Biomedical subjects

N Hamato

Publications and source records attributed to N Hamato.

At least 19 recordsLinked to original sources

PubMed2001

Expression and characterization of recombinant human antithrombin III in Pichia pastoris.

Antithrombin III (ATIII) is a member of the serpin superfamily and a major regulator of the blood coagulation cascade. To express recombinant human ATIII (rATIII) in the methylotrophic yeast Pichia pastoris, we constructed an rATIII expression plasmid which contained the ATIII cDNA encoding mature protein region connected with the truncated mAOX2 promoter and the SUC2 secretion signal, introduced it into the P. pastoris genome, and screened for a single copy transformant. The secretion of rATIII from the transformant reached a level of 320 IU/L in the culture broth at 169 h. From the culture-supernatant, rATIII was purified to over 99% by heparin-affinity chromatography and other column chromatography methods. We characterized rATIII and compared it with human plasma-derived ATIII (pATIII). The purified rATIII possessed correct N-terminal amino acid sequence, and its molecular weight by SDS-PAGE of 56,000 Da was slightly different from the 58,000 Da of pATIII. Sequence and mass spectrometry analysis of BrCN fragments revealed that posttranslational modifications had occurred in rATIII. O-linked mannosylation was found at Ser 3 and Thr 9, and in some rATIII molecules, modification with O-linked mannosyl-mannose had probably occurred at Thr 386, close to the reactive center. Although the heparin-binding affinity of rATIII was 10-fold higher than that of pATIII, its inhibitory activity against thrombin was only half. As the conformation of rATIII and pATIII by circular dichroism spectroscopy was similar, O-glycosylation in the reactive center loop was assumed to be mainly responsible for the decreased inhibitory activity. pATIII can inactivate thrombin through formation of a stable thrombin-ATIII complex, but rATIII modified with O-glycosylation in the reactive center loop may act as a substrate rather than an inhibitor of thrombin.

Antithrombin III↗

PubMed2000

Effect of P(2)' site tryptophan and P(20)' site deletion of Momordica charantia trypsin inhibitor II on inhibition of proteinases.

Momordica charantia trypsin inhibitor II (MCTI-II) inhibits the amidolytic activity of factor Xa with a K(i) value 10-100-fold smaller than those of other squash family inhibitors. It also inhibits factor X activation mediated by factor VIIa-tissue factor complex or factor IXa. Comparison of other squash family inhibitors reveal Trp at position 7 (P(2)') and a deletion at position 25 (P(20)') are characteristics of MCTI-II. In order to elucidate the effect of these positions on the inhibitory activity, we chemically synthesized three inhibitors: S-MCTI-II whose amino acid sequence is identical to natural MCTI-II, S-MCTI-II(7L) whose P(2)'(Trp) is substituted with Leu, and S-MCTI-II(25N) whose P(20)'(deletion) is filled with Asn. The dissociation constants of the complexes of human factor Xa with S-MCTI-II, S-MCTI-II(7L), and S-MCTI-II(25N) were 1.3x10(-6) M, 2.8x10(-5) M, and 7.3x10(-6) M, respectively. They inhibited factor X activation mediated by factor VIIa with the same degree. As in the case of natural MCTI-II, S-MCTI-II suppressed factor X activation mediated by factor IXa, while S-MCTI-II(7L) and S-MCTI-II(25N) did not. Both the Trp at the P(2)' position and deletion at the P(20)' position are thus likely required for the inhibition of factor Xa, trypsin, and factor IXa, while these two positions do not affect factor X activation initiated by the factor VIIa-tissue factor complex.

Amino Acid Sequence↗

PubMed1997

Change in hepatic arterial hemodynamics induced by hepatocellular carcinoma detected with Doppler sonography.

