Analysis of the essential sequence elements in the peroxisome proliferator-responsive enhancer of rat acyl-CoA oxidase gene.
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Acyl-CoA Oxidase↗
Biomedical subjects
T Tsukamoto
Publications and source records attributed to T Tsukamoto.
Confronting the minimal supersymmetric standard model with the study of scalar leptons at future linear e+e- colliders.
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Journal Article↗
Synthesis and biological evaluation of N alpha-(4-amino-4-deoxy-10-methylpteroyl)-DL-4,4-difluoroornithine.
N alpha-(4-Amino-4-deoxy-10-methylpteroyl)-DL-4,4-difluoroornithi ne (AMPte-DL-4,4-F2Orn, 4) was synthesized and evaluated as an inhibitor of human folypoly-gamma-glutamate synthetase (FPGS), dihydrofolate reductase (DHFR), and cell growth. Synthesis of 4 involved the use of a protected form of DL-4,4-difluoroornithine 9 which was derived from DL-4,4-difluoroglutamic acid. Biological activities of 4 were compared directly to those of the corresponding nonfluorinated compound N alpha-(4-amino-4-deoxy-10-methylpteroyl)-L-ornithine (AMPte-L-Orn, 3). Although the fluorinated analogue is a potent inhibitor of DHFR, it is a poor inhibitor of FPGS. However, the compound is transported across the cell membrane and inhibits cell growth, presumably due to the inhibition of DHFR. The data obtained with the fluorinated analogue are in contrast to those of the corresponding nonfluorinated compound 3, which is a potent inhibitor of both FPGS and DHFR but shows very low cytotoxicity due to poor transport.
Biological Transport↗
N-Ethyl-N-nitrosourea induces mammary cancers in the pituitary-isografted mouse which are histologically and genotypically distinct from those induced by N-methyl-N-nitrosourea.
N-Ethyl-N-nitrosourea (ENU) and N-methyl-N-nitrosourea (MNU) are alkylating agents which respectively ethylate or methylate nucleophilic centers in the cell such as DNA. In vitro studies with naked DNA and bacterial mutagenesis assays suggest that these two compounds induce different spectra of genetic lesions. In addition, the ethyl-DNA adducts induced by ENU persist longer than the methyl-DNA adducts induced by MNU. Since MNU is a known mammary carcinogen in the pituitary-isografted mouse, these data suggest that ENU may be an even more potent carcinogen than MNU. The purpose of this study was to determine whether ENU was a mammary carcinogen in the pituitary-isografted mouse and if so, to compare the genotype and phenotype of ENU-induced mammary tumors with those induced by MNU. Fifteen adult female virgin BALB/c mice were isografted with two pituitaries and subsequently treated with a single intravenous injection of ENU (50 micrograms/g body weight). Mammary adenocarcinomas arose in all of the survivors (n=12) with a median latency of 27 weeks and a mean frequency of 1.4 cancers per mouse. When tumor DNA was analyzed for mutations in the 12th and 61st codons of c-Ki-ras or c-Ha-ras protooncogenes, only wild type sequences were found. This is in contrast to MNU which causes a G to A transition mutation in the 12th codon of the c-Ha-ras proto-oncogene in about one of five mammary cancers induced in pituitary-isografted mice. Furthermore, the ENU-induced tumors were solid viable papillary adenocarcinomas, whereas MNU induced tumors are highly necrotic adenocarcinomas with squamous metaplasia. These results demonstrate that, in the pituitary-isografted mouse, ENU is as potent a mammary carcinogen as MNU and suggest that oncogenes other than c-Ki-ras or c-Ha-ras may be involved in ENU-induced mammary cancers.
Animals↗
Synthesis and biological evaluation of DL-4,4-difluoroglutamic acid and DL-gamma,gamma-difluoromethotrexate.
DL-4,4-Difluoroglutamic acid (DL-4,4-F2Glu) and its methotrexate analogue, DL-gamma,gamma-difluoromethotrexate (DL-gamma,gamma-F2MTX), were synthesized and evaluated as alternate substrates or inhibitors of folate-dependent enzymes. Synthesis of DL-4,4-F2Glu involved the nitroaldol reaction of ethyl nitroacetate with a difluorinated aldehyde ethyl hemiacetal as a key step. Attempted ligation of DL-4,4-F2Glu to methotrexate (MTX), catalyzed by human folylpoly-gamma-glutamate synthetase (FPGS), revealed that DL-4,4-F2Glu is a poor alternate substrate. DL-gamma,gamma-F2MTX was synthesized by a route proceeding through N-[4-(methylamino)benzoyl]-4,4-difluoroglutamic acid di-tert-butyl ester followed by alkylation with 6-(bromomethyl)-2,4-pteridinediamine hydrobromide. DL-gamma,gamma-F2MTX was found to be neither a substrate nor an inhibitor of human FPGS. The fluorinated analogue of MTX, however, inhibits DHFR and cell growth with the same potency as MTX.
