Biomedical subjects
Publications and source records attributed to S Mizuno.
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A QS pattern in the right precordial leads of the electrocardiogram was noted in a 9-year-old asymptomatic boy. Echocardiography revealed paradoxical movement of the interventricular septum, which showed interstitial myocardial fibrosis in the biopsy specimen and a defective uptake of thallium-201. Extensive myocardial fibrosis was probably a sequela of an attack of myocarditis in infancy.
Parathyroid hormone (PTH) rapidly decreases in patients with thyroid cancers, even after autotransplantation following total thyroparathyroidectomy. Simultaneously, serum calcium (Ca) and erythrocytic Ca levels also decrease, and in some cases, tetanic symptoms caused by hypocalcemia may occur. Assuming that magnesium (Mg) might participate in the alteration of Ca and PTH levels, the postoperative changes in intracytoplasmic Mg levels of erythrocyte were determined for several days. In the present paper, red blood cells (RBC) were used as a substitute for general somatic cells and controls were taken from patients with breast cancers. Although the erythrocytic Mg levels of the patients with thyroid cancers showed almost no change until day 7, an apparent decrease was noted in the patients with breast cancers during the period from day 1 to day 7, with a significant difference being seen between the two groups. Assuming that the operative invasions were compatible between these two groups of patients, the difference may be explained by a rapid decrease of postoperative serum PTH in the patients with thyroid cancers.
We studied the effectiveness of herbimycin A, an inhibitor of the function of the temperature-sensitive src oncogene, to reverse the morphologies of chicken and mammalian cells transformed by various oncogenes. It was found that the antibiotic was effective against the cells transformed by tyrosine kinase oncogenes src, yes, fps, ros, abl, erbB, but did not reverse the transformed morphologies induced by oncogenes raf, ras, and myc. Moreover, decreases in phosphotyrosine content of the total cellular proteins and in 36K protein phosphorylation by herbimycin treatment supported the selective inhibition of the antibiotic on the transforming activity of tyrosine kinase oncogenes.
Three homologous series of alpha-mycolic acids (dicyclopropanoyl acids, monocyclopropanoyl monoenoic acids and dienoic acids) from 16 rapidly growing and 14 slowly growing mycobacteria were separated by argentation thin-layer chromatography and analysed by gas chromatography/mass spectrometry of their trimethylsilyl ether derivatives. Mycobacterial species were separated into five groups. Strains of group A contained similar amounts of even and odd carbon-numbered dienoic acids, with a methyl branch on the odd acids and a C24-alpha-unit, as typified by Mycobacterium fortuitum and M. chitae. Group B strains possessed similar amounts of even carbon-numbered dicyclopropanoyl alpha-mycolic acids and odd carbon-numbered unsaturated acids with C22- and C24-alpha-units, as found in M. phlei and M. diernhoferi. Group C strains contained mainly even carbon-numbered dicyclopropanoyl acids with C22- and C24-alpha-units, as shown by M. vaccae and M. aurum. Group D strains possessed mainly odd carbon-numbered dienoic acids with a methyl branch and a C24-alpha-unit, as seen in M. triviale and M. nonchromogenicum. Group E strains had mainly even carbon-numbered dicyclopropanoyl acids with C24- or C26-alpha-units, as found in M. avium and M. tuberculosis. Many rapidly growing mycobacteria also produced alpha'-mycolic acids which were shorter in the length of the main carbon chain but whose alpha-units were the same as those in alpha-mycolic acids from the same species. These alpha'-mycolic acids had either one or two double bonds and showed variations in both their unsaturation and overall size, which may be useful in taxonomic studies.
We investigated the effect of heat shock on the development of thermotolerance using mouse FM3A cells and the temperature-sensitive mutant ts85. The shift-up incubation of FM3A from 33 to 39.5 degrees induced thermotolerance to subsequent heating at 44 degrees. In contrast, the similar treatment of ts85 at the nonpermissive temperature of 39.5 degrees could not induce thermotolerance. Furthermore, when ts85 cells were treated at 33 degrees after being heated at 44 degrees, they developed a reduced level of thermotolerance as compared with that developed in FM3A cells. Since ts85 cells are defective in ubiquitin-activating enzyme, these results suggest a role of the ubiquitin-protein conjugation system in the development of thermotolerance.
