Geographic distribution and environmental control of vertebral count in Ammodytes spp. along the northern Pacific coast of Japan.

To examine species composition and population structures in sand lance (Ammodytidae) along the northern Pacific coast of Japan, genetic analysis were carried out for specimens collected in 2014 from Otsuchi Bay, Iwate, Ishinomaki Bay, Miyagi, off Soma, Fukushima and Ise-Mikawa Bays, Aichi. The samples consisted of Ammodytes japonicus and Ammodytes heian, of which the latter is a recently described species. Neither species exhibited significant genetic differences among localities. Only A. japonicus was found in the most southern locality at Aichi, but it decreased northward to <90% in Miyagi and Fukushima and the two species occurred almost evenly in Iwate suggesting a latitudinal cline in their species composition along the northern Pacific coast of Japan, off Tohoku. The vertebral counts differed between A. japonicus and A. heian with modes of 65 and 63, respectively, but this characteristic did not differ significantly within a locality (Iwate). This suggests that the vertebral counts of Ammodytes spp. in Japanese waters are probably strongly determined by the environment than by a species-specific genetic trait.

Reproductive isolation between sympatric Anguilla japonica and Anguilla marmorata.

Species-specific restriction fragment length polymorphism in the intron of the androgen receptor gene (ar5) was found in glass to silver-stage individuals of Anguilla japonica (n = 51) and A. marmorata (n = 21). The sequence analysis of 16S rDNA from 328 anguillid leptocephali collected in the North Equatorial Current of the western North Pacific Ocean revealed the specimens to be A. japonica (n = 194), A. marmorata (n = 128), A. bicolor pacifica (n = 5) and A. luzonensis (n = 1). All leptocephali of A. japonica and A. marmorata were monomorphic and did not share an allele at the ar5 locus, indicating that the two species are reproductively isolated.

Rapid current ramp-up by cyclotron-driving electrons beyond runaway velocity.

The toroidal current has been rapidly ramped-up after the formation of an initial closed flux surface in an electron cyclotron heated discharge in the low aspect ratio torus experiment device. A current carrying fast electron tail is developed well beyond the runaway velocity against the reverse voltage from self-induction, suggesting a forward driving force on the tail by the cyclotron absorption of high N(parallel) electron Bernstein waves.

The effect of group-based lifestyle interventions on risk factors and insulin resistance in subjects at risk for metabolic syndrome: the Tabaruzaka Study 1.

AIM: The aim of this study was to evaluate the efficacy of two group-based lifestyle interventions in ameliorating the risk factors of metabolic syndrome (MS) and insulin resistance. METHODS: Ninety-eight subjects who had at least one component of MS were randomized into standard intervention (SI) (4-month intervention; n = 50) and extended intervention (EI) (10-month intervention; n = 48) groups, and 39 subjects were followed up for a control group. The effects of intervention were evaluated after 10, 22 and 34 months. RESULTS: At month 10, the standard and EI groups showed improved body mass index (BMI) (SI, -0.28; EI, -0.47; control, -0.09), high-density lipoprotein (HDL) cholesterol, fasting plasma glucose and A1c and a decreased mean number of components of MS (SI, -0.37; EI, -0.51; control, 0.08). At month 34, the effects on BMI (SI, -0.66; EI, -0.60; control, -0.05) and HDL-cholesterol were sustained for both the intervention groups. In controls, the increases in fasting plasma glucose and the mean number of components of MS from the baseline to month 34 were greater than those in the standard and EI groups. Whole body insulin sensitivity index and hepatic insulin resistance index were also improved at month 10. CONCLUSIONS: Group-based lifestyle intervention could be an efficient way to prevent MS. Its effects were sustainable, at least in part, for 2 years. These effects may be mediated by an improvement in insulin sensitivity.

Sympatric spawning of Anguilla marmorata and Anguilla japonica in the western North Pacific Ocean.

