Biomedical subjects

F Nakamura

Publications and source records attributed to F Nakamura.

At least 109 records · Page 6Linked to original sources

PubMed1996

Thromboxane A2 mediates the stimulation of inositol 1,4,5-trisphosphate production and intracellular calcium mobilization by bradykinin in neonatal rat ventricular cardiomyocytes.

Bradykinin is a mediator of the protection of myocardium by angiotensin I-converting enzyme/kininase II inhibitors. We reported that the activation of B2 bradykinin receptors in neonatal rat cardiac myocytes in primary culture was followed by hydrolysis of phosphatidylinositol 4,5-bisphosphate and formation of inositol 1,4,5-trisphosphate (IP3). Here we examine the regulation of IP3 formation stimulated by bradykinin. Activation of myocytes with 1 mu/L bradykinin increased IP3 production from 117 +/- 8.3 to 1011 +/- 48.6 pmol/mg protein. Treatment of the cells with 10 mu/L indomethacin or 1 mu/L dexamethasone partially blocked this bradykinin-induced response. Moreover, either U73122, a phospholipase C inhibitor, or (p-amylcinnamoyl) anthranilic acid, a phospholipase A2 inhibitor, blunted the IP3 response to bradykinin. Because thromboxane A2 stimulates inositol bisphosphate metabolism in guinea pig atria, we also investigated the effect of the thromboxane A2 receptor antagonist BM 13177 (1 mu/L), which strongly attenuated the stimulated IP3 production. Since thromboxane A2 appears to partly mediate the IP3 response to bradykinin, we examined the effect of the stable thromboxane A2 mimetic U46619. Control cultures were stimulated more by U46619 than by bradykinin (1629 +/- 14.5 versus 1011 +/- 48.6 pmol IP3/mg protein). This property of U46619 was selectively antagonized by BM 13177. Inhibition of either phospholipase C or phospholipase A2 blunted the IP3 response to U46619. Short-term (30 minutes) activation of protein kinase C with phorbol 12-myristate 13-acetate (10 pmol/L to 1 mu/L) attenuated the IP3 accumulation in response to bradykinin; the effect of phorbol 12-myristate 13-acetate was reversed with 1 mu/L staurosporine, a protein kinase C inhibitor. Treatment with 1 microgram/mL cholera toxin or pertussis toxin for 4 hours amplified the IP3 response to 10 nmol/L bradykinin from 570 +/- 20.0 to 1150 +/- 51.3 and to 1016.7 +/- 21.9 pmol/mg protein. Bradykinin mobilized 9.4% of intracellular calcium stores in cardiomyocytes as assessed by chlortetracycline-based fluorometry, and this effect of bradykinin was blocked by BM 13177 or the B2 bradykinin receptor blocker Hoe 140 by more than 70%. In functional studies, bradykinin (1 mu/L) increased by 12% the twitch contractile force of neonatal rat ventricular strips paced at threshold intensity, but this was unaffected by BM 13177. In conclusion, in cardiomyocytes, bradykinin enhances IP3 production mostly via phospholipase A2 stimulation and thromboxane A2 formation. This prostanoid in turn stimulates its receptor and activates phospholipase C, which then splits phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglycerol. The effect of bradykinin on phospholipase C, via thromboxane A2, is negatively regulated by protein kinase C activation.

Animals↗

PubMed1996

Antinociceptive effect of dihydroetorphine following various routes of administration: a comparative study with morphine.

Using various routes of administration, the antinociceptive effects of dihydroetorphine (DHE) and morphine were measured in mice. Regardless of the route of systemic and local administration, DHE (1-20 micrograms/kg, i.p.; 1-10 micrograms/kg, s.c.; 1-10 micrograms/kg, i.v.; 10-1000 micrograms/kg, p.o.; 10-100 ng/mouse, intracerebroventricularly (i.c.v.); and 10-100 ng/mouse, intrathecally (i.t.)) and morphine (1-20 mg/kg, i.p.; 1-10 mg/kg, s.c.; 1-10 mg/kg, i.v.; 10-100 mg/kg, p.o.; 1-10 micrograms/mouse, i.c.v.; and 0.5-3 micrograms/mouse, i.t.) produced an antinociceptive effect in a dose-dependent manner, as evaluated by the tail pinch method. However, the duration of the antinociceptive effect of DHE was shorter than that of morphine. The efficacy ratio of the antinociceptive effect between DHE and morphine was approximately 1000 to 1500: 1 by parenteral administration (i.p., s.c., or i.v.) and about 100: 1 by the oral route. Meanwhile, using direct application into the central nervous system (CNS) (i.c.v. or i.t.), the effect of DHE was only 10 to 20 times that of morphine. These data suggest that DHE has an ideal quality as an analgesic by systemic administration which is a more convenient application than local injection, since only a minimum dose of DHE is needed to induce suitable potency of antinociception, and the duration of the effect is short. Further, these unique characteristics of DHE might lead to the prevention of the development of dependence by avoiding accumulation of the drug in the CNS.

