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K O'Hara

Publications and source records attributed to K O'Hara.

At least 19 recordsLinked to original sources
PubMed2002

Volume increase in growth plate chondrocytes during hypertrophy: the contribution of organic osmolytes.

During the differentiation cascade of growth plate chondrocytes, cells undergo as much as a 10-15-fold increase in volume. This volume increase, which occurs to different extents in growth plates growing at different rates, has been demonstrated to be the single most significant variable in understanding the quantitative aspects of the cellular kinetics of long bone growth. Our hypothesis is that this volume increase, which occurs through cell swelling by water imbibition, requires intracellular accumulation of osmolytes through activation or upregulation of membrane transport mechanisms. Significant intracellular accumulation of inorganic osmolytes, such as Na+, K+, and Cl-, is potentially disruptive to normal cellular metabolism, whereas intracellular accumulation of organic osmolytes is considered to be more compatible with metabolic function. Thus, we concentrated on determining the contributions of organic osmolytes--betaine, amino acids, inositol, and sorbitol--to volume increase. Pooled cryostat sections of young bovine growth plates were extracted followed by automated analysis for their content of amino acids. Analysis for betaine and the sugar alcohols was done by extraction and derivatization, followed by high-performance liquid chromatography (HPLC). Parallel stereological analyses correlated osmolyte changes to stages of chondrocytic differentiation, specifically comparing intracellular concentration and amount in proliferative vs. hypertrophic chondrocytes. Calculations demonstrated that, maximally, these organic osmolytes, in total, account for 6%-7% of the intracellular osmolytes required to sustain the volume increase, and that the most significant contribution is from betaine. This suggests that intracellular accumulation of organic osmolytes is not a primary strategy used by growth plate chondrocytes during volume increase of their terminal differentiation. The data also suggest that there is a differential regulation of transporters of these osmolytes such that intracellular concentrations are constantly modified as cells proceed through the differentiation cascade.

Amino Acids↗
PubMed2001

CPT changes for 2001.

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Clinical Laboratory Techniques↗
PubMed2000

Evaluation of the effects of alpha-phenyl-N-tert-butyl nitrone pretreatment on the neurobehavioral effects of methamphetamine.

A relationship between formation of reactive oxygen species (ROS) and energy depletion has been proposed to play an important role in mediating methamphetamine (METH)-induced neurotoxicity. To evaluate this relationship, we examined the effect of the spin-trap agent, alpha-phenyl-N-tert-butyl nitrone (PBN) on hyperthermia and self-injurious behavior (SIB) and striatal dopamine (DA) depletion produced by METH (4 injections of 4 mg/kg, 2 hr intervals, s.c.) in BALB/c mice. Repeated administration of METH induced hyperthermia, incidence of SIB and striatal DA depletion (84% after 3 days). Pretreatment with PBN (4 injections of 60 or 120 mg/kg, i.p.) reduced METH-induced hyperthermia, but did not significantly attenuate METH-induced SIB or the striatal DA depletion. On the other hand, pretreatment with high doses of PBN (4 injections of 180 or 240 mg/kg, i.p.) protected against METH-induced hyperthermia and SIB, and PBN (180 mg/kg) also completely protected against the acute striatal DA depletion 60 min after the last injection of the drug. However, the long-lasting striatal DA depletion was only attenuated by 52 or 56%, respectively. These results indicate that METH-induced hyperthermia contributes to, but is not solely responsible for METH-induced neurotoxicity, and supports a role for formation of ROS and other mechanisms in the generation of METH-induced striatal dopaminergic neurotoxicity. In addition, the difference in the efficacy of PBN to protect against the acute or long-lasting striatal DA depletion induced by METH may indicate that both ROS formation and other mechanisms are required for METH-induced neurotoxicity to develop.

Animals↗
PubMed2000

Methamphetamine-induced striatal dopamine release, behavior changes and neurotoxicity in BALB/c mice.

