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Biomedical subjects

Y Fukui

Publications and source records attributed to Y Fukui.

At least 19 recordsLinked to original sources
PubMed1992

Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1.

Monoclonal antibodies raised against the 85-kDa subunit (p85) of bovine phosphatidylinositol (PI) 3-kinase were found to recognize uncomplexed p85 or p85 in the active PI 3-kinase. Immunoprecipitation studies of Chinese hamster ovary cells, which overexpress the human insulin receptor when treated with insulin, showed increased amounts of p85 and PI 3-kinase activity immunoprecipitable with monoclonal anti-p85 antibody and no increase in the tyrosine phosphorylation of p85. Insulin also induced an association of p85 with the tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and other phosphorylated proteins ranging in size from 100 to 170 kDa but not with the activated insulin receptor. In vitro reconstitution studies were used to show p85 in the active PI 3-kinase associated with the tyrosine-phosphorylated IRS-1 but not with the activated insulin receptor. Competition studies using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of IRS-1 revealed that phosphopeptides containing YMXM motifs inhibited this association with different potencies, whereas nonphosphorylated analogues and a phosphopeptide containing the EYYE motif had no effect. Src homology region 2 domains of p85 expressed as glutathione S-transferase fusion proteins also bound to tyrosine-phosphorylated IRS-1. These results suggest that insulin causes the association of PI 3-kinase with IRS-1 via phosphorylated YMXM motifs of IRS-1 and Src homology region 2 domains of p85.

Amino Acid Sequence↗
PubMed1992

Potent and specific inhibition of p60v-src protein kinase both in vivo and in vitro by radicicol.

A fungal metabolite, radicicol, with a macrocyclic ring induced the reversal of transformed phenotypes of v-src-transformed fibroblasts (Rous sarcoma virus-transformed 3Y1 rat fibroblast) at a quite low concentration of 0.1 microgram/ml. Actin stress fibers reappeared in the transformed cells after treatment with radicicol. Radicicol reduced the intracellular level of autophosphorylation of p60v-src as well as the level of other tyrosine-phosphorylated proteins in a dose-dependent manner. In vitro kinase assay revealed that radicicol effectively inhibited not only autophosphorylation but also transphosphorylation activities of purified p60v-src with a concentration producing 50% inhibition of 0.1 microgram/ml. However, radicicol showed no inhibitory effect on protein kinase C or protein kinase A. These results suggest that radicicol is a novel and specific protein-tyrosine kinase inhibitor and that the decreased level of tyrosine kinase activity of p60v-src causes reversion of transformed phenotypes of Rous sarcoma virus-transformed 3Y1 rat fibroblast. Furthermore, differentiation of Friend leukemia cells, which is one of the known characteristic phenomena associated with the inhibition of tyrosine kinase, was also induced in the concentration range of 0.05-0.5 microgram/ml, suggesting that the agent is useful for the analysis of differentiation as well as the kinase-mediated signal transduction.

Animals↗
PubMed1992

Osteoclasts express high levels of p60c-src, preferentially on ruffled border membranes.

Expression of p60c-src, the normal cellular counterpart of the transforming protein of Rous sarcoma virus (RSV), p60v-src, was examined in mouse and rat authentic osteoclasts and mouse osteoclast-like multinucleated cells (MNCs) formed in vitro. In co-cultures of mouse osteoblastic cells and spleen cells, the expression of p60c-src strikingly increased on day 5 in parallel with the appearance of MNCs in the presence of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). Immunohistochemical examination confirmed the high level expression of p60c-src in both mouse authentic osteoclasts and MNCs. Electron microscopic examination revealed that p60c-src was primarily localized on ruffled border membranes and vacuoles, but not on the clear zone in rat authentic osteoclasts. These results suggest that p60c-src is important in osteoclastic bone resorption.

Animals↗
PubMed1992

Insulin treatment stimulates the tyrosine phosphorylation of the alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase in vivo.

