Biomedical subjects

H Mihara

Publications and source records attributed to H Mihara.

At least 73 records · Page 4Linked to original sources

PubMed1997

Interaction of lipophilic peptides derived from mastoparan with phospholipid vesicles.

Various kinds of lipophilic peptides were prepared by acylation of an alpha-helical peptide, mastoparan, to investigate the effects of acyl groups on the interaction of peptides with phospholipid membranes. alpha-Helicity of the peptides was increased by introduction of long acyl groups. Acyl peptides showed different membrane-perturbation activities for neutral and acidic phospholipid vesicles, whereas a peptide with a dialkycarbamoyl group always exhibited a strong activity. High hemolytic activities were observed for the peptides with long acyls (single or double chain). These results indicate that lipophilic groups introduced to mastoparan contribute greatly to the interaction of the peptide with phospholipid membranes with lengthening of the acyl chain and that the structural character of the lipophilic group also influences the conformation of the peptide.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1997

Thrombomodulin levels in patients with coronary artery disease.

We investigated the association between the serum level of thrombomodulin and known coronary risk factors in 119 men who underwent coronary angiography. Total cholesterol level was significantly higher in patients with coronary atherosclerosis than in those without. Significantly higher frequency of hypertension was noted in patients with coronary atherosclerosis. Uric acid level and frequency of smoking tended to be higher in patients with coronary atherosclerosis but the differences were short short of the significant level. The serum level of thrombomodulin between patients with coronary atherosclerosis and those without was not statistically significant. Age, blood urea nitrogen, and creatinine were positively correlated and creatinine clearance was inversely correlated with the serum level of thrombomodulin. Serum levels of total cholesterol, triglyceride, high-density lipoprotein cholesterol, uric acid, and fasting blood sugar, plasma level of fibrinogen, and body mass index were not related to the serum level of thrombomodulin. There was no significant correlation between the severity of coronary atherosclerosis, hypertension, alcohol use, or smoking and the serum level of thrombomodulin. Restenosis was present in 8 of 16 patients who underwent percutaneous transluminal coronary angioplasty and had a follow-up angiogram at 6.0 +/- 3.0 months. Univariate analysis revealed no significant difference in the thrombomodulin level with and without restenosis. The present findings suggest that elevated thrombomodulin levels in patients with coronary artery disease may reflect retention of thrombomodulin due to decrease in thrombomodulin clearance in the kidney.

Adult↗

PubMed1996

Modulation of the receptor for urokinase-type plasminogen activator in macrophage-like U937 cells by inflammatory mediators.

Macrophages in the tissues have been shown to express receptor for urokinase-type plasminogen activator (uPAR) on their cell surface which plays an important role in cell invasion and attachment. We examined the effects of inflammatory mediators on the expression of uPAR employing U937 cells which have monocyte/macrophage-like characteristics. U937 cells were incubated with various mediators such as interleukins (IL), tumor necrosis factors (TNF), dexamethasone, thrombin, fibrin fragment D, bradykinin, complement C5a, and components of the extracellular matrix. The uPAR expression on the cell surface was then analyzed by radio-ligand binding assay using 125I-scuPA. The strongest enhancement of uPAR was observed in the cells stimulated by TNF alpha and TNF beta. IL-1 beta, IL-6, and C5a also increased the uPA binding sites with various patterns of affinity change. Dexamethasone decreased the uPA binding sites without changing the affinity. Fibrin fragment D and IL-3 reduced the affinity without changing the number of receptors. These findings suggest that the expression of uPAR in inflammatory cells could be modulated by various inflammatory mediators.

Bradykinin↗

PubMed1996

Alterations of fibrinolytic activity in human during and after hyperbaric oxygen exposure.

