Search PubMed⌕ Search

Biomedical subjects

R Ishikawa

Publications and source records attributed to R Ishikawa.

At least 37 records · Page 2Linked to original sources
PubMed1996

Modulatory role of drebrin on the cytoskeleton within dendritic spines in the rat cerebral cortex.

Morphological changes in the dendritic spines have been postulated to participate in the expression of synaptic plasticity. The cytoskeleton is likely to play a key role in regulating spine structure. Here we examine the molecular mechanisms responsible for the changes in spine morphology, focusing on drebrin, an actin-binding protein that is known to change the properties of actin filaments. We found that adult-type drebrin is localized in the dendritic spines of rat forebrain neurons, where it binds to the cytoskeleton. To identify the cytoskeletal proteins that associated with drebrin, we isolated drebrin-containing cytoskeletons using immunoprecipitation with a drebrin antibody. Drebrin, actin, myosin, and gelsolin were co-precipitated. We next examined the effect of drebrin on actomyosin interaction. In vitro, drebrin reduced the sliding velocity of actin filaments on immobilized myosin and inhibited the actin-activated ATPase activity of myosin. These results suggest that drebrin may modulate the actomyosin interaction within spines and may play a role in the structure-based plasticity of synapses.

Actins↗
PubMed1996

NK cell trafficking and cytokine expression in splenic compartments after IFN induction and viral infection.

Studies were undertaken to characterize mechanisms for NK cell cytokine delivery in vivo. Conditions of systemic IFN-beta expression elicited by polyinosinic-polycytidylic acid (poly(I:C)) treatment or IFN-alpha beta production during lymphocytic choriomeningitis virus or murine cytomegalovirus infections resulted in profound splenic histologic changes, with relocalization of nucleated cells from red to white pulp regions. Cell-trafficking experiments, with fluorescently labeled populations, showed that poly(I:C) induced T/B cell-dependent leukocyte migration into white pulp regions. Splenic leukocytes prepared from severe combined immunodeficient (SCID) and bone marrow cells prepared from SCID or normal C57BL/6 mice revealed a unique poly(I:C)-induced accumulation of non-T/non-B cells along splenic red and white pulp region borders characteristic of marginal zones. Lymphocytic choriomeningitis virus and murine cytomegalovirus infections also induced this trafficking pattern. Ab treatments of normal and SCID cell donor mice to eliminate specific cell subsets demonstrated that the unique migration of non-T/non-B cells was dependent upon cells with the NK phenotype, NK1.1+ AGM1+F4/80-. In situ hybridization showed that, at early times after infections, small proportions of cells along red and white pulp divisions expressed high levels of IFN-gamma mRNA, and that these cells were depleted by protocols eliminating NK cells. The results suggest an exciting model for induction of NK cell trafficking from bone marrow to secondary compartments. This migratory pattern may localize NK cells to receive additional activation signals and/or to precisely deliver NK cell-produced cytokines.

Animals↗
PubMed1996

Phylogenetic relationships and evolution of the Japanese Carabinae ground beetles based on mitochondrial ND5 gene sequences.

The phylogenetic relationships of the Japanese Carabinae ground beetles were analyzed by comparing 1,069 nucleotide sequences in the mitochondrial gene encoding NADH dehydrogenase subunit 5 (ND5). The ND5 phylogenetic tree revealed that the hind-wingless Carabina and the hind-winged Calosoma/Campalita (Calosomina) diverged from the common ancestor, and Cychrus (Cychrini) is the outgroup of them. Five distinct clusters (groups) can be recognized in the Carabina, i.e., CARABUS, HEMICARABUS, LEPTOCARABUS, APOTOMOPTERUS, and PROCRUSTES/DAMASTER. The ancestors of these lineages diverged almost at the same time more than 10 Myr ago. The Carabus cluster includes two subclusters, Carabus and Ohomopterus. Two species of Carabus examined are phylogenetically rather remote, while five species among Ohomopterus are closely related to each other. The results suggest that diversification of Carabus started much earlier than that of Ohomopterus, presumably in the Eurasian continent, and that of Ohomopterus in the Japanese archipelago. The branching order in the LEPTOCARABUS lineage was established, Authenocarabus/Pentacarabus being their outgroup. In the DAMASTER/PROCRUSTES lineage, Procrustes is placed as the outgroup of Damaster, with the branching order of Coptolabrus and Acoptolabrus/Damaster. The diversification of the Damaster subspecies appeared to have occurred in the Japanese archipelago earlier than Ohomopterus, and its phylogeny reflects their geographic distribution in the archipelago rather than the morphological characters.