After adding insulin to cells overexpressing the insulin receptor, the activity of phosphatidylinositol (PI) 3-kinase in the anti-phosphotyrosine immunoprecipitates was rapidly and greatly increased. This enzyme may therefore be a substrate for the insulin receptor tyrosine kinase and may be one of the mediators of insulin signal transduction. However, it is unclear whether or not activated tyrosine kinase of the insulin receptor directly phosphorylates PI 3-kinase at tyrosine residue(s) and whether insulin stimulates the specific activity of PI 3-kinase. We reported previously that the 85-kDa subunit of purified PI 3-kinase was phosphorylated at tyrosine residue(s) by the insulin receptor in vitro. To examine the tyrosine phosphorylation of PI 3-kinase and change of its activity by insulin treatment in vivo, we used a specific antibody to the 85-kDa subunit of PI 3-kinase. The activity of PI 3-kinase in immunoprecipitates with the antibody against the p85 subunit of PI 3-kinase was increased about 3-fold by insulin treatment of cells overexpressing insulin receptors. Insulin treatment also stimulated the tyrosine, serine, and threonine phosphorylation of the alpha-type 85-kDa subunit of PI 3-kinase in vivo. Phosphatase treatment of the immunoprecipitates abolished the increase in PI 3-kinase activity. The phosphorylation(s) of the kinase itself, tyrosine phosphorylation(s) of associated protein(s), or the complex formation of the phosphorylated PI 3-kinase with associated proteins may increase the activity of PI 3-kinase.

Animals↗
PubMed1992

Activation of Src-like protein-tyrosine kinase Lyn and its association with phosphatidylinositol 3-kinase upon B-cell antigen receptor-mediated signaling.

Crosslinking of membrane-bound immunoglobulins, which are B-cell antigen receptors, causes proliferation and differentiation of B cells or the inhibition of their growth. The receptor-mediated signaling involves tyrosine phosphorylation of cellular proteins. The Src-like protein-tyrosine kinase Lyn is expressed preferentially in B cells and is an intracytoplasmic constituent of the B-cell antigen receptor complex. Crosslinking of membrane-bound immunoglobulin M with antibody induced rapid increases in the kinase activities of Lyn and Lyn-associated phosphatidylinositol 3-kinase. Crosslinking of B-cell antigen receptor also induced association of Lyn with an 85-kDa noncatalytic subunit of phosphatidylinositol 3-kinase. Thus, Lyn is functionally associated with membrane-bound immunoglobulin M and seems likely to participate in B-cell antigen receptor-mediated signaling.

B-Lymphocytes↗
PubMed1992

Parthenogenetic development of bovine oocytes treated with ethanol and cytochalasin B after in vitro maturation.

The present study was conducted to investigate the effects of different culture durations (24-36 hr) on bovine oocyte maturation in vitro and the effect of the presence or absence of cumulus cells at the time of treatment to induce parthenogenetic activation (exposure to ethanol and cytochalasin B; CB) (experiment I). The effects of dosage (2.5 or 5.0 micrograms/ml) and incubation time (2.5, 5, or 10 hr) in CB (experiment II) on the subsequent development to the blastocyst stage in vitro was also investigated. In experiment I, cleavage and development to the blastocyst stage were not affected by the presence or absence of cumulus cells at the time of parthenogenetic activation. However, the 24-hr culture duration for in vitro maturation had a significantly lower rate of development to the blastocyst stage than the longer culture durations (27-36 hr). In experiment II, treatment with 5 micrograms/ml CB for 5 hr showed the highest percentage of development to blastocyst in the oocytes matured for both 27 and 30 hr. To determine the viability of the parthenogenetic embryos (morulae and blastocysts), four recipient heifers received two embryos each, and one heifer was found to be pregnant on day 35 following transfer. Although fetal heartbeat was not observed, the subsequent estrus was prolonged in all heifers. The present results demonstrate development of in vitro-matured, parthenogenetically activated bovine embryos up to the preimplantation stage.

Animals↗
PubMed1992

Cardiovascular simulation using a multiple modeling method on a digital computer--simulation of interaction between the cardiovascular system and angiotensin II.