To clarify the stage of fibrinolytic activation by hyperbaric oxygen (HBO) exposure, we examined its alterations in human during and after the HBO exposure. Eight healthy female volunteers breathed oxygen at 284 kPa (2.8 atmospheres absolute). Blood samples were collected before compression, shortly after compression to the pressure 284 kPa, shortly before the start of decompression, shortly after decompression, and then again 3 hours after decompression. We estimated the euglobulin fibrinolytic activity (EFA) and, the activities and antigens of both tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). The PAI-1 activity and PAI-1 antigen showed significant decrease after compression to a pressure 284 kPa, before the start of decompression, and after decompression. The EFA level and t-PA activity rose significantly shortly after decompression, and 3 hours later returned on baseline. These findings suggest that fibrinolytic activity is elicited after HBO rather than during HBO.

Adult↗

PubMed1996

[Measurement of the common carotid arterial flow during parabolic flight in the anesthetized rat].

To measure the blood flow of a common carotid artery (CCA) during parabolic flight in the rat, we developed an animal double hold box (ADHB) made of styrene expanded form for the anesthetized rat to keep the animal at a proper posture in an aircaft. Twelve anesthetized rats weighing 291-342 g were surgically operated to mount a ultrasound flowmeter probe (1 mm size,1RS:Transonic Systems Inc.) around the right CCA and to insert a catheter into the right axillar artery for blood pressure measurement. These animals were held comfortably in ADHBs which were placed on the rack installed in the aircraft (MU-300). A total of 27 parabolic flights was performed and the blood flow was measured accurately in 9 rats. This special animal holding facility is useful for various types of animal experiments in an aircraft.

Animals↗

PubMed1995

Prostate-specific antigen activates single-chain urokinase-type plasminogen activator.

Prostate-specific antigen (PSA) increases in the plasma of patients with prostate cancer, and has therefore been used as a reliable tumor marker. It has been demonstrated that prostate cancer cells over-express urokinase-type plasminogen activator (uPA), which plays an important role in tumor invasion and metastasis. We found that PSA converts the single-chain proform of urokinase-type plasminogen activator (scuPA) to an active 2-chain form. The active 2-chain uPA generated from scuPA by PSA was measured by hydrolyzation of S-2444, a synthetic substrate for uPA. PSA activated scuPA time- and dose-dependently. SDS-PAGE analysis revealed that, after incubation with PSA, the intensity of the 55-kDa band of scuPA decreased concomitantly with increases in the intensity of the 2 bands at 33 kDa and 22 kDa. Amino-acid-sequence analysis indicated that PSA cleaved Lys158-Ile159, which corresponds with the site cleaved by plasmin. PSA did not enhance or impair the activity of the 2-chain form of uPA. These findings imply that PSA could be an initiator of the protease cascade involved in prostate-cancer invasion and metastasis.

Amino Acid Sequence↗

PubMed1995

Enhancement of plasma fibrinolysis in vitro by jararhagin, the main haemorrhagic metalloproteinase in Bothrops jararaca venom.

Jararhagin, a haemorrhagic metalloproteinase from Bothrops jararaca venom, plays an important role in systemic as well as local haemorrhage. In this study, the effect of jararhagin on the fibrinolytic system was investigated. The fibrinolytic activity of various kinds of animal plasmas was measured by the fibrin plate method. No activity was detected in plasma alone. However, after mixing plasma with jararhagin, strong fibrinolytic activity was recorded in guinea-pig, horse, dog, rabbit and human plasmas. The mechanism of the increase of firbinolytic activity by jararhagin was studied further in guinea-pig plasma. Fibrin-zymographic studies indicated that jararhagin increased tissue-type plasminogen activator (tPA) activity by the dissociation of a complex of tPA with type 1 plasminogen activator inhibitor (PAI-1). alpha 2-Plasmin inhibitor (alpha 2-PI) activity in the plasma was measured using a synthetic chromogenic substrate method after incubation with jararhagin. The alpha 2-PI activity in the plasma decreased in both time-dependent and dose-dependent manners. These in vitro results suggest that, in some animal plasmas, jararhagin increases plasma fibrinolytic activity by causing dissociation of the tPA/PAI-1 complex and by the inactivation of alpha 2-PI. It is possible that this direct action of jararhagin on the enhancement of plasma fibrinolytic activity may contribute to the aetiology of systemic haemorrhage frequently observed in human victims of B. jararaca envenoming.

