Biomedical subjects

H Maki

Publications and source records attributed to H Maki.

At least 55 records · Page 3Linked to original sources

PubMed1994

Nuclear localization and transforming activity of human papillomavirus type 16 E7-beta-galactosidase fusion protein: characterization of the nuclear localization sequence.

A MAb 9F6 was capable of staining HPV16 E7 in a human cervical carcinoma line, CaSki, and rat 3Y1 cells stably expressing HPV16 E7 gene. Contrary to the current understanding of E7 as a nuclear protein, the site of staining was clearly cytoplasmic. The subcellular localization of E7 was further studied by using the beta-galactosidase (beta-gal) receptor method. A fusion protein composed of E7 and beta-gal was stably expressed in rat 3Y1 cells. The beta-gal activity in these cells was detected mostly in the nucleus, even though 9F6 still stained the cytoplasm of these cells. The fusion protein was also found to be oncogenic since transfected 3Y1 cells acquired transformed phenotypes such as increased saturation density and anchorage-independent growth. These results indicate that biologically active E7 exists mostly in the nucleus, but nuclear E7 is masked from 9F6. A series of deletion mutants of E7 further demonstrated that the amino acid sequence from 16 to 41 was enough to transport beta-gal into the nucleus. A mutation either at amino acid 24 or 26 which is known to disrupt the binding of E7 to RB, the retinoblastoma gene product, did not strongly affect the nuclear localization of the fusion protein, suggesting that the nuclear transportation of E7 is mostly independent of RB binding.

Amino Acid Sequence↗

PubMed1994

Effect of single DNA lesions on in vitro replication with DNA polymerase III holoenzyme. Comparison with other polymerases.

In the present work, we have studied in vitro replication of N-2-acetylaminofluorene (AAF) or cis-diamminedichloroplatinum II (cis-DDP) single modified DNA templates. We used the holoenzyme (pol III HE) or the alpha subunit of DNA polymerase III, which is involved in SOS mutagenesis, and other DNA polymerases in order to compare enzymes having different biological roles and properties. Single-stranded oligonucleotides (63-mer) bearing a single AAF adduct at one of the different guanine residues of the NarI sequence (-G1G2CG3CC-) have been used in primer extension assays. Site-specifically platinated 5'd(ApG) or 5'd(GpG) oligonucleotides were constructed and similarly used in primer extension assays. In all cases, irrespective of both the chemical nature of the lesion (i.e. AAF or cis-DDP) and its local sequence context (i.e. the 3 different sites for AAF adducts within the NarI site) replication by pol III HE and pol I Klenow fragment (pol I Kf) stops one base prior to the adduct site. Removal of the 3'-->5' proofreading activity alone was not sufficient to trigger bypass of DNA lesions. Indeed, when proofreading activity of pol I is inactivated by a point mutation (pol I Kf (exo-)), the major replication product corresponds to the position opposite the adduct site showing that incorporation across from the AAF adduct is possible. These results suggest that a polymerase with proofreading activity is actually found to stop one nucleotide before the adduct not because it is unable to insert a nucleotide opposite the adduct but most likely because elongation past the adduct is strongly impaired, giving thus an increased time frame for the proofreading exonuclease to remove the base inserted across from the adduct. These results are discussed in terms of their implications for error-free and error-prone bypass in vivo.

2-Acetylaminofluorene↗

PubMed1994

Roles of transcription and repair in alkylation mutagenesis.