Aminopterin↗
Exploitation of folate and antifolate polyglutamylation to achieve selective anticancer chemotherapy.
Synthesis of poly(gamma-glutamate) metabolites of natural folates and antifolates is a critical process. Folypolyglutamates are essential for cell proliferation. Polyglutamates of glutamate (Glu)-containing antifolates are often critical for their cytotoxic action and are relevant to antifolate resistance. However, the role of polyglutamate synthesis in selectivity is less clear. We have undertaken a research program to further define the significance of polyglutamate metabolism and to devise ways to exploit this metabolism to achieve greater therapeutic selectivity in cancer chemotherapy. This article briefly reviews several approaches tested thus far. Inhibition of folypolyglutamate synthesis should lead to cell death. Current ornithine (Orn)-containing folate-based inhibitors of the enzyme responsible for their synthesis, folypolyglutamate synthetase (FPGS), are poorly transported, apparently because of interference by the protonated delta-amine. Replacement of Orn with 4, 4-difluoroOrn, the delta-amine of which has a much lower pKa and is thus less protonated at physiological pH, was explored. Since it is unclear how polyglutamylation contributes to selectivity, we explored generic means either to eliminate or to enhance polyglutamylation. The data indicate that substitution for Glu in an antifolate by some Glu analogs in which the gamma-COOH is either altered or replaced (e.g., gamma-tetrazole-Glu) leads to loss of both FPGS substrate activity and binding; antifolate target specificity is unchanged, while uptake is actually enhanced. Substitution of 3,3-difluoroGlu for Glu leads to enhanced polyglutamylation (although probably only to the diglutamate), retention of target specificity, and at least equal uptake. Comparative studies of the same antifolate containing different replacements for Glu, such as gamma-tetrazole-Glu (no polyglutamylation) or 3,3-difluoroGlu (enhanced polyglutamylation), will be useful in exploring the role and significance of polyglutamylation.
Antineoplastic Agents↗
Positive and negative host factors for Sendai virus transcription and their organ distribution in rat.
In vitro mRNA synthesis by Sendai virus is almost entirely dependent on the addition of cellular proteins (positive host factors), one of which could be tubulin. In this study, we investigated the distribution of host factors in various rat organs. Extracts from the brain, thymus, heart, lung, testis, ovary, and uterus all supported in vitro Sendai virus transcription, among which the highest activity was obtained with the brain extract. On the other hand, little or no activity was detected in the liver, spleen, and kidney extracts. An inverse correlation between the apparent host factor activity to stimulate mRNA synthesis and RNase activity that hydrolyzes Sendai virus mRNAs was found, except in the liver extract. However, when a transcription initiation complex was isolated and subjected to RNA chain elongation reaction, all of the extracts including those from liver, spleen and kidney, were active. Immunoblotting showed that tubulin molecules were integrated in these initiation complexes, supporting the notion that tubulin is involved in the initiation complex formation. We also identified a transcription inhibitory activity without any detectable RNase activity in the liver extract. This negative host factor seemed to act on RNA chain elongation. It is likely that Sendai virus transcription is regulated by both positive and negative regulatory factors.
Animals↗
Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats.
This report deals with the mechanism involved in the cerebellar degeneration following experimental methylmercury poisoning of male Wistar rats. The cerebellar granule cells of animals that exhibited typical hind leg paresis were shrunken and displayed marked nuclear pyknosis. At the ultrastructural level, the nuclei of these cells were condensed and fragmented, features which are characteristic of apoptosis. In situ staining for DNA strand breaks revealed that the pyknotic nuclei were positively labeled. DNA fragmentation was confirmed by agarose gel electrophoresis; a ladder pattern of multiples of approximately 200-base pair fragments, typical of apoptosis, was observed with the cerebellar DNA of the methylmercury-treated animals. These observations suggest that the degeneration of cerebellar granule cells by alkyl mercury compounds involves an apoptotic process.
Animals↗
A unified nomenclature for peroxisome biogenesis factors.
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Animals↗
Cellular basis for protein C deficiency caused by a single amino acid substitution at Arg15 in the gamma-carboxyglutamic acid domain.