Extensive collections were made of the larvae of the temperate Japanese eel Anguilla japonica and the tropical giant mottled eel Anguilla marmorata in an overlapping area of the North Equatorial Current region of the western North Pacific Ocean. Collections of 189 A. marmorata and > 2500 A. japonica larvae during nine surveys from 1991 to 2007 showed that these two anguillid eels have similar spawning areas just west of the southern West Mariana Ridge. In July to August 2006 and August 2007, morphologically and genetically identified A. marmorata preleptocephali were mainly collected between 14.5-15 degrees N and 142-142.5 degrees E, where A. japonica preleptocephali were also caught in some of the same net tows. Fewer A. marmorata preleptocephali, however, were collected (n = 31) compared to those of A. japonica (n = c. 165), and fewer small larvae of A. marmorata were collected per tow than A. japonica (n = 1-10 and 1-294, respectively), suggesting relatively smaller spawning aggregations of A. marmorata. The distribution of preleptocephali and small larvae was wider in longitude in A. marmorata (131- 143 degrees E) than in A. japonica (137-143 degrees E), while the latitudinal range was almost the same (12-17 degrees N). Although spawning by these two species overlaps both spatially and temporally, the tropical eels of the North Pacific population of A. marmorata probably have a much longer spawning season with fewer spawners, at least in summer, and recruit to a much wider latitudinal range of growth habitats.

Neural stem cell transplantation in a model of fetal alcohol effects.

Neural stem cell (NSC) transplantation has been investigated and developed in areas such as brain injury, stroke and neurodegenerative diseases. Recently, emerging evidence suggest that many of clinical symptoms observed in psychiatric disease are likely related to neural network disruptions including neurogenesis dysfunction. In the present study, we transplanted NSCs into a model of fetal alchol effects (FAE) for the purpose of investigating the possibility of regenerative therapy for the FAE. We labeled NSCs with fluorescent dye and radioisotope which were transplanted into FAE rats by intravenous injection. The transplanted cells were detected in wide areas of brain and were greater in number in the brains of the FAE group compared to the control group. Furthermore NSC transplantation attenuated behavioral abnormalities in FAE animals. These results suggest NSC transplantation as a potental new therapy for human FAE.

Phosphoenolpyruvate carboxylase of Escherichia coli. Studies on multiple conformational states elicited by allosteric effectors with a fluorescent probe, 1-anilinonaphthalene-8-sulfonate.

Conformational change of phosphoenolpyruvate carboxylase (orthophosphate: oxaloacetate carboxy-lyase (phosphorylating), EC 4.1.1.31) induced by allosteric effectors was investigated using a hydrophobic probe, 1-anilinonaphthalene-8-sulfonate (ANS). Kinetic experiments suggested that ANS binds with the enzyme at the sites which are not involved in the catalytic and regulatory functions, though it partially inhibits the enzyme activity with half-saturation concentration (S0.5) of 38.5 muM. Binding experiments showed that a maximum of 2 mol of ANS are able to bind with 1 mol of the enzyme subunit presumably with an equal dissociation constant to each other (34.5 muM). Flourescence emission of ANS was markedly increased by binding with the enzyme. L-Aspartate, the allosteric inhibitor, and CoASAc and fructose 1,6-bisphosphate (Fru-1,6-P2) the allosteric activators, produced various degrees of change in fluorescence, when added singly or in combinations. The changes were shown to be attributable to the allosteric interactions between the enzyme and effectors from some criteria such as structural specificity, half-saturation concentrations, and heterotropic-homotropic interactions of the ligands. It was concluded from these analyses that the enzyme can be in at least four conformational states which are distinct from each other. Especially noteworthy is the finding that the enzyme, upon simultaneous binding of CoASAc and Fru-1,6-P2, takes a new conformation which is enterely different from those induced by sole binding of each effector. In addition, the heterotropic interaction between the activator and the inhibitor was observed through conformational change by the ANS method, as observed in the kinetic studies.

The role of zinc with special reference to the essential thiol groups in delta-aminolevulinic acid dehydratase of bovine liver.

delta-Aminolevulinic acid dehydratase (5-aminolevulinic acid hydro-lyase (adding 5-aminolevulinic acid and cyclizing), EC 4.2.1.24 purified from bovine liver in the presence of both SH-reducing reagent and zinc during the purification contained one zinc atom and eight SH groups/subunit. This preparation showed the full enzymatic activity even in the absence of thiol activator. It was found that two cysteine residues, one zinc atom and two histidine residues were involved in the active site. The enzyme was fullly active as long as two SH groups in the active site remained in the reduced form even in the absence of zinc. However, the enzymatic activity was completely lost, with a concomitant loss of bound zinc, upon oxidation of the SH groups to a disulfide bond, modification of SH groups with chemical reagents, or mercaptide formation by heavy metals. Thus, it is apparent that the activity depends on the essential SH groups. The zinc is not absolutely essential for the activity but may be required to prevent the essential SH groups from autooxidation by coordination. Binding experiments indicated that there was one binding site of zinc/subunit. Photooxidation of histidine residues diminished both enzymatic activity and bound zinc, suggesting that the histidine residues not only constituted the active site but also served as a possible ligand to zinc.