Mutations occurring in Escherichia coli cells exposed to alkylating agents have been analyzed using an assay for forward mutations in the E. coli rpsL gene cloned on a high copy number plasmid. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutations were recovered from wild-type and O6-methylguanine methyltransferase-deficient mutant (ada- ogt-) cells and their sequence alterations determined. We found that the mutations recovered from the wild-type strain were predominantly G:C to A:T transitions located at several hot spots in the rpsL sequence. A vast majority of the mutations were found at guanine residues preceded by thymine on the transcribed strand of the target gene. Although the methyltransferase mutant showed hypersensitivity to the alkylating reagent in terms of mutagenic effect and cell killing effects, the class and site distributions of the rpsL- mutations recovered from MNNG-treated ada- ogt- cells were similar to those observed with MNNG-treated wild-type cells. Therefore, the site preference of MNNG-induced rpsL- mutations seems to be due not to the specificity of methyl-transferring repair enzymes but probably to the distribution of the mutagenic lesions (O6-methylguanine) in the target sequence. Mutations induced by methyl methanesulfonate, an SN2 alkylating agent, showed similar class and site distributions in the rpsL system. The site preference of MNNG-induced mutations was significantly changed when the level of transcription of the rpsL gene was decreased to 120-fold lower than that promoted by the authentic rpsL promoter. Under these conditions, 78% of mutations were induced at the central guanine of 5'-GG(A or C)-3' and 2/3 of them were on the non-transcribed strand of the rpsL gene. These results suggested that the site preference of MNNG-induced mutations is determined by at least three factors: (i) a flanking-base effect on the chemical reactivity of a guanine residue, (ii) transcribed strand-specific repair, probably by the UvrABC system, and (iii) the effects of transcription of the target gene on the alkylation of DNA and the strand-specific repair.

Alkylation↗

PubMed1994

Degradation of alkylphenol ethoxylates by Pseudomonas sp. strain TR01.

An alkylphenol ethoxylate-degrading bacterium was isolated from activated sludge of a municipal sewage treatment plant by enrichment culture. This organism was found to belong to the genus Pseudomonas; since no corresponding species was identified, we designated it as Pseudomonas sp. strain TR01. This strain had an optimal temperature and pH of 30 degrees C and 7, respectively, for both growth and the degradation of Triton N-101 (a nonylphenol ethoxylate in which the average number of ethylene oxide [EO] units is 9.5). The strain was unable to mineralize Triton N-101 but was able to degrade its EO chain exclusively. The resulting dominant intermediate was identified by normal-phase high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry as a nonylphenol ethoxylate with 2 mol of EO units. A carboxylated metabolite, [(nonylphenoxy)ethoxy]acetic acid, was detected by gas chromatography-mass spectrometry. This bacterium also metabolized alcohol ethoxylates with various numbers of EO units but not polyethylene glycols whatever their degree of polymerization. By oxygen consumption assay, the alkyl group or arene corresponding to the hydrophobic part of alcohol ethoxylates or alkylphenol ethoxylates was shown to contribute to the induction of the metabolic system of the EO chain of Triton N-101, instead of the EO chain itself, which corresponds to its hydrophilic part. Thus, the isolated pseudomonad bacterium has unique substrate assimilability: it metabolizes the EO chain only when the chain linked to bulky hydrophobic groups.

Biodegradation, Environmental↗

PubMed1994

Cloning and characterization of a gene affecting the methicillin resistance level and the autolysis rate in Staphylococcus aureus.

Tn918 mutagenesis of a high-level methicillin-resistant Staphylococcus aureus (methicillin MIC, 800 micrograms/ml) led to the isolation of a low-resistance mutant. The Tn918 insert was transferred back to the parent to produce strain SRM563 (methicillin MIC, 12.5 micrograms/ml), which showed heterogeneous resistance. Twenty-two clinical isolates of methicillin-resistant S. aureus were transformed with DNA of SRM563. In the transformants of most strains, instances of reduced resistance were observed with concomitant increases of autolysis rate induced by Triton X-100 and were generally more profound in high-resistance strains. Two transformants exhibited a decrease of the autolysis rate and little reduction of resistance. In the transformant of methicillin-susceptible strain RN2677, an increase of the autolysis rate and little reduction of resistance were observed. The production of low-affinity penicillin-binding protein (PBP2') did not significantly decrease in the mutants. Insertion of Tn918 occurred within the 3'-terminal region of a novel gene designated llm, which was cloned and sequenced. RNA blot analysis demonstrated that the gene was transcribed. The encoded protein was composed of 351 amino acid residues with a molecular weight of 38,512 and was hydrophobic, suggesting its location on the membrane. The gene was detected by PCR in all S. aureus strains tested but not in the other 26 staphylococcal species. Comparison of the 3'-terminal sequences of the gene among several S. aureus strains showed that, whereas nucleotide substitutions occurred at the third position in seven of eight 3'-terminal codons, only C-terminal amino acid variation of glutamate or aspartate was observed.