Animals↗
PubMed1996

Parallel evolution in radiation of Ohomopterus ground beetles inferred from mitochondrial ND5 gene sequences.

Molecular phylogenetic analyses using mitochondrial NADH dehydrogenase subunit 5 (ND5) gene sequences representing all 15 species and the majority of subspecies or races of the Ohomopterus ground beetles from all over the Japanese archipelago have uncovered a remarkable evolutionary history. Clustering of the species in the molecular phylogenetic tree is linked to their geographic distribution and does not correlate with morphological characters. Taxonomically the "same" species or the members belonging to the same species-group fall out in more than two different places on the ND5 tree. Evidence has been presented against a possible participation of ancestral polymorphism and random lineage sorting or of hybrid individuals for the observed distribution of mitochondrial DNA haplotypes. The most plausible explanation of our results is that parallel evolution took place in different lineages. Most notably, O. dehaanii, O. yaconinus, and O. japonicus in a lineage reveal almost identical morphology with those of the "same" species (or subspecies) but belonging to the phylogenetically remote lineages.

Animals↗
PubMed1996

Inhibition by drebrin of the actin-bundling activity of brain fascin, a protein localized in filopodia of growth cones.

The purification of drebrin, an actin-binding protein that is specifically expressed in embryonic rat brain, was described previously. During the purification of drebrin, we found that an actin-binding protein of 54 kDa was also expressed at high levels in embryonic brain, and this protein was identified by immunoblotting as fascin. To explore the roles of fascin in brain development, we purified fascin from brains of infant rats and characterized it. We found that the actin-binding activity of fascin was strongly inhibited by drebrin. Fascin caused formation of actin bundles, a process that was inhibited in the presence of drebrin, as confirmed by electron microscopy and a low-speed centrifugation assay. In PC12 cells, fascin was localized in the filopodia of growth cones, whereas drebrin was localized in the basal region of growth cones. Our results suggest that fascin might play an important role in the organization of actin in filopodia and that this organization might be regulated by drebrin.

Actins↗
PubMed1996

[Factors related to ADL of stroke patients three months after discharge].

Focusing on the ADL of stroke patients, 94 hemiplegia cases in rehabilitation units of acute care general hospitals were studied to identify factors affecting ADL, measured using the Barthel Index, 3 months after discharge. The main results were as follows: 1. The 8 independent variables: age, gender, length of hospitalization, degree of paralysis of lower extremities at discharge, motivation toward rehabilitation, sight, Barthel scores at discharge, and the expression of one's intentions emerged as the 8 reliable predictive factors (R = .904, R2 = .817). 2. It may be deduced that in older patients in particular, a status of amyotrophy has a significant influence on ADL after discharge. The 94 stroke patients were divided into two age groups-44 patients aged 64 years or younger, and 50 patients aged 65 years or older. Barthel scores at 3 months after discharge were analyzed by multiple regression analysis in both groups. The result was that ADL, after discharge, of the aged 65 years or older group, was more affected by their status at discharge, than was it with the 64 years or younger group (64 years or younger; R = .871, R2 = .758, 65 years or older; R = .934, R2 = .873). 3. For patients in the 64 years or younger group, Barthel scores at 3 months after discharge were significantly related to age, length of hospitalization, sight, Barthel scores at discharge and expression of one's intentions. For patients in the 65 years or older group, the results were significantly related to sight, Barthel scores at discharge, and motivation for rehabilitation.

Activities of Daily Living↗
PubMed1996

Granulocyte-colony stimulating factor and stem cell factor are the crucial factors in long-term culture of human primitive hematopoietic cells supported by a murine stromal cell line.

