Search PubMed⌕ Search

Biomedical subjects

M Hirata

Publications and source records attributed to M Hirata.

At least 289 records · Page 16Linked to original sources
PubMed1994

Expression and characterization of an inositol 1,4,5-trisphosphate binding domain of phosphatidylinositol-specific phospholipase C-delta 1.

It was previously found that the 85-kDa protein purified from rat brain using an inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-immobilized matrix was the delta 1 isoform of phosphatidylinositol-specific phospholipase C (PLC). We expressed rat PLC-delta 1 in Escherichia coli as a fusion protein with glutathione S-transferase, and found that the bacterial lysate shows a significant amount of Ins(1,4,5)P3 binding. The lysate was applied to Ins(1,4,5)P3-immobilized column chromatography and the eluate with 2 M NaCl solution containing only a 100-kDa protein showed high Ins(1,4,5)P3 binding. The lysate was also purified to near homogeneity using a glutathione-Sepharose 4B affinity system. Bacterially-expressed enzyme thus purified showed essentially the same inositol phosphate binding characteristics as the brain-derived enzyme. PLC-delta 1 consists of the amino-terminal nonconserved region and two well-conserved regions among isozymes, designated as X and Y, which are thought to constitute a catalytic core of the enzyme. Using a combination of deletion mutants and proteolytic products of the enzyme, we were able to locate an Ins(1,4,5)P3 binding domain in the molecule. Deletion of 223 residues from the amino terminus completely abolished the binding activity, while deletion of X region only partially inhibited the binding and deletion of Y region did not affect the binding. A 76-kDa proteolytic product of the expressed PLC-delta 1 which lacked 60 amino acids at the amino terminus showed a minimal Ins(1,4,5)P3 binding activity. A peptide consists of 14 amino acids corresponding to residues 30-43 of PLC-delta 1, which contains 6 basic amino acids, binds to an Ins(1,4,5)P3-immobilized matrix. Moreover, Ins(1,4,5)P3 binding was blocked by phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. These results, taken together, indicate that the amino-terminal domain of PLC-delta 1 is important for the binding of both Ins(1,4,5)P3 and phosphatidylinositol 4,5-bisphosphate.

Amino Acid Sequence↗
PubMed1994

Ca2+ influx gated by inositol-3,4,5,6-tetrakisphosphate in NIH/3T3 fibroblasts.

The functional role of three inositol tetrakisphosphate isomers on Ca2+ influx was examined by intracellular application in NIH/3T3 mouse fibroblasts. Infusion of inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), inositol-1,3,4,6- tetrakisphosphate, and inositol-3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) from the patch pipette into the cytoplasm produced a transient increase in cytoplasmic free Ca2+ concentration by fura-2 recording in extracellular Ca(2+)-dependent fashion. Stimulation of NIH/3T3 cells with bradykinin increased the levels of Ins(1,3,4,5)P4 and Ins(3,4,5,6)P4. These results suggest that mouse fibroblast cells possess a Ca2+ influx pathway gated with naturally occurring inositol tetrakisphosphates in response to stimulation with bradykinin.

3T3 Cells↗
PubMed1994

Study on measurement of delta-aminolevulinic acid in plasma by high-performance liquid chromatography.

A method for the determination of delta-aminolevulinic acid in plasma of lead-exposed workers by high-performance liquid chromatography with fluorescence detection of a fluorescent delta-aminolevulinic acid derivative (2-methylidineamino-3,5-diacetyl-4,6-dimethylpropionic acid) was established. The detection limit of delta-aminolevulinic acid in plasma was 0.01 microgram/ml at a signal-to-noise ratio of 5:1. A linear correlation was obtained between the amounts of delta-aminolevulinic acid injected from 0.01 to 0.5 microgram/ml (r = 0.999). The recovery of 0.05 and 0.1 microgram/ml of delta-aminolevulinic acid added to plasma with various concentrations of delta-aminolevulinic acid in plasma ranged from 80.0 to 100.8%. This method, combined with the use of an automatic sampler, should facilitate the routine measurement of delta-aminolevulinic acid in plasma.