The behaviors associated with the neurotoxic effects of methamphetamine were evaluated in BALB/c mice. Hyperthermia and behavioral observations were measured 60 min after each subcutaneous injection of methamphetamine (4x4 or 8 mg/kg) or saline, each given 2 h apart. The behavioral observations included stereotyped behaviors, incidence of hemorrhage in breast, salivation and self-injurious behavior (SIB). Repeated administration of methamphetamine produced these behavioral changes and hyperthermia, but resulted in hypothermia by the final injection (8 mg/kg). In addition, the methamphetamine treatment induced a long-lasting dopamine depletion of similar magnitude in the 4 and 8 mg/kg-treated animals. In a time course study striatal monoamine levels were measured 60 min after each injection of these doses. The first and second injections of methamphetamine (8 mg/kg) produced a drastic increase in striatal 3-methoxytyramine; this failed to occur after the third or fourth injection of the same dose. In contrast, 4 mg/kg of methamphetamine also produced an increase in 3-methoxytyramine after the second and third injections of the drug and, in this case, these were maintained for the duration of the treatment. Striatal 3, 4-dihydroxyphenylacetic acid levels also drastically decreased following both doses of methamphetamine, suggesting inhibition of monoamine oxidase in striatum. Moreover, a single injection of methamphetamine increased striatal 2,3-dihydroxybenzoic acid formation. These results suggest that the incidence of hyperthermia, SIB and striatal dopamine neurotoxicity are closely linked to striatal dopamine release and inhibition of monoamine oxidase produced by methamphetamine in BALB/c mice.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2000

Amino acid substitutions in a variant of IMP-1 metallo-beta-lactamase.

In the course of surveying for the carbapenem-hydrolyzing metallo-beta-lactamase gene bla(IMP) in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-beta-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene bla(IMP-3) was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of bla(IMP-3) was identical to that of bla(IMP-1), except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from bla(IMP-3) and bla(IMP-1) by recombinant DNA techniques, and beta-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the bla(IMP-3) gene. This indicated that extension of the substrate profile in the metallo-beta-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene bla(IMP-3) is an ancestor of bla(IMP-1).

Amino Acid Substitution↗
PubMed2000

Functional analysis of the active site of a metallo-beta-lactamase proliferating in Japan.

An R-plasmid-mediated metallo-beta-lactamase was found in Klebsiella pneumoniae DK4 isolated in Japan in 1991. The nucleotide sequence of its structural gene revealed that the beta-lactamase termed DK4 was identical to the IMP-1 metallo-beta-lactamase which was mediated by a chromosomal gene of Serratia marcescens TN9106 isolated in Japan in 1991 (E. Osano et al., Antimicrob. Agents Chemother. 38:71-78, 1994). The dose effect of DK4 beta-lactamase production on the resistance levels indicated a significant contribution of the enzyme to bacterial resistance to all the beta-lactams except monobactams. The enzymatic characteristics of the DK4 beta-lactamase and its kinetic parameters for nine beta-lactams were examined. The DK4 beta-lactamase was confirmed to contain 2 mol of zinc per mol of enzyme protein. The apoenzyme that lacked the two zincs was structurally unstable, and the activities of only 30% of the apoenzyme molecules could be restored by the addition of 1 mM zinc sulfate. The substitution of five conserved histidines (His28, His86, His88, His149, His210) and a cysteine (Cys168) for an alanine indicated that His86, His88, and His149 served as ligands to one of the zincs and that Cys168 played a role as a ligand to the second zinc. Both zinc molecules contribute to the enzymatic process. Mutant enzymes that lack only one of these retained some activity. Additionally, a conserved aspartic acid at position 90 was replaced by asparagine. This mutant enzyme showed an approximately 1,000 times lower k(cat) value for cephalothin than that of the wild-type enzyme but retained the two zincs even after dialysis against zinc-free buffer. The observed effect of pH on the activity suggested that Asp90 functions as a general base in the enzymatic process.

Anti-Bacterial Agents↗
PubMed2000

Macrolide esterase-producing Escherichia coli clinically isolated in Japan.

Current Japanese clinical practice involves the usage of large amounts of new macrolides such as clarithromycin and roxithromycin for the treatment of diffuse panbronchiolitis, Helicobacter pylori and Mycobacterium avium complex infections. In this study, the phenotypes, genotypes, and macrolide resistance mechanisms of macrolide-inactivating Escherichia coli recovered in Japan from 1996 to 1997, were investigated. The isolation rate of erythromycin A highly-resistant E. coli (MIC > or = 1,600 microg/ml) in Japan slightly increased from 0.5% in 1986 to 1.2% in 1997. In six macrolide-resistant strains, recovered from the strains collected for this study during 1996 to 1997, the inactivation of macrolide could be detected with or without added ATP in the assay system. The appearance of erythromycin A-inactivating enzyme independent of ATP was novel from Japanese isolates, and the 1H NMR spectra of oleandomycin hydrolyzed by the three ATP-independent isolates were examined. It was clearly shown that the lactone ring at the position of C-13 was cleaved as 13-H signal in aglycon of oleandomycin upper shifted. These results suggested the first detection of macrolide-lactone ring-hydrolase from clinical isolates in Japan. These results suggested the first detection of an ATP-independent macrolide-hydrolyzing enzyme from Japanese clinical isolates. Substrate specificity of the macrolide-hydrolyzing enzyme was determined with twelve macrolides including the newer members of this group and it was found that not only erythromycin A but also the new macrolides, such as clarithromycin, roxithromycin, and azithromycin were inactivated. The NMR data, broad spectrum of activity, and independence of co-enzyme supported our naming of the enzyme as a macrolide esterase. PCR methodology was employed to detect an ereB-like gene from the 3 isolates producing macrolide esterase, and one of these was subsequently shown to contain both ereB-like and ermB-like genes. It was also clearly shown that the other three isolates, which inactivated macrolide in the presence of ATP, had an mphA-like gene.