The effect of chlorpromazine (0.5 and 4 mg/kg) on plasma catecholamines (adrenaline and noradrenaline) concentrations was investigated in rats. After an i.v. bolus administration of chlorpromazine, plasma adrenaline and noradrenaline concentrations showed a dose dependent increase. In order to clarify the pharmacokinetics of catecholamines in plasma, i.v. infusion of adrenaline or noradrenaline was also carried out. Plasma catecholamines after i.v. infusion showed typical characteristics of a one compartment model with a zero order production rate of endogenous catecholamines. From the data observed, a mathematical model was constructed to elucidate the relationship between the pharmacokinetics (brain concentrations) and the pharmacologic effect (plasma catecholamines concentrations) of chlorpromazine in rats. The results indicated that the time courses of plasma adrenaline and noradrenaline concentrations after an i.v. administration of chlorpromazine were described reasonably well by a simple pharmacokinetic-pharmacodynamic model.
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Genetic mechanisms involved in the kanamycin (KM)-hyper-resistance of Streptomyces griseus SS-1198PR and NP1-1PR generated by protoplast regeneration were investigated. Southern hybridization of Sph I-digested genomic DNA with a KM-resistance gene (kan) probe revealed that the strain SS-1198PR and its KM-sensitive parent (SS-1198) had the same copy number of a 4.2-kb Sph I fragment hybridizing to the kan probe, while the kan gene of the strain NP1-1PR was located on a highly amplified DNA sequence (100-200 copies/chromosome) consisting of the 6.7 kb amplifiable unit with Sph I site at the junction site region. There was no difference in the restriction endonuclease cleavage map of the kan gene region of the Sph I fragments from the three strains. However, the level (50 micrograms/ml) of KM-resistance conferred by the cloned NP1-1PR kan gene was much lower than that (1,000 micrograms/ml) conferred by the cloned SS-1198PR kan gene in Streptomyces lividans TK21 although the strain TK21 harboring these kan genes produced an aminoglycoside acetyltransferase, AAC(3)-V, with the same substrate specificity. It seemed, therefore, likely that a mutational alteration of the kan gene and a DNA amplification involving the kan region played major roles for the KM-hyper-resistance depending on AAC(3)-V in the strains SS-1198PR and NP1-1PR, respectively.
A mixed probe consisting of two synthetic deoxynucleotides (52 and 54 mers referred to as 50-mer) with arbitrarily chosen C or G for the third letters was prepared based on the amino acid sequences No. 31-48 and No. 72-90 of macromomycin (MCM) apoprotein and successfully used to clone the MCM apoprotein gene. Digestion with Sph I of total DNA of MCM-producing Streptomyces macromomyceticus M480-M1 yielded a 2.6-kb fragment that hybridized strongly to the probes. The hybridized probe was stable to washing with 3 x SSC at 75 degrees C. Radioactivity derived from the hybridized probe was comparable to that expected theoretically from hybridization between the probe and the true target sequence. The 2.6-kb fragment was cloned into Escherichia coli RR1 with pBR322 and subsequently subcloned into Streptomyces lividans TK21 with pIJ702. Nucleotide sequence analysis of the cloned fragment verified the existence of the sequence corresponding to the amino acid sequence of MCM apoprotein and about 90% homologies with the probes. Thus, the use of relatively long deoxynucleotide probes with arbitrarily chosen C or G for the third letters will be advantageous in cloning Streptomyces protein genes where more than 90% of the third letters have been known to be C or G. In addition, theoretical diagnosis of hybridization should be a great help to distinguish true positives from false ones.
The genetic and biochemical basis of a 200-fold increase in kanamycin (KM)-resistance shown in Streptomyces griseus SS-1198PR generated by protoplast regeneration was investigated. A 15-kb Bcl I-DNA segment responsible for the KM-resistance was cloned into pIJ61 with Streptomyces lividans TK21 as host. The KM-resistance segment was then subcloned into pIJ702 as a 1.8-kb BamH I-Bgl II fragment with a BamH I site essential for the KM-resistance. Both S. lividans TK21 containing the cloned segments and S. griseus SS-1198PR showed multiple resistance to KM, dibekacin and gentamicin C complex. Cell free extracts from these strains inactivated the antibiotics in the presence of acetyl CoA in agreement with their resistance pattern. The structure of the inactivated KM-A was determined as 3-N-acetyl-KM-A indicating acetylation by an aminoglycoside acetyltransferase, AAC(3). The substrate range of the enzyme was unique and was designated AAC(3)-V. No genetic linkage was found between the cloned 15 kb Bcl I segment and the separately cloned streptomycin resistance gene (str) segment (3.8 kb Sph I fragment). The str genes cloned from both the parent (SS-1198) and the strain SS-1198PR were identical in their size, restriction site and function. In addition, these strains showed no significant difference in the total DNA digestion pattern. These results indicate that protoplast regeneration may cause a critical change in a specific region of DNA resulting in a high activity of an AAC(3) with a novel substrate range.
