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Biomedical subjects

Y Kawakami

Publications and source records attributed to Y Kawakami.

At least 19 recordsLinked to original sources
PubMed1992

Association of protein-tyrosine kinase with phospholipase C-gamma 1 in bone marrow-derived mouse mast cells.

Bone marrow-derived mouse mast cells contain phospholipase C-gamma 1 (PLC-gamma 1), which is phosphorylated at tyrosine residues upon cross-linking of cell-bound IgE antibodies with multivalent antigen. It was found that immune complexes formed from digitonin lysates of the mast cells by monoclonal anti-PLC-gamma 1 antibodies contained protein-tyrosine kinase (PTK), which phosphorylated PLC-gamma 1 in vitro. The tyrosine kinase activity coprecipitated with PLC-gamma 1-anti-PLC-gamma 1 complexes markedly increased when the cell lysates were obtained immediately after antigen challenge. The results indicate that PTK is associated with PLC-gamma 1 in the mast cells and that the kinase is activated upon cross-linking of Fc epsilon RI. Neither beta nor gamma subunit of Fc epsilon RI nor src family PTK was coprecipitated with the PLC-gamma 1-anti-PLC-gamma 1 complexes. In situ denaturation/renaturation experiments, which detect autophosphorylated kinases, indicated that the PTK associated with PLC-gamma 1 was a 44-kDa protein.

Animals↗
PubMed1992

Activation of human CD4+CD45RA+ T cells by chrysotile asbestos in vitro.

Chrysotile asbestos stimulates T lymphocyte subsets. Cell surface CD4 or CD45RA expression in peripheral blood mononuclear cells (PBMC) was downregulated after incubation with chrysotile asbestos in vitro temporarily. The percentage of CD4+CD45RA+ cells and mean fluorescence intensity in CD4 or CD45RA decreased after incubation with asbestos and returned to the original level after 24 h of incubation, which suggests that chrysotile asbestos activates CD4+CD45RA+ cells. No change was observed in CD29 expression. An increased percentage of IL-2R positive cells and an elevated intracellular Ca++ level were also indicative of the activation of PBMC by chrysotile asbestos.

Antigens, CD↗
PubMed1992

Reduced drug accumulation in a newly established human lung squamous-carcinoma cell line resistant to cis-diamminedichloroplatinum(II).

A human lung squamous-carcinoma cell line resistant to cis-diamminedichloroplatinum(II) (CDDP), designated PC10-B3, has been established from the original cell line PC10 by a stepwise increment of the CDDP concentration. This is the first report, to our knowledge, to establish a CDDP-resistant lung squamous-carcinoma cell line. PC10-B3 has continued to proliferate in the presence of 0.5 micrograms/mL CDDP, whereas PC10 could not survive. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that PC10-B3 was 11.4-fold more resistant to CDDP than PC10 and cross-resistant to diammine(1,1-cyclobutanecarboxylate)platinum(II) (CBDCA) and 254-S, but not to doxorubicin or etoposide. PC10-B3 was characterized by a smaller DNA index and a larger cell size compared to PC10. The level of intracellular platinum accumulation was reduced by about 5- to 8-fold in PC10-B3 when compared with PC10, suggesting that reduced drug accumulation may be one of the important factors in contributing to CDDP resistance in PC10-B3.

Carcinoma, Squamous Cell↗
PubMed1992

Correlation between nucleolar organizer regions visualized by silver staining and the growth rate in lung adenocarcinoma.

BACKGROUND: The value of nucleolar organizer regions (NOR) visualized by silver staining (AgNOR) for the histologic differentiation, pathologic staging, and estimation of growth rate was assessed by the investigation of paraffin sections from 58 lung adenocarcinomas. AgNOR consist of NOR-associated proteins, and the number of AgNOR might be related to proliferative activity. METHODS: In lung adenocarcinoma, the growth rate can be measured by means of chest radiographs. Using this technique, the authors studied the correlation between the mean number of AgNOR and growth rate. RESULTS: The mean number of AgNOR ranged from 1.8 to 6.3 (mean +/- standard deviation, 4.0 +/- 0.8). Neither the degree of histologic differentiation nor the pathologic staging was related to the AgNOR count. The tumor growth rate was estimated on the basis of the doubling time in the chest radiographs of 13 patients. The doubling time ranged from 80 to 760 days. There was a high inverse correlation between the AgNOR count and the doubling time (r = -0.910; P less than 0.001). CONCLUSIONS: Thus, it appears to be possible to use the mean number of AgNOR as an index of proliferative activity.