The findings that murine marrow stromal cell line MS-5 supported the proliferation of human lineage-negative (Lin-) CD34+CD38- bone marrow cells in long-term culture have been reported. In this study, we analyzed this proliferating activity of MS-5-conditioned medium (CM) on human primitive hematopoietic cells. When Lin-CD34+CD38- cells of normal human cord blood cells were co-cultured with MS-5, colony forming cells (CFCs) were maintained over 7 weeks in vitro. Prevention of contact between MS-5 and Lin-CD34+CD38- cells by using membrane filter (0.45 micron) was negligible for this activity. This indicated that the activity of MS-5 on human primitive hematopoietic cells is a soluble factor(s) secreted from MS-5, which is not induced by the contact between MS-5 and Lin-CD34+CD38- cells. We tried to purify this soluble activity. An active material with a molecular weight of about 150 kDa, determined by gel filtration chromatography, solely supported the growth of Lin-CD34+CD38- cells and Mo7e, a human megakaryocytic cell line. This activity not only reacted with anti-mouse stem cell factor (mSCF) antibody on Western blots, but it was also neutralized in the presence of anti-mSCF antibody. Another active material with a molecular weight of about 20-30 kDa synergized with mSCF to stimulate the growth of Lin-CD34+CD38- cells but failed to do so alone, although this synergy was inhibited in the presence of soluble mouse granulocyte-colony stimulating factor (mG-CSF) receptor, which is a chimeric protein consisting of the extracellular domain of mG-CSF receptor and the Fe region of human IgG1. In addition, the latter molecule supported the growth of the G-CSF dependent cell line FD/GR3, which is a murine myeloid leukemia cell line, FDC-P2, transfected with mG-CSF receptor cDNA. Adding of anti-mSCF antibody and soluble mG-CSF receptor to the culture completely abrogated the activity of MS-5-CM. Recombinant (r) mSCF and rmG-CSF had synergistic activity on the growth of Lin-CD34+CD38- cells. These results indicated that the activity on Lin-CD34+CD38- cells included in MS-5-CM is based upon the synergistic effects of mSCF and mG-CSF.

ADP-ribosyl Cyclase↗
PubMed1995

The fastest actin-based motor protein from the green algae, Chara, and its distinct mode of interaction with actin.

The endoplasmic streaming in Characean cells is an actin-dependent movement. The motor protein responsible for the streaming was partially purified and characterized. It was soluble at low ionic strength, an ATPase of a molecular mass of 225 kDa and activated more than 100 times by muscle F-actin. Surprisingly, in an in vitro motility assay, the motor protein moved muscle F-actin at 60 microns/s, which is similar to the velocity of streaming in a living cell and 10 times faster than muscle myosin. Proteolytic cleavage of actin impaired movement crucially on muscle myosin, but did not affect movement at all on the Chara motor protein, suggesting that the Chara motor protein would interact with actin via a set of sites different from those of muscle myosin.

Actins↗
PubMed1995

Purification of an ATP-dependent actin-binding protein from a lower eukaryote, Physarum polycephalum.

A novel protein with a molecular mass of 55 kDa, as determined by SDS-PAGE, was purified from plasmodia of Physarum polycephalum. The protein bound to actin filaments with a stoichiometry of 0.27 moles per mole of actin with an apparent dissociation constant of 4 x 10(-8) M. In the presence of ATP, the protein dissociated from actin filaments. Adenosine 5-(gamma-thio)triphosphate and adenyl-5'-yl imidodiphosphate also abolished the actin-binding activity of the protein, but GTP did not. Because the cytoplasmic concentration of ATP oscillates in association with the shuttle streaming of the cytoplasm, it is possible that this protein might be involved in the actin-linked regulation of cytoplasmic streaming.

Actins↗
PubMed1995

Hematopoietic stem cells found in lineage-positive subsets in the bone marrow of 5-fluorouracil-treated mice.

It is known that treatment of mice with 5-fluorouracil (5-FU, 150 mg/kg) confers radioprotection. To investigate this effect, we performed bone marrow transplantation (BMT) using C57BL/6-Ly5 congenic mice treated with 5-FU five days prior to experiments. The mononuclear cells (MNC) in 5-FU-treated bone marrow (BM) were 10 times more radioprotective than those in untreated BM. Moreover, the number of BM MNC expressing c-kit on their surface from 5-FU-treated mice was markedly decreased relative to those from untreated controls. These results showed that the surface characteristics of cells that contributed to this radio-protective effect differ from those of stem cells as reported recently. BM MNC of mice treated with 5-FU were separated on the basis of expression of the lineage-specific antigens (Lin), c-kit, and Ly6A/E. When injected into lethally irradiated mice, 1,000 Lin+ and Lin-c-kit+Ly6A/E+ cells showed radioprotective effects such that 100% and 60% survived, respectively. Flow cytometric analysis 165 days after BMT showed that 88.8% and 65.1% of peripheral blood (PB) in mice transplanted with Lin+ and Lin-c-kit+Ly6A/E+ was derived from donor mice, respectively. After six months, donor-derived Lin-c-kit+Ly6A/E+ cells which showed radioprotective effects on a secondary irradiated host were detected from mice transplanted with Lin+ cells from 5-FU-treated mice. Taken together, these findings demonstrated that stem cells expressing Lin+ present in the BM of mice treated with 5-FU other than Lin-c-kit+Ly6A/E+ cells and these Lin+ cells play an important role in the recovery of myeloablative mice.