PURPOSE: To understand the hemodynamic differences between the hepatic arterial branches that supply hepatocellular carcinomas (HCCs) and those that do not, we compared the velocity waveforms of both types of arteries. METHODS: Using duplex color Doppler sonography, we examined 38 patients with HCC localized within a single lobe of the liver and 34 patients with chronic liver disease but without HCC (controls). We measured angle-corrected peak systolic velocity and the pulsatility index (PI) of color-coded hepatic arteries along the right anterior segmental portal branch and the vertical segment of the left portal vein. RESULTS: There was no significant difference in peak systolic velocity and PI between the arterial branches tested in the controls. In contrast, we found a significantly lower PI and a higher peak systolic velocity in the arterial branches supplying the tumor than in those not supplying the tumor in patients with an HCC 3 cm or larger in diameter (p < 0.05). The degree of these hemodynamic changes correlated with the tumor size and the presence or absence of tumor thrombus in the major portal branches. CONCLUSIONS: These results indicate that the hepatic arterial branch supplying an HCC has a lower impedance than the branch not supplying the tumor.

Adult↗

PubMed1997

Clinical application of hepatic venous hemodynamics by Doppler ultrasonography in chronic liver disease.

The phase differences of the hepatic vein (HV) Doppler waveform might be influenced by fibrosis and steatosis of the liver when using HV Doppler and pressure curve analysis. Because HV catheterization is invasive, we have advocated the noninvasive comparative index (QXs ratio) of the HV Doppler waveform phases recorded simultaneously with electrocardiography and phgonocardiography. Forty-three controls and 169 patients with a variety of liver diseases were enrolled in this study. The mean QXs ratio was 0.75 in the controls, 0.6 in the chronic hepatitis patients, 0.45 in the cirrhosis patients, and 0.90 in the fatty liver patients. There were significant differences between all pairs of these groups (p < 0.001). The QXs ratio may be used to assess the degree of hepatic fibrosis or steatosis.

Adult↗

PubMed1997

Phase shift of the hepatic vein flow velocity waveform in chronic liver disease: experimental and clinical studies.

We examined animal models and studied patients with chronic liver disease to evaluate phase shift of the flow-velocity waveform of the hepatic vein (HV). We decided the Doppler-Pressure (DP) ratio using electrocardiography, the HV Doppler waveform and HV pressure curves, and we calculated the DP ratios. In animal models, the mean DP ratio was 1.04 in controls, 0.68 in cirrhosis, and 1.22 in fatty liver. There were significant differences among these groups (p < 0.005). In the clinical study, the mean DP ratio was 1.03 in controls, 0.95 in chronic hepatitis and 0.79 in cirrhosis. The DP ratio in the Cirrhosis Group was significantly different from that in the Control Group (p < 0.001) and that in the Chronic Hepatitis Group (p < 0.05). We conclude that the DP ratio is decreased in certain hepatic disease states.

Adult↗

PubMed1995

Evaluation of protective effects of prostaglandin E1 on ischemic liver damage with in vivo 31P-MR spectroscopy.

The cytoprotective effect of prostaglandins (PG) was evaluated by in vivo 31P MR spectroscopy. Twenty rabbits were divided into two groups; the control group given physiological saline, and the PG group given prostaglandin E1 (0.5 microgram/kg/min). Each solution was infused for 8 min, after which complete hepatic ischemia was induced for 20 min, followed by reperfusion for 40 min. During ischemia, beta-ATP decreased to 23.6% and 42.3%, phosphomonoester increased to 260% and 200% of their initial values in the control and the PG groups, respectively. Inorganic phosphate also increased. After reperfusion, beta-ATP recovered to 65.2% and 96.5%, phosphomonoester to 130% and 110%, inorganic phosphate to 140% and 120% in the control and PG groups, respectively. The changes during ischemia were significantly smaller and the recoveries during reperfusion were more complete in the PG group. These results may confirm the cytoprotective effect of prostaglandins by in vivo 31P-MR spectroscopy.

Adenosine Triphosphate↗

PubMed1995

Vascular complications in living related liver transplantation detected with intraoperative and postoperative Doppler US.

