Electrophysiologic and histopathologic evaluation of the transplanted rat heart.
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Animals↗
Biomedical subjects
M Sekiguchi
Publications and source records attributed to M Sekiguchi.
Crystallization and preliminary X-ray diffraction studies of 3-methyladenine-DNA glycosylase II from Escherichia coli.
Single crystals of 3-methyladenine-DNA glycosylase II produced from the alkA gene of Escherichia coli have been obtained by the method of vapour diffusion with polyethylene glycol 6000 as precipitant at pH 8.5. The crystals belong to the monoclinic space group C2, with a = 58.6 A, b = 76.8 A, c = 62.2 A, beta = 110.3 degrees. They contain one molecule per asymmetric unit and diffract to 2.7 A resolution.
Crystallization↗
Proteolytic cleavage of Ada protein that carries methyltransferase and transcriptional regulator activities.
The 39-kDa Ada protein that carries two distinct methyltransferase activities and an activity to promote transcription of the ada and alkA genes is cleaved to smaller polypeptides in the case of incubation with a crude extract of Escherichia coli. A protease that specifically cleaves the Ada protein was partially purified and characterized. The enzyme showed maximal activity at pH 7.8-8.0 and did not require specific ions or ATP for the reaction. The activity was inhibited by p-chloromercuribenzoic acid, thereby suggesting that it is a thiol protease. When a purified preparation of Ada protein was incubated with the protease preparation, definite sizes of cleavage products were formed; at the initial stage of proteolysis, the 20- and 19-kDa proteins were formed as the major products. When each of these products was purified by successive column chromatography, the 20-kDa protein was found to catalyze transfer of a methyl group from methylphosphotriester of methylated polynucleotides, whereas the 19-kDa protein transfers a methyl group from O6-methylguanine of methylated DNA to each of the molecules. Neither the 20- nor 19-kDa protein, even after methyl acceptance, exhibited an activity to promote transcription of the ada gene. However, alkA transcription was promoted by the 20-kDa protein provided that it was methylated.
Animals↗
Activation of Ada protein as a transcriptional regulator by direct alkylation with methylating agents.
The adaptive response is a cellular process to induce DNA repair enzymes in response to a challenge of alkylating agents. In this process Ada protein, the product of the ada gene, plays a major role; it accepts the methyl groups of the methylated DNA at the cysteine residues of its own molecule, and the methylated form of Ada protein promotes transcription of its own gene, thereby triggering induction of the whole process. In addition to this DNA-mediated activation of Ada protein, we have proposed an alternative mechanism which involves direct methylation of Ada protein by methylating agents (Takahashi, K., and Kawazoe, Y. (1987) Biochem. Biophys. Res. Commun. 144, 447-453). Here we present evidence that Ada protein is activated as a transcriptional regulator through a direct methylation by certain methylating agents. A purified preparation of Ada protein was treated with various methylating agents, and the activity to promote transcription of the ada gene was determined using an in vitro reconstituted system. The ada-specific RNA was produced when the Ada protein treated with methyl methanesulfonate or with methyl iodide was present in the reaction mixture. The Ada protein treated with N-methyl-N-nitrosourea did not show such an activity. It is, therefore, suggested that the adaptive response induced by chemoselective methylating agents such as methyl iodide might be due, at least in part, to the direct methylation of the constitutive Ada protein present in the cell.
Alkylation↗
Expression of the ada gene of Escherichia coli in response to alkylating agents. Identification of transcriptional regulatory elements.
Ada protein plays a central role in the regulatory synthesis of DNA repair enzymes, following exposure of Escherichia coli to alkylating agents. Methyl groups of alkylated DNA are transferred to Ada protein by its own methyltransferase activity and the methylated Ada protein then acts as a positive regulator to overproduce the ada and related gene products. To elucidate regulatory mechanisms for the expression of the ada gene by its own product, we analyzed the ada promoter region by random and site-directed mutagenesis. A series of deletion analyses revealed that a sequence up to 53 nucleotides upstream from the transcription initiation site is required for the controlled expression of the ada gene. Libraries of base substitution mutants were constructed by synthesizing oligonucleotides corresponding to the ada promoter region in the presence of a small amount of all possible sets of nucleotides. Internal deletion and insertion mutants were also constructed with the use of synthetic oligonucleotides. Using these mutants, the -10 and the -35 boxes of the promoter as well as the ada regulatory sequence were identified, the latter being an eight-nucleotide sequence, AAAGCGCA. A six-nucleotide stretch between the regulatory sequence and the -35 box, also affected levels of expression of the gene. When the promoter DNAs derived from wild type or base substitution mutants that showed normal expression in vivo were used as templates for transcription in vitro, the ada-specific RNA was formed in the presence of a methylated form of Ada protein. With the DNAs derived from mutants of defective type as templates, no or relatively small amounts of the RNA were synthesized. Some base substitution mutants showed a constitutive expression of the gene in vivo, but this observation did not reconcile with findings in experiments in vitro.