Aminolevulinic Acid↗
PubMed1994

Ca2+ influx evoked by inositol-3,4,5,6-tetrakisphosphate in ras-transformed NIH/3T3 fibroblasts.

Infusion of inositol-3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4) from the patch pipette into the cytoplasm, produced a biphasic intracellular free Ca2+ concentration ([Ca2+]i) increase in ras-transformed NIH/3T3 (DT) cells. The Ins(3,4,5,6)P4-induced increase in DT cells depended upon extracellular Ca2+, and was enhanced by membrane hyperpolarization. Identical [Ca2+]i increases were observed with intracellular application of inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) and inositol-1,3,4,6-tetrakisphosphate but not with inositol-1,2,4,5-tetrakisphosphate, inositol-1,4,5-trisphosphate or inositol-1,3,4,5,6-pentakisphosphate. Stimulation of DT cells with bradykinin increased the levels of Ins(3,4,5,6)P4 and Ins(1,3,4,5)P4. These results suggest that Ins(3,4,5,6)P4 may serve as a second messenger for continuous Ca2+ influx along with other tetrakisphosphates downstream from bradykinin receptors in DT cells.

3T3 Cells↗
PubMed1994

A novel granulocyte-derived peptide with lipopolysaccharide-neutralizing activity.

Rabbit CAP18 (cationic antimicrobial protein, 18 kDa) is a leukocyte protein identified and purified using as an assay its capacity to bind and inhibit various activities of LPS. Oligonucleotide probes designed from the putative N-terminal protein sequence were used to obtain the corresponding cDNA from a rabbit bone marrow cDNA library. Examination of the cDNA sequence revealed that the protein fragment of the putative N-terminus was actually a 37-amino-acid C-terminal fragment. This fragment, designated CAP18(106-142), inhibits many activities of LPS. In the present studies, synthetic CAP18(106-142) is shown to: 1) bind to erythrocytes coated with diverse strains of LPS; 2) inhibit LPS-induced release of cytokines (TNF, IL-1, IL-6) and nitric oxide from macrophages; 3) inhibit LPS-induced LAL coagulation and 4) protect mice from LPS lethality. CAP18(106-142) may have therapeutic utility for conditions associated with elevated concentrations of LPS.

Amino Acid Sequence↗
PubMed1994

Seroepidemiology of hepatitis C virus infection in hemodialysis patients and the general population in Fukuoka and Okinawa, Japan.

In 1992, a seroepidemiologic study was carried out among hemodialysis patients and the general population in Fukuoka and Okinawa, Japan to determine the presence of hepatitis C virus (HCV) infection and HCV viremia. The markers used were antibody to HCV, determined by second-generation assay (anti-HCV), and HCV RNA, determined by the polymerase chain reaction. The prevalence of anti-HCV in Fukuoka was 3.3%, 73 per 2237 persons, significantly (P < 0.001) higher than the 0.4%, 5 per 1295, in Okinawa. The prevalence of anti-HCV in hemodialysis patients in Fukuoka was 51.9% (161 of 310 patients), significantly (P < 0.001) higher than the 9.1% (13 of 143 patients) in Okinawa. The ratio of HCV RNA-positive to anti-HCV-positive persons was significantly higher in hemodialysis patients (147/174, 84.5%) than in the general population (49/78, 62.8%) (P < 0.001). Elimination of HCV among hemodialysis patients appears to be difficult, as such patients have lower immune responses than the general population. In Fukuoka, but not in Okinawa, blood used for transfusion was supplied by paid donors at commercial blood banks from 1953 to 1969. This may explain why HCV infection is endemic in Fukuoka and not in Okinawa. Differences between the prevalence of anti-HCV in the hemodialysis patients in Fukuoka and Okinawa reflect differences in the prevalence in the general population in these two areas of Japan.

Adolescent↗
PubMed1994

Detection of HCV RNA in subjects with antibody to hepatitis C virus among the general population of Fukuoka, Japan.