A cardiovascular system model that simulates interactive responses to drugs has been developed on a small digital computer. The overall model basically consists of three models. The first is a momentum transport model that represents relations between blood pressure and flow in the cardiovascular system. In this model, the cardiovascular system is divided into 14 components and modeled by using equivalent electrical circuits. The second is a mass transport model comprising 14 compartments corresponding to the respective components of the cardiovascular system. This model represents the distribution of the administered drug in the various cardiovascular components. The third is an interaction model that represents the relationships between the momentum and mass transport models. This model causes variations in the resistance and capacitance parameters of the momentum transport model as a function of the current drug concentrations in the appropriate compartments of the mass transport model. The capacitances representing the ventricles are varied in a time-dependent fashion to simulate the beat of the heart. Simulation is performed by using the Euler method to solve a system of 28 ordinary differential equations governing the momentum and mass transport models on a 32-bit microcomputer, a Macintosh II. The model was assessed by performing two demonstrations of the cardiovascular response to the vasopressor angiotensin II (AT II). They first examined the interaction between the cardiovascular system and AT II. The effect of AT II on the cardiovascular system was incorporated into the interaction model. Administration of AT II as a constant infusion (200 micrograms/hr) resulted in an elevation of mean arterial pressure from approximately 100 to 150 mm Hg.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1992

Heart rate and body temperature sensitive diaphragm pacing.

Two ways of rate control for diaphragm pacing are proposed. One is rate control using only the patients' body temperature (method I). The other is rate control by both the patients' heart rate and body temperature (method II). To test the effectiveness of these methods, a diaphragm pacemaker which can be controlled by both heart rate and body temperature has been developed. It was applied to nine mongrel dogs. The pacing rate is controlled by atrial blood temperature (method I) or by both heart rate and temperature (method II). The animal's metabolism was elevated by the administration of a pyrogenic drug. It was found that method I is not suited to rapid changes in metabolism; however, it is useful in extreme metabolic elevation. An animal's metabolism was supported by using method II in all ranges of metabolism. This method proved more effective than method I for rate-responsive diaphragm pacing.

Animals↗
PubMed1992

Intrapancreatic duodenal duplication associated with pancreatic pseudocysts.

A rare case of intrapancreatic duodenal duplication causing pancreatitis is reported. At 2 years of age, the patient presented with a recurrent pancreatic pseudocyst. Intraoperative pancreatogram showed the presence of cystic duodenal duplication in the aberrant lobe of the pancreas communication with the pancreatic duct. Since the resection of the duplication, she has been free from recurrence of pancreatitis. In this case, intraoperative pancreatography was of great value.

Duodenum↗
PubMed1992

The superior cervical ganglion: origin of sympathetic fibers in the facial and hypoglossal nerves in the cat.

Location of superior cervical ganglion (SCG) neurons, sending axons into the facial and hypoglossal nerves, was investigated in the cat by means of retrograde axonal transport of horseradish peroxidase (HRP). After wheat germ agglutinin conjugated HRP (WGA-HRP) was injected into these nerves, many retrogradely labeled neurons were found widely in the ipsilateral SCG, particularly around the caudal half of the SCG. These neurons were round or oval in shape and 70-80% of these were medium in size. In fluorescent experiments, fast blue (FB) was used in combination with diamidino yellow (DY). After injections of FB into the facial nerve and DY into the hypoglossal nerve, a few FB-DY double-labeled neurons occurred in the SCG ipsilaterally.

Adrenergic Fibers↗
PubMed1992

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the carbodiimide method.

Copper(II) chloride was found to be an extremely efficient racemization-suppressing additive in the DCC method as compared with the hitherto known ones, by employing the model coupling Z-Gly-L-Val-OH + H-L-Val-OMe in DMF. Although some other copper salts also had a profound effect, copper(II) chloride was the best from the viewpoint of both racemization suppression and coupling efficiency. The effectiveness of copper(II) chloride was further confirmed by employing the EDC-mediated couplings of Z-Gly-containing dipeptides with amino acid esters or dipeptide esters, and those of Z-L-Ala (or L-Val)-L-Val-OH with amino acid esters or dipeptide esters. In almost all the cases studied, no detectable amount (less than 0.1%) of epimer was observed by the HPLC analysis in the presence of copper(II) chloride. This was also the case even with an extremely stringent coupling system Z-L-Pro-L-Val-OH + H-L-Pro-OMe. With reference to the mechanism of racemization suppression, it was found that copper(II) chloride has a strong ability to suppress the racemization of the 5(4H)-oxazolone, which may be formed from an activated carboxyl component during the coupling.

