-20% of the cells.     

Food preferences and nematode parasitism in mycophagousDrosophila

Food preferences and nematode parasitism were studied in natural populations of mycophagousDrosophila in and near Sapporo, northern Japan. Species which preferred fresh mushrooms showed species-specific responses toPleurotus mushrooms:D. pirka bred only onPleurotus cornucopiae, D. trivitata onP. cornucopiae andP. ostreatus, D. trilineata on these twoPleurotus mushrooms and some other mushrooms, whileD. sexvittata bred on a wide variety of mushrooms but seldom onPleurotus mushrooms. Species which preferred decayed mushrooms (D. quadrivittata, D. histrioides, D. testacea and species of thequinaria species-group) showed host preferences different from those of the above species. The rate of parasitism by nematodes was generally higher in species which prefer decayed mushrooms than in species which prefer fresh mushrooms. Among species which prefer fresh mushrooms, onlyD. trilineata was parasitized frequently by nematodes. It was not clear what factors determine the rate of parasitism in these mycophagousDrosophila. D. pirka, D. trivittata andD. trilineata passed through three or four generations per year and entered reproductive diapause in early September in and near Sapporo. Bionomics of Drosophilidae (Diptera) in Hokkaido, IX.     

Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Incomplete paternal inheritance of chloroplast DNA recognized in Chamaecyparis obtusa using an intraspecific polymorphism of the trnD-trnY intergenic spacer region

Using a fluorescence-based PCR-SSCP (single-strand conformation polymorphism), we verified imperfectibility in the paternal inheritance of chloroplast DNA (cpDNA) in Chamaecyparis obtusa (Cupressaceae) controlled crosses. An intraspecific sequence polymorphism of the intergenic spacer region between the trnD and trnY genes was utilized as a molecular marker. Of 361 progenies, in which the cpDNA haplotypes of their female and male parents were different, 352 (97.5%) possessed the same haplotypes as their male parents, and nine (2.5%) the same haplotypes as their female parents. The parentage of the nine progenies with female parental types was diagnosed using DNA fingerprinting based on fluorescence-based RAPD profiles. Their parentage showed convincing evidence of the low frequency of maternal inheritance. Moreover, heteroplasmy was observed in the open-pollinated seeds collected in a seed orchard. The confirmation of maternal plastid transmission in the full-sib families and the observation of heteroplasmy in seeds reveal that the paternal inheritance of cpDNA is not an exclusive phenomenon and that the mode of its inheritance is biparental in C. obtusa. Received: 15 April 2000 / Accepted: 13 July 2000     

Two-dimensional electrophoresis on the layers of cellulose acetate membrane

A new type of two-dimensional electrophoresis for analysis of protein using cellulose acetate membrane has been developed. Prior to the separation, proteins in a sample are concentrated to a narrow zone on a strip of cellulose acetate according to “steady-state stacking” of isotachophoresis. Electroendosmotic counterflow on cellulose acetate membranes is advantageous for the isotachophoretic concentration of large sample volumes. The concentrated protein zone is then subjected to electrophoretic separation on the same strip. This first-dimensional separation including the concentrating process is named “concentrating electrophoresis.” Iso-electric focusing on several layers of cellulose acetate membrane is performed in the second-dimensional step. Many kinds of detection methods can be applied to the layers among which proteins are distributed. The novel two-dimensional electrophoresis takes only 5 h to perform.     

Design and synthesis of new potent dipeptidyl peptidase IV inhibitors with enhanced ex vivo duration

A series of 5beta-methylprolyl-2-cyanopyrrolidine analogs were synthesized and evaluated as DPP-IV inhibitors, and the duration of their ex vivo activity was assessed. Comparison of their potency and duration of action was done among three different species. The mode of binding was investigated, and the effect on the plasma glucose level was evaluated. Structure-activity relationships are also presented.