Biomedical subjects

Y Kudo

Publications and source records attributed to Y Kudo.

At least 19 recordsLinked to original sources

PubMed1992

The expression of presynaptic t-ACPD receptor in rat cerebellum.

The expression of a receptor subtype for one type of excitatory amino acid agonist, t-ACPD, was examined in developing Purkinje cells of cerebellar slices. The t-ACPD-induced responses were compared with those induced by QA in current response, single cell Ca2+ imaging and changes in the miniature currents in the same preparation. It was found that t-ACPD induced a single component of inward current, and an increase in the frequency of miniature currents associated with the presence of external Ca2+, but was ineffective at mobilizing intracellular Ca2+ even in the presence of external Ca2+. The present study suggests the expression of at least two types of metabotropic receptors in the Purkinje cell region, one of which, expressed in the Purkinje cell dendrites, is highly sensitive to QA, but relatively insensitive to t-ACPD, and the other of which is a t-ACPD-sensitive receptor expressed on the presynaptic terminals of the neurons making synapses onto Purkinje cells.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1992

Calpain may produce a Ca(2+)-independent form of kinase C in long-term potentiation.

Both an enhancement of Ca(2+)-independent kinase activity in the supernatant fraction and enhanced breakdown of type beta kinase C (PKC-beta) were observed in the hippocampus after induction of tetanus-induced long-term potentiation (LTP) in the hippocampal CA1 region of rat. The enhanced activity was inhibited by the PKC-specific inhibitor, PKC19-36. Both phenomena were also observed simultaneously in the in vitro model system in which hippocampal homogenate was treated with CaCl2, and both enhancements were inhibited by the addition of calpain inhibitors, leupeptin and benzyloxycarbonyl-Leu-Met-H. The results suggest that Ca(2+)-independent kinase activity enhanced in the supernatant fraction during LTP derives from the catalytic fragment of PKC-beta released by calpain.

Animals↗

PubMed1992

Chemiluminescence-HPLC assay of phosphatidylcholine hydroperoxide generated by ischemia-reperfusion in the liver of rats.

To determine cellular damage due to "oxidative stress", we developed a sensitive and specific quantitative assay for phosphatidylcholine hydroperoxide (PCOOH) by coupling HPLC with detection of chemiluminescence (CL). The qualitative and quantitative detection limits of PCOOH by this assay were 0.5 and 2 pmol (based on active oxygen from hydroperoxide). Using this CL-HPLC method, we determined PCOOH levels caused by ischemia-reperfusion in rat livers. The PCOOH levels in livers of control, sham-operated and operated rats with only ischemic treatment were approximately 2 nmol/g wet liver weight. The PCOOH level and several serum parameters of liver injury increased with an increase in the duration of ischemia, and also increased in proportion to the duration of reperfusion. The determination of PCOOH in liver caused by ischemia-reperfusion could be a useful method for investigating liver damage induced by free radicals.

Animals↗

PubMed1992

Block of synapse formation between cerebral cortical neurons by a protein kinase inhibitor.

Synchronized Ca2+ transients in cultured hippocampal neurons reflect the pattern of underlying electrical activity. Here we demonstrate a similar synchronization of cerebral cortical neurons in culture, and show that this functional coupling is correlated to the appearance of morphologically identified synapses using electron microscopy. During screening of a series of drugs for inhibition of in vitro synaptogenesis, the continuous presence of a protein kinase inhibitor (K-252b) in the culture medium was found to block the synchronous firing and to decrease significantly the number of morphologically identifiable synapses. Since K-252b does not permeate the cell membrane, the results strongly suggest that phosphorylation of cell surface protein(s) by a K-252b sensitive-protein kinase is an essential process in synapse formation.

Animals↗

PubMed1992

Antibody specific for the Thr-286-autophosphorylated alpha subunit of Ca2+/calmodulin-dependent protein kinase II.

