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Biomedical subjects

T Kuga

Publications and source records attributed to T Kuga.

At least 109 records · Page 6Linked to original sources
PubMed1991

Bovine brain coated vesicles contain guanine nucleotide regulatory proteins.

Binding of [3H]guanosine triphosphate (GTP) with a high affinity was found to be present in the coated vesicle fraction prepared from bovine cerebral cortex. The binding was saturable and displaced by 1 microM of GTP, guanosine diphosphate and guanosine 5'-(3-O-thio)triphosphate. Incubation of the vesicles with islet-activating protein and [32P]NAD resulted in ADP-ribosylation of a 39,000-41,000-dalton polypeptide. Antibodies to the alpha-subunit of stimulatory guanine nucleotide regulatory proteins (G-proteins) immunoblotted 52,000- and 45,000-dalton polypeptides. The results indicated that stimulatory and inhibitory G-proteins are contained in a fraction of the bovine brain coated vesicles.

Adenosine Diphosphate Ribose↗
PubMed1990

In vitro and in vivo hematopoietic effect of mutant human granulocyte colony-stimulating factor.

About 100 derivatives of human recombinant granulocyte colony-stimulating factor (rhG-CSF) were created by various gene-mutagenic techniques, and KW-2228, in which amino acids were replaced at five positions of N-terminal region of intact rhG-CSF, was picked up and evaluated for its biologic and physicochemical properties in comparison with intact rhG-CSF. KW-2228 showed two to four times higher specific activity than that of intact rhG-CSF in mouse and/or human bone marrow progenitor cells by colony-forming unit assay in soft agar, and by cell-proliferation assay in liquid culture. KW-2228 showed a potency to increase peripheral neutrophil counts when it was administered to normal C3H/He mice by single intravenous injection. Increase of total leukocyte count and neutrophils was observed, with peak level at 8 to 12 hours at low doses (0.5 to 1.0 micrograms/mouse), and the highest level was maintained for 24 to 30 hours at high doses (5 to 10 micrograms/mouse). The granulopoietic effect of KW-2228 was examined by several doses of single course (once daily for 10 days) or multiple courses (twice daily injection for 5 days followed by cessation for 9 days on one cycle, 3 cycles in total) of treatment. KW-2228 showed higher activity than that of rhG-CSF, especially at sub-optimal doses of multiple courses of treatment. Furthermore, KW-2228 was found to be more stable physicochemically and biologically than intact rhG-CSF, especially under thermal conditions at 56 degrees C and in the human plasma at 37 degrees C, suggesting a protease resistancy. Pharmacokinetic study showed that plasma concentration of KW-2228 assayed for its bioactivity maintained a higher level than that of intact rhG-CSF for 60 minutes after intravenous injection of this protein to normal mice. Those results suggest that KW-2228 might show a superior in vivo hematopoietic effect to intact rhG-CSF due to its high specific activity to progenitor cells, and also due to its improved physicochemical, biologic, and pharmacokinetic stability in host animals.

Animals↗
PubMed1990

Smooth muscles from spastic coronary artery segments show hypercontractility to histamine.

An animal model of coronary spasm was produced in Göttingen miniature pigs by a selective endothelial denudation of the coronary artery. Five months after the denudation, intracoronary bolus administration of 10 micrograms/kg histamine reduced the luminal diameter angiographically by 57 +/- 16 and 17 +/- 10% (P less than 0.01) in the previously denuded and contralateral control coronary arteries. Muscle fibers of 0.08-0.1 mm wide were prepared from circumferential bundles of the medial smooth muscle in the spastic and nonspastic coronary arteries. Upward shifts of either dose-tonic contraction relationships in Ca2(+)-containing solution or dose-monophasic contraction relationships in Ca2(+)-free solution were noted in muscle fibers taken from the spastic site compared with those from the nonspastic site with no difference between the mean effective dose values. After skinning the muscle fibers with saponin, there was no significant difference in the Ca2+ concentration-tension relationships between the two fibers. These findings suggest that an increased number of histaminergic receptors and/or augmentation of signal transduction, but not Ca2+ sensitivity of the contractile proteins in the medial smooth muscle cells, cause histamine-induced coronary hypercontraction.