Analgesics, Opioid↗

PubMed1996

Local delivery of antithrombotic drug prevents restenosis after balloon angioplasty in atherosclerotic rabbit artery.

We investigated the ability of various antithrombotic drugs, delivered locally, to prevent restenosis after angioplasty in hypercholesterolemic rabbits. After dilating atherosclerotic iliac stenoses by balloon angioplasty, a low dose of heparin or a new antithrombotic drug, such as low molecular weight heparin (fragmin), argatroban, or batroxobin, was delivered locally using the balloon double-occlusion technique. In 1 group, high-dose heparin was administered intravenously. Animals that received no drugs served as a control group. After angioplasty, the stenotic segment was dilated and the mean percentage luminal stenosis fell from 89% to 9% in the group that received locally delivered heparin, from 88% to 7% in the group that received locally delivered argatroban, from 87% to 11% in the group that received locally delivered fragmin, from 88% to 15% in the group that received locally delivered batroxobin, from 82% to 18% in the group that received i.v. heparin (p < 0.0001 compared with before angioplasty in each case), and from 84% to 17% in the control group (p < 0.005 compared with before angioplasty). Twenty-eight days after angioplasty, the percentage luminal stenosis remained at 14% in the group that received locally delivered argatroban, 15% in the group that received locally delivered fragmin, and 28% in the group that received locally delivered batroxobin, whereas it increased to 45% in the group that received i.v. heparin, 30% in the group that received locally delivered heparin and 72% in the control group (p < 0.05 compared with after angioplasty in each case). Thus, local delivery low doses of new antithrombotic drugs prevents restenosis after angioplasty without affecting systemic coagulability; heparin, whether administered locally or intravenously, was less effective than the new drugs in preventing restenosis.

Angiography↗

PubMed1996

A potent mu-opioid receptor agonist, dihydroetorphine, fails to produce the conditioned place preference in mice.

Reinforcing effects of dihydroetorphine (DHE) and morphine were evaluated by the conditioned place preference paradigm. Both DHE and morphine produced an antinociceptive effect in a dose-dependent manner. On the other hand, DHE (0.1, 1, 3 and 10 micrograms/kg, i.p.) failed to induce the conditioned preference, while morphine (0.1, 1, 3 and 10 mg/kg, i.p.) caused a dose-dependent preference for the drug-paired place. Thus, these characteristic properties of DHE make it attractive for development as a novel potent analgesic compound that has less dependence liability.

Analgesics, Opioid↗

PubMed1996

Bradykinin B2 receptors on skeletal muscle are coupled to inositol 1,4,5-trisphosphate formation.

