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Biomedical subjects

R Ishikawa

Publications and source records attributed to R Ishikawa.

At least 73 records · Page 4Linked to original sources
PubMed1991

Appearance of nerve growth factor and acidic fibroblast growth factor with different time courses in the cavity-lesioned cortex of the rat brain.

Time-dependent changes in both nerve growth factor (NGF) and acidic fibroblast growth factor (aFGF) levels in the rat brain after cortical cavity lesioning were examined, by using sensitive enzyme immunoassays (EIA) specific for each factor. In the cavity fluid, the NGF level increased rapidly and temporarily with a sharp peak 16 h after lesioning. A relatively high level was sustained during the next 3-6 days. Contrary to NGF, aFGF was first detectable only 10 days after lesioning, and its level increased gradually until 30 days. These results suggest that NGF and aFGF would play some roles for neuronal repair in different ways.

Animals↗
PubMed1991

Purification of a novel Ca-binding protein that inhibits myosin light chain kinase activity in lower eukaryote Physarum polycephalum.

Myosin light chain kinase (MLCK) was partially purified from the lower eukaryote Physarum polycephalum. The activity to phosphorylate Physarum myosin was maximal in the absence of Ca2+ and decreased with an increase in Ca2+ concentration with a microM-level Kd. The Ca-binding protein contained in the MLCK preparation was purified to homogeneity. The native protein had a molecular mass of 75 kDa, while under denaturing conditions, it was 38 kDa. Ca-dependent changes in the intensities of intrinsic fluorescence showed that the Kd of the protein for Ca2+ was also in the microM-range. Our results suggest that the Ca-binding protein would play a key role in the effects of Ca2+ in the MLCK preparation.

Calcium↗
PubMed1991

In vitro movement of actin filaments on gizzard smooth muscle myosin: requirement of phosphorylation of myosin light chain and effects of tropomyosin and caldesmon.

ATP-dependent movement of actin filaments on smooth muscle myosin was investigated by using the in vitro motility assay method in which myosin was fixed on the surface of a coverslip in a phosphorylated or an unphosphorylated state. Actin filaments slid on gizzard myosin phosphorylated with myosin light chain kinase (MLCK) at a rate of 0.35 micron/s, but did not slide at all on unphosphorylated myosin. The movement of actin filaments on phosphorylated myosin was stopped by perfusion of phosphatase. Subsequent perfusion with a solution containing MLCK, calmodulin, and Ca2+ enabled actin filaments to move again. The sliding velocities on monophosphorylated and diphosphorylated myosin by MLCK were not different. Actin filaments did not move on myosin phosphorylated with protein kinase C (PKC). The sliding velocity on myosin phosphorylated with both MLCK and PKC was identical to that on myosin phosphorylated only with MLCK. Gizzard tropomyosin enhanced the sliding velocity to 0.76 micron/s. Gizzard caldesmon decreased the sliding velocity with increase in its concentration. At a 5-fold molar ratio of caldesmon to actin, the movement stopped completely. This inhibitory effect of caldesmon was relieved upon addition of excess calmodulin and Ca2+.

Actins↗
PubMed1991

Characterization of calcium-binding light chain as a Ca(2+)-receptive subunit of Physarum myosin.

Physarum myosin is uniquely under an inhibitory Ca(2+)-regulation in the ATP-dependent interaction with actin [Kohama (1990) Trends Pharmacol. Sci. 11, 433-435, for review]. Calcium-binding light chain (CaLc) has been suggested to be of primary importance to the control from its amino acid sequence [Kobayashi et al. (1988) J. Biol. Chem. 263, 305-313]. To provide a biochemical basis for this suggestion, the Ca-binding capacity of CaLc and its Kd for Ca2+ were measured. The Ca-binding properties of CaLc allowed those of Physarum myosin to be explained in terms of CaLc. However, the mode of Ca(2+)-regulation by CaLc differs according to the enzyme upon which Ca-sensitivity is confered by CaLc, i.e., CaLc activated bovine phosphodiesterase activity and inhibited Physarum myosin ATPase activity, with the same Kd in microM levels. Thus, CaLc appears to work as a mere Ca-receptive subunit in Physarum myosin, with the secret of the inhibition lying in other subunits. CaLc was also shown to belong to a family of alkali light chains (AlLc) by allowing it to bind skeletal myosin as a substitute for its AlLc. Therefore, present study is the first biochemical indication that the AlLc family is involved in regulating the myosin function.