Amino Acid Sequence↗

PubMed1994

[A case of eosinophilic pneumonia with elevated levels of carcino-embryonic antigen (CEA)].

A 70-year-old woman was admitted for productive cough and infiltrative shadows in the right lower lung field on chest X-ray film. Eosinophilia (17%) in blood, an abnormally high percentage of eosinophiles (7%) in bronchoalveolar lavage fluid and eosinophilic infiltration with proliferated goblet cells in transbronchial lung biopsy specimens led to the diagnosis of eosinophilic pneumonia. Laboratory data on admission also revealed a high level of CEA (17.1 ng/ml) in serum. After administration of prednisolone (30 mg/day), the symptoms ameliorated and the CEA levels were normalized. The proliferated goblet cells were immunohistochemically positive for CEA, which suggests that the high levels of CEA were caused by excessive CEA secretion from the goblet cells associated with eosinophilic pneumonia. These studies showed that serum CEA also may be a marker for disease activity in eosinophilic pneumonia.

Aged↗

PubMed1993

Cloning and expression of cDNA for a human enzyme that hydrolyzes 8-oxo-dGTP, a mutagenic substrate for DNA synthesis.

8-Oxoguanine (8-oxo-7, 8-dihydroguanine) is produced in DNA, as well as in nucleotide pools of cells, by active oxygen species normally formed during cellular metabolic processes. 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies, and transversion mutation ensues. Human cells contain enzyme activity, which hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, and this enzyme is responsible for preventing misincorporation of 8-oxoguanine into DNA. We purified this particular human enzyme to physical homogeneity and determined a partial amino acid sequence. We then cloned the cDNA for human 8-oxo-dGTPase and examined its nucleotide sequence. The human protein comprises 156 amino acid residues and has some sequence homology with the Escherichia coli MutT protein, which has a distinct 8-oxo-dGTPase activity. When the human cDNA was expressed in E. coli mutT- mutant cells, there was a significant amount of 8-oxo-dGTPase activity. In such cells, the frequency of spontaneous mutation was greatly reduced. We propose that the human 8-oxo-dGTPase protects genetic information from the untoward effects of endogenous oxygen radicals.

Amino Acid Sequence↗

PubMed1993

Emergency laparotomy in uncontrolled thyrotoxic patient with preoperative fulminant hepatic failure.

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Journal Article↗

PubMed1993

Determination of the target volume of HeLa cells treated with platinum-195m radiolabeled trans-diaminedichloroplatinum(II): a comparison with cis-diaminedichloroplatinum(II).

HeLa S-3 cells were treated with 195mPt-radiolabeled trans-diaminedichloroplatinum(II) (TDDP) under various conditions, and the relationship between lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentrations for the cells treated with TDDP at 37 degrees C for 1, 2 and 3 h were 163.7, 65.8 and 24.9 microM, respectively. By using identically treated cells, the number of Pt atoms combined with DNA, RNA and protein molecules was determined after the cells were fractionated using the method of Schneider. In this way, the D0 values given as the drug concentration were substituted for the number of Pt atoms combined with each fraction, then the target volumes, expressed as the reciprocals of the D0 values, were calculated for each fraction. The results suggested that DNA and high molecular weight RNAs (except t-RNA), under some limited condition, could be the target molecules for cell killing by TDDP. The target volumes for DNA were 1.31 x 10(3), 3.01 x 10(3) and 6.26 x 10(3) nucleotides for 1, 2 and 3 h treated cells, respectively. Cell killing effects of TDDP were lower than CDDP by a factor of 39.5, 19.0 and 16.5 for 1, 2 and 3 h treatments at 37 degrees C, respectively, when viewed from the stand point of the target volume, while those from the mean lethal dose (D0) were 17.6, 9.8 and 6.7, respectively.

