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Biomedical subjects

A Kawamura

Publications and source records attributed to A Kawamura.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of BiP/Kar2 protein from Saccharomyces cerevisiae.

Using specific anti-BiP/Kar2 antibody as the probe, we have developed an efficient purification method of BiP/Kar2 protein from the total cell extract of Saccharomyces cerevisiae. Overproduction of BiP/Kar2 protein was achieved by the cloning of the KAR2 gene on multicopy plasmids and the treatment of cells harboring the cloned KAR2 gene with tunicamycin. Freeze-thaw treatment, hydroxyapatite high pressure liquid chromatography, and ATP-agarose column chromatography of crude extract yielded homogeneous BiP/Kar2 protein (including less than 0.2% of degradative derivative) with a 430-fold purification and 28% recovery. Edman degradation of purified BiP/Kar2 suggests that the mature protein corresponds to a processed product with the removal of a 42-amino acid presequence. It is active as a homodimer and exhibits ATPase activity with a specific activity of 2 pmol/min/micrograms of protein. Protease susceptibility indicated that the ADP form of BiP/Kar2 is more resistant than the ATP form to the chymotrypsin digestion and that BiP/Kar2 required the presence of ATP to avoid the irreversible denaturation. Synthesis of BiP/Kar2 was induced by the inducible expression of an aberrant heterologous protein, yeast killer prepro-signal mouse alpha-amylase fusion protein.

Adenosine Triphosphatases↗
PubMed1992

Site-specific dissection of E. coli chromosome by lambda terminase.

We have succeeded the targeted cleavage of chromosomes by lambda terminase that introduces double-strand cleavages in DNA recognizing the lambda cos sequence. When chromosomal DNAs of various Escherichia coli K-12 strains were subjected to terminase digestion, all were found to contain two common cleavage sites. Therefore, DNAs from lambda lysogens in which lambda DNA was inserted at different chromosomal sites were specifically cleaved at one more additional site. The two sites, termed ecos1 and ecos2, were mapped at approximately 35.1' and 12.7' of E. coli genetic map. The ecos1 and ecos2 sites were included in qin and qsr' regions, respectively. Therefore, the cleavage sites were associated with cryptic prophages. Sequences at the ecos1 and ecos2 sites showed 98% homology to the lambda cos sequence, indicating high fidelity of sequence recognition by the terminase. Since the strategy for integration of a DNA segment into chromosomal DNA through homologous recombination has been established, the dissection method that uses lambda terminase should be applicable for gene mapping as well as construction of macrophysical maps of larger genomes.

Base Sequence↗
PubMed1992

Structure of yeast pGKL 128-kDa killer-toxin secretion signal sequence. Processing of the 128-kDa killer-toxin-secretion-signal-alpha-amylase fusion protein.

The linear double-stranded DNA plasmid pGKL1 in yeast encodes a killer toxin consisting of 97-kDa, 31-kDa and 28-kDa subunits. A 128-kDa protein precursor of the 97-kDa and 31-kDa subunits, was first synthesized with a 29-amino-acid extension at its NH2-terminus as a secretion signal sequence. In the present study, the property of this signal sequence was studied by the analysis of a fusion protein with mouse alpha-amylase. Using the secretion signal sequence of the killer protein, the mouse alpha-amylase was successfully secreted into the culture medium. An intracellular precursor form of alpha-amylase was identified and purified. Analysis of the NH2-terminal sequence of this precursor molecule indicated that it corresponded to the secretory intermediate (pro form) of alpha-amylase with the removal of the hydrophobic segment (Met1-Gly16) of the secretion signal. Both the secretion of alpha-amylase into the culture medium and the detection of the pro-alpha-amylase species in the cells were prohibited by a sec 11 mutation, or by the conversion of Gly to Val at the 16th position of the secretion signal. These results strongly suggest that the cleavage occurs between Gly16 and Leu17 by a signal peptidase, and that this cleavage is required for the secretion of alpha-amylase into the medium. Based on the data from the NH2-terminal amino acid sequences of secreted alpha-amylases, we conclude that the 29-amino-acid secretion signal present in the 128-kDa killer toxin precursor protein is a prepro structure.

Amino Acid Sequence↗
PubMed1992

Changes in the plasma abnormal prothrombin level following treatment of hepatocellular carcinoma.