To determine the presence of bradykinin receptors in skeletal muscle, we examined in both displacement and saturation studies the binding of [125I-Tyr8]bradykinin or [3H]bradykinin in three types of skeletal muscle preparations: membrane fractions from guinea pig hindlimb quadriceps, dog semimembranosus and semitendinosus muscles, and L8 rat skeletal muscle myoblasts. Scatchard analysis of [125I-Tyr8]bradykinin x bradykinin competition binding demonstrated specific bradykinin binding of 4.9 and 3.2 fmol/mg protein in dog and guinea pig skeletal muscle preparations, respectively. Unlabeled bradykinin specifically displaced [125I-Tyr8]bradykinin with IC50 values of 36.5 +/- 6 and 118.0 +/- 16.0 pmol/l from dog and guinea pig muscle membranes, respectively. The B2 bradykinin receptor antagonist HOE 140 and the B1 bradykinin receptor antagonist des-Arg9[Leu8]bradykinin displaced the binding of [3H]bradykinin from dog membranes with IC50 values of 0.38 and 217.3 nmol/l, respectively, suggesting that bradykinin binds to a B2-type receptor. In addition, unlabeled bradykinin competed with [3H]bradykinin for binding to dog skeletal muscle membrane preparations in a biphasic manner. To assess whether this represents multiple bradykinin receptor subtypes present in skeletal muscle homogenates or several affinity states of a single binding site, we examined bradykinin receptors on a pure skeletal muscle system, the L8 neonatal rat skeletal muscle myoblast cell line. These myoblasts also contain specific [3H]bradykinin-binding sites with a Bmax of 271 fmol/mg protein and a Kd of 0.83 nmol/l. Competitive agonist binding curves were biphasic (high-affinity IC50 = 3.9 pmol/l, low-affinity IC50 = 22.6 nmol/l) in the absence of guanosine 5'-O-(3-thio-trisphosphate) (GTP gamma S); they shifted to a model of one affinity (8.1 nmol/l) in the presence of GTP gamma S. Because the enzyme neutral endopeptidase 24.11 is an important kininase in skeletal muscle, we examined the effect of the neutral endopeptidase inhibitor phosphoramidon on the binding of bradykinin to dog skeletal muscle membranes. We found that phosphoramidon decreased the apparent Bmax from 7.3 to 5.8 fmol/mg protein. In addition, in this cell line we investigated the action of bradykinin on phosphoinositide hydrolysis. Inositol 1,4,5-trisphosphate (IP3) was measured with a radioreceptor assay. Bradykinin (0.1 nmol/l to 1 mumol/l) induced IP3 formation in a dose-dependent manner (EC50 = 1.42 nmol/l) from a basal level of 72.8 +/- 16 pmol/mg protein to 433 +/- 35.5 at the highest (1 mumol/l) concentration. We conclude that bradykinin B2 receptors are expressed in skeletal muscle. Phosphoinositide hydrolysis upon stimulation of this receptor is an indicator of intracellular signal transduction. Part of the bradykinin binding in skeletal muscle is due to interaction with the enzyme neutral endopeptidase.

Animals↗

PubMed1996

Coexpression of cell-surface immunoglobulin (sIg), terminal deoxynucleotidyl transferase (TdT) and recombination activating gene 1 (RAG-1): two cases and derived cell lines.

While it is generally agreed that in the lymphoid differentiation of B lineage cells there is no stage in which cell-surface immunoglobulin (sIg) and terminal deoxynucleotidyl transferase (TdT) are expressed simultaneously, a few B cell acute lymphoblastic leukemia (B-ALL) cases with this phenotype have been reported. Two such cases and the derived cell lines are reported here, in which the expression of recombination activating gene-1 (RAG-1) was also detected. One case was a CD19+ CD22+ HLA-DR+ sIg+ (gamma, kappa) B-ALL. The cell line (Bay9I) also expressed CD10. Karyotypic analysis revealed t(14;18)(q32;q21) and additional aberrations. In the other case, the fresh leukemia cells expressed CD19, CD24 and HLA-DR antigen. The derived cell line (Tree92) also expressed CD22 and sIg (mu, lambda). The karyotype of the Tree92 cells was t(8;14)(q24;q32) with additional aberrations. Tree92 is the first established cell line having both t(8;14)(q24;q32) and TdT. TdT was detected by Northern blotting as well as indirect immunofluorescence analysis. In addition, both Bay9I and Tree92 expressed RAG-1, as detected by Northern blot analysis. Cross-linking of sIg on Tree92 cells with anti-mu antibody led to significant down-regulation of RAG-1 expression. It seems that there is a sIg+ TdT+ RAG-1+ B lineage differentiation stage, and that signaling through sIg can modulate RAG-1 expression.

Adult↗

PubMed1996

[Markedly elevated serum fructosamine in a non-diabetic patient with IgA-kappa type multiple myeloma].

We reported a non-diabetic patient with IgA-kappa multiple myeloma whose serum fructosamine value was markedly elevated. The M-protein from this patient was shown to be conjugated to serum albumin confirmed by immunoelectrophoresis and immunofixation. The fructosamine activity was shown in the high molecular weight fraction by S-300 superfine gel chromatography. Although serum fructosamine values of other three non-diabetic patients with IgA type multiple myeloma were elevated, patients with IgG type multiple myeloma and primary macroglobulinemia had low or normal serum fructosamine values. These findings suggested that glycation of monoclonal IgA of multiple myeloma was much more increased than that of other types of immunoglobulins and monoclonal IgA in this patient was conjugated to serum albumin resulting in the elevated serum fructosamine.