Actins↗
PubMed1991

Developmental changes in distribution of acidic fibroblast growth factor in rat brain evaluated by a sensitive two-site enzyme immunoassay.

We developed a sensitive two-site enzyme immunoassay (EIA) system for acidic fibroblast growth factor (aFGF), using a polyclonal antibody raised in rats. This assay is based on the sandwiching of the antigen between anti-aFGF antibody immunoglobulin G (IgG) coated on plates and biotinylated anti-aFGF antibody IgG; the detection of biotinylated IgG was performed by enzyme reaction of streptavidin-conjugated beta-D-galactosidase (beta-D-galactoside hydrolase; EC 3.2.1.23). Our system was specific for aFGF, because basic fibroblast growth factor, which shares a 55% homology of amino acid sequence with aFGF, hardly cross-reacted at all. The sensitivity of this system (0.2 ng/ml) enabled us to quantify endogenous immunoreactive aFGF in the CNS. Using this two-site EIA system, we examined the levels of aFGF in various regions of rat brain and their developmental changes. At the early stage of neonatal development, i.e., 2 days after birth, all brain regions registered low aFGF levels (less than 10 ng/g tissue). However, at the young adult stage (21- to 49-day-old animals), an extremely high level of aFGF (75-90 ng/g tissue) was found in the ponsmedulla; relatively high levels (30-40 ng/g tissue) were found in the diencephalon and mesencephalon; and comparatively low aFGF levels (5-15 ng/g tissue) were found in various other brain regions such as the frontal cortex, piriform cortex, hippocampus, olfactory bulb, cerebellum, and striatum. This marked change in the regional distribution of aFGF in the rat brain during postnatal development from 2 to 21 days after birth suggests that this factor plays a significant role in the brain during this period.

Aging↗
PubMed1991

A myosin-like protein from smooth muscle.

A protein was purified from chicken gizzard smooth muscle. It bound ATP and actin. Actin activated the Mg2(+)-ATPase activity of this protein. The Ca2(+)-ATPase activity was lower than K(+)-EDTA ATPase activity. Thus, it appears that this protein is akin to myosin I rather than to conventional myosin. However, ATPase activities of the protein were much lower than those of myosin I. A protein cofactor, such as protein kinase, which would enhance these activities remains to be purified from the smooth muscle.

Actins↗
PubMed1991

Inhibitory Ca(2+)-regulation of myosin light chain kinase in the lower eukaryote, Physarum polycephalum: role of a Ca(2+)-dependent inhibitory factor.

ATP-dependent interactions between myosin and actin in the lower eukaryote, Physarum polycephalum, are inhibited by micromolar levels of Ca2+. This inhibition is mediated by the binding of Ca2+ to myosin, the phosphorylation of which is required if Ca2+ is to inhibit the activities of myosin (Kohama, K., Trends Pharmacol. Sci. 11, 433-435 (1990)). As the first step to examine whether Ca2+ also regulates phosphorylation in the actomyosin system, we purified myosin light chain kinase (MLCK) of 55 kDa almost to homogeneity. The MLCK activity was high whether or not Ca2+ was present. However, a Ca(2+)-dependent inhibitory factor (CIF) purified from Physarum (Okagaki et al., Biochem. Biophys. Res. Commun. 176, 564-570 (1991)) was shown to reduce the MLCK activity in a Ca(2+)-dependent manner. Using crude preparations, not only MLCK but also myosin heavy chain kinase and actin kinase were shown to be inhibited by Ca2+ half-maximally at micromolar levels. Since CIF is the only Ca(2+)-binding protein in the preparations, we propose that this inhibitory Ca(2+)-regulation of the kinases for actomyosin is mediated by CIF.