Protein C is a zymogen of an anticoagulant vitamin K-dependent serine protease. Inherited protein C deficiency is often associated with a high risk for venous thromboembolism. It is characteristic of protein C deficiency that most single amino acid replacements result in type I (secretion defect) deficiency. To determine the molecular and cellular bases of protein C deficiency, we expressed recombinant human protein C mutants in which Arg15 was mutated to either Gly, Trp, Gln, Leu, or Pro by a single base exchange. Arg15 is one of the conservative residues in the gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent coagulation factors, and is also one of the high frequency multiple mutation sites in protein C deficiency. In transient expression studies using human kidney 293 cells, the relative amounts of Arg15 mutants secreted into the medium and determined by enzyme-linked immunosorbent assay (ELISA) were as follows: Gly, 42%; Trp, 14%; Gln, 54%; Leu, 22%; and Pro, 13%, the amount of wild-type (Wt) protein C being taken as 100%. Thus, the order of the secreted amounts of the recombinant mutants was determined to be Wt > Gln > Gly > Leu > Trp, Pro. Pulse-chase experiments using both transiently-transfected and a pool of stably-transfected 293 cells, and stably-transfected BHK cells showed the same order of secretion efficiency. Since this order correlated well with that of the hydrophobicity scale of amino acid side chains, a conformational alteration of the Gla domain resulting in impaired secretion may be dependent on the hydrophobicity of the replaced amino acid. In transient cells, the relative radioactivities of pulse-labeled bands of all recombinant protein C were almost equal, suggesting that the same translational efficiency for Wt and all Arg15 mutants. All of the Arg15-mutated protein C precursors were shown to be located in the same organelle as protein disulfide isomerase (PDI), an endoplasmic reticulum-resident protein, and were sensitive to endoglycosidase H digestion. These results suggest that mutations of the highly conserved Arg15 in the Gla domain of protein C caused a secretion defect to variable degrees depending on replaced amino acid residue.
Amino Acid Sequence↗
Synchronization of a chaotic laser pulsation with its prerecorded history.
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Journal Article↗
Typing of verotoxins by DNA colony hybridization with poly- and oligonucleotide probes, a bead-enzyme-linked immunosorbent assay, and polymerase chain reaction.
To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.
Animals↗
Estimate criteria for diagnosis and severity in benign prostatic hyperplasia.
BACKGROUND: Standard severity criteria for benign prostatic hyperplasia (BPH) are needed to facilitate clinical and epidemiologic investigations in the common terminology. PATIENTS AND METHODS: A total of 692 BPH patients from 8 institutions were judged by specialized physicians for their overall disease severity and severity in 4 domains: symptom, function, anatomy, and quality of life (QOL). The items measuring severity in these 4 domains were the International Prostate Symptom Score (I-PSS) questionnaire, uroflowmetry, transrectal ultrasonography (TRUS), and questionnaires on bother and I-PSS QOL index, respectively. By Spearman's rank correlation and multiple regression analysis using physicians' judgement as the criterion variable (gold standard) the items and their cut-off levels were selected so that high predictivity, practicality and clinical significance were maintained, while the number of items in use was minimized. RESULTS: The item(s) and their ranges of values for each severity grade were summarized as follows; the evaluation domain (the selected item(s): range of mild, moderate, severe): symptom (I-PSS: 0-7, 8-19, 20-35), function (maximum flow rate and residual urine volume: > or = 15 mL/s and < 50 mL, > or = 5 mL/s and < 100 mL, < 5 mL/s or > or = 100 mL), anatomy (prostate volume on TRUS: < 20 mL, < 50 mL, > or = 50 mL), QOL (QOL index: 0 and 1, 2 to 4, 5 and 6). The overall severity was efficiently predicted when defined according to the combinations of severity grades of the 4 domains. These criteria predicted physicians' judgement with accuracy of about 80% for overall severity and severity of individual domains. CONCLUSION: The proposed criteria are valid, practical, and would be useful for the common scale of the BPH severity in clinical and epidemiologic studies.
Humans↗
Estimate criteria for efficacy of treatment in benign prostatic hyperplasia.
BACKGROUND: Various treatment modalities for benign prostatic hyperplasia (BPH) have emerged and are now in use or await evaluation of clinical usefulness. It is difficult, however, to compare their efficacies on a single scale, because standardized criteria for therapeutic efficacy of BPH treatments have not been established. PATIENTS AND METHODS: A total of 692 BPH patients from 8 institutions in Japan received various treatments, and were judged by specialized physicians for overall efficacy, and for efficacy in 4 domains: symptom, function, anatomy, and quality of life (QOL). Efficacy of treatment was graded as excellent, good, fair or poor, and assessed using items based on conventional clinical measurements. These items included 1) the difference (post-pretreatment value), 2) relative ratio (post/pre) and, 3) the individual values of pre- or posttreatment measurements. The cut off levels for each grade were heuristically selected by Spearman's rank correlation and multiple regression analysis so that the results accurately predicted physicians' judgement, while the feasibility was maintained. RESULTS: The results for each efficacy grade (range of excellent, range of good, range of fair, range of poor) were summarized as follows: Symptom: (post/pre treatment ratio of I-PSS) < or = 0.25, < or = 0.5, < or = 0.75, > 0.75. Function: (post-pre of Qmax) > or = 10 mL/s, > or = 5 mL/s, > or = 0.25 mL/s, < 0.25 mL/s. Anatomy: (post/pre ratio of prostate volume) < or = 0.5, < or = 0.75, < or = 0.9, > 0.9. QOL: (pre-post of QOL index) > or = 4, 3, 2 and 1, < or = 0. The overall efficacy grade was defined as the median of efficacy grades of 3 domains: symptom, function and QOL. The agreement rates between the criteria and physicians' judgement on the dichotomous efficacy (either excellent plus good, or fair plus poor) were approximately 80% in individual domains and overall estimate, and consistent among various treatments. CONCLUSION: The proposed criteria are fairly accurate, simple, and practical, and thus may be useful as a standard method for assessing the clinical efficacy of BPH treatments.