Diabetes Mellitus↗

PubMed1996

Sensitivity to dexamethasone and absence of bcl-2 protein in Burkitt's lymphoma cell line (Black93) derived from a patient with acute tumor lysis syndrome: comparative study with other BL and non-BL lines.

An Epstein-Barr virus (EBV)-negative Burkitt's lymphoma (BL) cell line, designated Black93, was established in culture from a patient who developed acute tumor lysis syndrome (ATLS). Growth inhibition in vitro by dexamethasone (DXM) and the expression of bcl-2 protein (Bcl-2) were investigated in Black93 and 17 other cell lines derived from EBV-negative or -positive BL, pre-B acute lymphoblastic leukemia (ALL), follicular lymphoma (FL), and EBV-positive lymphoblastoid cell lines of normal B cell origin (B-LCL), assuming an inherent susceptibility of Black93 to cell death. The most marked growth inhibition by DXM was observed in Black93, two other BL, two pre-B-ALL and two FL lines. The other cell lines were less sensitive or were resistant. DNA extracted from the Black93 cells treated with DXM showed a ladder of oligo-nucleosomal DNA on electrophoresis. On testing of fixed smears by indirect immunofluorescence, bcl-2 protein (Bcl-2) was undetectable in Black93 and three BL lines but was detected in all the other cell lines at varying intensity. Western blot analysis showed mostly the same results. In the BL lines, the most DXM-sensitive cell lines lacked Bcl-2 expression, and the DXM-resistant cell lines always expressed Bcl-2. While none of the DXM-resistant cell lines lacked Bcl-2 expression, several pre-B or FL lines that expressed [correction of expessed] Bcl-2 were sensitive to DXM. Black93 is the first reported cell line established from a patient with ATLS. The positive sensitivity to DXM and the lack of Bcl-2 expression observed in Black93 are a major characteristic exhibited frequently by BL lines and, probably, by fresh BL cells. These properties may contribute to the precipitation of ATLS.

Acute Disease↗

PubMed1996

[Epstein-Barr virus (EBV)-positive NK cell line YT, and ALL/LBL of NK-lineage].

The YT cells, already known as natural killer (NK) line, were tested for Epstein-Barr virus (EBV). The simply maintained YT cells (YT-O) and the two different subclones showed and identical length of junctional DNA of terminal repeats in Southern blot with LMP-1 probe, indicating that the 3 had already been positive for EBV before subcloning. YT-O expressed limited amount of EBNA2 or LMP-1 mRNA, whereas the 2 subclones expressed abundant EBNA2 or LMP-1 mRNA in the Northern blot analysis with EBNA2 or LMP-1 probe. Thus, the ex vivo cells were positive for EBV, since the BL (Burkitt lymphoma) type EBV gene expression in the YT-O does not generally occur in in vitro infection. The remaining clinical record indicated that the lymphoma was extranodal (angiocentric lymphoma), involving mediastinum and liver, but not nodal or lymphoblastic lymphoma (LBL). Acute lymphoblastic lymphoma (ALL)/LBL of the NK-lineage has not been defined, although such neoplasms should exist. Since T- and NK-lineages are so close in immature stages of differentiation that ALL/LBL of NK may have been sorted into T-lineage. The phenotypic records of the T-ALL/LBL in our laboratory indicated that CD7+CD5+CD2- is much higher in incidence than CD7+CD5-CD2+. This may reflect the size difference of physiological populations of T- and NK-lineage cells. Furthermore, the latter is of CD45RO type in contrast to the rest of the early-thymic (pro-thymic) T-ALL/LBL groups of CD45RA type. A CD56+ case of 4 CD7+CD5-CD2+ cases have been published as a case of LBL of NK-lineage. It is necessary to scrutinize CD7+CD5-CD2+ cells in order to clarify the phenotype of neoplastic and physiological NK cells in immature stages.

Adolescent↗

PubMed1996

Membrane currents of murine osteoclasts generated from bone marrow/stromal cell co-culture.