Anti-Bacterial Agents↗
PubMed2000

[The resistance of recent clinical isolates against isepamycin, other aminoglycosides and injectable beta-lactams].

Clinical isolates collected from clinical facilities across Japan in 1998 were tested against five aminoglycosides and three beta-lactams. The resistance of 50 strains each of methicillin sensitive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, Citrobacter freundii, Klebsiella pneumoniae, Enterobacter sp., Serratia sp., Pseudomonas aeruginosa and Proteus sp. (P. mirabilis 25 strains and P. vulgaris 25 strains) to the aminoglycosides isepamicin (ISP), amikacin (AMK), gentamicin, tobramycin and dibekacin, and to the beta-lactams imipenem, ceftazidime and piperacillin (all three known to be effective against P. aeruginosa) were investigated using a micro liquid dilution method with the following results: 1. ISP was effective against all strains except for 14% of MRSA, 2% of Proteus sp., and 4% of P. aeruginosa. 2. Six strains of MRSA were resistant to all eight drugs; however, in these cases ISP exhibited a relatively low minimum inhibitory concentration (MIC) compared to the other compounds. 3. Four strains of MRSA were resistant to all drugs except ISP. MRSA was the only isolate to demonstrate a resistance to seven or more drugs. 4. Twenty-one strains of MRSA and 1 strain of P. aeruginosa were resistant to six drugs; however, all of these were susceptible to both ISP and AMK. 5. Against all strains tested, ISP generally exhibited a lower MIC compared to AMK. These results suggest that, even ten years after its entering the market, ISP is still an aminoglycoside having a high anti-bacterial activity against a wide range of clinical isolates.

Amikacin↗
PubMed1999

Identification of functional amino acids in the macrolide 2'-phosphotransferase II.

Macrolide 2'-phosphotransferase [MPH(2')] transfers the gamma phosphate of ATP to the 2'-OH group of macrolide antibiotics. The role of aspartic acids in the putative ATP-binding site of MPH(2')II was investigated through the substitution of alanine for aspartate by site-directed mutagenesis. D200A, D209A, D219A, and D231A mutant strains were unable to inactivate the substrate oleandomycin, while a D227A mutant retained 7% of the activity of the original enzyme.

Adenosine Triphosphate↗
PubMed1999

Appearance in Japan of highly macrolide-resistant Escherichia coli producing macrolide 2'-phosphotransferase II.

Escherichia coli CU1, a clinical isolate recovered in Japan in 1997, was found to be highly-resistant to both 14-membered and 16-membered ring macrolide antibiotics. A crude extract prepared from strain CU1 inactivated 14-, 15- and 16-membered ring macrolides in the presence of ATP and the Rf value of inactivated oleandomycin was identical to that of oleandomycin 2'-phosphate. This suggested that strain CU1 produced the enzyme macrolide 2'-phosphotransferase [MPH(2')]. Substrate specificity of the crude enzyme from strain CU1 against 14-, 15- and 16-membered ring macrolides was basically similar to that of MPH(2')II from strain BM2506, differing in that the former more effectively inactivated roxithromycin and tylosin. Subsequent attempts were made to clone the novel mph gene encoding for MPH(2') in strain CU1. The mph gene carried by strain CU1 was located on nontransmissible plasmid DNA, designated pCU001. Its molecular weight, estimated by agarose electrophoresis, was approximately 57 kD. The DNA sequence of the cloned mph gene from the Japanese isolate CU1 was identical to that of mphB, which until now had only been recovered in France. The variance in the substrate specificity of MPH(2')II from each strain led us to speculate that other factors in the reaction affect the enzymatic inactivation activity.

Anti-Bacterial Agents↗
PubMed1998

Identification of Escherichia coli clinical isolates producing macrolide 2'-phosphotransferase by a highly sensitive detection method.

