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Biomedical subjects

K Ubukata

Publications and source records attributed to K Ubukata.

At least 37 records · Page 2Linked to original sources
PubMed1996

Directly repeated insertion of 9-nucleotide sequence detected in penicillin-binding protein 2B gene of penicillin-resistant Streptococcus pneumoniae.

We investigated the molecular mechanism of 50 penicillin-resistant Streptococcus pneumoniae strains (penicillin: MIC, > or = 0.125 microgram/ml) having neither class A nor class B mutations in the penicillin-binding protein 2B gene (pbp2b). An analysis of the nucleotide sequences of the pbp2b genes from seven strains revealed an unique direct repeat of 9 nucleotides (TGGTATACT) between active-site serine (residue 385) and Ser-X-Asn (residues 442 to 444) motifs. The same insertion was detected in 13 strains.

Amino Acid Sequence↗
PubMed1996

Combinational detection of autolysin and penicillin-binding protein 2B genes of Streptococcus pneumoniae by PCR.

PCR was used to identify penicillin resistance in 1,062 clinical isolates of Streptococcus pneumoniae. Three sets of primers were designed to amplify (i) a 240-bp fragment of the penicillin-binding protein (PBP) 2B gene (pbp2b) of penicillin-susceptible S. pneumoniae (PSSP), (ii) a 215-bp fragment of the class A mutations of the pbp2b gene present in penicillin-resistant S. pneumoniae, and (iii) a 286-bp fragment of the class B mutation. In addition, a set of primers that amplify 273 bp of the autolysin (lytA) gene was applied in combination with the above to identify S. pneumoniae. Of 621 isolates for which MICs of penicillin were < or = 0.06 mu g/ml, 614 (98.9%) were ascertained as having DNA fragments amplified by the PSSP primers. Of 441 isolates for which MICs of penicillin were > or = 0.125 mu g/ml, a class A mutation was detected in only 8 (1.8%), a class B mutation was detected in 310 (70.3%), and neither class A nor class B mutations were found in the remaining 123 (27.9%). However, when analysis was limited to isolates for which MICs of penicillin were > or = 1.0 mu g/ml, 247 isolates (89.8%) of 275 were found to possess a class B mutation. When PBPs were analyzed in 12 isolates with unclear mutations of the pbp2b gene by using [3H]benzylpenicillin, low affinity to PBP 2B was observed in them all. These findings suggest that a pbp2b mutation other than class A or class B is present in these isolates. These results also indicate that it may be possible to identify PSSP and penicillin-resistant S. pneumoniae by applying PCR using a combination of primers to detect the susceptible pbp2b gene, resistant pbp2b gene mutations, and the lytA gene.

Aminoacyltransferases↗
PubMed1996

An autopsy case of abandoned infant body with severe destruction.

One case of infanticide and abandonment by the natural mother is presented. The body was extensively damaged, evidently by a fox. Fortunately, since the body showed less advanced putrefaction due to cold weather, it was possible to narrow down the cause of death and to detect the drug which had been forced down the victim's throat before the crime. However, our estimates of age and postmortem interval were erroneous. We comment as to why we were misled. Pathological problems associated with "aspiration of vomitus" are also discussed.

Age Determination by Teeth↗
PubMed1992

A new form of cytochrome P-450 responsible for mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline in human livers.

Antibodies to P-450IA2 strongly inhibited the mutagenic activation of 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate but not aflatoxin B1 in human liver microsomes. The anti-rat P-450IA2 antibodies were capable of recognizing two proteins which show different mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of human liver microsomes. A new form of cytochrome P-450 (designated P-450-HM4) cross-reactive with anti-rat P-450IA2 antibodies showing that the smaller molecular weight was purified from human liver microsomes by means of the fast-performance liquid chromatography system. The molecular weight of P-450-HM4 was estimated to be 49,000, which was apparently different from that of P-450PA (human P-450IA2). The antibodies to P-450-HM4 did not cross-react with P-450PA (human P-450IA2) but inhibited to various extents the mutagenic activation of IQ in microsomes from human livers. In addition, P-450-HM4 showed significant mutagen-producing activity from IQ in a reconstituted system. Together with these and other results reported previously, it is concluded that at least two forms of cytochrome P-450 [P-450-HM4 and P-450PA (human P-450IA2)] are involved in the mutagenic activation of IQ in human liver.