Cell Survival↗

PubMed1993

Cloning, sequence analysis and expression in Escherichia coli of a gene encoding an alginate lyase from Pseudomonas sp. OS-ALG-9.

A gene (aly) encoding alginate lyase (ALY; EC 4.2.2.3) was isolated from a library constructed with the cosmid vector pHC79 and Sau3AI-digested genomic DNA of Pseudomonas sp. OS-ALG-9. Successive subcloning of the aly-containing cosmid enabled us to locate the gene on a 2.3 kb HpaI fragment. Nucleotide sequencing of this fragment revealed a single open reading frame (ORF) of 1365 bp. The directly determined N-terminal amino acid sequence of the ALY protein purified from Pseudomonas sp. OS-ALG-9 was found in the amino acid sequence deduced from this ORF between nucleotides 282 and 366. Expression of aly was induced by IPTG in Escherichia coli and leakage of the enzyme into the extracellular milieu was significantly enhanced by addition of glycine to the growth medium. The ALY enzyme had a greater specificity for the homopolymer of mannuronate than for that of guluronate.

Amino Acid Sequence↗

PubMed1993

Effect of reperfusion by direct percutaneous transluminal coronary angioplasty on ventricular late potentials in cases of total coronary occlusion at initial coronary arteriography.

The relationship between the time required for reperfusion and the incidence of late potentials was studied in 94 patients who survived a first acute myocardial infarction (AMI) and who showed total occlusion of the infarct-related artery at an initial coronary arteriography. Sixty-three patients who successfully underwent direct percutaneous transluminal coronary angioplasty (PTCA), and 31 who were treated conventionally (controls), underwent signal-averaged electrocardiography. Direct PTCA patients were classified into 5 groups according to the time required for reperfusion: < or = 4, 4-6, 6-8, 8-10, and > or = 10 h. The incidence of late potentials in these groups was 8%, 12%, 14%, 33%, and 43%, respectively, and 48% in the controls. Late potentials were recorded more frequently as the period until successful reperfusion increased: the incidence of late potentials was significantly lower in the < or = 4 and 4-6 h groups than in the controls (p < 0.005 and p < 0.05, respectively). Therefore, reperfusion achieved within 6 h reduced the incidence of late potentials in AMI patients and may be effective for preventing malignant ventricular arrhythmias.

Acute Disease↗

PubMed1992

Hydrolytic elimination of a mutagenic nucleotide, 8-oxodGTP, by human 18-kilodalton protein: sanitization of nucleotide pool.

8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies. Once 8-oxodGTP is formed in the cellular nucleotide pool, this mutagenic nucleotide is incorporated into DNA and would cause transversion mutations. The MutT protein of Escherichia coli possesses enzyme activity to hydrolyze 8-oxodGTP to the corresponding nucleoside monophosphate and thus may be responsible for preventing the occurrence of such mutations. Here we show that the human cell has an enzyme specifically hydrolyzing 8-oxodGTP in a fashion similar to that seen with MutT protein. The human 8-oxodGTPase has been found in cell-free extracts from Jurkat cells and purified > 400-fold. Analyses by gel filtration and gel electrophoresis revealed that the molecular mass of the native form of human 8-oxodGTPase is 18 kDa. Mg2+ ion is required for the enzyme action and the optimum pH for the reaction is pH 8.0. The enzyme hydrolyzes 8-oxodGTP to 8-oxodGMP with a Km value of 12.5 microM. dGTP and dATP are also degraded to dGMP and dAMP, respectively, with Km values 70 times greater than that for 8-oxodGTP. dTTP and dCTP are not hydrolyzed. These properties of the human 8-oxodGTPase are similar to those observed with the E. coli MutT protein, suggesting that the function of protecting the genetic information from the threat of endogenous oxygen radicals is widely distributed in organisms.