Volunteer blood donors and aged people who came to hospitals for a thorough physical checkup were surveyed to evaluate the exact prevalence of hepatitis C virus (HCV) infection in the general population of Fukuoka, Japan. We tested for antibody to HCV (anti-HCV) by second-generation assay and, to distinguish active HCV infection from past resolved infection, we tested for HCV RNA in reactive serum samples by polymerase chain reaction. The prevalence of anti-HCV was 286 (2.0%) of 14,341 subjects, increasing with advancing age, from 0.4% in the under-29 age group to 12.0% in the over-70 age group. There were no differences between sexes. HCV RNA was detected in 170 of 286 (59.4%) anti-HCV-positive subjects. The ratio of HCV RNA-positive to anti-HCV-positive subjects was higher in males than in females (P < 0.05) and decreased with advancing age, from 72.2% to 46.5%. The prevalence of elevated alanine aminotransferase (ALT) was only 15.9% in subjects with HCV RNA, higher in males (21.4%) than in females (8.3%) (P < 0.05). This study revealed that the prevalence of anti-HCV was high in the aged population, but that the ratio of HCV RNA-positive to anti-HCV-positive subjects was low, and most of the HCV RNA-positive subjects had normal ALT levels.

Adult↗
PubMed1994

The role of angiography in the assessment of blunt liver injury.

Angiography and therapeutic embolism (TE) were studied retrospectively in cases of blunt liver injury with regard to their indications and usefulness. The management of patients fell into three groups distinguished by the clinical evidence of the severity of the liver injuries. The most severe 42 cases (39.6 per cent) were managed surgically and promptly, the least severe 38 cases were not subjected to angiography and the intermediate group (26 cases; 24.5 per cent) underwent angiography and 12/26 cases underwent TE. However, haemodynamic stability on admission was not significantly different between these groups. In addition, all patients who underwent angiography and TE had more severe parenchymal injury on imaging studies while their haemodynamic instability was not identified on admission. Angiography and TE for blunt liver injury were most strongly indicated in patients with good haemodynamic responses to intravenous fluid administration during the acute phase and/or in cases of severe parenchymal injury on imaging.

Algorithms↗
PubMed1994

Prevalence and role of hepatitis C viraemia in haemodialysis patients in Japan.

A second generation assay for antibody to hepatitis C virus (anti-HCV) was used in order to establish the exact prevalence of HCV infection in haemodialysis patients. HCV RNA was sought by the polymerase chain reaction in order to determine whether haemodialysis patients with anti-HCV had been infected with HCV in the past or were presently infected. Of 357 patients, 198 (55.5%) were positive for anti-HCV, compared to 113 (31.7%) positive for original antibody to c100-3 protein (P < 0.001). HCV RNA was detected in 171 (86.4%) of the 198 patients with anti-HCV. Liver dysfunction was found in 63 (17.6%) of all haemodialysis patients. Of these, 55 (87.3%) had HCV infection, one (1.6%) hepatitis B virus infection while, in the remaining seven, the origin was unknown. Thus, in almost all anti-HCV-positive patients, HCV viraemia was present. We conclude that HCV is an important cause of liver disease in haemodialysis patients.

Adolescent↗
PubMed1994

HBsAg subtypes among HBsAg carriers in Okinawa, Japan. Evidence of an important relationship in seroconversion from HBeAg to anti-HBe.

In Okinawa, Japan 1261 hepatitis B surface antigen (HBsAg) carriers were investigated for clinical differences among HBsAg subtypes. Among the 854 for whom they could be determined, subtype adw was found in 604 (70.7%), adr in 232 (27.2%) and others in 18 (2.1%). The overall prevalence of hepatitis B e antigen (HBeAg) was significantly lower in subtype adw (11.9%) than in adr (17.7%) (P < 0.01). The mean age of HBeAg-positive carriers was significantly lower in adw (20.4 years) than in adr (26.9 years) (P < 0.05). Seroconversion from HBeAg to antibody to HBeAg (anti-HBe) occurred in 43 carriers. The seroconversion rate was higher in adw (43.1%) than in adr (29.3%) and the seroconversion age was younger in adw (22.7 years) than in adr (27.9 years). These results suggest that carriers with subtype adw tend to seroconvert from HBeAg to anti-HBe at a higher rate and a younger age than do those with adr. HBsAg subtypes may be closely associated with the HBeAg/anti-HBe status.

Adolescent↗
PubMed1994

Carbon sources for D-lactate formation in rat liver.