Macrolide is inactivated with ATP plus crude extract of Escherichia coli producing macrolide 2'-phosphotransferase (MPH(2')), but not by living cells. Therefore, a convenient method for detection of MPH(2') using intact cells is needed. In this report, we determine that the modified lysozyme-DNase-RNase (LDR) method (named ELDR method) is at least one hundred times more sensitive for the detection of MPH(2') activity than the LDR method and, in addition, highly sensitive for the detection of aminoglycoside-modifying enzymes. Therefore, three new MPH(2')-producing strains were found in clinically isolated E. coli in Japan in 1997 by this method. It suggests that MPH(2')-producing E. coli have been spread in Japanese clinical fields.

Anti-Bacterial Agents↗
PubMed1998

Novel metallo beta-lactamase mediated by a Shigella flexneri plasmid.

Novel carbapenem-hydrolyzing beta-lactamase (newly named MET-1) encoded on a transferable plasmid pMS390 from Shigella flexneri JS19622 was purified. The molecular weight was 28,000 by SDS-PAGE and the isoelectric point was higher than 9.3. This beta-lactamase favorably hydrolyzed classical cephalosporins and oxyimino-cephalosporins rather than penicillins and carbapenems, but did not hydrolyze monobactams. The enzymatic activity was inhibited by EDTA, and the enzyme was found to contain two moles of zinc per mole of enzyme protein by means of atomic absorption spectrophotometry. These results indicated that the enzyme is a zinc beta-lactamase which differs from known metallo beta-lactamases, especially in its cephalosporinase-type substrate profile.

Bacterial Proteins↗
PubMed1998

Novel antibiotic susceptibility tests by the ATP-bioluminescence method using filamentous cell treatment.

Antimicrobial susceptibility testing by the ATP-bioluminescence method has been noted for its speed; it provides susceptibility results within 2 to 5 h. However, several disagreements between the ATP method and standard methodology have been reported. The present paper describes a novel ATP method in a 3.5-h test which overcomes these deficiencies through the elimination of false-resistance discrepancies in tests on gram-negative bacteria with beta-lactam agents. In our test model using Pseudomonas aeruginosa and piperacillin, it was shown that ATP in filamentous cells accounted for the false resistance. We found that 0.5% 2-amino-2-methyl-1,3-propanediol (AMPD) extracted ATP from the filamentous cells without affecting normal cells and that 0.3 U of adenosine phosphate deaminase (APDase)/ml simultaneously digested the extracted ATP. We used the mixture of these reagents for the pretreatment of cells in a procedure we named filamentous cell treatment, prior to ATP measurements. This novel ATP method with the filamentous cell treatment eliminated false-resistance discrepancies in tests on P. aeruginosa with beta-lactam agents, including piperacillin, cefoperazone, aztreonam, imipenem-cilastatin, ceftazidime, and cefsulodin. Furthermore, this novel methodology produced results which agreed with those of the standard microdilution method in other tests on gram-negative and gram-positive bacteria, including P. aeruginosa, Escherichia coli, Staphylococcus aureus, and Enterococcus faecalis, for non-beta-lactam agents, such as fosfomycin, ofloxacin, minocycline, and aminoglycosides. MICs obtained by the novel ATP method were also in agreement with those obtained by the agar dilution method of susceptibility testing. From these results, it was shown that the novel ATP method could be used successfully to test the activities of antimicrobial agents with the elimination of the previously reported discrepancies.

Adenosine Triphosphate↗
PubMed1998

Isolation and characterization of two plasmids that mediate macrolide resistance in Escherichia coli: transferability and molecular properties.

Escherichia coli BM2506 is highly resistant to macrolide antibiotics; it produces macrolide 2'-phosphotransferase II [MPH(2')II] which inactivates such drugs. We investigated the localization and the transfer of the macrolide-resistance determinant that encoded the mphB gene for MPH(2')II in strain BM2506. Although we detected no clear band of plasmid DNA after agarose gel electrophoresis, transformation analysis using satellite DNA that corresponded to plasmid DNA after CsCl-ethidium bromide gradient centrifugation and restriction analysis of plasmid DNA in transformants showed that strain BM2506 harbored two plasmids, pTZ3721 (84 kb) and pTZ3723 (24 kb), that specified resistance to macrolides, ampicillin, streptomycin, tetracycline and sulfonamide and to macrolides and ampicillin, respectively. Southern hybridization showed that the mphB gene hybridized to both plasmids. Furthermore, pTZ3721 was transferred by conjugation to another strain of E. coli and pTZ3723 was mobilized with a self-transferable plasmid RP1 to other strains of E. coli. Therefore, it appears that the mphB gene is located on two plasmids in BM2506 and can be transferred to other strains of E. coli by conjugation or mobilization.

Anti-Bacterial Agents↗