Animals↗
PubMed1990

Actions of Ca2+ antagonists on two types of Ca2+ channels in rat aorta smooth muscle cells in primary culture.

Mechanisms of blockade of two types of Ca2+ channels by the organic Ca2+ antagonists, nicardipine, diltiazem, verapamil, and flunarizine, were examined in rat aorta smooth muscle cells in primary culture by using the whole-cell voltage-clamp method. T-type Ca2+ current (T-type ICa) was isolated by an internal perfusion of 5 mM F-, which irreversibly suppressed the L-type ICa, without affecting T-type ICa. L-type ICa was isolated by setting a holding potential at -60 mV, at which most of the T-type Ca2+ channels were inactivated. L-type ICa is halved by 0.1 microM nicardipine, 3.0 microM diltiazem, 0.6 microM verapamil, and 0.1 microM flunarizine, whereas T-type ICa is halved by the same drugs at 0.6, 30, 30, and 0.1 microM, respectively. Diltiazem and verapamil accelerated the decay of L-type ICa and cumulatively blocked L-type ICa during repetitive step depolarizations elicited every 30 seconds ("use-dependent block"). Diltiazem and verapamil neither changed the decay of T-type ICa nor showed a use-dependent block of T-type ICa. Nicardipine and flunarizine blocked both L- and T-type ICa from the first depolarization step after drug treatment ("tonic block") and shifted their steady-state inactivation curves to the left. The estimated binding constants of nicardipine and flunarizine for the inactivated state of T-type Ca2+ channels (48 and 19 nM, respectively) were smaller than those for the resting state of L-type Ca2+ channels (160 and 90 nM, respectively). A low concentration (0.1 microM) of nicardipine initially potentiated T-type ICa and then reduced it. We conclude from these results that 1) nicardipine and flunarizine block not only the resting state but, more preferentially, the inactivated state of both the L- and T-type Ca2+ channels; 2) verapamil and diltiazem preferentially act on the open state of the L-type Ca2+ channel and on the resting and inactivated state of the T-type Ca2+ channel; and 3) the T-type Ca2+ channel of the rat aorta smooth muscle cells appears to be more sensitive to nicardipine and flunarizine than does the L-type Ca2+ channel at around the resting membrane potential.

Animals↗
PubMed1990

Central effects of the neurotropic mycotoxin fumitremorgin A in the rabbit. (II). Effects on the brain stem.

In order to determine the main site of the convulsant action of the neurotropic mycotoxin fumitremorgin A, the role of the brain stem reticular formation was studied. In rabbits lightly anesthetized with urethane and chloralose, electrical stimulation of the reticular formation elicited burst discharges in the common peroneal nerve and in the tibial nerve. This facilitatory effect of electrical stimulation was markedly potentiated by intravenous administration of a small dose of FTA, before onset of actual convulsion. Under the influence of FTA, a remarkable increase in the spontaneous electrical activity of the midbrain reticular formation was observed. The firing pattern of some neurons in the reticular formation corresponded very well with abnormal burst discharges in the common peroneal nerve. These effects of FTA were inhibited by chlorpromazine (0.1-1.0 mg/kg, i.v.), diazepam (0.1-1.0 mg/kg, i.v.), mephenesin (5-10 mg/kg, i.v.) and pentobarbital (5-15 mg/kg, i.v.). It was concluded that FTA might activate some neurons in the midbrain reticular formation and that convulsive burst discharges in peripheral motor nerves resulted from abnormal activation of these neurons, although the role of the medullary reticular formation could not be excluded.

Animals↗
PubMed1989

Mutagenesis of human granulocyte colony stimulating factor.

To define the structure-function relationship, we have made a number of mutants of human granulocyte colony-stimulating factor (hG-CSF) by in vitro mutagenesis. The results indicate that most of the mutations located in the internal and C-terminal regions of the molecule abolished the activity, whereas the mutants without N-terminal 4, 5, 7, or 11 amino acids retained the activity. N-terminal amino acids were also altered by cassette mutagenesis using a synthetic oligonucleotide mixture. Among them, KW2228, in which Thr-1, Leu-3, Gly-4, Pro-5 and Cys-17 were respectively substituted with Ala, Thr, Tyr, Arg and Ser, showed more potent granulopoietic activity than that of intact hG-CSF both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1989

Low-voltage-activated calcium current in rat aorta smooth muscle cells in primary culture.