Antibodies↗
PubMed1992

Rapid detection of the mecA gene in methicillin-resistant staphylococci by enzymatic detection of polymerase chain reaction products.

In order to identify methicillin-resistant staphylococci from clinical sources with ease and reliability, enzymatic detection of polymerase chain reaction (ED-PCR) was applied. ED-PCR is based on the capture of amplified products via biotin-streptavidin affinity and the detection of an incorporated hapten in amplified products with an enzyme-linked antibody. In order to identify methicillin-resistant staphylococci of all species, a 150-bp fragment of the mecA gene was targeted for ED-PCR. After PCR was performed with a pair of biotin and dinitrophenol 5'-labeled primers, the reaction mixture was applied to a microtiter well precoated with streptavidin. Thereafter, bound PCR products were detected colorimetrically with alkaline phosphatase-conjugated anti-dinitrophenol antibody. The extraction of DNA from staphylococcal cells for PCR was simplified so that it could be performed within one tube. The total assay, including PCR, took less than 3 h. The sensitivity of mecA gene detection ranged from greater than 5 x 10(2) CFU per tube for Staphylococcus aureus to greater than 5 x 10(3) CFU per tube for Staphylococcus epidermidis. Genotyping results obtained by ED-PCR of 161 tested strains from the colonies (97 strains of S. aureus and 64 strains of coagulase-negative staphylococci) were compared with the phenotypic susceptibilities of the strains to oxacillin. The results of ED-PCR showed excellent agreement with the MICs of oxacillin with very few exceptions; only one strain of S. aureus and two strains of coagulase-negative staphylococci were found to possess the mecA gene, which was discrepant with their phenotypes. Fifty-five blood culture samples were also tested by ED-PCR. For staphylococcal isolates in 33 of the cultures, oxacillin MICs were >4 microgram/ml; 31 of the 33 staphylococcal isolates were determined by ED-PCR to be mecA gene positive. These results suggest that ED-PCR can be used with reasonable confidence in the clinical microbiological laboratory.

Alkaline Phosphatase↗
PubMed1990

Homology of mecA gene in methicillin-resistant Staphylococcus haemolyticus and Staphylococcus simulans to that of Staphylococcus aureus.

A penicillin-binding protein of molecular weight 76,000 inducible by beta-lactams was detected in methicillin-resistant Staphylococcus haemolyticus and Staphylococcus simulans. DNA from these strains hybridized to the mecA gene from Staphylococcus aureus; however, the chromosomal HindIII fragments containing the mecA genes were 3.4 kilobases in S. haemolyticus and 4.3 kilobases in S. simulans.

Bacterial Proteins↗
PubMed1990

In vitro activity of LJC10,627, a new carbapenem antibiotic with high stability to dehydropeptidase I.

The in vitro activity of LJC10,627, a new carbapenem, was compared with those of imipenem and ceftazidime. LJC10,627 had broad-spectrum activity against gram-positive and gram-negative clinical isolates. The MICs of this compound for 90% of members of the family Enterobacteriaceae tested (MIC90s), including strains resistant to ceftazidime, ranged from 0.1 to 25 micrograms/ml. LJC10,627 inhibited Pseudomonas aeruginosa at an MIC90 of 3.13 micrograms/ml; it thus was twofold more active than imipenem. This compound inhibited Haemophilus, Neisseria, and Branhamella species at MIC90s of 3.13, 0.1, and 0.1 micrograms/ml, respectively. LJC10,627 was two- to fourfold less active than imipenem against methicillin-susceptible Staphylococcus aureus and Staphylococcus epidermidis at MIC90s of 0.1 and 0.39 microgram/ml. However, the compound was found to be twofold more active than imipenem against Bacteroides fragilis at an MIC90 of 1.56 microgram/ml. LJC10,627 was very stable to various beta-lactamases except for Xanthomonas maltophilia oxyiminocephalosporinase type II. LJC10,627 was minimally hydrolyzed by swine renal dehydropeptidase I; its residual activity was 93.0% after 2 h. Killing kinetics of this compound for Escherichia coli and Pseudomonas aeruginosa showed that bactericidal action occurred at concentrations above the MIC (0.05 and 0.39 microgram/ml, respectively). LJC10,627 had a high affinity for penicillin-binding proteins 2, 4, and 1B(s) of Escherichia coli and Pseudomonas aeruginosa and penicillin-binding proteins 1 and 4 of Staphylococcus aureus.