BACKGROUND/AIMS: The purpose of this study was to clarify changes in the graft hemodynamics induced by vascular complications in living related liver transplantation. METHODS: This study included 46 pediatric recipients who underwent partial liver transplantation from living related donors. The blood flow was evaluated in the portal system, the hepatic artery and the hepatic vein with serial intra- and post-operative Doppler ultrasound (US). RESULTS: In 12 patients, intraoperative Doppler US showed a decrease in portal venous inflow (< 9 ml.min-1.kg-1) toward the liver graft and could act as a guide for ligation of collaterals in seven patients, portal re-construction in two, thrombectomy in one and relief of hepatic venous outflow obstruction in two for increasing the portal venous inflow. In five patients, intraoperative Doppler US showed poor arterial inflow, i.e. dampened arterial waveforms which involved both low pulsatility index (< 0.90) and low peak-systolic velocity (< 31 cm/s). In three of them, the waveform was more pulsatile after re-anastomosis or relief from stretching of the hepatic artery. The remaining two patients developed hepatic artery thrombosis. Most of the hepatic venous outflow obstruction (four of five patients) had flat waveforms, low flow velocity (< 10 cm/s) of the hepatic vein, and poor portal inflow (flow velocity < 14 cm/s). Postoperative Doppler US showed hepatic venous outflow obstruction in three patients, hepatic artery thrombosis in three (twice in one patient), portal vein stenosis in two and portal vein thrombosis in one. These complications were successfully managed with surgical procedures in three patients, transhepatic angioplasty in three and conservative treatments in four. Six patients died of non-vascular complications. CONCLUSIONS: Serial intra- and post-operative Doppler US was a useful technique for making an early diagnosis of abnormal hemodynamics of the graft circulation. Furthermore, intraoperative Doppler US could assess reconstructed vessels objectively and would reduce the incidence of vascular complications following transplantation.

Adolescent↗

PubMed1995

Trypsin and elastase inhibitors from bitter gourd (Momordica charantia LINN.) seeds: purification, amino acid sequences, and inhibitory activities of four new inhibitors.

Serine proteinase inhibitors of the squash family were isolated from bitter gourd (Momordica charantia LINN.) seeds by the conventional purification method. Heat treatment of the extract of the seeds allowed removal of large amounts of protein without loss of trypsin and elastase inhibitory activities. From the supernatants thus obtained, the inhibitors were isolated to homogeneity by ion-exchange chromatography, gel filtration, and reversed phase chromatography. One trypsin inhibitor (Momordica charantia trypsin inhibitor-III; MCTI-III) and three elastase inhibitors (Momordica charantia elastase inhibitor-II, -III, and -IV; MCEI-II, -III, and -IV) were newly isolated in addition to trypsin inhibitors MCTI-I and -II and elastase inhibitor MCEI-I previously reported [Hara, S. et. al. (1989). J. Biochem. 105, 88-92]. The primary structures of the four new inhibitors were determined as follows. [sequence: see text] The dissociation constants, Ki, of MCTI-III complex with bovine beta-trypsin, and of MCEI-II, -III, -IV with porcine elastase were determined to be 1.9 x 10(-7) M, 9.4 x 10(-9) M, 4.0 x 10(-9) M, and 4.7 x 10(-9) M, respectively. Although MCTI-III differed from MCTI-I in only two amino acids, having Gly(3) and Gln(13) in place of Arg(3) and Arg(13), the Ki value of MCTI-III was 20-fold larger than that of MCTI-I. Addition of an amino terminal Glu residue, a dipeptide (Glu-Glu-), and a tripeptide (Glu-Glu-Glu-) to MCEI-I strengthened its elastase inhibitory activity by 200-fold.

Amino Acid Sequence↗

PubMed1995

Azygos venous blood flow: measurement with direct bolus imaging.

PURPOSE: To evaluate a magnetic resonance (MR) imaging technique for measurement of azygos blood flow volume, which is an important indicator of superior collateral circulation in patients with portal hypertension. MATERIALS AND METHODS: Azygos blood flow was measured by means of direct bolus (DB) imaging, an MR flow measurement technique. A presaturation pulse was applied in combination with electrocardiographic gating. RESULTS: The measured flow volume was 320 mL/min +/- 30 (mean +/- standard error of mean) in the control subjects, 550 mL/min +/- 130 in patients with cirrhosis of the liver and without varices, and 660 mL/min +/- 60 in patients with cirrhosis of the liver and with varices. The flow volume was significantly higher in each of the groups with cirrhosis of the liver than in the control group (P < .01). Furthermore, a significant correlation was shown between DB imaging and continuous thermodilution measurements (r = .707, P < .01). CONCLUSION: DB imaging, which is noninvasive, was useful in the monitoring of the effect of treatment and in determination of the prognosis in patients with portal hypertension.