Mouse coproporphyrinogen oxidase is a copper-containing enzyme: expression in Escherichia coli and site-directed mutagenesis.

We previously isolated cDNA for mouse coproporphyrinogen oxidase (CPO) and provided evidence for the induction of mRNA during differentiation of murine erythroleukemia cells (Kohno et al. (1993) J. Biol. Chem. 268, 21359-21363). To better understand the structure and the mechanisms of reaction of the enzyme, we expressed mouse CPO in Escherichia. coli and purified it to a homogeneity. Analysis of the metal content revealed that the recombinant mouse CPO contains one copper atom per polypeptide chain. When the bacterial cells were treated with D-penicillamine, a copper chelator, formation of the active CPO was partially reduced. Addition of Cu2+ in minimal medium resulted in 6-fold higher level of CPO activity. These results suggest that expression of active mouse CPO in E. coli depended on the presence of Cu2+ in the culture medium. To elucidate the apparent involvement of Cu2+ in enzyme function, a series of mutant enzymes, whose highly conserved histidine and cysteine residues were individually converted to alanine residue, were prepared by site-directed mutagenesis. Mutant enzymes were expressed in E. coli and their activities examined. Mutation at histidine 158 resulted in a complete loss of enzyme activity, yet the enzyme protein was expressed at a comparable level. Concomitantly, only a trace amount of Cu2+ was detected in the purified H158A enzyme. We propose that mouse CPO is copper-containing enzyme and Cu2+ interacts with a conserved histidine residue.

Molecular cloning, sequencing and expression of cDNA encoding human coproporphyrinogen oxidase.

A complete cDNA clone encoding human coproporphyrinogen (coprogen) oxidase, the sixth enzyme in the heme biosynthetic pathway, has been isolated from a human placenta cDNA library. The cDNA had an open reading frame of 1062 base pairs encoding a protein of 354 amino acid residues (M(r) 40,291). Amino acid sequencing showed that the mature enzyme consists of 323 amino acid residues (M(r) 36,842) with a putative leader peptide of 31 amino acid residues. The human enzyme showed an 86% identity to the mouse enzyme. In addition, the recombinant enzyme which did not contain leader peptide was actively expressed in Escherichia coli. The isolation and expression of cDNA for human coprogen oxidase should facilitate studies of the structure of the gene as well as characterization of molecular lesions causing hereditary coproporphyria.

Sequential activation of genes for heme pathway enzymes during erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells.

Changes in the level of transcripts encoding enzymes of the heme biosynthetic pathway as well as those encoding ubiquitous proteins were examined in murine Friend virus-transformed erythroleukemia cells during erythroid cell differentiation induced by chemicals including dimethyl sulfoxide (DMSO). Early changes following DMSO treatment were marked decreases in mRNAs for three ubiquitous proteins, i.e., a 70 kDa heat shock protein (less than 6 h), heme oxygenase and nonspecific delta-aminolevulinate synthase (ALAS) (less than 12 h). These changes were followed by sequential increases in mRNAs for enzymes in the heme biosynthetic pathway. Namely, mRNAs for the erythroid-specific ALAS, delta-aminolevulinate dehydratase, porphobilinogen deaminase and uroporphyrinogen decarboxylase started to increase at 12, 18, 18-24 and 24 h, respectively. Nuclear runoff studies revealed that these changes are largely transcriptional. Treatments with other inducers of erythroid differentiation, e.g., hexamethylene bisacetamide, n-butyric acid and N'-methylnicotinamide, also showed similar effects on mRNAs as those following DMSO. These findings suggest that both suppression of ubiquitous genes and activation of heme pathway enzyme genes are associated with erythroid differentiation, and the former occurs preceding changes in the latter.

The human 32-kDa stress protein induced by exposure to arsenite and cadmium ions is heme oxygenase.

Exposure of HeLa and HL60 cells to sodium arsenite or cadmium chloride led to marked increases in cellular heme oxygenase activity. SDS-polyacrylamide gel electrophoresis of [35S]methionine-labeled cellular proteins indicated that these treatments also resulted in the induction of a 32-kDa protein. Immunoblot analysis further showed that the 32-kDa protein reacted with anti-bovine heme oxygenase antibodies. Treatment of the cells with cobaltic chloride or heat induced neither the 32-kDa protein nor heme oxygenase activity. It is concluded that the 32-kDa stress protein induced by arsenite and cadmium ions in these human cells is heme oxygenase.