Animals↗
PubMed1994

Drebrin, a development-associated brain protein from rat embryo, causes the dissociation of tropomyosin from actin filaments.

Drebrin is a development-associated neuroprotein whose cDNA into fibroblasts causes the formation of dendrite-like structures (Shirao, T., Kojima, N., and Obata, K. (1992) Neuroreport 3, 109-112). To explore molecular functions of drebrin during brain development, we purified drebrin from brains of rat embryos. Drebrin bound to actin filaments at a stoichiometry of 1:5 with a dissociation constant (Kd) of 1.2 x 10(-7) M. It strongly inhibited the actin binding activity of tropomyosin. Excess amounts of tropomyosin also inhibited the drebrin binding to actin filaments, suggesting that drebrin and tropomyosin competitively bind to actin filaments. Further, drebrin inhibited not only the actin binding activity of alpha-actinin but also the actin cross-linking activity of alpha-actinin. Gene transfection experiments revealed that tropomyosin was dissociated from actin filaments in drebrin-overexpressing fibroblasts. Thus we hypothesize that drebrin may destabilize actin filaments by dissociating tropomyosin and alpha-actinin from actin filaments, resulting in the formation of axon and dendrites during neuronal development.

Actin Cytoskeleton↗
PubMed1994

Stimulation of the ATP-dependent interaction between actin and myosin by a myosin-binding fragment of smooth muscle caldesmon.

We reported previously that smooth muscle caldesmon stimulates the ATP-dependent interaction between actin and phosphorylated smooth muscle myosin, as monitored by ATPase measurement and in vitro motility assay. Furthermore, this effect changes from stimulatory to inhibitory with increasing concentrations of caldesmon [Ishikawa et al., 1991: J. Biol. Chem. 266:21784-21790]. The N-terminal (myosin-binding) fragment and the C-terminal (actin-binding) fragment were purified from digests of caldesmon. The effects of the myosin-binding fragment and the actin-binding fragment on the interaction were stimulatory and inhibitory, respectively, indicating that stimulatory and inhibitory domains are localized in the myosin-binding domain and actin-binding domain of caldesmon, respectively. The effect of the myosin-binding fragment on the interaction was exclusively stimulatory when the interaction was challenged by caldesmon, both at lower and higher concentrations. However, the actin-binding fragment had no effect on the interaction at lower concentrations and inhibited the interaction at higher concentrations. Thus, the stimulatory effect of caldesmon that is observed at lower concentrations can be explained by the hypothesis that the stimulatory effect of the myosin-binding domain predominates over the inhibitory effect of the actin-binding domain when the concentration of caldesmon is low. With uncleaved caldesmon, we also emphasized the role of the myosin-binding domain in the stimulation as follows; the stimulatory effect of caldesmon became obscured when binding of caldesmon to myosin was competed by the exogenous caldesmon-binding fragment of myosin.

Actins↗
PubMed1994

Formation of thick, curving bundles of actin by drebrin A expressed in fibroblasts.

Drebrin A is a neuron-specific protein, the expression of which is regulated during development. Upon transfection of fibroblasts with drebrin A cDNA, the protein is expressed at high levels in fibroblasts and the outgrowth of highly branched, neurite-like cell processes is induced. In this report, we describe a biochemical examination of the binding of drebrin A to actin filaments. We also demonstrate by an immunocytochemical method that, when drebrin A is expressed in transfected cells, it binds to actin filaments and is concentrated in cell processes. Furthermore, we provide evidence that thick, curving bundles of actin together with drebrin are formed in some of the transfected cells. Our results suggest that the actin filaments that bind drebrin might be a novel class of actin filaments and might play a role in neuronal morphogenesis.

Actins↗
PubMed1994

Local environmental change from the G- to F-form of the actin molecule detected on anisotropy decay measurement.

