Kinetic properties and metal content of the metallo-beta-lactamase CcrA harboring selective amino acid substitutions. (1/158)

The crystal structure of the metallo-beta-lactamase CcrA3 indicates that the active site of this enzyme contains a binuclear zinc center. To aid in assessing the involvement of specific residues in beta-lactam hydrolysis and susceptibility to inhibitors, individual substitutions of selected amino acids were generated. Substitution of the zinc-ligating residue Cys181 with Ser (C181S) resulted in a significant reduction in hydrolytic activity; kcat values decreased 2-4 orders of magnitude for all substrates. Replacement of His99 with Asn (H99N) significantly reduced the hydrolytic activity for penicillin and imipenem. Replacement of Asp103 with Asn (D103N) showed reduced hydrolytic activity for cephaloridine and imipenem. Deletion of amino acids 46-51 dramatically reduced both the hydrolytic activity and affinity for all beta-lactams. The metal binding capacity of each mutant enzyme was examined using nondenaturing electrospray ionization mass spectrometry. Two zinc ions were observed for the wild-type enzyme and most of the mutant enzymes. However, for the H99N, C181S, and D103N enzymes, three different zinc content patterns were observed. These enzymes contained two zinc molecules, one zinc molecule, and a mixture of one or two zinc molecules/enzyme molecule, respectively. Two enzymes with substitutions of Cys104 or Cys104 and Cys155 were also composed of mixed enzyme populations.  (+info)

The interaction and transport of beta-lactam antibiotics with the cloned rat renal organic anion transporter 1. (2/158)

In the present study, we investigated the interactions between antibiotics, especially beta-lactam antibiotics, and rat renal organic anion transporter 1 (OAT1). [(14)C]p-Aminohippurate (PAH) uptake via OAT1 expressed in Xenopus laevis oocytes was inhibited by all of the penicillins and cephalosporins tested. Penicillin G, carbenicillin, cephaloridine, cephalothin, cefazolin, and cephalexin inhibited [(14)C]PAH uptake via OAT1 in a competitive manner (K(i) = 0.29-2.33 mM). Cinoxacin, a quinolone gyrase inhibitor, also inhibited PAH uptake via OAT1. Other antibiotics, such as gentamicin, streptomycin, and vancomycin, which do not contain anionic moieties, did not interact with OAT1. [(3)H]Penicillin G and [(14)C]cephaloridine were demonstrated to be transported via OAT1. Using the cells that stably expressed OAT1, we analyzed the cytotoxicity of several beta-lactam antibiotics. Cells expressing OAT1 showed higher susceptibility to cephaloridine (a potentially nephrotoxic beta-lactam antibiotic) toxicity than did control cells. The present study suggests that OAT1 is the major organic anion transporter in the kidney that is responsible for the renal secretion of antibiotics, especially that of beta-lactam antibiotics. Furthermore, the culture cell system expressing OAT1 was revealed to be useful for the prediction of the nephrotoxicity of beta-lactam antibiotics.  (+info)

Inducible oxacillin-hydrolyzing penicillinase in Aeromonas hydrophila isolated from fish. (3/158)

An inducible penicillinase was shown to be present in a strain of Aeromonas hydrophila subsp. hydrophila isolated from freshwater fish. Enzyme induction was observed with benzylpenicillin or 6-aminopenicillanic acid, and the enzyme was cell bound. The penicillinase was purified 50-fold from a crude cell extract. The molecular weight was estimated to be 23,000 by gel filtration. The pH and temperature optima for the enzyme activity were 8.0 and 35 degrees C, respectively. The penicillinase showed a unique substrate profile by hydrolyzing oxacillin about twice as rapidly as benzylpenicillin. The enzyme activity was weakly inhibited by sodium chloride but was not affected by p-chloromercuribenzoate. The property of penicillinase production by the A. hydrophila strain could not be transferred to Escherichia coli and also could not be eliminated from the bacteria by ethidium bromide treatment.  (+info)

Role of organic anion transporter 1 (OAT1) in cephaloridine (CER)-induced nephrotoxicity. (4/158)