Animals↗

PubMed1995

QT dispersion in acute myocardial infarction with special reference to left ventriculographic findings.

QT and QT dispersion, which is the time difference between QT maximum and QT minimum, were evaluated in 22 patients with anterior myocardial infarction approximately one month after onset. The purpose of this study was to observe how LV wall motion abnormally is related to these variables. Twenty age-matched patients without overt heart disease were also studied as a control group. QT and QT max in patients with acute myocardial infarction (AMI) were markedly prolonged compared to those in normal controls (472.8 +/- 48.0, 483.2 +/- 32.1 vs 390.2 +/- 18.8, and 418.0 +/- 21.0 msec, respectively). QT dispersion and QTc dispersion in patients with AMI were significantly more prolonged than in normal controls (111.2 +/- 33.9, (113.4 +/- 32.9 vs 54.3 +/- 15.0, and 60.3 +/- 17.2 msec, respectively). QT dispersion has a positive correlation with QT max in AMI patients. Ejection fraction (EF) of the left ventricle was relatively well maintained in cases where only one segment of the left anterior ventricular wall was impaired in its motion. It decreased, however, in accordance with the extent of wall motion abnormality QT max and QTc max were prolonged as the number of LV wall segments with impairment increased. This, however, was not statistically significant. QT dispersion and QTc dispersion had no relation to the extent of LV wall motion abnormality nor to EF of the left ventricle In conclusion, no definite relationships between QT dispersion (QTc dispersion) and EF of the left ventricle, or between these variables and the extent of left ventricular wall motion abnormality were found in patients with anterior myocardial infarction in our study. Although both QT max and QT dispersion were prolonged in patients with myocardial infarction, this suggests that electrical heterogeneity or regional variation in electrical ventricular recovery did not always parallel the severity of mechanical abnormality of the left ventricle.

Aged↗

PubMed1995

Oral low-dose etoposide therapy for refractory multiple myeloma with extramedullary involvement.

A 65-year-old man was hospitalized with IgG kappa-type multiple myeloma (MM) and enormous subcutaneous plasmacytomas. Two different combination chemotherapy regimens (MMCP and AVPP) and alpha-interferon therapy were ineffectual. Oral administration of etoposide at 50 mg/day was subsequently started, the tumors completely disappeared after 5 months. The blood level of monoclonal protein became undetectable after 8 months of continuous treatment. The side effect noted was loss of hair. The course in this patient suggests that long-term daily low-dose administration of etoposide should be attempted in patients with refractory MM and extramedullary plasmacytoma.

Administration, Oral↗

PubMed1995

[Pharmacological and biochemical investigation of intracavital fluorinated pyrimidine chemotherapy].

We investigated the biochemical and molecular pharmacological parameters of fluorinated pyrimidines using malignant cells in pleural effusion or ascites from patients with malignant disease both before and after fluorinated pyrimidine chemotherapy, either systemically or intravesically. In four out of fifteen cancer patients, we could analyze the alteration of thymidylate synthase (TS) catalytic activity both before and after the treatment, showing that there was a decrease ranging 28% through 96.9%. We also analyzed a level of TS messenger RNA and TS protein using molecular biotechniques. None of these mutual correlations has so far been demonstrated, indicating that the data were analyzed through samples from patients not responding to the fluorinated pyrimidines. In a responding patient with advanced breast cancer, pre-treatment total TS protein was 130 fmol/mg and the TS activity was 3.27 pmol/mg/min. After intrapleural instillation of a combination of 5-FU 250 mg and leucovorin 3 mg, the total amount and the catalytic activity of TS could be measured. On the 11th day of treatment, the TS protein level decreased to 26 fmol/mg and the TS catalytic activity also decreased to 0.1 pmol/mg/min resulting in a TS inhibition rate of 92.3 percent. On the 17th day, the patient's malignant pleural effusion disappeared almost completely, suggesting that substantial TS inhibition may reflect the clinical evidence. This particular data showed that it would be predictable for clinical outcome to evaluate these parameters before and after fluorinated pyrimidine chemotherapy. Further study is warranted to evaluate the exact role of analyzing these parameters in clinical practice.