The Hokkaido Liver Cancer Study Group focused on the changes in PIVKA-II levels observed in 61 HCC patients after several regimens of treatment in comparison with the AFP levels and the pathophysiological characteristics of HCC. The overall positivity rate for PIVKA-II was 47%, and there was no correlation between the PIVKA-II values and the AFP levels. Accordingly, the HCC detection rate was increased by about 20% by the measurement of both markers. In all, 13 patients underwent hepatic resection, and nonsurgical therapy was carried out in the other 48 subjects. Of the 6 surgically treated patients, 5 (83%) showed a fall in PIVKA-II levels to the normal range immediately after surgery, whereas 14/29 (48%) subjects receiving nonsurgical treatment showed a decrease in PIVKA-II values. Although inconsistency between these tumor markers was detected in four treated cases, we concluded that assay for both of these two parameters may expand their clinical utility for the diagnosis of HCC and monitoring of patients after treatment.

Adult↗
PubMed1992

Comparative effects of diltiazem, nifedipine, and verapamil on large and small coronary artery constriction induced by intracoronary acetylcholine in pigs.

The in vivo protective effects of diltiazem, nifedipine, and verapamil on large and small coronary artery constriction induced by intracoronary injection of acetylcholine were compared by coronary arteriography in pigs. The percent narrowing of the epicardial major right coronary artery was used as an indicator of large coronary artery constriction, and the time required for contrast medium to reach the posterior descending coronary artery from the ostium of the right coronary artery was used as an indicator of small coronary artery constriction. Doses of 12.5, 25, 50, 100, and 200 micrograms of acetylcholine were administered into the right coronary artery under left ventricular pacing to keep the systemic hemodynamics constant. Marked prolongation of the flow time of contrast medium to greater than or equal to 8.1 s (control of less than or equal to 1.8 s) with mild narrowing of the epicardial major right coronary artery (less than or equal to 35%) was observed at doses of 12.5-50 micrograms of acetylcholine and was accompanied by myocardial ischemia. Over 50% narrowing of the epicardial major coronary artery plus markedly slow flow of contrast medium were induced in 12 of the 15 pigs by 100-200 micrograms of acetylcholine. Narrowing of the epicardial major coronary artery and the delay time of contrast medium flow induced by acetylcholine were both significantly reduced to 12-33% (control: 36-81%) and to 4.3-16.8 s (control: 16.2-37.7 s) after intracoronary injection of 100 micrograms of diltiazem.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1992

[Spotted fever group rickettsiosis in Chiba Prefecture].

In Chiba Prefecture, the first patient of infection with spotted fever group Rickettsia was found in 1987, thereafter nine patients were detected serologically by the end of 1990. Patients were found in the villages of Amatsukominato, Katsuura and Ootaki, which were located in southern part of Chiba Prefecture. The illness occurred from June to October. On the other hand, patients with Rickettsia tsutsugamushi were seen from October to September. Difference of prevalent seasons of these two types of rickettsiosis is important to make a clinical diagnosis and serological identification of this spotted fever group rickettsiosis. Antibody of the patients showed the highest titer to YH strain of Rickettsia and showed high cross-reactivity to other spotted fever group rickettsiae. For the diagnosis of the patient serologically, it was confirmed that any strain of spotted fever group Rickettsia were useful.

Adult↗
PubMed1992

[Bietti's crystalline retinopathy].

The authors previously reported four cases with crystalline retinopathy and suggested that it be classified into three stages: 1) retinal pigment epithelium (RPE) atrophy in the posterior pole, 2) choriocapillaris atrophy appearing in the enlarged areas of RPE atrophy, and 3) prominent diffuse atrophy of both the RPE and choriocapillaris. We followed up 4 cases, including 2 cases which were previously been reported, and have now evaluated the clinical features of this disease. The results indicated that the above classification was clinically useful. Enlargement of the RPE atrophy from stage 2 to 3 was due to enlargement and confluence of the RPE atrophy between the posterior pole and the periphery. In addition, disturbances in ERG and color vision were thought to correlate with secondary dysfunction at the level of the photoreceptor cell layer within the diseased lesions.

Adult↗
PubMed1991

Purification and determination of the NH2-terminal amino acid sequence of mouse alpha-amylase secreted from Saccharomyces cerevisiae: correct processing of the secretion signal from pGKL killer 28 kDa precursor protein.