Animals↗
PubMed1990

Mitosis-specific phosphorylation causes 83K non-muscle caldesmon to dissociate from microfilaments.

At mitosis in eukaryotic cells there are profound changes of shape and structure whose causes are almost entirely obscure. What is known is that there are changes in the organization of microfilaments, including the disassembly of microfilament bundles during prophases and the accompanying rounding-up of cultured cells; the formation of transient contractile rings during cytokinesis; and, subsequently, the reassembly of microfilament bundles and the respreading of the two daughter cells. As an initial step towards the biochemical understanding of these events, in which the disassembly and reassembly of microfilaments appear to play an important part, we searched for alterations of the molecular constitution of microfilaments during mitosis. We found that non-muscle caldesmon, a protein with a relative molecular mass (Mr) of 83,000 (83K) which binds to actin and calmodulin, is dissociated from microfilaments during mitosis, apparently as a consequence of phosphorylation. This process may contribute to the changes of shape and structure of cells in mitosis, as caldesmon inhibits actomyosin ATPase.

Actin Cytoskeleton↗
PubMed1990

Sex hormone modulation of interferon (IFN) alpha/beta and gamma production by mouse spleen cell subsets following picornavirus infection.

Replication of the diabetogenic variant of encephalomyocarditis virus (EMCV-D) in spleen cells and its association with subpopulations of spleen cells (L3T4+, Lyt-2+, Mac 1+, 33D1+ and AGM1+ cells) from both sexes of ICR Swiss mice were examined. Virus replication was limited to less than 0.5 log in suspensions of whole spleen cells, nonadherent cells or a B cell subfraction from both sexes of ICR Swiss mice following infection with EMCV-D at an MOI of 10; no virus replication was seen in adherent spleen cells from either sex. After 1 hour adsorption of EMCV-D onto spleen cells at a multiplicity of infection (MOI) of either 10 or 0.1, virus-associated cells were isolated using a monoclonal murine anti-EMCV-D and anti-mouse IgG conjugated to magnetic beads. Using an MOI of 0.1, less than 1% of spleen cells bound virus particles after 1 hour adsorption at 4 degrees C. Among the virus-positive cells, relatively higher percentages of adherent cell populations (Mac 1+ and 33D1+ cells) of both sexes bound virus particles within the first hour post-infection (PI) than did the other spleen cell subpopulations. Interferon (IFN) alpha/beta production was detected as early as 4 hours PI in female spleen cell cultures infected with EMCV-D at an MOI of 0.1 while no IFN alpha/beta activity was found in comparably infected male spleen cell cultures. Inhibiting IFN alpha/beta activity in the virus-infected spleen cell cultures during the first 20 hours of infection using polyclonal rabbit anti-mouse IFN alpha/beta serum eliminated production of IFN gamma as well as IFN alpha/beta. Spleen cell cultures depleted of adherent cells were unable to produce IFN alpha/beta or IFN gamma in the first 24 hours PI. The capacity to produce IFN gamma at 12 hours after virus infection of spleen cells from both sexes of mice was restored to adherent cell-depleted cultures by addition of mouse IFN alpha/beta at the time of infection. These results suggest that IFN alpha/beta and adherent cells play critical roles in the early production of IFN gamma (less than 16 hours PI) characteristic of the infected spleen cell cultures of females. Production of IFN alpha/beta and IFN gamma by spleen cells from both sexes of ICR Swiss mice was enhanced by administrating estrone to donor mice during the week before harvesting spleen cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Annealing of gelsolin-severed actin fragments by tropomyosin in the presence of Ca2+. Potentiation of the annealing process by caldesmon.