Chromatography, Affinity↗

PubMed1992

MutT protein specifically hydrolyses a potent mutagenic substrate for DNA synthesis.

Errors in the replication of DNA are a major source of spontaneous mutations, and a number of cellular functions are involved in correction of these errors to keep the frequency of spontaneous mutations very low. We report here a novel mechanism which prevents replicational errors by degrading a potent mutagenic substrate for DNA synthesis. This error-avoiding process is catalysed by a protein encoded by the mutT gene of Escherichia coli, mutations of which increase the occurrence of A.T----C.G transversions 100 to 10,000 times the level of the wild type. Spontaneous oxidation of dGTP forms 8-oxo-7,8-dihydro-2'-dGTP (8-oxodGTP), which is inserted opposite dA and dC residues of template DNA with almost equal efficiency, and the MutT protein specifically degrades 8-oxodGTP to the monophosphate. This indicates that elimination from the nucleotide pool of the oxidized form of guanine nucleotide is important for the high fidelity of DNA synthesis.

Bacterial Proteins↗

PubMed1992

Giant condyloma acuminatum in a baby boy.

A giant condyloma acuminatum developed on the penis of a one and a half-year-old Japanese boy in two months. The histological features of this tumor were compatible with those of ordinary condyloma acuminatum. Although we detected the presence of human papilloma virus (HPV) type 16 by using the polymerase chain reaction system, we could not rule out the possibility that this HPV was present concurrently with other HPV types that cause condyloma acuminatum. The lesion was successfully treated with cryotherapy and topical application of 5% fluorouracil ointment under occlusion.

Condylomata Acuminata↗

PubMed1992

The number of platinum atoms binding to DNA, RNA and protein molecules of HeLa cells treated with cisplatin at its mean lethal concentration.

HeLa S-3 cells were treated with 195mPt-radiolabeled cis-diamminedichloroplatinum(II) (CDDP) under various conditions, and the relationship between lethal effect and the number of Pt atoms binding to DNA, RNA and proteins was examined. The mean lethal concentrations for the cells treated with CDDP at 37 degrees C for 1, 2 and 3 h were 2.8, 2.0 and 1.1 microgram/ml, respectively. By using identically treated cells, the number of Pt atoms combined with DNA, RNA and protein molecules were determined after fractionation of the cells using the method of Schneider. In this way, the D0 values given as the drug concentration were substituted for the number of Pt atoms combined with each fraction, then the target volumes expressed as the reciprocals of D0 values were calculated for each fraction. The results provide strong support for the idea that DNA is the primary target for cell killing by CDDP, and the target volumes were 5.17 x 10(4), 5.71 x 10(4) and 1.03 x 10(5) nucleotides for 1, 2 and 3 h treated cells, respectively.

Cell Survival↗

PubMed1992

Molecular mechanisms for controlling spontaneous and induced mutagenesis.

Alkylation↗

PubMed1992

[Detection of ventricular late potentials comparison of 4 commercial high-resolution systems].