Alkylating Agents↗
Functional sites of the Ada regulatory protein of Escherichia coli. Analysis by amino acid substitutions.
Specific cysteine residues at possible methyl acceptor sites of the Ada protein of Escherichia coli were converted to other amino acids by site-directed mutagenesis of the cloned ada gene of E. coli. Ada protein with the cysteine residue at 321 replaced by alanine was capable of accepting the methyl group from the methylphosphotriester but not from O6-methylguanine or O4-methylthymine of alkylated DNA, whereas the protein with alanine at position 69 accepted the methyl group from the methylated bases but not from the methylphosphotriester. These two mutants were used to elucidate the biological significance of repair of the two types of alkylation lesions. Introduction of the ada gene with the Ala69 mutation into an ada- cell rendered the cell more resistant to alkylating agents with respect to both killing and induction of mutations, but the gene with the Ala321 mutation exhibited no such activity. Replacement of the cysteine residue at position 69, but not at position 321, abolished the ability of Ada protein to promote transcription of both ada and alkA genes in vitro. These results are compatible with the idea that methylation of the cysteine residue at position 69 renders Ada protein active as a transcriptional regulator, whilst the cysteine residue at position 321 is responsible for repair of pre-mutagenic and lethal lesions in DNA. The actions of mutant Ada proteins on the ada and alkA promoters in vivo were investigated using an artificially composed gene expression system. When the ada gene with the Ala69 mutation was introduced into the cell, there was little induction of expression of either the ada or the alkA genes, even after treatment with an alkylating agent, in agreement with the data obtained from studies in vitro. With the Ala321 mutation, however, a considerable degree of ada gene expression occurred without adaptive treatment. The latter finding suggests that the cysteine residue at position 321, which is located near the C terminus of the Ada protein, is involved in regulating activity, as the transcriptional activator.
Alanine↗
Immunohistocytochemical localization of alpha human atrial natriuretic peptide in human atrium obtained from endomyocardial biopsy and cardiac surgery.
To demonstrate the localization of atrial natriuretic peptide (ANP), we were able to apply the indirect and avidin-biotin peroxidase complex (ABC) methods for immunohistocytochemical analysis on sections with routine fixation and embedding for conventional histopathological and ultrastructural examinations. Human right and/or left atrial myocardial specimens were obtained at cardiac surgery (n = 12) or by right atrial endomyocardial biopsy, employing Konno's catheter bioptome (n = 30). In both specimens, ANP-like immunoreactivity was observed mainly around the nuclei and in specific granules in atrial myocytes. These new techniques are, therefore, considered to be useful for clinical analysis employing minute human atrial specimens in various heart diseases.
Atrial Natriuretic Factor↗
Structural requirement for the induction of the adaptive response in Escherichia coli among N-(substituted alkyl)-N-nitrosoureas.
Using E. coli CSH26 transformed with a plasmid carrying an alkA'-lacZ' fused gene, a series of N-(substituted alkyl)-N-nitrosoureas were subjected to a colorimetric assay to evaluate their capacity to induce the adaptive response, an inducible DNA-repair network in E. coli. Some of these derivatives induced the response in greater or lesser degrees, while others did not. Several structural requirements for the induction were disclosed. The capacity of these derivatives to induce the SOS response, which is another inducible DNA-repair network, was also evaluated using E. coli transformed with a plasmid carrying a umuC'-lacZ' fused gene. Since all the derivatives induced the SOS response, the structural requirements for the adaptive response disclosed in this study are substantially related to the molecular mechanism involved in the adaptive response.
Alkylating Agents↗
Production of beta-human chorionic gonadotropin by germ cell tumors in vivo and in vitro.
Production of the beta-subunit of human chorionic gonadotropin (beta-hCG) by human germ cell tumors was studied in surgical specimens, cultured cells and xenografted tumors. beta-hCG was identified most frequently in the syncytiotrophoblastic and intermediate trophoblastic components of choriocarcinoma, and it was also detected in syncytiotrophoblastic giant cells, occasionally in association with embryonal carcinoma, and less frequently with yolk sac tumor, seminoma and dysgerminoma. Our experimental data suggest that beta-hCG production by syncytiotrophoblastic giant cells in association with embryonal carcinoma is a result of trophoblastic differentiation of embryonal carcinoma. Moreover, it was proved that the production of beta-hCG by human embryonal carcinomas is regulated by more factors than is the case for human choriocarcinomas.