We report the production of an antibody specific for Ca2+/calmodulin-dependent protein kinase II (CaM-KII) autophosphorylated only at Thr-286 of the alpha subunit. Peptide Y-66 [sequence MHRQETVDC (Met-281 to Cys-289 of alpha subunit of CaM-KII)] was synthesized and phosphorylated by the CaM-KII endogenous to synaptic cytoskeleton (postsynaptic density-enriched fraction); the phosphorylated amino acid residue threonine corresponds to Thr-286 in the kinase alpha subunit. The phosphorylated Y-66 peptide was separated from the unphosphorylated peptide by HPLC and used as an immunogen after being coupled to hemocyanin. The antibodies that reacted with hemocyanin and unphosphorylated Y-66 peptide were adsorbed, and then IgG was purified. ELISA proved that the IgG obtained reacted specifically with phosphorylated Y-66 peptide. Immunoblot analysis showed that the antibody reacted specifically to the autophosphorylated CaM-KII both in purified and synaptic cytoskeleton-associated form. Appearance of CaM-KII subunits immunoreactive to anti-phosphorylated Y-66 antibody paralleled the generation of Ca(2+)-independent kinase activity. Immunocytochemical experiments clearly showed expression of the Thr-286- or Thr-287-autophosphorylated form of CaM-KII in cultured hippocampal cells treated with N-methyl-D-aspartate. Thus, this antibody could be extremely useful for studying the biological functions of CaM-KII.

Amino Acid Sequence↗

PubMed1992

Delayed appearance of G-protein coupled signal transduction system in developing cerebellar Purkinje cell dendrites.

To investigate the relation between the function of Ca(2+)-activated K+ channels and phosphoinositide turnover, we have examined the physiological and pharmacological characteristics of ionotropic and metabotropic quisqualate (QA) receptors in rat cerebellar Purkinje cells during development using the slice-patch method combined with Ca2+ imaging. The typical response to QA obtained from a rat on postnatal day (PND) 21 consisted of three components: (1) a fast inactivating inward current, (2) a slow inward current, and (3) a slow outward current. The slow inward current was abolished in Ca(2+)-free medium, while the fast inactivating inward current and the slow outward current remained unaffected. The slow outward current which appeared to be activated via a metabotropic receptor was not observed in the Purkinje cell of PND 7 rat, in which dendrites were poorly developed but its amplitude increased linearly with PND. QA caused significant increases in [Ca2+]i in the fully developed dendritic region of the Purkinje cells even in Ca(2+)-free medium, suggesting a dendritic localization of the metabotropic receptors.

Animals↗

PubMed1992

Further evidence for calcium permeability of non-NMDA receptor channels in hippocampal neurons.

We have previously suggested that kainic acid (KA) increases the fluorescence of a fluoroprobe for Ca2+ via activation of the non-NMDA (N-methyl-D-aspartic acid) receptor in cultured hippocampal cells. The present study provides further evidence to support this suggestion. KA and NMDA increased the fluorescence occasionally in different subcellular loci of a single cell for each agonist, excluding the possibility that KA activated the NMDA receptor. KA elevated fluorescence in the cell whose membrane was voltage-clamped, thus eliminating possible Ca2+ entry through voltage-dependent channels. A monophasic response to KA was found to occur in the cells identified immunochemically as neurons. Even when the cells were cultured at different embryonic stages, KA responsiveness reached a peak at a rather fixed timepoint through the total age of neurons, while NMDA responsiveness continued to increase. This suggests that the Ca(2+)-permeable non-NMDA receptor is expressed according to a genetic schedule, but not to an artifactual effect of cell culture.

Animals↗

PubMed1992

A single optical fiber fluorometric device for measurement of intracellular Ca2+ concentration: its application to hippocampal neurons in vitro and in vivo.

We developed a new system to measure the intracellular Ca2+ concentration in the deep region of the central nervous system with a single optical fiber (300 microns in diameter), used for both excitation and detection of the fluorescence of previously loaded fura-2. With this system, a brain region loaded with fura-2 was illuminated by a rotating disc bearing three different interference filters of 340, 360 and 380 nm at a rate of 600 rpm. The emitted fluorescence was collected by the same fiber connected to a photomultiplier whose output was fed into a computer which regulates the timing of illumination and detection. The time course of the change in the fluorescence due to 340, 360 or 380 nm excitation was measured simultaneously at the maximum sampling rate of 10 points/s. Ratios of fluorescence intensities were obtained after the experiment. After confirming that this system was sensitive enough to detect the change of intracellular Ca2+ concentration in cultured hippocampal neurons and hippocampal slices during depolarization by high potassium medium (50 mM), we applied this system to anesthetized rats. In the hippocampus preloaded with fura-2, characteristic changes in fluorescence intensities ascribed to an increase in intracellular Ca2+ concentration were detected after asphyxia. The system is potentially useful for investigating the physiological and pathological roles of Ca2+ in the brain.