Evaluation Studies as Topic↗
Bilateral deoxycorticosterone-secreting adrenocortical adenoma.
A case of a 58-year-old man with bilateral deoxycorticosterone (DOC)-secreting adrenocortical adenoma is reported. Before surgery, plasma levels of DOC and corticosterone were markedly elevated, but both adrenal hormone levels normalized after the surgical removal of the bilateral adrenal tumors. The histologic examination revealed bilateral adrenocortical adenoma, but curiously, the tissue concentrations of DOC and corticosterone were elevated only in the right adrenal gland.
Adrenal Cortex Neoplasms↗
[PCR methods for detection of enteropathogenic Escherichia coli (localized adherence) and enteroaggregative Escherichia coli].
A PCR method was developed to detect enteropathogenic Escherichia coli (localized adherence) and enteroaggregative Escherichia coli. A portion of the structural gene (bfpA) encoding bundle-forming pilus (BFP), was amplified by the PCR for the detection of enteropathogenic Escherichia coli. It proved to be sensitive and specific in detecting enteropathogenic Escherichia coli. For the detection of enteroaggregative Escherichia coli, a portion of aggR (a transcriptional activator of aggA coding a major fimbrial subunit) was amplified. Although few strains showing non-aggregative adherence gave positive result, the PCR method was shown to have greater sensitivity than the probe assay in detecting enteroaggregative Escherichia coli.
Bacterial Adhesion↗
Persistent bacteriuria caused by uropathogenic Escherichia coli.
The predominant strain of Escherichia coli, the most frequently associated causative agent of urinary tract infections (UTI), in the rectal flora of patients with UTI has been reported to match the strain associated with the etiologic agent of UTI. Since acquisition of UTI may depend on the immediate surrounding flora of the patient, the chromosomal DNAs of strains of E. coli isolated from 3 patients suffering from recurrent cystitis and asymptomatic bacteriuria were examined by multiple polymerase chain reaction using 6 sets of primers for all the known urovirulence factors and by pulsed-filed gel electrophoresis. Genetically identical or similar strains were continuously isolated from these patients and each strain carried the identical urovirulence factors. The findings strongly supported the previous notion that the acquisition of UTI is dependent upon the occurrence of the strain carrying the urovirulence factors in patients or in the environment.
Adult↗
International comparison of the community prevalence of symptoms of prostatism in four countries.
We conducted an international comparison of the prevalence of urinary symptoms of prostatism in 4 countries, using a community-based random sampling of subjects, similar study procedures, and a single definition of cases that was based on a standardized symptom questionnaire. In Scotland 1,994 medically eligible men aged 40-79 years agreed to participate from 3 communities of the Forth Valley. In France, a nation-wide survey was conducted cross-sectionally in a representative sample of 2,011 French men aged 50-84 years. In the USA, the Olmsted County (OC) study recruited an age- and urban/rural-stratified random sample of 2,115 county residents drawn from medically eligible men aged 40-79 years. In Japan, 290 men aged 40-79 years from a fishing village participated in the study. Response rates were 55, 53, 55, and 43% in Scotland, France, OC and Japan, respectively. Urinary symptoms were assessed by the International Prostate Symptom Score (I-PSS), after metrologic validation in English and cross-cultural adaptation of the questionnaire. The prevalence of moderate to severe symptoms (I-PSS > 7) were 14, 18, 38, and 56% in France, Scotland, OC and Japan, respectively. This pattern was consistent within decades of age, and was found for most of the individual urinary symptoms. The proportion of men in Japan reporting very low I-PSS (0 or 1) was approximately 2, 4 and 8 times less frequent, than in OC, Scotland, and France, respectively. Differences in the prevalence of reported urinary symptoms might reflect between-country differences in the true prevalence of benign prostatic hyperplasia. However, cross-cultural differences in the perception and/or willingness to report urinary symptoms may play an important role in the observed differences. Further study will be required to elucidate the underlying causes of the observed differences.
Adult↗