Aged↗

PubMed1994

Multiple proline substitutions cumulatively thermostabilize Bacillus cereus ATCC7064 oligo-1,6-glucosidase. Irrefragable proof supporting the proline rule.

Nine residues of Bacillus cereus ATCC7064 oligo-1,6-glucosidase were replaced stepwise with proline residues. Of the nine residues, Lys121, Glu208 and Glu290 were at second sites of beta turns; Asn109, Glu175 and Thr261 were at N-caps of alpha helices; Glu216, Glu270 and Glu378 were in coils within loops. The replacements were carried out in the order, Lys121-->Pro, Glu175-->Pro, Glu290-->Pro, Glu208-->Pro, Glu270-->Pro, Glu378-->Pro, Thr261-->Pro, Glu216-->Pro and Asn109-->Pro. The resultant nine active mutant enzymes contained 1-9 more proline residues than B. cereus oligo-1,6-glucosidase. The thermostability of these mutants was additively enhanced with the increase in the number of proline residues introduced. The increase in the thermostability was most remarkable when proline residues were introduced at second sites of beta turns or at N-caps of alpha helices. The above results afforded irrefragable proof for the proline rule as an effective principle for increasing protein thermostability [Suzuki, Y., Oishi, K., Nakano, H. & Nagayama, T. (1987) Appl. Microbiol. Biotechnol. 26, 546-551].

Amino Acid Sequence↗

PubMed1994

Enhancement of the expression of urokinase-type plasminogen activator from PC-3 human prostate cancer cells by thrombin.

The presence of procoagulants and fibrin deposition have been demonstrated in malignant tumors. Although thrombin, a key enzyme in coagulation, has other various biological functions, the significance of its presence in tumors is not known. We studied the effects of thrombin on the expression of urokinase-type plasminogen activator (uPA) which is known to play a role in tumor invasion, using a human prostate cancer cell line PC-3. Human alpha-thrombin added to cultures of PC-3 produced a dose-dependent and time-dependent increased secretion of uPA that was greatest at 3-6 h after exposure to thrombin. Increase in uPA antigen paralleled the increase in mRNA level, which reached a maximum at 4 h. Thrombin showed the maximum effect on uPA expression at a concentration 1-2 units/ml. Zymography showed that transient exposure to thrombin induced an increase in fibrinolytic activity which could be quenched by anti-uPA antibody. The thrombin receptor-activating peptide also caused an increase in uPA protein and mRNA level, indicating the presence of the same thrombin specific receptor on PC-3 cells as on platelets and endothelial cells. Thrombin did not affect the expression of other components of the plasminogen activation system, tissue-type plasminogen activator and type-1 plasminogen activator inhibitor, and uPA receptor. These results indicate that thrombin increases uPA expression selectively by the stimulation of a functional thrombin receptor on PC-3 cells. Since uPA is known to play a role in pericellular proteolysis of extracellular matrix, thrombin may be involved in the regulation of tumor invasion and metastasis.

Adenocarcinoma↗

PubMed1994

Design, synthesis, and conformation of a model peptide of endothelin with cystine-stabilized alpha-helix motif.

A model 16-peptide of endothelin-1 (MET-1), which has the minimized sequence homology to the corresponding part of endothelin-1 (ET-1), was designed to confirm the cystine-stabilized alpha-helix motif. The model structure consists of an extended structure, a beta-turn part, and an alpha-helix structure that is stabilized by two disulfide bonds. The alpha-helix segment was designed to emphasize the amphiphilic nature. In order to combine the extended structure and the alpha-helix segment, a D-Ala-Pro sequence was selected to fix the beta-turn. The model endothelin 16-peptide amide was synthesized by solid-phase synthesis on a 4-methylbenzhydrylamine resin. Its conformation was examined by CD and two-dimensional (2D) 1H-nmr measurements. MET-1 showed similar CD patterns to ET-1 in both buffer and 50% aqueous trifluoroethanol solution. The 2D nmr experiments in 50% aqueous ethylene glycol revealed that MET-1 closely resembles the conformation of ET-1 with an extended structure, an alpha-helix, and a beta-turn unit in the same position of the sequence. Furthermore, model peptides without disulfide bond(s) could not assume a stable structure in aqueous solution, while they did have similar alpha-helical content in 50% trifluoroethanol with MET-1. When the two disulfide bridges were simultaneously formed, the peptide with the correct disulfide bonds (MET-1) was obtained in threefold excess to the isomer (apamin type, MET-2). These findings obtained by the modeling of ET-1 showed an important role for the stabilization of peptide conformation with disulfide bonds.