Whole cell clamp recordings were conducted to examine the general behavior of ion channels in osteoclasts which were generated from bone marrow/stromal cell co-culture. In the resting state, inwardly and outwardly rectifying (IR and OR) currents were observed in the osteoclasts cultured for 5-18 days and the whole cell currents were classified into three types according to the conductance ratio between the OR and IR currents, such as, the IR type (OR/IR conductance < or = 0.2), the IR-OR type (0.2 < OR/IR < or = 5), and the OR type (OR/IR > 5). The IR type was found in 73% of cells (n = 78), the IR-OR type in 26% and the OR type in 1%. No relation was found between the current types and the number of nuclei or cultured days. The conductances varied among cells, but the IR conductance was larger than the OR conductance. The IR current was characterized by voltage-dependent kinetics, a negative conductance region and Ba(2+)-sensitivity. The reversal potential depended on the extracellular K+ concentration, indicating that K+ mediates the IR current. On the other hand, the OR current was reversibly reduced by decreasing extracellular Cl- concentration but not affected by K+, suggesting that the OR current depends on Cl-. High intracellular Ca2+ (1-10 microM) transiently activated a different class of outward current. These electrophysiological features resemble those in freshly isolated osteoclasts. We suggest that the ion channels involved in osteoclastic functions are expressed in the in vitro-generated osteoclasts and that the channels develop early in differentiation and are maintained for at least up to 18 days. Thus the co-culture system provide a useful model to examine roles of ion channels in osteoclastic functions.

Animals↗

PubMed1996

Diverse properties of human t(8;14) neoplasms: [1] ATLS and absence of BCL-2 [2] modulation of RAG-1 expression with S-Ig ligation.

An EBV(-) BL (Burkitt lymphoma) line (Black93), established from a patient exhibiting glucocorticoid-induced ATLS (acute tumor lysis syndrome), was highly sensitive to dexamethazone (DX) in vitro in the studies including 18 lymphoid cell lines (10 BL lines). In the BL lines, the highly sensitive ones always lacked Bcl-2(bcl-2 protein), while the DX resistant ones expressed Bcl-2. Black93 is the first BL cell-line derived from a ATLS patient, proving that cell lines can be established in vitro from ATLS patients. Since some pre-B ALL lines expressing Bcl-2 were DX-sensitive, the relationship between Bcl-2 and DX-sensitivity is not straight-forward. In the BL cells, however, the absence of Bcl-2 appears to be responsible for the DX-sensitivity. The DX-sensitivity and the absence of Bcl-2 is a major characteristic carried by t(8;14) neoplasms. In addition, there may be a stage of B-lineage differentiation without Bcl-2. While rare BL cases have been reported to express TdT (terminal desoxynucleotidyl transferase), Tree92 is the first such line, expressing S-Ig(mu, lambda), TdT and RAG (recombination activating gene)-1. When surface mu is ligated with antibody, RAG-1 was suppressed in expression, indicating that the signal through S-Ig can modulate the expression of RAG-1 in the Tree92 cells. Chromosome translocation is known to be associated with a specific stage of differentiation. Such specific stage for t(8;14), however, is broad enough to cover S-Ig(+), TdT(+) and RAG-1(+) stage, too. The phenotypic classification of leukemia/lymphoma and the delineation of differentiation scheme of normal hematopoietic cells, are dependent on each other. The documentation of the properties such as DX-sensitivity, the absence of Bcl-2, the expression of RAG-1 and its modulation by the signal through S-Ig is an example in which the diverse properties of human t(8;14) neoplasms can contribute for delineating the differentiation scheme of normal hematopoietic cells more precisely.

Burkitt Lymphoma↗

PubMed1995

Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets.

The phosphorylation and localization of the membrane-linking protein moesin was analyzed during early activation of platelets with thrombin. Activated platelets elaborate filopodia and spread to assume flat pancake-like shapes, and moesin is localized in filopodia and cell body. In resting platelets, approximately 25% of moesin molecules are phosphorylated as shown by metabolic labeling with 32P(i) and by isoelectric focusing. Within seconds after exposure to thrombin, phosphorylation increases, reaching a maximum of 35% labeled molecules by 1 min, followed by a decrease to a new basal level within 5 min. This modification affects a single residue, Thr558, which is located within or close to a binding site for F-actin. Rapid shifts (0-100%) in the number of phosphorylated molecules are observed in the presence of inhibitors of serine/threonine kinases and phosphatases. Inhibitors affecting tyrosine phosphorylation also modulate phosphorylation at this site suggesting that the enzymes involved in the modification of Thr558 are regulated by tyrosine phosphorylation. Platelets respond to both extremes of modification by forming extremely long filopodia and the absence of spreading on glass. Completely phosphorylated moesin is concentrated together with F-actin in the center of the cell. The rapid modification of moesin at or near its actin-binding domain suggests a model for regulated membrane-cytoskeleton interaction during cell activation.