Aged↗

PubMed1994

Inhibition of serine proteases of the blood coagulation system by squash family protease inhibitors.

Squash family inhibitors are the smallest protein serine protease inhibitors, being composed of approximately 30 amino acid residues. We isolated 8 squash family inhibitors from the seeds of bitter gourd, squash, gourd and luffa and examined their effect on serine proteases of the blood coagulation system. Five of them prolonged the activated partial thromboplastin time of human plasma to various extents, but three did not. Only Momordica charantia (bitter gourd) trypsin inhibitor-II prolonged the prothrombin time of human plasma. All inhibitors inhibited the amidolytic activities of factor XIIa, plasma kallikrein, factor Xa, but did not inhibit significantly those of factor XIa, factor IXa, factor VIIa, and thrombin. Ki values for factor XIIa, plasma kallikrein, and factor Xa were in the order of 10(-6)-10(-9), 10(-4)-10(-5), and 10(-4)-10(-6)M, respectively. The prolongation of the activated partial thromboplastin time by inhibitors appeared to correspond to their inhibitory potencies for factor XIIa. Momordica charantia trypsin inhibitor-II, which has the strongest inhibitory potency toward the amidolytic activity of factor Xa, with a Ki value 10-100 times smaller than those of other inhibitors, inhibited the activation of factor X by factor VIIa-tissue factor complex or factor IXa, while others did not.

Amino Acid Sequence↗

PubMed1994

Effects of dobutamine on hepatosplanchnic hemodynamics in patients with chronic liver disease.

BACKGROUND: It is said that catecholamines increase hepatic blood flow in patients without liver diseases, although several reports have suggested a blunted response to catecholamines in patients with liver cirrhosis. METHODS: We investigated changes in splanchnic blood flow distribution induced by the infusion of dobutamine into peripheral veins of healthy adults (NC group), patients with chronic hepatitis (CH group), and patients with liver cirrhosis (LC group), using a Doppler duplex system (protocol 1). We also investigated changes in hepatic hemodynamics induced by dobutamine infusion in patients with liver cirrhosis (cirrhosis group) and patients without liver diseases (control group), using hepatic catheterization (protocol 2). RESULTS: In protocol 1 the average increase in portal venous blood flow during dobutamine infusion was significant in the NC and CH groups but was not significant in the LC group. Changes in the blood flow in the splenic artery and vein, superior mesenteric artery and vein, and femoral artery were similar to those in the portal vein in each of the three groups. Infusion did not cause a change in the common hepatic arterial flow in any of the three groups. In protocol 2 the portal venous flow, cardiac index, and hepatic venous pressure gradient increased significantly during dobutamine infusion in both the cirrhosis and the control groups. Hepatic vascular resistance in the cirrhosis group increased slightly, whereas, in contrast, that in the control group increased significantly. The rate of change in almost all variables was lower in the cirrhosis group than in the control group. CONCLUSION: These results indicate that dobutamine has less effect on hepatic circulation in patients with liver cirrhosis than in those without liver diseases, indicating that the value of dobutamine in increasing hepatic blood flow in cirrhotic patients is very limited.

Aged↗

PubMed1993

Distribution of mesenteric and splenic blood flow in cirrhosis of the liver as estimated by radionuclide angiography.