Amino Acid Sequence↗
PubMed1992

Simultaneous use of 1-hydroxybenzotriazole and copper(II) chloride as additives for racemization-free and efficient peptide synthesis by the carbodiimide method.

In the carbodiimide mediated coupling of Z-Gly-L-Val-OH with H-L-Val-OMe in DMF, the simultaneous use of HOBt and copper(II) chloride as additives was found to give the desired peptide in a high yield without racemization. In the presence of HOBt, reducing the amount of copper(II) chloride produced a higher yield. Besides improving the coupling efficiency as compared with the case using copper(II) chloride alone as an additive, the present procedure offered another advantage for racemization suppression. Thus, even for the couplings where a low level of racemization was observed in the presence of copper(II) chloride, the simultaneous addition of HOBt and copper(II) chloride resulted in the elimination of racemization. The effectiveness of this new procedure using the two carbodiimide additives in the synthesis of biologically active peptides was assessed by the preparation of a protected Leu-enkephalin. In the 4 + 1 segment condensation using HOBt and copper(II) chloride simultaneously as additives, no racemization was detected and the yield was high enough. The elimination of racemization and improvement of coupling efficiency produced by the present procedure can be attributable to a reduced tendency for the activated forms of the carboxyl component to form a 5(4H)-oxazolone by the action of HOBt, and to the prevention of racemization by copper(II) chloride of the small amount of the oxazolone formed which is not eliminated by the action of HOBt alone.

Amino Acid Sequence↗
PubMed1992

Quantitative immunocytochemical analysis of the induction of cytochrome P450IIB in rat hepatocytes.

We examined whether induction of the phenobarbital (PB)-inducible form of cytochrome P450 (P450IIB) in rat hepatocytes could be analyzed quantitatively by immunogold electron microscopy. Rats received intraperitoneal injections of PB every 24 hr and livers at the various stages of PB induction were fixed by perfusion with a mixture of paraformaldehyde (4%) and glutaraldehyde (0.1%) and embedded in LR White. Ultra-thin sections were cut and labeled by the protein A-gold procedure using affinity-purified anti-P450IIB antibody which was previously immunoabsorbed with liver microsomes from a control rat (not treated with PB). We counted the number of gold particles per micron of the rough ER membranes (particle density). Before PB treatment, the particle density of the rough ER in rat hepatocytes was practically zero and increased markedly at 48 and 72 hr after PB treatment. The rough microsomes were prepared from these PB-treated rat livers. The amount of P450IIB was estimated by immunoblot analysis and the number of gold particles bound to the rough microsomal membrane was determined by the same post-embedding immunogold procedure. The particle density of the rough microsomes increased in parallel with the increase in the amount of P450IIB, indicating good correlation of the two variables. Thus, the induction of cytochrome P450IIB can be quantitatively and reliably investigated by immunogold electron microscopy.

Acrylic Resins↗
PubMed1992

Effects of hyperthermia induced by microwave irradiation on brain development in mice.

Pregnant mice were exposed to 2.45 GHz of microwave radiation for 15 or 20 min on day 13 of gestation. The highest maternal core temperature during the exposure did not exceed 42.5 degrees C. Pregnant females also were immersed in hot water at 42 degrees C for 15 min to compare thermal effects on brain development. Animals were killed 9 hours after treatment, and the pyknotic cells in the ventricular zone of telencephalon were counted. The respective incidences of these cells in the groups exposed to microwaves for 15 and 20 min were 1.83% and 3.06%. Microwave radiation for 20 min had an effect that was comparable to that of immersion in 42 degrees C hot water for 15 min. In addition, some animals were examined on day 18 of gestation, and some of their offspring were examined at 6 weeks of age in an examination of long-term effects. Brain weight for the group exposed to microwaves for 20 min was significantly lower than for the control group, and the numerical density of the neurons in the cerebrum was higher. We concluded that microwave radiation at the dose tested mainly has a thermal effect.