Previous results from our laboratory have shown that 1) cultured rat cells contain two classes of tropomyosin (TM), one (high Mr TMs) with higher Mr values and greater affinity for actin than the other (low Mr TMs); 2) presaturation of F-actin with high Mr TMs, but not with low Mr TMs, inhibits both actin-severing and actin binding activities of gelsolin; and 3) nonmuscle caldesmon not only enhances the inhibitory effects of high Mr TMs but also makes low Mr TMs capable of inhibiting the severing activity of gelsolin (Ishikawa, R., Yamashiro, S., and Matsumura, F. (1989) J. Biol. Chem. 264, 7490-7497). These results suggest that gelsolin has much lower affinity for F-actin-TM-caldesmon complexes than for pure F-actin. We have therefore examined whether addition of TM and/or caldesmon to gelsolin-severed actin filaments can make gelsolin dissociate from barbed ends of actin filaments, resulting in annealing of short actin filaments into long ones. Flow birefringence and electron microscopic studies have suggested that high Mr TMs slowly and partially anneal gelsolin-severed actin fragments in 3 h, whereas low Mr TMs have no effects. Nonmuscle caldesmon greatly potentiates the effects of high Mr TMs and accelerates the process to 20 min, whereas nonmuscle caldesmon alone shows no effects. Furthermore, nonmuscle caldesmon makes low Mr TMs capable of reversing gelsolin-severing action. Actin binding assay has shown that gelsolin (or a gelsolin-actin complex) is dissociated from these annealed actin filaments. Smooth muscle TM and smooth muscle caldesmon also appear to anneal gelsolin-severed actin fragments as do high Mr TMs and nonmuscle caldesmon. Calmodulin decreases the potentiation effects of caldesmon as calmodulin inhibits actin binding of caldesmon. These results suggest that tropomyosin and caldesmon may regulate both capping and severing activities of gelsolin.

Actins↗
PubMed1989

Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. Potentiation of protective ability of tropomyosins by 83-kDa nonmuscle caldesmon.

Multiple isoforms of tropomyosin (TM) of rat cultured cells show differential effects on actin-severing activity of gelsolin. Flow birefringence measurements have revealed that tropomyosin isoforms with high Mr values (high Mr TMs) partially protect actin filaments from fragmentation by gelsolin, while tropomyosins with low Mr values (low Mr TMs) have no significant protection even when the actin filaments have been fully saturated with low Mr TMs. We have also examined effect of nonmuscle caldesmon on the severing activity of gelsolin because 83-kDa nonmuscle caldesmon stimulates actin binding of rat cell TMs (Yamashiro-Matsumura, S., and Matsumura, F. (1988) J. Cell Biol. 106, 1973-1983). While nonmuscle caldesmon alone or low Mr TMs alone show no significant protection against fragmentation by gelsolin, the low Mr TMs coupled with 83-kDa protein are able to protect actin filaments. Further, high Mr TMs together with 83-kDa protein appear to block the severing activity completely. Electron microscopic analyses of length distribution of actin filaments have confirmed the results. The average length of control actin filaments is measured as 1.46 +/- microns, and gelsolin shortens the average length to 0.084 +/- 0.039 micron. Similar short average lengths are obtained when gelsolin severs actin complexed with low Mr TMs (0.080 +/- 0.045 micron) or with nonmuscle caldesmon (0.11 +/- 0.072 micron) while longer average length (0.22 +/- 0.18 micron) is measured in the presence of high Mr TMs. The simultaneous addition of nonmuscle caldesmon makes the average length considerably longer, i.e. 0.61 +/- 0.37 micron in the presence of low Mr TMs and 1.57 +/- 0.97 micron in the presence of high Mr TMs. Furthermore, the actin binding of gelsolin is strongly inhibited by co-addition of high Mr TMs and nonmuscle caldesmon. These results suggest that TM and gelsolin share the same binding site on actin molecules and that the differences in the actin affinities between TMs are related to their abilities of protection against gelsolin.

Actin Cytoskeleton↗
PubMed1989

[Prognosis for the deciduous molars of juvenile dogs after root canal filling using apatite root sealer].