Actins↗

PubMed1995

Local treatment with antithrombotic drugs can prevent thrombus formation: an angioscopic and angiographic study.

OBJECTIVES: This study was designed to evaluate the efficacy of local versus systemic treatment of thrombosis with various antithrombotic drugs. BACKGROUND: Local use of low dose antithrombotic drugs has been proposed as being effective and safe. METHODS: Heparin (30 U/kg), an antithrombin agent (argatroban, 0.05 mg/kg body weight) or a defibrinogenating drug (batroxobin, 0.05 U/kg) was locally infused into one side of the canine iliac artery after injury by balloon inflation. The other side was injured as a control. The efficacy of systemic delivery of high dose (heparin [300 U/kg] and argatroban [0.5 mg/kg]) and low dose drugs was also assessed. RESULTS: Sixty minutes after local treatment in 22 dogs, no thrombotic stenosis was observed by angiography in locally treated arteries (p < 0.005 vs. mean thrombotic stenosis of 27% in control segments for heparin, 25.3% in control segments for argatroban and 32% in control segments for batroxobin). Angioscopy demonstrated the same trend. In locally treated arteries, thrombus weight was significantly lower in the treated than control side. In the systemic high dose group (n = 10), angiographic thrombotic stenosis was < 5% after high dose drug delivery (p < 0.05 vs. control segments, 37.4% for heparin, 43% for argatroban). In another 10 dogs, low dose systemic delivery was not effective in inhibiting thrombus formation. Activated partial thromboplastin time and fibrinogen levels did not change with local treatment. CONCLUSIONS: Compared with systemic administration of antithrombotic drugs, local treatment is a safer and more effective method of preventing thrombosis.

Angiography↗

PubMed1995

Potentiation of excitatory postsynaptic potentials by a metabotropic glutamate receptor agonist (1S,3R-ACPD) in frog spinal motoneurons.

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg2+, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 microM ACPD and in 7 of 12 cells with 0.5 microM ACPD. The EPSP amplitudes with 5 and 0.5 microM ACPD were 142 +/- 10% (mean +/- S.E.M., n = 19) and 130 +/- 13% (n = 12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 microM ACPD were not significantly different in cells perfused with the low Ca(2+)-high Mg2+ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence of D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Collapsin-induced growth cone collapse mediated by an intracellular protein related to UNC-33.

Collapsin, a member of the newly recognized semaphorin family, contributes to axonal pathfinding during neural development by inhibiting growth cone extension. The mechanism of collapsin action is poorly understood. Here we use a Xenopus laevis oocyte expression system to identify molecules involved in collapsin signalling, because several experiments have raised the possibility that heterotrimeric GTP-binding proteins might participate in these events. A collapsin response mediator protein of relative molecular mass (M(r)) 62K (CRMP-62) required for collapsin-induced inward currents in X. laevis oocytes is isolated. CRMP-62 shares homology with UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. CRMP-62 is localized exclusively in the developing chick nervous system. Introduction of anti-CRMP-62 antibodies into dorsal root ganglion neurons blocks collapsin-induced growth cone collapse. CRMP-62 appears to be an intracellular component of a signalling cascade initiated by an unidentified transmembrane collapsin-binding protein.

Amino Acid Sequence↗

PubMed1995

Characterization of Gq family G proteins GL1 alpha (G14 alpha), GL2 alpha (G11 alpha), and Gq alpha expressed in the baculovirus-insect cell system.