Anesthesia↗

PubMed1992

Platelet-activating factor (PAF) receptor in rat brain: PAF mobilizes intracellular Ca2+ in hippocampal neurons.

Platelet-activating factor (PAF), an alkylether phospholipid, is produced in the brain when it is subjected to various stimuli. Using a Xenopus oocyte expression system, we obtained evidence for functional PAF receptor mRNA expression in rat brain. The presence of the PAF receptor was confirmed and shown to be quite ubiquitous in the CNS by RNA blot and radioligand binding studies. To investigate the neuronal functions of PAF, intracellular Ca2+ increase elicited by nanomolar PAF application was analyzed in cultured rat hippocampal cells. Fractions of NMDA-responsive cells and non-NMDA-responsive cells were shown to respond to PAF, suggesting a potential role for PAF in the Ca2+ signaling pathway in the hippocampus.

Animals↗

PubMed1992

Kinetics of Escherichia coli destruction by microwave irradiation.

The kinetics of destruction of Escherichia coli cells suspended in a solution by microwave irradiation with a microwave oven were studied. During radiation at several powers, the temperature of 0.01 M phosphate buffer (PB), pH 7.0, in a glass beaker increased linearly at a rate of A (degrees Centigrade per second) according to the exposure time. When E. coli cells suspended in PB were exposed in the same beaker, the number of viable cells decreased according to the exposure time and the power used. The survival curve was approximated to a set of three linear parts. For each part, a rate constant of destruction (k) and an extrapolated starting temperature (T0) at several powers were estimated. Thereafter, the relationships between A and k and between A and T0 were studied. When a flat petri dish was used, the A value of exposed PB was lower and bacterial destruction was inhibited; the survival curve was similar to a curve predicted from the A value by using the relationships between the parameters. As the concentration of salt in the solution increased (from 0 to 1.35 M), the A value decreased and bacterial destruction was more suppressed. No remarkable difference between the destruction profiles for microwave exposure and conventional heating, which had the potential to generate an equal A value, was detected. These results showed that the parameter A of an irradiated solution is essential when kinetics of bacterial destruction by microwave exposure are studied and that the destruction profile can be interpreted mostly by means of thermal effects.

Escherichia coli↗

PubMed1992

Augmented contractile response to endothelin and blunted endothelium-dependent relaxation in post-ischemic reperfused coronary arteries.

To elucidate the pathogenesis of the post-ischemic vascular injury in reperfused coronary arteries, the left anterior descending coronary artery (LAD) was occluded for 30 min or 60 min in 26 dogs. After 120 min of reperfusion, vascular strips were prepared from LAD and the left circumflex coronary artery (LCX) as control, and suspended in organ chambers containing Krebs-Henseleit solution and vascular reactivity was evaluated pharmacologically. In a separate experiment, LCX-strips from 12 dogs were subjected ex vivo to blood cell-free simulated ischemia by the substitution of perfusate to hypoxic, low pH and high K+ for 60 min and the following 60 min of reoxygenation. Vascular responses to various agents were compared prior to and after simulated ischemia. Vascular injury was also investigated histologically with electron microscopy. As the results; significantly blunted endothelium-dependent relaxations to acetylcholine (10(-6) M) and Ca-ionophore (A-23187: 10(-6) M) in LAD-strips compared to LCX were noticed (43.7 +/- 4.8 vs 61.6 +/- 7.3%, 22.7 +/- 5.2 vs 47.9 +/- 8.4% in % relaxation, respectively). Augmented contractile response selective to endothelin was also observed in reperfused vessels (LAD) compared to control vessels (10(-9) M: 105.4 +/- 19.5 vs 42.4 +/- 12.3% of 20 mM-KCl induced contraction, p less than 0.01). Electron microscopy revealed partial detachment and blebbing of endothelial cells in reperfused coronary arteries. Similar changes were also observed in the simulated ischemia and reoxygenation study, but the augmented response to endothelin was seen only when polymorphonuclear leukocytes (PMN) activated with phorbol myristate acetate were added. Our results suggest that endothelial injury does not essentially depend on PMN, but PMN promote augmented response to endothelin. These changes indicate that enhanced spasmogeneity is present in reperfused arteries, which may contribute to post-infarction angina and prolonged myocardial dysfunction after reperfusion.

Animals↗

PubMed1992

[Effects of Gomishi and Shosaiko-to on lipid peroxidation of rat brain].