Amino Acid Sequence↗

PubMed1994

Immunohistochemical demonstration of bikunin, a light chain of inter-alpha-trypsin inhibitor, in human brain tumors.

The presence and localization of bikunin (HI-30, or acid-stable protease inhibitor), a light chain of inter-alpha-trypsin inhibitor, was examined in 30 brain tumors employing immunohistochemical methods. The brain tumors involved 13 kinds of histological diagnosis. Bikunin immunoreactivity was detected in all of the brain tumors examined. Fibrillary staining of the glial processes was observed in astrocytoma, oligodendroglioma, and schwannoma. Intracytoplasmic staining in the interstitial cells, reactive astrocytes, and macrophages was noted in medulloblastoma, ependymoma, and meningioma. Metastatic tumors demonstrated intense immunoreactivity in the tissues surrounding the tumor cells. Neuronal cells revealed no bikunin immunoreactivity. There was no correlation between the intensity of staining and histologic type or grading of malignancy. In view of our earlier report that bikunin was present in the connective tissues around the site of cancer invasion, the above findings suggest that bikunin may play an important role in defense or repair at the tissue destruction and degeneration site.

Brain Neoplasms↗

PubMed1994

Clearance and distribution of a haemorrhagic factor purified from Bothrops jararaca venom in mice.

We previously purified two fibrinolytic/haemorrhagic enzymes (jararafibrase-I and II) from Bothrops jararaca venom. In the present study, the clearance, organ distribution and local absorption rate were examined in mice using 125I-labelled jararafibrase-I. Following intravenous injection of 125I-labelled jararafibrase-I, a complex was rapidly formed with the plasma protein and the radioactivity quickly disappeared from the circulation with a half-life of about 3 min for the initial part of the curve. The highest level of the radioactivity (59.5%) was seen in the liver at 5 min after dosing, and the next highest level of radioactivity (14.4%) was seen in the kidney at 60 min after dosing. At 60 min after dosing, 36.8% of the total injected radioactivity was seen in the contents of the small intestine, and 11.4% of the total injected radioactivity was seen in the contents of the large intestine at 120 min after dosing. It is assumed that the jararafibrase-I was metabolized mainly in the liver, to small mol. wt products, and excreted in the intestine via the bile duct. Also, a small amount of jararafibrase-I appeared to be metabolized in the kidney. Following subcutaneous injection, a high-dose group revealed a low local absorption rate. The low local absorption rate was apparently due to a diminished blood flow caused by subcutaneous haemorrhage.

Absorption↗

PubMed1993

Application of tryptic condensation strategy for the synthesis of alpha-melanocyte stimulating hormone.

The use of trypsin in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) paired with N,N-dimethylformamide (DMF) has been proposed for the enzymatic condensation of peptides. The tryptic condensation strategy was applied to the synthesis of the 13-peptide, alpha-melanocyte stimulating hormone (alpha-MSH), which contains two susceptible points to trypsin, in a low-water-containing solvent system, 4% H2O in HFIP/DMF (1/1, v/v). The N-terminal 8-peptide segment (S1) ending with Arg and C-terminal 5-peptide with the side-chain protections at Lys and Trp (S2) were prepared by the stepwise coupling on p-nitrobenzophenone oxime resin and by a conventional method, respectively. Both segments were condensed by the aid of trypsin. The acid component was converted into the partially protected alpha-MSH at as high as 95% conversion determined by reversed-phase HPLC. When the side-chain of Lys at the 11-position was not protected, alpha-MSH was obtained only in 35% yield, and was contaminated with products of secondary hydrolysis. Although the Lys11-Pro12 sequence was very poorly susceptible to trypsin, the side-chain of Lys had to be protected in order to be inert to trypsin under the synthetic conditions. HFIP is demonstrated as a good solvent with DMF to allow the efficient tryptic condensation of peptides. The strategy increased the value of the enzymatic condensation as practical method by avoidance of secondary hydrolysis, high dissolution of peptides and retention of activity of enzyme.