Plausible phosphoenolpyruvate binding site revealed by 2.6 A structure of Mn2+-bound phosphoenolpyruvate carboxylase from Escherichia coli.

We have determined the crystal structure of Mn2+-bound Escherichia coli phosphoenolpyruvate carboxylase (PEPC) using X-ray diffraction at 2.6 A resolution, and specified the location of enzyme-bound Mn2+, which is essential for catalytic activity. The electron density map reveals that Mn2+ is bound to the side chain oxygens of Glu-506 and Asp-543, and located at the top of the alpha/beta barrel in PEPC. The coordination sphere of Mn2+ observed in E. coli PEPC is similar to that of Mn2+ found in the pyruvate kinase structure. The model study of Mn2+-bound PEPC complexed with phosphoenolpyruvate (PEP) reveals that the side chains of Arg-396, Arg-581 and Arg-713 could interact with PEP.

Homozygosity for the transthyretin-Met30 gene in three Japanese siblings with type I familial amyloidotic polyneuropathy.

We report the cases of three siblings homozygous for a mutated transthyretin (TTR) gene that causes type I familial amyloidotic polyneuropathy (FAP), in whom we made the diagnosis by identifying both the mutated TTR gene and a variant TTR in their sera. Their serum levels for the variant TTR are twice those of patients heterozygous for the gene, but two have late-onset FAP and the third is an elderly asymptomatic carrier. TTR abnormality is a necessary condition for the development of FAP, but there may be other factors that retard or prevent its clinical development.

Reduced amount of secretory component of IgA secretion in tears of patients with atopic dermatitis.

Coupled with the previous finding that sIgA excretion was reduced onto the surface of the skin, we demonstrated that sIgA secretion in the tears of patients with atopic dermatitis (AD) was significantly lower than that of normal subjects, using a small stick made of nitrocellulose membrane. In the bacterial cultures, we have also detected a higher frequency of Staphylococcus aureus in the tears from patients with AD compared to normal subjects. These findings suggested reduced sIgA secretion on the mucous membrane might play a crucial role in the pathomechanisms of the ocular lesions, such as abnormal bacterial flora and ocular complications as well as the establishment of skin lesions in AD.

Structural specificity of the allosteric inhibitor of phosphoenolpyruvate carboxylase of Escherichia coli.

The structural specificity of the allosteric inhibitor of phosphoenolpyruvate carboxylas [EC 4.1.1.31] of Escherichia coli W was investigated using native enzyme and photooxidized enzyme which was desensitized to L-aspartate. Inhibitory activity was expressed in terms of the concentration of the compound required for 50% inhibition (I0.5). For the native enzyme, L-aspartate and L-malate were the strongest inhibitors with I0.5 values of about 0.10-0.15 mM among about 20 componds tested. For the photooxidized enzyme, oxaloacetate and L-malate were relatively strong inhibitors wiht I0.5 values of about 11-16 mM. The results obtained suggest that the inhibition of the native enzyme mainly reflects allosteric inhibition.

Phosphoenolpyruvate carboxylase of Escherichia coli. The role of lysyl residues in the catalytic and regulatory functions.

Phosphoenolpyruvate (PEP) carboxylase [EC 4.1.1.31] of E. coli was inactivated by 2,4,6-trinitrobenzene sulfonate (TNBS), a reagent known to attack amino groups in polypeptides. When the modified enzyme was hydrolyzed with acid, epsilon-trinitrophenyl lysine (TNP-lysine) was identified as a product. Close similarity of the absorption spectrum of the modified enzyme to that of TNP-alpha-acetyl lysine and other observations indicated that most of the amino acid residues modified were lysyl residues. Spectrophotometric determination suggested that five lysyl residues out of 37 residues per subunit were modified concomitant with the complete inactivation of the enzyme. DL-Phospholactate (P-lactate), a potent competitive inhibitor of the enzyme, protected the enzyme from TNBS inactivation. The concentration of P-lactate required for half-maximal protection was 3 mM in the presence of Mg2+ and acetyl-CoA (CoASAc), which is one of the allosteric activators of the enzyme. About 1.3 lysyl residues per subunit were protected from modification by 10 mM P-lactate, indicating that one or two lysyl residues are essential for the catalytic activity and are located at or near the active site. The Km values of the partially inactivated enzyme for PEP and Mg2+ were essentially unchanged, though Vmax was decreased. The partially inactivated enzyme showed no sensitivity to the allosteric activators, i.e., fructose 1,6-bisphosphate (Fru-1,6-P2) and GTP, or to the allosteric inhibitor, i.e., L-aspartate (or L-malate), but retained sensitivities to other activators, i.e., CoASAc and long-chain fatty acids. P-lactate, in the presence of Mg2+ and CoASAc, protected the enzyme from inactivation, but did not protect it from desensitization to Fru-1,6-P2, GTP, and L-aspartate. However, when the modification was carried out in the presence of L-malate, the enzyme was protected from desensitization to L-aspartate (or L-malate), but was not protected from desensitization to Fru-1,6-P2 and GTP. These results indicate that the lysyl residues involved in the catalytic and regulatory functions are different from each other, and that lysyl residues involved in the regulation by L-aspartate (or L-malate) are also different from those involved in the regulation by Fru-1,6-P2 and GTP.