Gomishi and Shosaiko-to were administered to the rats at a dose of 10-100 mg/kg daily for 2 weeks, and their effects on lipid peroxidation of rat brain were compared with that of alpha-tocopherol. Administration of Gomishi and Shosaiko-to showed almost the same suppressive action on the lipid peroxidation. Gomishi and Shosaiko-to exhibited a radical-trapping action on a stable free radical, 1, 1-diphenyl-2-picrylhydrazyl (DPPH), which was estimated photometrically. The effects of Gomishi or Shosaiko-to at concentrations of 10(-3) to 10(-5)g/ml on lipid peroxidation of rat brain homogenates were investigated. The lipid peroxidation was inhibited by the addition of these drugs, and the suppressive effect was also dependent on the concentration. These suppressive effects with Shosaiko-to were stronger than those of Gomishi. These results suggest that the radical trapping action of Gomishi or Shosaiko-to is the likely mechanism suppressing brain lipid peroxidation; Particularly, the suppressive effect on the brain's lipid peroxidation by Shosaiko-to is at least in part due to its radical trapping action and inhibition of O2-. production.

Administration, Oral↗

PubMed1992

[An injury of the liver caused by ischemia-reperfusion in rat liver. Report 2: Relationship between the damage of the liver and during the period of reperfusion].

Liver injury by 30-min ischemia following reperfusion was examined biochemically and histopathologically. A greater increase in the level of LDH was observed after 1-hr reperfusion. However, the level of LDH decreased in proportion to the period of reperfusion, while the levels of GOT and GPT were also increased rapidly and reached its peak at 12 hr following reperfusion and were almost restored to the control level by 48 hr. A similar increase was obtained in the lipid peroxides of the liver. In addition, cyt. P-450 content and NADPH cyt. c reductase activity decreased in proportion to the period of reperfusion up to 12 hr and then recovered by 96 hr. On the other hand, heme oxygenase activity was significantly increased by ischemia-reperfusion. The ischemia-reperfused liver resulted in various morphological changes with the period of reperfusion. The destruction of Disse's space, vacuolization of the cytoplasm and nonviable hepatocytes were observed after 12-hr reperfusion. These results indicate the greatest damages of the liver induced by 30-min ischemia following reperfusion is observed after 12-hr or 24-hr reperfusion. The liver injury by ischemia-reperfusion could be a useful experimental model to develop for future studies.

Alanine Transaminase↗

PubMed1992

Attenuation of dysfunction in the ischemia-reperfused liver by glycyrrhizin.

The present study evaluated the effect of glycyrrhizin (GR) on an injury of the liver caused by ischemia-reperfusion in rats. In the liver ischemia-reperfusion model, levels of serum GOT, GPT and LDH activities and lipid peroxides in the liver tissue were significantly increased. On the contrary, total glutathione content in the liver tissue and NADPH cytochrome P-450 reductase activity of liver microsomes were decreased. Pretreatment with GR 20 mg/kg, i.v. 10 min before induction of ischemia resulted in significant decreases in serum GOT, GPT, LDH activities and the lipid peroxide level and a higher tissue glutathione content during the period of reperfusion. Electron microscopic studies revealed various hepatocellular damages with an almost intact sinusoidal endothelium in ischemia-reperfused livers. However, the degree of damage was less severe in the livers from the rats pretreated with 20 mg/kg GR. The results indicate that GR is able to provide partial protection against ischemia-reperfused damage.

Animals↗

PubMed1992

Protective effect of ulinastatin against liver injury caused by ischemia-reperfusion in rats.

The effects of ulinastatin (ULN), a human urinary protease inhibitor, on liver injury caused by ischemia-reperfusion were studied in rats. In the liver ischemia-reperfusion model, ULN suppressed the elevation of serum transaminase levels and tissue lipid peroxide levels in the liver. ULN did not exhibit a radical-trapping action on the superoxide and hydroxyl radicals as measured by electron spin resonance (ESR). ULN suppressed formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA)-induced superoxide production from polymorphonuclear leukocytes (PMNs) as measured by the cytochrome c assay. ULN did not inhibit either xanthine oxidase (XO) activity or the conversion of xanthine dehydrogenase (XDH) to XO during the ischemic period. ULN also strongly protected against the hypotonic hemolysis of rat erythrocytes. These results suggest that ULN's membrane stabilizing action and suppressive effect against PMNs superoxide production might be attributed to its suppressive effect on the liver's lipid peroxidation caused by ischemia-reperfusion.