We have previously reported the construction of recombinant mouse salivary alpha-amylase secretion vector in Saccharomyces cerevisiae utilizing novel yeast secretion signal derived from killer 28 kDa precursor protein. Here, we have first purified recombinant mouse alpha-amylase to homogeneity from the culture medium of S. cerevisiae, and determined its NH2-terminal amino acid sequence. The sequencing data indicated that the 28 kDa killer secretion signal-alpha-amylase fusion protein was cleaved accurately at its native processing site, and that both the core-glycosylated and non-glycosylated alpha-amylases possessed the same NH2-terminal amino acid sequences.

Amino Acid Sequence↗
PubMed1991

Failure to reduce infarct size by intracoronary infusion of recombinant human superoxide dismutase at reperfusion in the porcine heart: immunohistochemical and histological analysis.

Failure to Reduce Infarct Size by Intracoronary Infusion of Recombinant Human Superoxide Dismutase at Reperfusion in the Porcine Heart: Immunohistochemical and Histological Analysis. Journal of Molecular and Cellular Cardiology (1991) 23, 1287-1296. We quantitatively determined the extent of infarction and contraction band necrosis in porcine hearts, and analyzed the distribution of administered recombinant human superoxide dismutase (h-SOD) in the myocardium using a polyclonal antibody to h-SOD. After 1 hour of occlusion, h-SOD was infused for the first 30 min of reperfusion in SOD group, while pigs received only arterial blood in control group. The extent of infarction or contraction band necrosis was not significantly different between SOD group and control group. Positive staining by polyclonal antibody to h-SOD was detected only in the infarcted area in SOD group. Thus, h-SOD only entered irreversibly damaged myocytes and neither diminished reperfusion injury nor reduced infarct size in pigs.

Animals↗
PubMed1991

Reperfusion injury in dog hearts with permanent occlusion of a coronary artery, probably due to reperfusion via collateral vessels.

To clarify whether or not reperfusion injury occurs in the permanent occlusion of a coronary artery, we analyzed quantitatively contraction band necrosis as an indicator of early recanalization, coagulation necrosis, infarct size and measured regional blood flow in dog hearts with collateral circulation. Fifty mongrel dogs were divided into four groups: 15 dogs with a 24-hour occlusion of the left anterior descending coronary artery just distal to the first diagonal branch (permanent occlusion group): 15 dogs a with 3-hour occlusion followed by 24-hour recanalization (recanalization group); 10 dogs with a 2-hour occlusion without recanalization (transient occlusion group); 10 dogs with a 4-hour occlusion without recanalization (transient occlusion group). The regional blood flow in the subepicardium and subendocardium determined by the generated hydrogen gas clearance method was greatly decreased 30 minutes after occlusion (14 + 8%/12 +/- 9%) and was relatively restored from 180 minutes (31 +/- 21%/21 +/- 14%) to 24 hours later (41 + 19%/26 + 16%) in spite of complete occlusion of the coronary artery. The percentage infarct area in the risk area was significantly greater in the permanent occlusion group (60 +/- 26%) than in the recanalization group (35 +/- 31%). Although most of the infarct was occupied by contraction band necrosis in the recanalization group (86 +/- 12%), contraction band necrosis was diffusely seen even in the permanent occlusion group (54 +/- 27%). In both the permanent and recanalization groups, contraction band necrosis was the main histological feature of small infarcts occupying less than 30% of the risk area, while coagulation necrosis was the main feature in very large infarcts occupying more than 80% of the risk area. In the occlusion groups without recanalization, the percentage area of contraction band necrosis in the risk area was 6 +/- 8% after the 2-hour occlusion, 23 +/- 17% after the 4-hour occlusion and 31 +/- 21% after permanent occlusion; the difference between the 4-hour and permanent occlusion groups was not significant. In the permanent occlusion group, the percentage infarct area in the risk area was inversely correlated with regional blood flow during occlusion, an indicator of collateral flow. It was concluded that reperfusion injury occurs even in hearts without recanalization. The pathogenesis may involve reperfusion in the risk area via collateral circulation. Protection against reperfusion injury is important to minimize the infarct size even in hearts with permanent occlusion, although the presence of collateral flow is an important factor in limiting infarct size.

Animals↗
PubMed1991

Hypertrophy of surviving myocytes overlying the infarct in human old myocardial infarctions with abnormal Q waves.