Adenocarcinoma↗
PubMed1992

Determination of free N-acetylamino acids in biological samples and N-terminal acetylamino acids of proteins.

N-Acetylamino acids were derivatized with 9-anthryldiazomethane to the corresponding esters. The anthryl esters were separated by high-performance liquid chromatography and detected fluorimetrically (excitation at 365 nm; fluorescence emission measured at 412 nm). N-Acetyl derivatives of Asn, Gln, Ser, Thr, Gly, Ala, Tyr, Pro, Met, Val, Ile and Leu as well as N-formyl-Met could be separated and identified in the same chromatographic run. The detection limit was from 0.10 pmol for AcGln to 5.5 pmol for AcIle and AcLeu. When the acetylamino acids listed above were added to the 700 g supernatant of a rat liver homogenate, the mean recovery was 72%. AcAla and AcTyr were found in free form in baker's yeast. Proteins with an acetylated N-terminus were digested by a protease, and the peptides formed were treated with an N-acylamino acid-releasing enzyme. This method was applied to end-group determination of four proteins (each 0.5 nmol).

Acetamides↗
PubMed1992

Shared human melanoma antigens. Recognition by tumor-infiltrating lymphocytes in HLA-A2.1-transfected melanomas.

Three predominantly CD8+ CTL lines, TIL 501, TIL 620, and TIL 660, were generated from three HLA-A2+ melanoma patients by culturing tumor-infiltrating lymphocytes in 1000 U/ml IL-2. These tumor-infiltrating lymphocytes lysed 12 of 18 HLA-A2+ autologous and allogeneic melanomas, but none of 20 HLA-A2-negative melanomas. They also did not lyse the MHC class I negative lymphoma-leukemia cell lines, Daudi, K562, or HLA-A2+ non-melanoma cell lines including PHA or Con A-induced lymphoblast, fibroblast, EBV-transformed B cell, Burkitt's B cell lymphoma, and colon cancer cell lines. Autologous and allogeneic melanoma lysis was inhibited by anti-CD3, by anti-MHC class I, and by anti-HLA-A2 mAb, indicating recognition of shared tumor Ag among melanoma cell lines in a TCR-dependent, HLA-A2-restricted manner. Six HLA-A2-negative melanoma cell lines obtained from five HLA-A2-negative patients were co-transfected with the HLA-A2.1 gene and pSV2neo. All 17 cloned transfectants expressing cell surface HLA-A2 molecules, but none of 12 transfectants lacking HLA-A2 expression, were lysed by these three HLA-A2-restricted, melanoma-specific CTL. Lysis of the HLA-A2+ transfectants was inhibited by anti-CD3, by anti-MHC class I, and by anti-HLA-A2 mAb, indicating recognition of shared tumor Ag on transfectants in a TCR-dependent, HLA-A2-restricted manner. These results identify the HLA-A2.1 molecule as an Ag-presenting molecule for melanoma Ag. They also suggest that common melanoma Ag are expressed among melanoma patients regardless of HLA type. These findings have implications for the development of melanoma vaccines that would induce antitumor T cell responses.

Antigens, Neoplasm↗
PubMed1992

Prognostic significance of the expression of ras oncogene product in non-small cell lung cancer.