Induction in mouse peritoneal macrophages of 34 kDa stress protein and heme oxygenase by sulfhydryl-reactive agents.

The synthesis of 34-kDa stress protein was enhanced, with a simultaneous increase in heme oxygenase activity, when mouse macrophages were exposed to diethylmaleate or sodium arsenite. After 7 h of exposure to the sulfhydryl agents, the 34-kDa protein was the most actively synthesized protein. Immunoblot analysis showed that the induced 34-kDa protein reacted with an antibody raised against bovine heme oxygenase. Cadmium ions or 1-chloro-2,4-dinitrobenzene also induced the 34-kDa protein which reacted with the antibody. Treatments of the cells with buthionine sulfoximine or hydrogen peroxide weakly induced the protein, while diamide treatment or heat shock was without effect. These results are consistent with our previous findings that heavy metal ions including arsenite and cadmium ions induce heme oxygenase (32-kDa stress protein) in human cell lines [Taketani, S., Kohno, H., Yoshinaga, T., & Tokunaga, R. (1989) FEBS Lett. 245, 173-176], and also suggest that the formation of glutathione conjugate with sulfhydryl-reactive agents may mediate the induction of the stress protein in mouse peritoneal macrophages.

Differential nociceptive responses in mice lacking the alpha(1B) subunit of N-type Ca(2+) channels.

The role of N-type Ca(2+) channels in nociceptive transmission was examined in genetically engineered mice lacking the alpha(1B) subunit of N-type channels and in their heterozygote and wild-type littermates. In alpha(1B)-deficient mice, N-type channel activities in dorsal root ganglion neurons and spinal synaptoneurosomes were eliminated without compensation by other types of voltage-dependent Ca(2+) channels. The alpha(1B)-deficient mice showed a diminution in the phase 2 nociceptive responses more extensively than in the phase 1 nociceptive responses of the formalin test. The alpha(1B)-deficient mice exhibited significantly increased thermal nociceptive thresholds in the hot plate test, but failed to increase mechanical nociceptive thresholds in the tail pinch test. These results suggest a crucial role of N-type channels in nociceptive transmission, especially for persistent pain like phase 2 of the formalin test and for nociception induced by thermal stimuli.

Myonuclear domain and myosin phenotype in human soleus after bed rest with or without loading.

After 2 or 4 mo of bed rest (6 degrees head-down tilt) and 1 mo of ambulation, there was a tendency toward a higher percentage of fibers expressing fast myosin heavy chain (MHC) isoforms and a de novo appearance of fibers coexpressing type I+IIa+IIx and IIa+IIx MHC in human soleus fibers. After 2 and 4 mo of bed rest, the mean size of type I fibers decreased by 12 (P > 0.05) and 39%, respectively. Because myonuclear number/mm of fiber length was unchanged, myonuclear domain was smaller after bed rest than before. The mean size and myonuclear domain of type I fibers were largest after 1 mo of recovery. The effects of wearing an antigravity device (Penguin suit), which had a modest but continuous resistance at the knee and ankle (Penguin-1) or knee resistance without loading on the ankle (Penguin-2), for 10 consecutive h/day were determined during 2 mo of bed rest. Mean fiber sizes in Penguin-1, but not Penguin-2, group were maintained at or above pre-bed-rest levels, whereas neither group showed phenotype changes. Myonuclear domain in type I fibers was larger in Penguin-1 and smaller in Penguin-2 group post- compared with pre-bed rest, indicating that a single daily 10-h bout of modest muscle loading can prevent bed-rest-induced soleus fiber atrophy but has minimal effect on myosin phenotype. The specific adaptive cellular strategies involved may be a function of the duration and magnitude of the adaptive stimulus as well as the immediate activity history of the fiber before the newly changed functional demands.