Animals↗

PubMed1992

[A study of the humoral immunity of mice injected with beryllium chloride].

We studied changes of humoral immunity, such as complement pathway activity, C3 contents and contents of immunoglobulin, in mice injected subcutaneously with BeCl2 or CuCl2 once a week for 12 weeks. Mean body weights of JCL: ICR female mice were approximately 30g in control mice (control group; n = 7), in mice injected with Be (Be group; n = 8) and in mice injected with Cu (Cu group; n = 8). Values of classical complement pathway activity (CH50) were 18.8 +/- 1.4 U per ml, 15.3 +/- 1.8 U per ml and 16.7 +/- 1.3 U per ml in the control group, Be group and Cu group, respectively. The CH50 values of Be and Cu groups were significantly lower than that of the control group (P < 0.01). In contrast, values of alternative complement pathway activity (ACH50) and contents of C3 were almost constant in the three groups. The immunoglobulin content in the Be group tended to increase. The activity of alanine aminotransferase in the Be group was markedly higher than that in the control group (P < 0.05), and the aspartate aminotransferase activity was also high. The CH50 value of mice injected with a small amount of Be once a week over a 12-week period decreased markedly, although either the ACH50 value or C3 content was the same as in the control group. The immunoglobulin content somewhat increased in the Be group. These results suggest the possibility that immune complex is induced by Be.

Animals↗

PubMed1992

The use of bovine anti-PMSG serum in beef cattle after PMSG-superovulation.

Thirteen beef cows were superovulated using 4,000 i.u. of pregnant mare serum gonadotrophin (PMSG) on days 9 to 14 of the estrous cycle, followed by two injections of 500 micrograms prostaglandin F2 alpha analogue (PGF2 alpha) 48 and 55 hrs later. Seven of them were injected intramuscularly with bovine anti-PMSG serum 12 hrs after the first signs of estrus. The remaining 6 cows were served as controls and received no antiserum. Peripheral blood concentrations of progesterone (P) and estradiol-17 beta (E2) were compared in relation to the superovulatory responses. The injection of anti-PMSG serum did not significantly affect the numbers of the corpora lutea (CL), the anovulatory follicles and the transferable embryos at 7 to 8 days after superovulatory estrus, but increased the ratio of embryos classified as excellent or good quality. Although the plasma P concentration showed no significant differences between the anti-PMSG-treated and control cows, the plasma E2 concentration displayed a characteristic difference, suppressing the second E2 peak in the anti-PMSG-treated cows. It is concluded that the use of bovine anti-PMSG serum for PMSG/PGF2 alpha-treated cows at 12 hrs after the beginning of the estrus improves the quality of embryos recovered, probably due to inhibition of high estrogenic environment following ovulation.

Animals↗

PubMed1992

[Determination of protoporphyrins in blood using HPLC. Standardization of protoporphyrins and interlaboratory comparison of analyses].

1) In order to compare the results of determination of protoporphyrin IX (PP) and zinc protoporphyrin IX (ZP) using HPLC, the preparation procedures for standard solutions of PP and ZP were studied at four laboratories. According to the methods mutually agreeable to the four laboratories, the absorption values using UV-Vis spectrometer from the 4 laboratories were found to be identical. 2) The total of 28 whole blood samples obtained from the same workers whose Pb-B levels ranged from 2.9 to 87.9 micrograms/dl were sent to the 4 laboratories for determination of PP and ZP. The laboratories were allowed to use their own HPLC apparatus, column, mobile phase and fluorometric detectors, although the same procedures were used to prepare the standard solutions of PP and ZP. The results showed that significant biases in ZP and PP levels were found between two out of the 4 laboratories, but the ZP values corrected by the recovery rate showed no statistical difference among the laboratories. 3) Blood levels of ZP, total protoporphyrin (TP, calculated by PP+ZP/1.1), and PP determined by HPLC method correlated well with free erythrocyte protoporphyrin (FEP) determined by the method of extraction into the acidic solvent. The correlation coefficients between Pb-B and log TP, ZP, FEP or PP were 0.82-0.84, 0.80-0.83, 0.786, or 0.70-0.73, respectively. 4) The coincident ratios of PP/TP obtained at 4 laboratories ranged from 12.1 to 19.0%. These values were within a relatively narrow range, although two different kinds of organic solvents (DMF: N',N'-dimethylformamide or Methanol/triton-X 100) were used in the extraction steps.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