We used radionuclide angiography following an injection of 185 MBq Tc-99m tin colloid as a non-invasive method for measuring the mesenteric and portal components of the total hepatic circulation, according to the method of Fleming et al (J Nucl Med 24; 1983). Fifty-seven patients with cirrhosis and 33 controls were examined. The relative mesenteric flow (MF) and the relative portal flow (PF) were 68.6 +/- 8.5% and 78.6 +/- 5.9%, respectively, in the control group. These values were 42.0 +/- 16.4% and 70.9 +/- 8.9%, respectively in the cirrhosis group, and were significantly lower than in the controls. In the cirrhosis group, the reduction in PF was small, while that in MF was large. Consequently, we found that portal blood flow was relatively well maintained in the cirrhosis patients, while the contribution of splanchnic blood flow to the portal flow was relatively reduced.

Adult↗

PubMed1992

Use of proton nuclear magnetic resonance spectroscopy of plasma in screening for malignant disease.

In 1986, narrow plasma proton nuclear magnetic resonance (NMR) methyl and methylene line widths were reported to be associated with malignant disease, but more recent studies have not confirmed this relationship. The authors analyzed 106 plasma samples from healthy control subjects and patients with cirrhosis, hepatocellular carcinoma, metastatic liver tumors, other untreated cancers, and hyperlipidemia. NMR spectroscopy was performed using a proton NMR spectrometer operating at 399.65 MHz. A significant difference was found between the mean line widths of the plasma methyl resonances in control subjects and those in patients with cancer or hyperlipidemia. However, no significant difference was found between the mean plasma methylene line widths in control subjects and patients with cancer. Plasma samples from patients with liver disease or hyperlipidemia showed a characteristic methylene spectral pattern. The methylene pattern could be separated into three types: type A had a small peak on the right shoulder of the main peak; type B was a sharp single peak; and type C was a broad single peak. All control subjects had type A pattern; patients with liver disease had type C pattern; and patients who had hyperlipidemia had type B pattern, and hyperlipidemia may affect methyl and methylene line widths in NMR spectra. Because the methyl and methylene levels and their average line widths correlated inversely with triglyceride levels, considering the spectral patterns that indicate hyperlipidemia should decrease false-positive results and make the methyl line width useful for cancer screening.

Adult↗

PubMed1992

Trypsin inhibitors from bottle gourd (Lagenaria leucantha Rusby var. Depressa Makino) seeds. Purification and amino acid sequences.

Two almost identical trypsin isoinhibitors, LLDTI-I and LLDTI-II, from bottle gourd (Lagenaria leucantha Rusby var. Depressa Makino) seeds were purified by acetone precipitation, gel filtration and reversed phase chromatography. LLDTI-I and LLDTI-II consist of 30 and 29 amino acid residues, respectively, and have identical sequences, except that LLDTI-I has one additional pyroglutamic acid residue at N-terminus. Both proteins are strong inhibitors of bovine trypsin, with Ki values of 2.4.10(-10) M (LLDTI-I) and 9.6.10(-11) M (LLDTI-II). Amino acid sequences are as follows: [sequence: see text]

Amino Acid Sequence↗

PubMed1992

Purification and characterization of serine proteinase inhibitors form gourd (Lagenaria leucantha Rusby var. Gourda Makino) seeds.

Gourd seed inhibitors were purified in the following manner: gourd seeds were ground and extracted with 10 mM ammonium carbonate, pH 7.8. The extract was precipitated with 65-90% acetone and the acetone precipitates were gel filtered in a Cellulofine GCL-90-m column. Fractions of 3000 Da showing trypsin inhibitory activity were combined and purified further by ion exchange and reversed phase chromatographies. Three inhibitors, LLTI-I, II, and III were thus purified to homogeneity and the amino acid sequences of these inhibitors were: [sequence: see text] The exact sequences are unique but very similar to proteinase inhibitors belonging to the squash family. Based on the sequence, it is assumed that the peptide bond (Arg-Ile) found in the three inhibitors is the reactive site for trypsin. The Ki values estimated for complexes of LLTI-I, II, and III with bovine trypsin were 3.6 x 10(-10) M, 6.5 x 10(-11) M, and 3.0 x 10(-11) M, respectively.

Amino Acid Sequence↗

PubMed1991

Mucin-histochemical and immunohistochemical profiles of epithelial cells of several types of hepatic cysts.