Carbon sources for D-lactate formation were investigated in vitro using 6,000 x g supernatant of rat liver homogenate and by rat liver perfusion in situ. As carbon sources, L-threonine, glucose, glycerol, acetone, and acetoacetate were tested. Glycerol was the best substrate for D-lactate formation via methylglyoxal in rat liver. Glucose was the second most preferred substrate, while L-threonine, acetone, and acetoacetate were poor substrates for D-lactate formation. Glycerol was several times more effective than normal as a substrate of D-lactate in the supernatants of liver homogenates of diabetic and starved rats, while it was less effective as a substrate of L-lactate. The glycerol kinase [EC 2.7.1.30] activities in livers increased in the diabetic and starved states. These and other results can explain why the plasma concentration of D-lactate increases several-fold after running and why the D-lactate contents in plasma, liver, and skeletal muscle are markedly increases in diabetic and starved rats.

Acetoacetates↗
PubMed1994

D-myo-inositol 1,4,5-trisphosphate-binding proteins in rat brain membranes.

Rat brain membrane fractions obtained using Triton X-100 were applied to a D-myo-inositol 1,4,5-trisphosphate [D-Ins(1,4,5)P3] immobilized column, followed by gel filtration and anion-exchange chromatography. Two proteins with molecular masses of 130 and 85 kDa, as assessed by SDS-polyacrylamide gel electrophoresis, were purified to apparent homogeneity as D-[3H]Ins(1,4,5)P3-binding proteins with no D-Ins(1,4,5)P3-metabolizing activity. Partial amino acid sequence determinations of these proteins revealed that the 130 kDa protein appears to be a new D-Ins(1,4,5)P3-binding protein and the 85 kDa protein is a delta 1-isozyme of phospholipase C. We have previously purified 130 and 85 kDa proteins as D-[3H]Ins(1,4,5)P3-binding proteins, from rat brain cytosol fraction. Antibodies against the 130 kDa protein from the cytosol cross-reacted with the membrane 130 kDa protein purified in this study, suggesting that the membrane 130 kDa protein is likely to be the same as the protein from the cytosol fraction. The inhibition of D-[3H]Ins(1,4,5)P3 binding by D-isomers of inositol phosphates available clarified that the 130 kDa protein has a similar affinity for D-Ins(1,4,5,6)P4 to that for D-Ins(1,4,5)P3, while the 85 kDa protein is specific to D-Ins(1,4,5)P3.

Amino Acid Sequence↗
PubMed1994

Characterization of a rabbit cationic protein (CAP18) with lipopolysaccharide-inhibitory activity.

Cationic antibacterial proteins (CAP) were purified from rabbit granulocytes, and the effects of CAP on lipopolysaccharide (LPS)-induced tissue factor generation by murine peritoneal macrophages and human blood monocytes were studied. CAP were purified from rabbit peritoneal leukocytes by using as an assay the agglutination of erythrocytes coated with Re-LPS. Two proteins with CAP activity, CAP18 (18 kDa) and CAP7 (7 kDa), were isolated by acid extraction, ethanol precipitation, affinity chromatography, gel filtration, and reverse-phase high-pressure liquid chromatography. On the basis of protein sequencing, CAP7 was identified as the C-terminal fragment of CAP18, designated CAP18(106-142). Various forms of LPS (S-LPS, Re-LPS, and lipid A) activate murine macrophages and human blood monocytes to generate tissue factor (tissue thromboplastin). Incubation of LPS for 18 h with partially purified CAP (heparin-Sepharose fraction) inhibited the capacity of LPS to induce tissue factor; however, purified CAP18 inhibited about 75% of the activity of S-LPS after 1 h of incubation. CAP more effectively inhibited S-LPS than Re-LPS or lipid A. Synthetic CAP18(106-142) inhibited LPS-induced tissue factor generation by murine macrophages. CAP18(106-142) has greater LPS-binding and LPS-neutralizing activities than CAP18. We hypothesize that CAP18 and the derivative peptide, CAP18(106-142), bind to LPS and alter the capacity of LPS to initiate disseminated intravascular coagulation. In this regard, CAP may have therapeutic potential for sepsis and endotoxin shock.

Amino Acid Sequence↗