Multivariate analysis by Cox's proportional hazard model was performed on 71 bladder cancer patients treated by radical cystectomy to clarify factors affecting survival and to evaluate the efficacy of postoperative adjuvant chemotherapy. Clinicopathologic data included in the analysis were age, sex, interval from onset of symptoms to first consultation, prior tumor history, year of operation and tumor characteristics: size, number, growth pattern, stage, grade, mode of infiltration, lymphatic invasion, lymph-node metastasis and lymphocyte infiltration around tumors. Analysis revealed that stage is the most statistically significant factor for survival (p less than 0.0001); followed by year of operation (p less than 0.05) and lymph node metastasis (p less than 0.1). The above three determinants yielded hazard ratios of 16.6 for stage (pT3a-pT4 vs. pTis-pT2), 2.7 for year of operation (1978-1982 vs. 1983-1987) and 2.4 for lymph node metastasis (positive vs. negative). Postoperative adjuvant chemotherapy consisting of cisplatin, peplomycin and mitomycin C (PPM therapy) seemed to improve survival, when the influence of the major prognostic factors were adjusted by the proportional hazards model. The authors conclude that stage and lymph node metastasis should be considered when postoperative adjuvant treatment is selected for patients with bladder cancer treated by radical cystectomy, and that further prospective randomized trials are mandatory to confirm the efficacy of postoperative adjuvant PPM therapy suggested by the present retrospective multivariate analysis.
Flow cytometric (FCM) determinations of DNA index were found to be insufficient to distinguish the presence of tumor cells from normal ones in neoplastic tissues obtained from 29 patients with lung cancer. Therefore, the DNA and tumor-associated antigen (TAA) contents of cultured human lung cancer cells were simultaneously analyzed using FCM to assess whether this dual technique would help in distinguishing tumor cells from normal ones. For the study, cells from PC-10 (a squamous cell carcinoma line), PC-3 (an adenocarcinoma line) and PC-6 (a small cell carcinoma line) were mixed with normal peripheral lymphocytes. The TAAs studied were carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCCA) and neuron-specific enolase (NSE). The alcohol-fixed cells were treated with the respective primary TAA, followed by fluorescein-isothiocyanate-conjugated secondary antibody; the cellular DNA was then stained using propidium iodide. Red and green fluorescences were measured simultaneously by FCM. The results showed CEA mainly in PC-3 cells, SCC in PC-10 cells and NSE in PC-6 cells; thus, each cell type had a relatively specific TAA. DNA content and cell size analyses differentiated neoplastic cells from normal lymphocytes for PC-3 and PC-10 cells, but not for PC-6 cells. Simultaneous FCM analyses of DNA and the TAA specific for the individual cell type made it possible to distinguish all tumor cell types from normal lymphocytes.
We determined by flow cytometry the proportion of cells in cycle with the monoclonal antibody Ki-67 and also in S-phase after incorporation of bromodeoxyuridine (BrdUrd) with monoclonal antibody to BrdUrd. The monoclonal antibody Ki-67 was useful to detect a nuclear antigen present only in proliferating cells but not expressed in resting (Go) cells. Cell preparation to measure BrdUrd amount incorporated into cellular DNA was difficult but this anti-BrdUrd antibody was useful for measuring the rate of DNA synthesis and for the analysis of precious cell kinetics. These antibodies may provide useful information of cell kinetics.
The relationship between alpha 1-acid glycoprotein (alpha 1-AG) and liver fibrosis was studied. Immunoperoxidase staining of liver specimens from patients with chronic hepatitis showed large amounts of alpha 1-AG to be located primarily in hepatocytes adjacent to areas of piecemeal necrosis, bridging fibrosis and fibrous septa. In patients with severe chronic active hepatitis, hepatocytes throughout the liver stained similarly to those adjacent to areas of piecemeal necrosis. In cell cultures of human embryonal lung (HEL) fibroblasts, addition to the culture medium of alpha 1-AG promoted cell growth. It is known that alpha 1-AG is also produced in the liver. It thus appears likely that alpha 1-AG is a promoter of hepatic fibrosis in chronic hepatitis.
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