UNLABELLED: The purpose of this study was to evaluate the methodologic problems of noninvasive registration of ventricular late potentials. METHODS: we compared the results obtained in the same patients with four different systems which were Marquette MAC1, MAC12, ART LP101 and FUKUDA VCM 3000. Filtering was employed digital forward direction, 100-300 Hz in MAC1, digital FFT, 40-250 Hz in MAC12, digital, bidirectional, 40-250 Hz in ART and analogue, forward direction 40-300 Hz in FUKUDA VCM 3000. DEFINITION OF LATE POTENTIALS (LPs): Filtered QRS duration (FQRSD) was 120 msec or more, root mean square voltage in the last 40 msec (RMSV) was 15 microV or less in MAC12 and ART. FQRSD was 130 msec or more, RMSV was 15 microV or less in FUKUDA. FQRSD was 20 msec or more after QRS obtained from low resolution leads in MAC1. SUBJECTS: 163 patients [control 13, PVCs 23, myocardial infarction 55, others 51, IVCD (RBBB, LBBB, IVCD) 21] were included in this study. RESULTS: 17.6% of all patients (except for IVCD) showed LPs with MAC12, 20.0% with ART, 30.3% with FUKUDA, and 14.1% with MAC1. 23.1% of the control group showed LPs with MAC12 and FUKUDA, but no cases showed LPs with ART and FUKUDA. 4.3% of the PVC group showed LPs with MAC12, 13.0% with ART and FUKUDA, but no cases showed LPs with MAC1. 25.5% of the myocardial infarction group showed LPs with MAC12 and MAC1, 30.9% with ART and 36.3% with FUKUDA. All four methods gave corresponding results in 65.4% of patients, 7.0% positive and 58.4% negative findings. FQRSD of MAC12, ART and FUKUDA was 110.2 +/- 18.1 msec, 105.2 +/- 20.4 msec and 125.8 +/- 22.8 msec respectively. Correlation coefficient (r value) in FQRSD was 0.90 between ART and MAC12, 0.74 between ART and FUKUDA, and 0.75 between FUKUDA and MAC12. RMSV of MAC12, ART and FUKUDA was 49.2 +/- 30.2 microV, 43.7 +/- 37.8 microV and 22.5 +/- 10.6 microV respectively. R value in RMSV was 0.59 between ART and MAC12, 0.38 between ART and FUKUDA and 0.43 between FUKUDA and MAC12. R value in high frequency low amplitude signals duration under 40 microV of LPs positive patients in MAC1 was 0.51 between ART and MAC 12, 0.18 between MAC12 and MAC1, and 0.32 between ART and MAC1. Most of the differences result from a different interpretation of the tracings. Especially LPs far from QRS offset could be registered with neither MAC12, ART nor FUKUDA.

Action Potentials↗

PubMed1991

Mutational specificity of the dnaE173 mutator associated with a defect in the catalytic subunit of DNA polymerase III of Escherichia coli.

We developed a system to examine forward mutations that occurred in the rpsL gene of Escherichia coli placed on a multicopy plasmid. Using this system we determined the mutational specificity for a dnaE173 mutator strain in which the editing function of DNA polymerase III is impeded. The frequency of rpsL- mutations increased 32,000-fold, due to the dnaE173 mutator, and 87 independent rpsL- mutations in the mutator strain were analyzed by DNA sequencing, together with 100 mutants recovered from dnaE+ strain, as the control. While half the number of mutations that occurred in the wild-type strain were caused by insertion elements, no such mutations were recovered from the mutator strain. A novel class of mutation, named "sequence substitution" was present in mutants raised in the dnaE173 strain; seven sequence substitutions induced in the mutator strain occurred at six sites, and all were located in quasipalindromic sequences, carrying the GTG or CAC sequence at one or both endpoints. While other types of mutation were found in both strains, single-base frameshifts were the most frequent events in the mutator strain. Thus, the mutator effect on this class of mutation was 175,000-fold. A total of 95% of the single-base frameshifts in the mutator strain were additions, most of which occurred at runs of A or C bases so as to increase the number of identical residues. Base substitutions, the frequency of which was enhanced 25,000-fold by the mutator effect, occurred primarily at several hotspots in the mutator strain, whereas those induced in the wild-type strain were more randomly distributed throughout the rpsL sequence. The dnaE173 mutator also increased the frequency of duplications 28,000-fold. Of the three duplications recovered from the mutator strain, one was a simple duplication, the region of which was flanked by direct repeats. The other duplications were complex, one half part of which was in the inverted orientation of a region containing two sets of inverted repeats. The same duplications were also recovered from the wild-type strain. The present data suggest that dnaE173 is a novel class of mutator that sharply induces sequence-directed mutagenesis, yielding high frequencies of single base frameshifts, duplications with inversions, sequence substitutions and base substitutions at hotspots.

Base Sequence↗