1. Electrical and pharmacological properties of the low-voltage-activated Ca2+ current (ICa, LVA) in rat aorta smooth muscle cells (SMC) in primary culture were examined, particularly in comparison with the high-voltage-activated Ca2+ current (ICa, HVA). Both types of Ca2+ currents were recorded in external solution containing 20 mM-Ca2+, using the whole-cell voltage-clamp technique. 2. ICa, LVA was evoked by step depolarizations to potentials more positive than -60 mV from a holding potential of -100 mV, and reached a peak in the current-voltage (I-V) relationship around -30 mV. ICa, HVA was activated at -20 mV, and reached a peak at +20 mV. 3. The intracellular dialysis of 5 mM-F- irreversibly suppressed ICa, HVA, with time, while it has little effect on the ICa, LVA. The ICa, LVA could be separated from the ICa, HVA by either selecting the holding and test potential levels or by perfusing intracellularly with F-. 4. The ratio of peak amplitude of Ba2+, Sr2+ and Ca2+ currents in the respective I-V relationship was 1.6:1.2:1.0 for high-voltage-activated Ca2+ channels and was 1.0:1.4:1.0 for low-voltage-activated ones. 5. The inactivation phase of ICa, HVA was fitted by a sum of double-exponential functions, the time constants of which were larger when the current was carried by Ba2+ than by Ca2+. The inactivation time course of ICa, LVA was fitted by a single-exponential function, and the time constant was practically the same when the current was carried by Ba2+ or by Ca2+. Activation and inactivation processes of ICa, LVA were potential-dependent. 6. The steady-state inactivation curve of ICa, LVA was fitted by the Boltzmann equation, having a mid-potential of -80 mV and a slope factor of 5.0. The recovery time course from steady-state inactivation was fitted by a sum of two exponential functions. The time constants of the faster phase were 230 and 380 ms, and those of slower phase were 2.8 and 1.8 s at the repolarization potentials of -120 and -100 mV, respectively. 7. The amplitude of ICa, LVA depended on the external Ca2+ concentration ([Ca2+]o), approaching saturation at 95 mM [Ca2+]o. 8. Various polyvalent cations blocked both types of Ca2+ current reversibly in the order (IC50 in M): La3+ (8 x 10(-8)) greater than Cd2+ (6 x 10(-6)) greater than Ni2+ (1 x 10(-5)) greater than Zn2+ (2 x 10(-5)) for ICa, HVA, and La3+ (6 x 10(-7)) greater than Zn2+ (3 x 10(-5)) greater than Cd2+ (4 x 10(-4)) greater than Ni2+ (6 x 10(-4)) for ICa, LVA.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Aminopyridine↗
PubMed1989

Histamine-induced calcium transients in vascular smooth muscle cells: effects of verapamil and diltiazem.

We investigated the effects of verapamil and diltiazem on histamine-induced Ca2+ transients in vascular smooth muscle. 1) With the use of quin2 microfluorometry, cytosolic Ca2+ concentrations were directly measured in cultured vascular smooth muscle cells of the rat aorta. In the presence of extracellular Ca2+, histamine induced an elevation of cytosolic Ca2+ concentrations of a peak and plateau type. The peak component is due to a release of Ca2+ from cellular store sites, and the plateau component depends on extracellular Ca2+. Verapamil and diltiazem inhibited the plateau component, and the 50% inhibitive concentration (IC50) of verapamil and diltiazem for 10 microM histamine was 0.09 and 0.18 microM, respectively. Only at high concentrations did verapamil (IC50 = 8.7 microM) and diltiazem (IC50 = 95.7 microM) inhibit the Ca2+ release from the cellular store sites, as induced by 10 microM histamine. 2) Histamine, verapamil, and diltiazem competed with [3H]mepyramine for binding to the porcine aortic membranes, the order of potency being verapamil (Ki = 7.1 microM) greater than histamine (Ki = 18 microM) greater than diltiazem (Ki = 114 microM). From these results, we conclude that verapamil and diltiazem strongly inhibit the histamine-mediated, extracellular Ca2+-dependent intracellular [Ca2+] increase. In addition, verapamil and diltiazem seem to inhibit the release of Ca2+ from intracellular store sites, only at high concentrations, and probably by competing with histamine for binding to the H1-receptor. The inhibitory effects of Ca2+ antagonists on the histamine-induced contraction or spasm of vascular smooth muscle may well relate to these mechanisms.