The alpha subunits of Gq family G proteins, GL1 alpha (G14 alpha), GL2 alpha(G11 alpha), and Gq alpha were expressed with G protein beta 1 and gamma 2 subunits in insect cells using a baculovirus system. The trimeric forms of G proteins, GL1 (GL1 alpha beta gamma), GL2 (GL2 alpha beta gamma), and Gq (Gq alpha beta gamma), were solubilized by 1% sodium cholate and purified by sequential chromatography on three kinds of columns. GL1, GL2, and Gq activated phospholipase C-beta purified from bovine brain in the presence of aluminum fluoride to the same extent. Muscarinic acetylcholine receptor m1 subtype stimulated the guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) binding to GL1, GL2, and Gq in the presence of similar concentrations of carbamylcholine. When m1 receptor, G protein, and phospholipase C-beta were reconstituted in lipid vesicles, each subtype of Gq family G proteins mediated the activation of phospholipase C-beta by carbamylcholine in the presence of either 1 microM GTP gamma S or 1 mM GTP. Phospholipase C-beta stimulated the GTPase activity of GL1, GL2, and Gq in the presence of m1 receptor and carbamylcholine but did not stimulate the GTPase activity of GO. Protein kinase C phosphorylated m1 receptor and phospholipase C-beta, but the phosphorylation did not significantly affect the ability of the m1 receptor to stimulate phospholipase C-beta in the reconstitution system of purified proteins.

Animals↗

PubMed1995

Expression pattern of CD45 RA/RO isoformic antigens in T-lineage neoplasms.

The expression of CD45 RA/RO antigen was investigated in neoplasms including cases expressing CD7 antigen as the sole pan-T antigen (n = 8), T-lineage acute lymphoblastic leukemia (ALL)/lymphoblastic lymphoma (LBL) at various stages of differentiation (n = 32), peripheral stage T-lineage leukemia (n = 10) and adult T-cell leukemia (ATL) (n = 14). The p56lck gene expression was also investigated in selected cases. The expression pattern of CD45 RA/RO antigen was defined as of RA, mixed, or RO type. All but one CD7+ CD5- CD2- case were of the RA type. The CD7+ CD5+ CD2- prothymic stage included seven RA and one mixed type cases. One CD7+ CD5- CD2+ case was of the RA type, but the other was of the RO type. The CD7+ CD5+ CD2+ prothymic stage included three RA and four mixed type cases. All seven CD3- CD4+ CD8+ (double-positive) thymic cases were of the RO type. The CD3+ CD4+ CD8+ (triple-positive) stage included two RO and three mixed-type cases. One CD3+ CD4+ CD8- late thymic case was of the mixed type. The peripheral stage cases included five RA, three RO, and two mixed type cases. All ATL cases were of the RO type. The expression of p56lck gene in the prothymic stage was less marked than that in the thymic stage. On the basis of these results, the following sequence of pattern of the CD45 RA/RO antigen expression along with T-lineage differentiation was reconstructed: prothymic stage [RA and mixed type]-->double-positive thymic stage [RO type]-->triple-positive thymic stage [RO and mixed type]-->peripheral stage [RA, mixed, and RO type]. While one RO-type CD7+ CD5- CD2- and one RO-type CD7+ CD5- CD2+ cases were not in accord with this sequence, the pattern of CD45 RA/RO antigen expression in most of T-lineage neoplasms could be determined by the respective stage of differentiation. The poor expression of the p56lck gene by the prothymic blasts compared with the thymic blasts may be related to the expression pattern of the CD45 RA/RO molecules, which exhibits phosphatase activity. The consistent RO-type expression in the ATL cases may reflect the activated status of the neoplastic T cells due to the presence of the HTLV-I gene. Alternatively, the target cells for HTLV-I-induced neoplastic transformation may possible be of the RO type.

Adolescent↗

PubMed1995

Rheological significance of tandem lesions of the coronary artery.

It is not known whether the individual lesions that constitute tandem lesions of the coronary artery are developmentally or rheologically related. Luminal changes and their rheological significance were examined by percutaneous angioscopy in 44 tandem lesions of 21 patients with ischemic heart disease. Angioscopically, individual narrowing of angiographically documented tandem lesions appeared only as a tangentially expressed prominent portion of an atherosclerotic spiral fold. The directions of the fold were counterclockwise in the proximal to middle segments and clockwise in the distal segment of the right coronary artery, clockwise in the proximal to middle segments and counterclockwise in the distal segment of the left anterior descending artery, and counterclockwise in the proximal to middle segments of the left circumflex artery. The bloodstream always ran along the spiral folds in the tandem lesions. The results suggest that angiographically documented tandem coronary lesions are merely a tangential expression of atherosclerotic spiral folds and that they may act to prevent blood turbulence by generating a spiral laminal flow.

Adult↗