Adult↗
Cloning and sequence of the gene encoding the major structural component of mannose-resistant fimbriae of Serratia marcescens.
Serratia marcescens US46, a human urinary tract isolate, exhibits mannose-resistant hemagglutination and agglutinates yeast cells, thereby indicating that it has two types of adhesins. We constructed a cosmid library for the DNA of this organism and isolated DNA clones carrying genes for mannose-sensitive (MS) and mannose-resistant (MR) fimbriae. On introduction of the cloned genes into Escherichia coli K-12, MS and MR fimbriae were formed. These fimbriae were functionally and morphologically indistinguishable from those of S. marcescens. Subcloning of these gene clusters revealed that the genes encoding MS fimbriae reside on a 9-kilobase (kb) DNA fragment, while those encoding MR fimbriae are present on a 12-kb fragment. Transposon insertion and maxicell analyses revealed that formation of MR fimbriae is controlled by several genes which reside on the 9-kb fragment. The nucleotide sequence of smfA, the gene encoding the major structural component of MR fimbriae, revealed that this gene encodes a 174-amino-acid polypeptide with a typical procaryotic signal peptide. The primary structure of the smfA product showed significant homology with the primary structure of the E. coli fimbrial subunit.
Amino Acid Sequence↗
Regulation and expression of the adaptive response to alkylating agents.
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Adaptation, Biological↗
An integrated study employing histopathological, immunohistocytochemical and radioimmunoassay analyses of atrial natriuretic peptide in the right and left atria in patients with mitral valve disease.
To clarify the production mechanism of atrial natriuretic peptide (ANP) in right (RA) and left atria (LA) in mitral valve disease, histopathological and immunohistocytochemical analyses were performed and ANP levels were investigated by radioimmunoassay (RIA) in 28 patients. Atrial tissues were obtained during mitral valve replacement. ANP-like immunoreactivity of the myocytes applied by the avidin-biotin peroxidase complex method was observed around the nuclei of the atrial myocytes. Electronmicroscopically, immunoreactivity was observed in atrial specific granules. Light-microscopically determined intensity of the immunoreactivity was classified into 4 grades and the intensity in 100 myocytes was expressed by adding the scores of each myocyte. Mean right atrial pressure was positively correlated with the activity score in RA (r = 0.80). Pulmonary capillary wedge pressure was not correlated with the score in LA. The score in RA was significantly higher than that in LA. The ANP level in RA investigated by RIA was also higher than that in LA. Histopathological findings such as myocyte hypertrophy, degeneration and interstitial fibrosis were more severe in LA than in RA. In conclusion, longstanding atrial overloading, especially in LA, caused severe pathological damage, resulting in a smaller production of ANP. Much more ANP may be produced from RA in long-standing mitral valve disease.
Adult↗
DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness) syndrome associated with myocardial disease.
A 30 year old man with DIDMOAD (diabetes insipidus, diabetes mellitus, optic atrophy and deafness) syndrome associated with myocardial disease is reported. Echocardiographic study revealed a marked symmetric left ventricular hypertrophy. Histology of the endomyocardial biopsy specimen from the right ventricle showed severe glycogen deposition in the myocytes. This case may indicate that DIDMOAD syndrome is a hereditary systemic disease affecting multiple organs, including the myocardium.
Adult↗
Pharmacokinetics of nilvadipine, a new dihydropyridine calcium antagonist, in mice, rats, rabbits and dogs.
1. The pharmacokinetics of nilvadipine in male and female rats, and in male mice, rabbits and dogs were studied after i.v. and oral dosing. 2. After i.v. dosing (0.1 mg/kg), the plasma concentrations of nilvadipine declined two- or three-exponential with terminal half-lives of 0.73 h in mice, 1.2 h in male and female rats, 3.7 h in rabbits and 5.0 h in dogs. Sex difference in pharmacokinetics after i.v. dosing in rats was not found. The systemic plasma clearance was in the order of mice greater than rats greater than rabbits greater than dogs, and nearly equalled the hepatic blood flow in each species. The volume of distribution at steady-state was high (greater than 4 L/kg) in all species. 3. After oral dosing, plasma concentrations of nilvadipine peaked within 1 h in all species except for middle and higher doses (4 and 16 mg/kg) in dogs. The area under the plasma concentration-time curves in male rats (3.2-100 mg/kg) and dogs (1-16 mg/kg) increased in proportion to the dose. Bioavailability was low in male rats (3-4%) and rabbits (2%), but in other species was 29-44%. The oral clearance in male rats was about 8 times higher than in female rats. 4. The free fraction of nilvadipine in plasma was 1.94% in mice, 1.89% in rabbits and 0.85% in dogs, with no dependence on plasma concentration over a range of 10-100 ng/ml.