Animals↗
PubMed1990

Nucleotide sequence and characterization of the Staphylococcus aureus norA gene, which confers resistance to quinolones.

The norA gene cloned from chromosomal DNA of quinolone-resistant Staphylococcus aureus TK2566 conferred relatively high resistance to hydrophilic quinolones such as norfloxacin, enoxacin, ofloxacin, and ciprofloxacin, but only low or no resistance at all to hydrophobic ones such as nalidixic acid, oxolinic acid, and sparfloxacin in S. aureus and Escherichia coli. The 2.7-kb DNA fragment containing the norA gene had a long open reading frame coding for 388 amino acid residues with a molecular weight of 42,265, which was consistent with the experimental value of about 49,000 obtained on DNA-directed translation. The deduced NorA polypeptide has 12 hydrophobic membrane-spanning regions and is partly homologous to tetracycline resistance protein and sugar transport proteins. The uptake of a hydrophilic quinolone, enoxacin, by S. aureus harboring a plasmid carrying the norA gene was about 50% that by the parent strain lacking the plasmid, but it increased to almost the same level as that by the latter strain with carbonyl cyanide m-chlorophenyl hydrazone. On the other hand, the uptake of a hydrophobic quinolone, sparfloxacin, was similar in the two strains. These results suggest that the NorA polypeptide may constitute a membrane-associated active efflux pump of hydrophilic quinolones.

Amino Acid Sequence↗
PubMed1989

Cloning and expression of the norA gene for fluoroquinolone resistance in Staphylococcus aureus.

The norA gene for fluoroquinolone resistance in Staphylococcus aureus TK2566 was cloned from chromosomal DNA into fluoroquinolone-susceptible Escherichia coli TG1. The resulting transformant, NY11, contained a recombinant plasmid, designated pTUS1, with a cloned 5.5-kilobase (kb) HindIII fragment of staphylococcal DNA. The MIC of norfloxacin for the strain increased from 0.1 to 3.13 micrograms/ml. Furthermore, when the fragment was recloned into S. aureus, the transformant NY12, containing recombinant plasmid pTUS20, had the same level of resistance to norfloxacin as did the original strain, although it was less resistant to ofloxacin and ciprofloxacin. A single KpnI-HaeIII fragment was found to be the minimum size able to express norfloxacin resistance, suggesting that the norA gene is located within the 2.6- to 3.2-kb region of the original 5.5-kb fragment. The 5.5-kb fragment hybridized to DNA from a fluoroquinolone-susceptible S. aureus strain.

Anti-Infective Agents↗
PubMed1989

Restriction maps of the regions coding for methicillin and tobramycin resistances on chromosomal DNA in methicillin-resistant staphylococci.

Chromosomal BamHI DNA fragments containing both the mecA gene encoding the penicillin-binding protein responsible for methicillin resistance and the aadD gene encoding 4',4"-adenylyltransferase responsible for tobramycin resistance were cloned from three methicillin- and tobramycin-resistant strains of Staphylococcus aureus and one strain of Staphylococcus epidermidis. Physical maps of the fragments were similar, suggesting their unique origin.

Blotting, Southern↗
PubMed1989

Expression and inducibility in Staphylococcus aureus of the mecA gene, which encodes a methicillin-resistant S. aureus-specific penicillin-binding protein.

A beta-lactam-sensitive strain of Staphylococcus aureus could be converted to methicillin resistance by the introduction of a plasmid carrying the 4.3-kilobase HindIII chromosomal DNA fragment which encoded the mecA gene from a methicillin-resistant S. aureus. Transformant cells produced methicillin-resistant S. aureus-specific penicillin-binding protein constitutively, and additional insertion of an inducible penicillinase plasmid caused production of the pencillin-binding protein to become inducible.