The clinical significance of ras oncogene expression in non-small cell lung cancer was evaluated in 116 surgically treated patients. Archival paraffin sections of the tumors were analyzed immunohistochemically using anti-ras p21 monoclonal antibody (MoAb) rp-35, and p21 staining was correlated with clinicopathologic parameters and survival. Positive reactions (+ and ++) were observed in 72.5% of the adenocarcinomas and 55.6% of the squamous cell carcinomas studied. The T1 tumors showed a ++ reaction less frequently than T2 and T3 tumors (P less than 0.05). Stage I tumors also were less reactive with MoAb rp-35 than tumors in more advanced stages (P less than 0.05). Survival analysis showed that patients with p21-negative tumors had significantly longer survival times (a 5-year survival rate of 64.1%) than those with p21 + tumors (38.0%, P less than 0.05) or those with p21 ++ tumors (11.5%, P less than 0.005). The significant correlation between p21 staining and patient survival was independent of histologic type, stage of disease, tumor or node status, and the resectability of tumors. On Cox's multivariate analysis, p21 staining was a major and independent prognostic determinant of survival. These results suggest that enhanced ras p21 expression may be one of the important biologic and clinical markers indicating the malignant potential of non-small cell lung cancer.

Carcinoma, Non-Small-Cell Lung↗
PubMed1992

Accuracy of the bronchoscopic DNA content analysis of non-small-cell lung carcinoma.

To assess the accuracy of the bronchoscopic DNA content analysis, samples of non-small-cell lung carcinomas (NSCLC) were investigated by means of flow cytometry. Samples were dissociated using the detergent Triton X-100. In 58 NSCLC cases, 39 (67%) had DNA aneuploid tumors. We compared the DNA indices of bronchoscopic brushing samples with 21 corresponding surgical samples. In 16 (76%) cases, DNA ploidy of both bronchoscopic and surgical samples were in concordance. In 3 (14%) cases, both bronchoscopic and surgical sample showed DNA aneuploidy, but the number of the DNA aneuploid stem cell lines was different. The cause of these differences was ascribed to the intratumor DNA heterogeneity. In 2 (10%) cases, the bronchoscopic sample showed DNA diploidy, but the surgical sample showed DNA aneuploidy. In these cases, tumor cells obtained by bronchoscopic brushing were so few that the small DNA aneuploid peak was undetectable in the DNA histogram. But the tumor DNA ploidy was evaluated correctly in 90% of 21 cases using bronchoscopic samples. Consequently, despite some drawbacks, the DNA ploidy diagnosis using bronchoscopic samples in this relatively small study, was almost as reliable as surgical samples.

Adenocarcinoma↗
PubMed1992

Secretion of genetically-engineered dihydrofolate reductase from Escherichia coli using an E. coli alpha-hemolysin membrane translocation system.

Secretion of fusion proteins composed of cytoplasmic protein dihydrofolate reductase (DHFR) and the Escherichia coli alpha-haemolysin (HlyA) C-terminal sequence was examined through the haemolysin secretion machinery of E. coli. DHFR of various lengths was combined with the HlyA C-terminal region, and both secretion and DHFR activity of the fusions were measured. The secretion was found to be inversely correlated with the intracellular DHFR activity. Moreover, when one amino acid (Ile155) in a beta-sheet of the DHFR C-terminal region was replaced with Lys, the enzymatically active DHFR fusion protein was secreted into the medium. We discuss the possibility of a relationship between folding and secretion of HlyA-fused protein in the HlyA secretion system.

Amino Acid Sequence↗
PubMed1992

Reliability of measurement of oxygen uptake by a portable telemetric system.

The purpose of the present study was to check the reliability of measurements of oxygen uptake (VO2) using a newly developed portable telemetry system. This system (K2) consisted of a face mask, a flow meter, a gas analyser with a transmitter, and a receiver. The total mass for the subject to carry was about 850 g. Three experiments were carried out, firstly to check the reliability and reproducibility of the flow meter and the K2 gas analyser, secondly to check the accuracy of K2 by comparing it with the Douglas bag method (DB), and thirdly to apply K2 to sports activities. In the first experiment, the flow meter was highly accurate up to 180 l.min-1 with good reproducibility. The measurement error of the gas analyser was less than 2%. In the second experiment, there was no significant difference in the calculated ventilation between K2 and DB. The VO2 showed no significant difference between K2 and DB with some exceptions. In the third experiment, we succeeded in the measurement of VO2 during rowing on water. The measurement of VO2 during running and playing soccer was also possible. It would seem that the present system could well be a powerful tool in the field measurement of VO2 during various sports activities.

Exercise↗
PubMed1992

Concentrations of D-lactate and its related metabolic intermediates in liver, blood, and muscle of diabetic and starved rats.