The time course of hypertrophy of surviving myocytes overlying the infarct after the onset was examined and the hypertrophy was analyzed in relation to the transmural extent of infarct in 34 autopsied hearts with Q wave infarction. The 34 hearts were divided into 4 groups according to the length of time between the onset of infarction and death. This was less than 5 days in group 1 (n = 10), 20-30 days in group 2 (n = 7), 40-60 days in group 3 (n = 7), and 12-24 months in group 4 (n = 10). To clarify the regional hypertrophy of myocytes overlying the infarct, the size of the surviving myocytes in the outer third of the left ventricular wall in the 1-cm wide central zone of the infarct was compared with that of the myocytes in the outer third of the left ventricular wall without infarction (control wall) in the same heart. To exclude factors which stimulate the hypertrophy of the whole left ventricle, the ratio of the monocyte diameter in the infarcted wall to that in the control wall was examined. It was 1.0 +/- 0.0 (mean +/- SD) in group 1, 1.0 +/- 0.1 in group 2, 1.2 +/- 0.1 in group 3, and 1.3 +/- 0.1 in group 4. The ratio was significantly higher in group 3 than in group 1 and 2, and was highest in group 4. In group 4, the corrected percentage transmural extent of infarct indicating the original transmural extent of infarct at the acute stage was 63 + 8%, and this transmural extent correlated positively with the ratio of myocyte diameter.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1991

Acceleration of the hemopoietic reconstitution in mice undergoing bone marrow transplantation by recombinant human granulocyte colony-stimulating factor.

We have investigated the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on hemopoietic reconstitution after bone marrow transplantation (BMT) following lethal irradiation in mice. Mice received a daily administration of 10 micrograms/kg rG-CSF or control vehicle one through 21 days after BMT. Spleen colony-forming units (CFU-S), granulocyte-macrophage colony-forming units (CFU-GM), megakaryocyte colony-forming units (CFU-Meg), and erythroid burst-forming units (BFU-E) increased in both bone marrow and spleen of the rG-CSF-treated mice as compared with the control. This increase was evident during the administration period. In spite of the increase in the progenitor cells in bone marrow and spleen, only a recovery of neutrophils was accelerated in peripheral blood. Thus rG-CSF accelerated granulopoietic recovery in the BMT mice, with an enhanced recovery of the stem cells and the progenitors for erythrocytes and megakaryocytes. These results indicate the potential clinical usefulness of rG-CSF in the treatment of patients undergoing BMT.

Animals↗
PubMed1991

Simple in vivo bioassay for erythropoietin.

A new method of in vivo bioassay for erythropoietin (EPO) is described. This method is based on the measurement of immature reticulocytes in EPO-treated mice using an automatic microcell counter, and is simpler and more precise than the existing methods of polycythaemic mouse assay and starved rat assay. Normal mice were injected subcutaneously for 3 successive days with EPO at the doses between 0 and 9.6 IU/mouse. On the following day, 20 microliters of peripheral blood from each EPO-treated mouse was collected and haemolysed with a stromatolysing agent. Quicklyser. Residual particles derived from immature reticulocytes in the stromatolysed blood cells were counted using a microcell counter (Sysmex CC-180A) and a cell monitor (Sysmex CM-5). The number of the residual particles increased in a dose-dependent manner in EPO-treated mice. The mean of correlation coefficients of five log dose-response lines was 0.924, and the mean of precision indices was 0.138. A good correlation was also observed between the residual particle counts and reticulocyte counts obtained from smears.

Animals↗
PubMed1991

Specific amplification of Rickettsia tsutsugamushi DNA from clinical specimens by polymerase chain reaction.

Polymerase chain reaction (PCR) was used to detect Rickettsia tsutsugamushi-specific DNA in clinical specimens. The primer pair used for PCR was designed from the nucleotide sequence of the gene encoding the 56-kDa antigen of the Gilliam strain. Theses primers led to a 78-bp fragment by amplifying the genomic DNAs from five serovariants, i.e., the Gilliam, Karp, Kato, Kawasaki, and Kuroki strains of R. tsutsugamushi, and also the DNA from blood clots of patients with scrub typhus, even at the early stage of onset of the disease. This indicates that this method is suitable for the diagnosis of scrub typhus.

Base Sequence↗
PubMed1991

Use of monoclonal antibodies against Rickettsia tsutsugamushi Kawasaki for serodiagnosis by enzyme-linked immunosorbent assay.