Animals↗
PubMed1992

[Trace analysis for drugs and poisons in human tissues].

Despite continuous developments of analytical techniques in terms of sensitivity and accuracy, uncountable increase in number of chemical substances are brought into the field of forensic sciences to be analyzed. Based on the idea that progress in analytical technique to cope with the change of situation is always required, a research team including 10 members was organized to set up the most advanced methods at present to analyze the important drugs and poisons in biological materials for forensic purposes. Stimulant drugs such as methamphetamine and amphetamine were studied on the improvement of extraction procedure prior to mass spectrometric analysis. A conventional solvent extraction method was replaced by an extraction technique using Extrelut column. The technical procedure was simplified and the accuracy of measurement was improved. The changes in CO-Hb concentration in the whole blood in storage was examined with regard to the lapse of time and temperature, where a differential spectrophotometry was used. Useful information could be obtained from practical aspects. Analytical conditions of gas chromatography were revised on volatiles including alcohols, especially as to column conditions. The use of capillary column was recommended for sensitivity and peak separation. The optimum conditions for detecting barbiturates in the blood were examined. The combination of Sep-pak C18 cartridge with a capillary column for gas chromatography using nitrogen phosphorus detection was found preferable. Mass spectrometry of various kinds of local anesthetics was studied. Quantitative analysis of the drugs was examined on gas chromatography with a surface ionization detector. Three types of insecticides including organophosphorus, chlorinated and fluorine compounds were analyzed by gas chromatography/mass spectrometry in order to establish a sensitive and selective method. The detection limits, calibration and reproducibility were examined. Herbicide, paraquat, was examined on the sensitivity, recovery, required time and costs in connection with methods of pretreatment and analytical procedure. A secondary spectrophotometry was found useful practically. Antimony in biological tissues was analyzed, using a flameless atomic absorption spectrometer with carbon tube atomizer. The time for analysis was reduced, and sensitivity was improved. Immunoassay method was examined from general aspects on drugs and hormones. Using antibodies specific to haptens, the dynamics of such antigens as drugs and hormones in the body tissues were observed. Interfering substances at the time of toxicological analysis were checked in order to obtain reliable information. Exact identification was found possible by using gas chromatography/mass spectrometry. As described above, the research was made from the practical aspects of forensic toxicology, and meaningful results could be obtained from each study.

Animals↗
PubMed1992

Can death at a low COHb concentration frequently observed in fire victims be explained by hypoxic hypoxia?

Fire deaths at low COHb (carboxyhemoglobin) concentrations are frequently observed, but it is difficult to determine the causative factors. Participation of hypoxic hypoxia was examined using rats and rabbits exposed to various low-O2 and CO gas mixtures. The ranges of O2 and CO concentration were 3.0-22.2% and 0.3-2.9%, respectively. The concentration of CO2 was fixed at about 5%. Rats were individually exposed to the test gas in a plastic chamber. Rabbits inhaled the test gas through a tracheal cannula. The survival time of rats ranged from 3.9-7.7 min, and that of rabbits was 8.0-22.5 min. Rats exposed to the most hypoxic gas mixture (O2 3%-CO 0.3%) died with COHb values below 40%. Rabbits also died with a low COHb concentration under this condition, but the COHb concentration was not below 50%. Rabbits were considered to be more resistant to hypoxic hypoxia than rats. From the literature on the relationship between the grade of burns and the concentration of COHb, as well as the findings obtained in the present experiment, hypoxic hypoxia did not appear to be a main factor causing death at low COHb concentrations. In the rabbit the COHb concentration increased exponentially and reached plateau levels within 10 min in many groups. The time required to reach a plateau COHb level was shorter when the concentration of CO was high and that of O2 was low.

Animals↗