This is a report investigating the methylglyoxal (MG) bypass in animals, by which D-lactate is produced from triosephosphate via MG. Rats were made diabetic using streptozotocin or starved for 72 h. D-Lactate and various metabolites related to it, such as L-lactate, pyruvate, methylglyoxal, glucose, and inorganic phosphate, were measured in the blood plasma, liver, and skeletal muscle of the rats. Diabetic and starved rats had significantly higher levels of D-lactate in plasma, liver, and skeletal muscle compared with the control group. In contrast, pyruvate levels in plasma, liver, and skeletal muscle was markedly lower than normal in diabetic and starved rats. L-Lactate level lowered markedly in plasma, liver, and skeletal muscle of starved rats and elevated in liver of diabetic rats. Differences between plasma L-lactate level for diabetes and control were not significant. MG level was significantly elevated in plasma and depressed in livers and muscles of starved rats as well as livers of diabetic rats. Hepatic glycerol content was markedly increased in those states. Enzyme activities related to D- and L-lactate, such as pyruvate kinase, phosphofructokinase, aldolase, and glyoxalase I, were measured in the livers of these rats. Pyruvate kinase activity decreased in these states, but other enzyme activities showed no significant changes. D-Lactate was much more excreted than L-lactate in the urine of diabetic and fasted rats compared with normal rats.

Animals↗
PubMed1992

Single photon emission computed tomography using 201Tl chloride in pulmonary nodules: comparison with 67Ga citrate and 99mTc-labeled hexamethylpropyleneamine-oxime.

A single photon emission computed tomography (SPECT) with 201Tl chloride (Tl-201) was carried out prospectively in 50 patients with pulmonary nodules and its diagnostic value was compared with those of 67Ga citrate (Ga-67) and 99mTc-labeled hexamethylpropyleneamine-oxime (Tc-99m-HMPAO). Tl-201 SPECT provided 88% (early)-91% (delayed) sensitivity, 85% (early and delayed) specificity and 87% (early)-89% (delayed) accuracy. The sensitivity of the Tl-201 planar image was 56 (early)-62% (delayed), which was significantly lower than that of SPECT. Delayed SPECT images at 2 hour postinjection were more preferable to disclose the malignant pulmonary nodule than early SPECT images at 15 minutes postinjection. The application of SPECT with Ga-67 failed to improve the sensitivity of planar imaging for malignant pulmonary nodules. Tc-99m-HMPAO was concentrated in 62% of 13 patients with malignant pulmonary nodules, which was slightly higher than Ga-67 in 54% of 28 patients. In an analysis of the histologic types of lung cancer, the sensitivity of Tl-201 was not significantly different in all types. On the other hand, Ga-67 was positive only in 25% of 12 patients with adenocarcinoma. A combination of SPECT and Tl-201 is the best choice among routine scintigraphic techniques for depicting malignant pulmonary nodules. The Tl-201 SPECT image may play a complementary role in the characterization of pulmonary nodules which are revealed on a plain radiograph and computed tomography.

Adult↗
PubMed1992

Effects of fasting, refeeding, and fasting with T3 administration on Na-K,ATPase in rat skeletal muscle.

It is known that Na-K,adenosine triphosphatase (ATPase) in cell membranes represents an important consumer of cellular energy, eg, adenosine triphosphate (ATP), and that the concentration and activity of this enzyme change in a dose-dependent manner with serum thyroid hormone levels. To examine the hypothesis that low triiodothyronine (T3) syndrome represents a cellular adaptation in generalized severe illnesses that saves tissue energy expenditure, we measured the muscle Na-K,ATPase concentration and its activity in rats that led to low T3 syndrome induced by fasting. The Na-K,ATPase concentration was measured by 3H-ouabain binding to soleus muscle, and its activity was measured by 42K uptake in the contralateral soleus muscle. The effects of refeeding or T3 administration on Na-K,ATPase in soleus muscle in fasted rats were also examined. Na-K,ATPase concentration and activity were both increased in hyperthyroid rats and decreased in hypothyroid rats. In the fasting state, they were decreased to as low as the levels seen in hypothyroidism. Furthermore, with fasting + refeeding or fasting + T3 administration, Na-K,ATPase in soleus muscle returned to the normal level. These results suggest that tissue energy expenditure, as assessed by Na-K,ATPase, in skeletal muscles of fasted rats with low T3 syndrome is actually decreased to levels seen in hypothyroidism, due at least partly to the decrease in serum T3 concentrations, and that there exist some adaptation mechanisms in the peripheral tissues for the accommodation of energy metabolism in the body through decreased thyroxine (T4) to T3 conversion.