There are various restrictions on the root canal filling of deciduous roots after root canal treatment unlike in the case of permanent teeth, because deciduous molars undergo physiological root resorption with aging. Among the restrictions, it is most important that the root canal filling material be similarly absorbed and disappear along with the deciduous root so that it does not prevent the eruption of the permanent successors. Hardly any of the root canal filling materials currently on the market, however, are suitable for deciduous molars because some of them disappear earlier than the deciduous root without becoming indurated in the root canal, while some delay tooth replacement because they are resorbed with great difficulty, and continue strongly to stimulate the surrounding tissue of the root apex. Root canal filling materials utilizing alpha-TCP and hydroxyapatite with excellent biocompatibility have recently been put on the market by Sankin Kygyo Co. Type-III, in particular, contains only 5% iodoform, causing only very slight stimulation, and is a relatively good product from a practical standpoint, i.e., filling the root canal using a lentulo. Root canal filling was performed immediately after dental pulp extraction from the deciduous mandibular molars of juvenile dogs to determine the feasibility of filling deciduous teeth with root canal filling materials. Three dogs were then observed for sequential changes in the resorption of the deciduous roots, presence/absence of adverse effects on permanent successors, and influence on the process of eruption by means of radiographic examination every week. The results obtained were as follows: 1. The type-III apatite root sealar or Vitapex used for root canal filling was not absorbed, and there was almost no resorption of the root. Thus, the deciduous teeth on the experimental side were ejected and shed. 2. There was no significant difference in the amount of tooth germ formed between the experimental and control sides. 3. The amount of eruption tended to be delayed because of insufficient resorption of the root of the deciduous predecessor, but this tendency was considered to be due to the influence of the timing of root canal filling and individual difference. In the final analysis, no definite conclusions could be drawn.

Animals↗
PubMed1986

Structural polypeptides of the murine coronavirus DVIM.

The structural polypeptides of the murine coronavirus DVIM (diarrhoea virus of infant mice) have been analysed in comparison with other strains MHV-2, MHV-3, MHV-4 (JHM) and MHV-S by SDS-PAGE. In the presence of 2-mercaptoethanol, three major glycopolypeptides, gp180, gp69, gp25 (as a group of similar species) and one major non-glycosylated polypeptide p58 were detected. The gp69 is a DVIM specific glycopolypeptide, in which the glycosidic moieties are linked to the core polypeptide through N-glycosidic bonds, and hence may be correlated with the short projections of the viral envelope. Further gp140, which appears in the absence of reducing agents, is apparently a dimer of gp69 held together by disulfide linkages. The gp25 family, on the other hand, consists of four polypeptides, two of which are not metabolically inhibited by tunicamycin suggesting that they are O-linked glycopolypeptides. DVIM seems to be serologically closely related to the MHV-S strain as shown by neutralization.

Animals↗
PubMed1986

[A case of downhill esophageal varices--Behçet disease associated with abdominal aortic aneurysm and occlusions of the superior and inferior vena cava].

A case of Behçet disease which vascular lesions progressed remarkably and had massive gastrointestinal bleeding is reported. A 39 year old man was admitted to the hospital complaining of intractable abdominal pain and angiography revealed impending rupture of a saccular aneurysm just above the bifurcation of the aorta. Aneurysmectomy and patch closure of the defect were performed. Four months later, the patient was admitted again complaining of severe abdominal pain and rerupture of the patch graft was recognized by RI angiography. Aorto-iliac bypass was carried out. Although postoperative course was excellent, the sign of superior vena cava syndrome appeared gradually. Venography revealed occlusion or stenosis of the bilateral subclavian and brachiocephalic veins and the superior vena cava. Femoral venography revealed occlusion of the inferior vena cava, too. Two years and three months later of the first operation, the patient had hematemesis and the extensive varicose vein of the entire esophagus was seen by the upper gastrointestinal examination. Five months later of the examination the patient suddenly had massive gastrointestinal hemorrhage and died in spite of vigorous resuscitative therapy. This esophageal varices was considered as downhill esophageal varices caused by superior vena caval occlusion, not by portal hypertension. Downhill esophageal varices due to mediastinal tumor is seen sporadically, but one due to superior vena caval occlusion secondary to Behçet disease is very rare.

Adult↗