Aminoquinolines↗
PubMed1989

Central effects of the neurotropic mycotoxin fumitremorgin A in the rabbit (I). Effects on the spinal cord.

Effects of a potent neurotropic mycotoxin, fumitremorgin A (FTA), on the spinal cord were studied, using rabbits lightly anesthetized with urethane and chloralose. Spontaneous discharges of L7 spinal ventral roots and common peroneal nerves were increased after intravenous injection of 100-200 micrograms/kg of FTA. Their abnormal discharge pattern represented the convulsive effect of FTA. Spinal monosynaptic reflexes became irregular in amplitude, with a slight predominance of smaller reflexes. Polysynaptic reflexes were inhibited in many cases. These FTA-induced changes in ventral root discharges and spinal reflexes were abolished by spinal transection at a segment of the upper level. These results suggest that FTA may have no direct facilitatory action on spinal motoneurons and that its remarkable motor effect has its origin in the supraspinal central nervous system.

Animals↗
PubMed1989

Detection and characterization of 3H-clonidine binding sites in coated vesicles isolated from bovine brain.

The binding of 3H-clonidine to bovine brain coated vesicles was studied. The binding was reversible and saturable. Saturation studies revealed a one-site interaction: Kd was 8.7 nM and Bmax was 24.5 fmol/mg protein. Noradrenaline, yohimbine and phenoxybenzamine displaced 3H-clonidine binding from the binding sites at concentrations of 10(-7)-10(-4) M. The results indicate that the coated vesicles from the bovine cerebral cortex contain 3H-clonidine binding sites comparable to the low affinity sites of alpha 2-adrenergic receptors in bovine brain membranes.

Adrenergic alpha-Agonists↗
PubMed1988

A gag-env hybrid protein of human T-cell leukemia virus type I and its application to serum diagnosis.

A fused gene of the gag and env sequences of human T-cell leukemia virus type I (HTLV-I), the causative agent of adult T-cell leukemia, was constructed in vitro and expressed in Escherichia coli. The gag-env hybrid protein accumulated as insoluble granules with a yield of approximately 12% of the total proteins. In an enzyme-linked immunosorbent assay done with the use of the gag-env hybrid protein, all 57 seropositive sera gave positive signals, and none of the sera from normal persons did. This system can produce large quantities of the gag-env hybrid protein, which can be used for mass screening of human sera for HTLV-I infection.

Epitopes↗
PubMed1988

Involvement of GABAergic systems and benzodiazepine receptors in the jumping behavior induced by harmine and apomorphine in rats.

Harmine (HA) induces a jumping behavior in rats when the central dopaminergic function has been activated. Possible involvement of the GABAergic systems and the benzodiazepine receptors (BZA-R) in the jumping behavior induced by HA and apomorphine (APO) was investigated. Pretreatment with GABAergic agonists, muscimol (0.05-0.2 mg/kg) and diazepam (0.2-1.0 mg/kg), and antagonists, picrotoxin (0.1-0.5 mg/kg) and bicuculline (0.1-0.5 mg/kg), suppressed the jumping behavior in a dose-dependent manner in rats treated with HA (10 mg/kg) and APO (2 mg/kg). After treatment with 2, 5 or 10 mg/kg of HA in combination with 2 mg/kg of APO, a decrease in 3H-diazepam binding to the brain regional membranes was observed in the corpus striatum (CS) in a dose-dependent manner, but not in the other brain regions. The decrease in the 3H-diazepam binding to the CS membranes was proportional to the increase in the HA levels in the CS, and the HA levels were correlated with the intensity of jumping behavior. Pretreatment with Ro 15-1788, an antagonist of BZA-R, suppressed the jumping behavior induced by HA and APO. These results indicated the involvement of the GABAergic systems in the jumping behavior and suggested that HA induced the jumping behavior partly as a result of interacting with the BZA-R.

Alkaloids↗