Adenosine Triphosphate↗
PubMed1992

Effect of insulin on impaired antioxidant activities in aortic endothelial cells from diabetic rabbits.

The defense system of aortic endothelial cells against oxidative stress was studied in alloxan-induced diabetic rabbits, and the effect of insulin on the antioxidant activities was estimated. Endothelial cells were prepared from 10 diabetic rabbits, 18 diabetic rabbits treated with insulin, and 10 age-matched controls after 17 days of diabetes. These cells were used for the estimation of glutathione (GSH) levels and its related enzyme activities. The antioxidant activities in these endothelial cells from diabetic rabbits were compared with those from control subjects. The concentration of GSH decreased in diabetic rabbits (1.6 +/- 0.2 nmol/mg protein [mean +/- SD] v 3.7 +/- 0.6 nmol/mg protein). Decreases in the activities of Cu, Zn-superoxide dismutase (Cu,Zn-SOD) (62.7 +/- 11.0 U/mg protein v 172.9 +/- 20.2 U/mg protein), catalase (7.6 +/- 2.1 U/mg protein v 12.3 +/- 3.2 U/mg protein), and GSH peroxidase (134.0 +/- 27.0 mU/mg protein v 179.1 +/- 26.2 mU/mg protein) were observed. The activities of other GSH-related enzymes such as GSH S-transferase or GSH reductase did not change in endothelial cells from diabetic rabbits. Most of these antioxidant activities were prevented when diabetic rabbits were treated with insulin (1 to 2 U/kg/d). These antioxidant activities were also determined in the diabetic liver and kidney. Similar decreases in the cellular defense activities and prevention of the decrease in activities by insulin were observed in the diabetic liver, while these antioxidant enzyme activities in the kidney were resistant to diabetic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Genomic analysis of Theileria sergenti stocks in Japan with DNA probes.

Restriction fragment length polymorphisms of Theileria sergenti DNA from 18 different infections of cattle in 14 locations in Japan were analyzed by Southern blotting using T. sergenti genomic DNA fragments as probes. Probe pTs 2 hybridized with four fragments in BamHI digested piroplasm DNA, at 8.0, 7.3, 6.0 and 3.4 kb. Probe pTs 11-D1 hybridized with multiple fragments. With each probe, polymorphisms were observed among stocks from different locations. However, there was no correlation between the patterns of hybridization bands and the locations where parasites were collected. Analysis of the hybridization patterns of stocks obtained from individual cattle in the same grazing areas showed an almost identical pattern.

Animals↗
PubMed1992

Purification and characterization of formaldehyde dehydrogenase from rat liver cytosol.

Formaldehyde dehydrogenase was purified to electrophoretic and column chromatographic homogeneity from rat liver cytosolic fraction by a procedure which includes ammonium sulfate precipitation, DEAE-cellulose-, hydroxyapatite-, Mono Q-chromatography, and gel filtration. Its molecular mass was estimated to be 41 kDa by gel filtration and SDS-PAGE, suggesting that it is a monomer. It utilized neither methylglyoxal nor aldehydes except formaldehyde as a substrate. It has been reported that liver class III alcohol dehydrogenase and formaldehyde dehydrogenase are the same enzyme and oxidize formaldehyde and long chain primary alcohols. However, the enzyme examined here did not use n-octanoi as a substrate. The Km values for formaldehyde and NAD+ were 5.09 and 2.34 microM at 25 degrees C, respectively. The amino acid sequences of 10 peptides obtained from the purified enzyme after digestion with either V8 protease or lysyl endopeptidase were determined. From these results, the enzyme was proved to be different from the previously described mammalian formaldehyde dehydrogenase and is the first true formaldehyde dehydrogenase to be isolated from a mammalian source.

Aldehyde Oxidoreductases↗