The fluorescence intensity has been reported to increase 10 to 25 times when N-(1-pyrene)-iodoacetamide (PIAA)-conjugated actin polymerizes from the G- to the F-form. To elucidate the molecular mechanism underlying this process, we measured the time-averaged anisotropy of PIAA-actin in both the G- and F-forms. The anisotropy ratio of PIAA-G-actin (0.137 +/- 0.008) was smaller than that of PIAA-F-actin (0.221 +/- 0.008). Similar results were obtained when N-(1-pyrene)-3-iodopropionamide (PIPA), a PIAA analogue with an extra carbon-chain in its reactive group, was conjugated with actin. The anisotropy ratio increased from 0.096 +/- 0.042 to 0.199 +/- 0.035 when PIPA-actin was transformed from the G- to the F-form. Further more, we measured the anisotropy decay of PIAA-actin in both the G- and F-forms. Least-square fitting revealed that the decay pattern was well fitted the wobbling-in-cone model. In the G-form, the pyrene of PIAA actin diffused in a cone region with a vertical half angle of 33.4 degrees. This value decreased to 25.9 degrees when the actin was transformed to the F-form. Because PIAA and PIPA were conjugated at Cys-374 of actin, our results suggest that a small cleft exists in the actin molecule in the vicinity of Cys-374, and this cleft becomes narrow upon polymerization, resulting in an increase in fluorescence intensity.

Actins↗
PubMed1994

Evaluation of urinary enzymes in dogs with early renal disorder.

We investigated urinary N-acetyl-beta-D-glucosaminidase NAG (EC 3.2.1.30), gamma-glutamyl transpeptidase gamma-GTP (EC 2.3.2.2) and glycyl-prolyl dipeptidyl aminopeptidase GP-DAP (EC 3.4.14.5) in dogs with heartworm disease and renal failure. In the renal failure dogs, the NAG, gamma-GTP and GP-DAP index were significantly higher than those in the healthy dogs. In the heartworm disease dogs with normal chest X-rays (HW I), none of the enzyme values was significantly different from those of the healthy controls. In the dogs with heartworm disease showing abnormal heart shadows on their chest X-rays (HW II), enzyme values were significantly higher than those in the healthy dogs (P < 0.01) and the HW I dogs (P < 0.01). Thus, these urinary enzymes tests are available for the early detection of renal disorders.

Acetylglucosaminidase↗
PubMed1993

Transforming growth factor-beta expression and natural killer cell responses during virus infection of normal, nude, and SCID mice.

The experiments presented here assess the contribution of T cells to NK cell regulation and transforming growth factor-beta (TGF-beta) production. The kinetics of NK cell activation in the absence of T cells was evaluated by comparing responses in athymic nude and SCID mice to those in normal C3H/HeN, C57BL/6, and BALB/c mice during lymphocytic choriomeningitis virus (LCMV) infection. T cell-deficient mice exhibited high NK cell activity for longer periods of time post-infection. To determine whether the reduction in NK cell activation at later times post-infection of normal mice resulted from induction of negative regulatory elements, mice were treated with the chemical IFN and NK cell inducing agent polyinosinic-polycytidylic acid (poly I:C). Although poly I:C induced plasma IFN in both uninfected and LCMV-infected normal mice, NK cell activity was elevated in the uninfected mice only. In vivo expression of TGF-beta protein in normal and T cell-deficient mice was evaluated by immunohistochemical studies on splenic sections. TGF-beta proteins were not detected in sections from uninfected mice but were induced in all strains during infection. High levels of TGF-beta proteins were localized to discrete cells, and diffuse staining was observed along marginal zones. The ability of the splenic leukocytes to release TGF-beta into conditioned media was evaluated in the Mv 1 Lu biologic assay. Transient acid treatment to release active factor from the latent form and use of specific neutralizing antibodies demonstrated that leukocytes from all strains of infected mice had released latent TGF-beta 1 into conditioned media. However, cells from infected nude or SCID mice produced substantially less active TGF-beta than those isolated from normal mice. Production of biologically active TGF-beta 1 was dependent upon the presence of T cells in normal mice, as in vivo depletion of CD4+ and CD8+ T cells by antibody treatment resulted in augmented NK cell activity and loss of active factor production. The lack of TGF-beta production was accompanied by increased NK cell numbers; at late times post-infection, T cell-deficient C57BL/6-nude mice had almost twice as many NK1.1+CD3- cells per spleen compared with T cell-containing C57BL/6 littermate control mice. Treatment with active TGF-beta 1 reduced the numbers of NK cells per spleen in infected nude mice to the levels found in infected littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