Animals↗
Effects of repeated exposure to cyclophosphamide on drug resistance and biological properties of a newly autonomy-acquired mouse mammary tumor (T4-OI320).
T4-OI320 tumors, the autonomous but estrogen receptor (ER-)-positive subline recently established from the pregnancy-dependent TPDMT-4 mouse mammary tumor, were characterized by moderate sensitivity to 1 mg cyclophosphamide (CY) and high sensitivity to 20 micrograms mitomycin C (MMC) and 1.5 mg 5-fluorouracil (5FU) twice weekly, the modal chromosome number of 40, the mean ER level of 20.8 fmol/mg protein, and a population of cells with one exogenous mouse mammary tumor virus (MMTV) genome. Serial tumor passages under CY treatment were conducted to investigate the effects of continued chemotherapy on growth habit, drug resistance, ER, karyotype, proviral MMTV genome and 22 oncogenes including int-1 and int-2. The growth rate was 2.9- and 4.5-fold increased after 10 and 20 passages, respectively. Along with this, the tumors acquired greater or complete resistance to CY and MMC similar to each other in antitumor mechanism, but maintained similarly high sensitivity to 5FU different from CY in antitumor mechanism. The ER level remained the same as but declined to half of the initial level after 10 and 20 passages, respectively. Tumor cells became more heterogenous in karyotype and the proportion of hyperdiploid cells with 41 to 47 chromosomes increased, resulting in shift of the modal number to 42 after 20 passages: Cells with 42 chromosomes accounted for 41% of the population. The exogenous MMTV bands in the Southern blotting became more intense after CY treatment accompanying neither amplification nor rearrangement of any cellular oncogenes. The tumors transplanted under no treatment for 20 generations had 1.5-fold higher growth rate, slightly stronger resistance to CY and MMC, similarly high sensitivity to 5FU, 4-fold lower ER level, the same distribution of chromosome numbers compared to the original tumors and similar intensity of the exogenous MMTV bands. Thus, acquisition of higher growth potential and greater drug resistance in the course of CY treatment appeared to be associated with chromosomal changes and selective overgrowth of the particular cell population with exogenous MMTV information in this model.
Animals↗
[Application of logistic curve fitting to cell-mediated cytotoxicity tests].
Blood mononuclear cells from 6 healthy adults were examined for LAK activity against 16 cell lines derived from human malignant tumors. The cytotoxicity curves obtained from a linear plot of the log of E/T ratio versus the percentage of the target cells killed were found to well fit the logistic curves. As the logistic distribution is known to approximate to the normal distribution, and the K/2 value is the function value for the representative, the K/2 value is reasonably selected as the % lysis value for the lytic unit calculation. Values of the three parameters, K as the maximum cytotoxicity, A as the scale parameter and B as the shape parameter, were analyzed using the experimental data. Eleven targets had three or more significantly fitted curves. Six among them were selected as highly sensitive cell lines to LAK activity, because the average values for K were higher than 80% (coefficients of variation were from 0.02 to 0.11) and analyzed further. The average values for B were relatively constant (range: 1.09-1.56) and the average values for A varied from 3.5 to 20.3 (conefficients of variation were from 0.26 to 0.71). The shape of the curves were nearly the same. These results indicate that the 6 cell lines are useful as the targets for the LAK activity.
Cytotoxicity Tests, Immunologic↗
Abdominal surgery in patients over 80 years of age.
Out of 179 surgical admissions in 1987, 8 (4.5%) were over 80 years old; of these, 7 had malignant disease. Careful clinical assessment indicated that the benefits of operation outweighed the surgical risks. Surgery was performed under general anesthesia. All 8 patients recovered, despite numerous complications, and were symptom- free up to 11 months after treatment. Provided that such patients are in the care of an experienced, skilled surgical team, major operative procedures should be carried out more frequently in the elderly, especially when the patient anticipates a successful outcome.
Abdomen↗
Subacute toxicity of muroctasin in mice and dogs.
The subacute toxicity of N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine (MDP-Lys (L18), muroctasin), a synthetic muramyl dipeptide, was investigated in mice and dogs. Both species exhibited leukocytosis, immunologically activated appearances of the axillary lymphnode and splenic white pulp, and irritatic lesions at the injection site, when treated with MDP-Lys(L18) for 4 weeks. Anemia, and synovitis were also found in dogs. These findings reflected main pharmacological action of MDP-Lys(L18), though there was some species difference.
Acetylmuramyl-Alanyl-Isoglutamine↗