Bacterial Proteins↗
PubMed1986

Molecular cloning of the gene of a penicillin-binding protein supposed to cause high resistance to beta-lactam antibiotics in Staphylococcus aureus.

A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus.

Anti-Bacterial Agents↗
PubMed1985

Occurrence of a beta-lactam-inducible penicillin-binding protein in methicillin-resistant staphylococci.

The mechanism of methicillin resistance was investigated in methicillin-resistant staphylococci (MRS) and in variants which had lost methicillin resistance. Phase-contrast microscopy showed that cells swelled at low concentrations of beta-lactam antibiotics in both MRS and variants which had lost methicillin resistance. Cells of variants which had lost methicillin resistance were lysed easily when higher concentrations of antibiotic were used. In contrast, MRS cells remained swollen at even higher concentrations of antibiotics. Furthermore, bacterial growth was inhibited at antibiotic concentrations much lower than MICs for MRS. Examination of the penicillin-binding proteins (PBPs) in MRS revealed that a new PBP-2' (molecular weight, 74,000) was induced in large quantity by exposure to beta-lactams. PBP-2' was produced constitutively in variants of MRS which had lost a penicillinase plasmid. The induction of PBP-2' by beta-lactams was not detected in variants which had lost methicillin resistance. High concentrations of beta-lactam were required for saturation of PBP-2'. The optimum antibiotic concentration for the induction of PBP-2' varied with the beta-lactam used as the inducer, and PBP-2' was produced in a larger amount at 32 degrees C than at 37 degrees C. From these results, we suggest that the mechanism of methicillin resistance depends on the induction of PBP-2', which may function as a detour enzyme for PBP-2 or PBP-3 or may be a particular enzyme involved in peptidoglycan synthesis.

Anti-Bacterial Agents↗
PubMed1984

Purification and characterization of aminoglycoside-modifying enzymes from Staphylococcus aureus and Staphylococcus epidermidis.

Several strains of Staphylococcus aureus and Staphylococcus epidermidis, exhibiting characteristic resistance patterns to aminoglycoside antibiotics, were examined. The aminoglycoside-modifying enzymes from these strains were purified by DEAE-Sephadex A-50 chromatography, affinity chromatography, and Sephadex G-100 gel filtration. Three enzymes, a 3'-phosphotransferase III (molecular weight, 31,000; pI 4.1), a bifunctional enzyme having 6'-acetyltransferase and 2"-phosphotransferase (molecular weight, 56,000; pI 4.1) activity, and a 4'4"-adenylytransferase (molecular weight, 34,000; pI 4.7), were isolated from crude extracts of the resistant strains. Aminoglycoside-modifying enzymes with identical enzymatic properties derived from S. aureus and S. epidermidis were also immunologically identical.

Acetyltransferases↗
PubMed1979

[Morphological studies on antibacterial activities of cefotiam (author's transl)].

1. Cefotiam was demonstrated to be more potent than cefazolin in its antibacterial activities against clinical isolates of E. coli, Klebsiella, Serratia, Proteus mirabilis, Proteus morganii and Proteus inconstans. MICs of cefotiam with 10(6) cells/ml inoculum size were considerably lower than those with 10(8) cells/ml. 2. Organisms lysed when exposed to cefotiam at concentrations higher than the MICs with 10(8) cells/ml. Morphological changes of organisms into filament occurred even at concentrations lower than the MICs with 10(6) cells/ml. This indicates that cefotiam is incorporated into organisms at remarkably low concentrations and exerts its antibacterial activities. 3. Cefotiam showed a high affinity for penicillin-binding proteins (PBP) 1A, 1Bs and 3. The formation of filament at low concentrations of cefotiam is possibly attributable to the high affinity of cefotiam for PBP 3 in addition to its high permeability through outer cell membrane. 4. As the antibacterial activities of cefotiam are displayed at lower concentrations, it is reasonable to consider that doses of cefotiam on clinical use can be reduced in comparison with those of conventional cephalosporins.

Cefazolin↗