1-Propanol↗

PubMed1993

Purification and characterization of low molecular weight fibrinolytic/hemorrhagic enzymes from snake (Bothrops jararaca) venom.

Two low molecular weight fibrinolytic/hemorrhagic enzymes, jararafibrase III and jararafibrase IV, were purified from Bothrops jararaca venom using a fast protein liquid chromatography system. The purified jararafibrase III and jararafibrase IV were single chain proteins with molecular weights of 20,400 +/- 500 and 21,200 +/- 400, respectively, by SDS-PAGE. The isoelectric points of jararafibrase III and jararafibrase IV were 9.4 and 6.9, respectively. The activity of the enzyme was inhibited by 1,10-phenanthroline and EDTA, suggesting that both enzymes were metalloproteinases. The specific fibrinolytic activities of jararafibrase III and jararafibrase IV were 7.5 +/- 0.4 and 6.5 +/- 1.6 units/mg protein, respectively. The enzymes induced local hemorrhage in the skin of rats. The minimal hemorrhagic doses of jararafibrase III and IV were 31.0 and 34.0 micrograms/rat, respectively. The enzymes displayed broad substrate specificities like the previously purified jararafibrases I and II. Jararabrases III and IV degraded type-IV collagen, gelatin, laminin and fibronectin into smaller fragments. The specific activities of jararafibrase III for type-IV collagen and gelatin were 7.6 +/- 0.3 and 43 +/- 11 units/mg protein, respectively. The specific activities of jararafibrase IV for type IV-collagen and gelatin were 16.5 +/- 1.2 and 112 +/- 9 units/mg protein, respectively.

Animals↗

PubMed1993

High sensitivity to salt in kininogen-deficient brown Norway Katholiek rats.

Brown Norway Katholiek rats, which have very low levels of plasma kininogens, excreted a much smaller amount of kinin in the urine than normal rats of the same strain. The systolic blood pressure of 7-week-old kininogen-deficient rats fed low (0.3%) NaCl diets (131 +/- 4 mm Hg, n = 12) was not different from that in normal rats. Two percent NaCl diets given from 7 weeks of age for 4 weeks caused rapid increases in blood pressure (167 +/- 4 mm Hg, n = 12, 9 weeks old) in deficient rats, although the same diets induced no blood pressure increase in normal rats. Urinary excretion of active kallikrein and prokallikrein remained constant in both rat groups throughout NaCl loading. During this period, the deficient rats secreted less urine (9 weeks old, P < .05) and less urinary sodium (11 weeks old, P < .05). Serum levels of sodium in deficient rats were higher (P < .05) than in normal rats at 9 weeks of age. Intracellular concentrations of sodium in the erythrocytes of deficient rats were higher (P < .05) than in normal rats throughout NaCl loading. Subcutaneous infusion of bovine low molecular weight kininogen with an osmotic pump in NaCl-loaded deficient rats induced a reduction (P < .01) in blood pressure and increases (P < .05) in urine volume and urinary sodium and kinin levels. By contrast, subcutaneous infusion of the bradykinin antagonist Hoe 140 or of aprotinin in NaCl-loaded normal rats induced a hypertensive response. This antagonist treatment reduced urine volume and urinary sodium. These results indicate that the lack of kinin generation observed in the kininogen-deficient rats was related through sodium retention to the hypertensive response to NaCl loading.

Aging↗