Monoclonal antibodies (MAbs) against Rickettsia tsutsugamushi Kawasaki were prepared. The crossreactivity tests of the MAbs performed by using antigenically distinct strains of R. tsutsugamushi in immunofluorescence and immunoblotting analyses indicated that the Kawasaki strain contains a strain-specific epitope and also contains a common epitope on the 56-kDa polypeptide cross-reactive with the Gilliam strain, group- and subgroup-specific epitopes on the 46-kDa polypeptide, and a subgroup-specific epitope on the 25-kDa polypeptide. By using the strain-specific MAb for serodiagnosis of tsutsugamushi disease (or scrub typhus fever), we have established a method which was designated the inhibition enzyme-linked immunosorbent assay. The principle of the method is to measure the percentage of inhibition of antigen absorption on a MAb-coated plate by antibody-positive sample sera which were mixed with the antigen suspension. The advantages of this test for practical use are that (i) crude antigen can be used, i.e., purification of the antigen is not required; (ii) the test is more sensitive than immunofluorescence; (iii) the final judgment of plus or minus is clear-cut; and (iv) rickettsial antigenic types in the patients can be distinguished by this test.

Antibodies, Bacterial↗
PubMed1991

Expression and distribution of atrial natriuretic peptide in human hypertrophic ventricle of hypertensive hearts and hearts with hypertrophic cardiomyopathy.

To investigate the ventricular expression of atrial natriuretic peptide (ANP) in human hypertrophic hearts, we conducted an immunohistochemical study of 130 endomyocardial biopsy specimens obtained from the right side of the ventricular septum (RVB), left ventricular free wall (LVB), or both from a total of 80 patients: 44 patients with hypertrophic cardiomyopathy (HCM), 14 with apical hypertrophic cardiomyopathy (APH), 13 with hypertensive hearts (HHD), and nine without hypertrophy (controls). No patients had apparent congestive heart failure. ANP was not seen in ventricular myocytes in controls but was identified in biopsy specimens of hypertrophic hearts, and its distribution was characteristic in each hypertrophic group: 15 RVB (37%) and two LVB (7%) of the HCM group, one RVB (7%) and two LVB (18%) of the APH group, and zero RVB (0%) and five LVB (46%) of the HHD group. Clinical data (including echocardiographic, hemodynamic, and angiographic data) were not directly related to ventricular ANP expression in HCM, APH, or HHD with one exception. In HHD patients, LVB specimens with ANP showed greater ventricular wall thickness than LVB specimens without ANP. According to histological data, however, the ANP-present RVB specimens of HCM or ANP-present LVB specimens of HHD had greater myocyte size than did the ANP-absent specimens. In addition, in HCM patients, the ANP-present RVB specimens showed more severe fibrosis and myofiber disarray than did the ANP-absent specimens. We conclude that a failing state and hemodynamic overload are not likely to be indispensable for ANP expression in human hypertrophic ventricles and that ventricular ANP expression occurs as a response to disease-specific changes: hemodynamic overload in HHD and histological changes such as myocardial fiber disarray, hypertrophy of myocytes, and fibrosis in HCM, which may reflect the characteristic distribution of intraventricular ANP.

Adult↗
PubMed1991

Relation between myoglobin and cardiac dysfunction in myocarditis--immunohistochemical study of endomyocardial biopsy specimens.

To investigate the mechanism of cardiac dysfunction in myocarditis, myoglobin, an intracellular oxygen-transport, was immunohistochemically examined in biopsy specimens obtained from the right side of the ventricular septum and left ventricular free wall in 58 patients with myocarditis and 19 controls. Sections 4 microns thick were stained by the indirect immunoperoxidase method using a polyclonal antibody to human myoglobin as the primary antibody. Under light microscopy, the intensity of myoglobin immunoreactivity in the tissue section was semiquantitatively classified from grade 0 to grade 3. Then, the grade of myoglobin staining was compared with clinical, hemodynamic and histopathologic parameters. In right and left ventricular specimens, the grade of myoglobin staining was positively correlated with ejection fraction, but inversely with left ventricular end-diastolic and end-systolic volume indices. The percentage of myocytes with grade 0 was correlated with the number of mononuclear cells in the specimens. In addition, the grade of myoglobin staining in right ventricular specimens was positively correlated with the duration of illness but inversely correlated with the number of mononuclear cells. In 4 patients who had serial biopsies, the ejection fraction was improved and the grade of myoglobin staining was increased in the convalescent stage. These results indicate that myoglobin staining reflects the intensity of myocarditis and a decrease of myoglobin may be important as one of the pathogenetic factors of cardiac dysfunction in myocarditis.

Adolescent↗