Epithelial cells of several types of hepatic cysts were examined by mucin histochemistry and immunohistochemically. There were some differences in mucus and antigenic expression among the hepatic cysts examined. Epithelial cells of non-parasitic simple cysts and adult-type polycystic liver showed similar mucin-histochemical and immunohistochemical features, and were characterized by little mucin and weak immunoreactivities to several antibodies examined. Epithelial cells of hepatic hilar cysts were characterized by much mucin and moderate immunoreactivities to carbohydrate antigen 19-9 (CA 19-9), carcinoembryonic antigen (CEA) and epithelial membrane antigen (EMA). Epithelial cells of ciliated hepatic foregut cysts were characterized by much mucin and immunoreactivities to actin and tubulin which were positive in cilia. Epithelial cells of biliary cystadenoma were characterized by much mucin and moderate to strong immunoreactivities to cytokeratins CAM5.2 and AE1 and 3 as well as to CA 19-9, CEA, EMA and DU-PAN-2. Epithelial cells of biliary cyst-adenocarcinoma were characterized by much mucin and moderate to strong immunoreactivities to cytokeratins CAM5.2 and AE1 and 3 as well as to CA 19-9, CEA, EMA and DU-PAN-2. These differences in epithelial mucus and antigenic expression among several types of hepatic cysts may reflect differences in their origin and biological characteristics. These differences may be helpful in the differential diagnosis of hepatic cysts in small biopsy specimens.

Antigens↗

PubMed1991

Photodynamic therapy using pheophorbide-a and Q-switched Nd:YAG laser on implanted human hepatocellular carcinoma.

To evaluate whether administration of pheophorbide-a, a new photosensitizer, followed by use of Q-switched Nd:YAG laser produces a photodynamic reaction, we administered pheophorbide-a to female nude mice (BALB/c-nu) that had been implanted with human hepatocellular carcinoma. Intra-tumoral concentrations of pheophorbide-a were measured by high-performance liquid chromatography. 3 hours after peroral administration of 1 mg/kg body weight, the intra-tumoral concentration was too low to reveal photodynamic effects. Peroral administration of 250 mg/kg body weight, intra-peritoneal administration of 5 mg/kg body weight, and intra-tumoral injection of 200 micrograms yielded 0.24 micrograms/g, 0.83 micrograms/g and 3.68-108 micrograms/g tumor concentrations, respectively. All tumors were irradiated interstitially using a Q-switched Nd:YAG laser at 1064 nm. Only tumors that had been intra-tumorally injected had areas of necrosis larger than those in control tumors. The results suggest that the injection of pheophorbide-a followed by interstitial irradiation using a Q-switched Nd:YAG laser does not induce sufficient photodynamic reaction if the intra-tumoral pheophorbide-a concentration is less than 0.83 micrograms/g tumor tissue, and that photodynamic therapy may be useful if the pheophorbide-a tumor concentration is within the range of 0.83-108 micrograms/g.

Animals↗

PubMed1990

[Evaluation of photodynamic therapy using pheophorbide-a as a photosensitizer].

Intra-tumoral concentrations of pheophorbide-a, a new photosensitizer, were measured by high-performance liquid chromatography after administration of pheophorbide-a to nude mice (BALB/c-nu) that had been implanted with human hepatocellular carcinoma. The results were as follows: 1) 0.009 +/- 0.007 microgram/g tissue in 1 mg/kg body p.o. administration group, 2) 0.22 +/- 0.06 microgram/g tissue in 250 mg/kg body p.o. administration group, 3) 0.85 +/- 0.14 microgram/g tissue in 5 mg/kg body intra-peritoneal administration group, 4) 3.42 +/- 2.84 microgram/g tissue in not-injected side, and 116 +/- 24 microgram/g tissue in injected side of 200 micrograms intra-tumoral administration group. Tumors in each group were irradiated using an Nd:YAG laser (Q-switch; mean power 0.5 W, duration 10 min.). Areas of tumor necrosis were larger than in the control group only in the intra-tumoral administration group. These results suggest that no photodynamic reaction occurs if the intra-tumoral pheophorbide-a concentration is less than 0.85 microgram/g tissue.

Animals↗