Role of organic anion transporter 1 (OAT1) in cephaloridine (CER)-induced nephrotoxicity. BACKGROUND: Cephaloridine (CER) has been used to elucidate the mechanisms of cephalosporin antibiotic-induced nephrotoxicity. Organic anion transporters have been thought to mediate CER uptake by the proximal tubule. The purpose of this study was to elucidate the possible involvement of organic anion transporter 1 (OAT1) in CER-induced nephrotoxicity. METHODS: A mouse terminal proximal straight tubule (S3) cell line stably expressing rat OAT1 (S3 rOAT1) was established and used in this study. The cellular uptake of [14C]-para-aminohippuric acid (PAH), a prototype organic anion, and that of [14C]-CER were measured. The effects of CER on the viability of the cells and the amount of lipid peroxidation were estimated. RESULTS: S3 rOAT1 expressed a functional organic anion transporter in the cytoplasmic membrane, and exhibited CER uptake activity. CER treatment resulted in a more significant decrease in the viability and a more significant increase in the amount of lipid peroxidation in S3 rOAT1 than in S3 cells transfected with an expression vector lacking the rOAT1 insert. Probenecid, an inhibitor of organic anion transport, and probucol, an antioxidant, significantly suppressed the decrease in viability and increase in the amount of lipid peroxidation in S3 rOAT1 treated with CER. The effects of various cephalosporin antibiotics on the uptake of [14C]PAH were correlated significantly with the effects of these drugs on cell viability. CONCLUSIONS: These results suggest that rOAT1 is, at least in part, responsible for the cellular uptake of CER and therefore CER-induced nephrotoxicity.  (+info)

Antibiotics and the Aberdeen typhoid outbreak in 1964. (5/158)

This paper gives an abbreviated account of part of a research programme which followed the Aberdeen typhoid outbreak in 1964. Chloramphenicol, the main antibiotic used in treatment, was shown to have a minimum inhibitory concentration (MIC) of between 5 and 2-5 mug./ml. for the S. typhi phage type 34 of the outbreak. The MIC for methacycline was between 5 and 2-5 and 2 mug./ml. Whereas the deep and shallow broth techniques used gave similar results with these antibiotics, the MIC for ampicillin, and also cephaloridine, was less in the deep than in the shallow broths. Serum assays in patients given ampicillin or cephaloridine yielded abnormally high concentrations of both antiboitics when S. typhi phage type 34 was the test organism whereas, with other test organisms, the concentrations were within expectation. These abnormally high values fell within expected values when the sera under investigation had first been heated to 56 degrees C. for 30 min. before assay against the S. typhi of the outbreak. The findings with ampicillin suggested that dosages given were satisfactory. With cephaloridine the concentrations found in patients' sera seemed to show that twice daily doses of 0-5 g. fell short of adequacy.  (+info)

beta-lactam antibiotics as substrates for OCTN2, an organic cation/carnitine transporter. (6/158)

Therapeutic use of cephaloridine, a beta-lactam antibiotic, in humans is associated with carnitine deficiency. A potential mechanism for the development of carnitine deficiency is competition between cephaloridine and carnitine for the renal reabsorptive process. OCTN2 is an organic cation/carnitine transporter that is responsible for Na(+)-coupled transport of carnitine in the kidney and other tissues. We investigated the interaction of several beta-lactam antibiotics with OCTN2 using human cell lines that express the transporter constitutively as well as using cloned human and rat OCTN2s expressed heterologously in human cell lines. The beta-lactam antibiotics cephaloridine, cefoselis, cefepime, and cefluprenam were found to inhibit OCTN2-mediated carnitine transport. These antibiotics possess a quaternary nitrogen as does carnitine. Several other beta-lactam antibiotics that do not possess this structural feature did not interact with OCTN2. The interaction of cephaloridine with OCTN2 is competitive with respect to carnitine. Interestingly, many of the beta-lactam antibiotics that were not recognized by OCTN2 were good substrates for the H(+)-coupled peptide transporters PEPT1 and PEPT2. In contrast, cephaloridine, cefoselis, cefepime, and cefluprenam, which were recognized by OCTN2, did not interact with PEPT1 and PEPT2. The interaction of cephaloridine with OCTN2 was Na(+)-dependent, whereas the interaction of cefoselis and cefepime with OCTN2 was largely Na(+)-independent. Furthermore, the Na(+)-dependent, OCTN2-mediated cellular uptake of cephaloridine could be demonstrated by direct uptake measurements. These studies show that OCTN2 plays a crucial role in the pharmacokinetics and therapeutic efficacy of certain beta-lactam antibiotics such as cephaloridine and that cephaloridine-induced carnitine deficiency is likely to be due to inhibition of carnitine reabsorption in the kidney.  (+info)

R-factor mediated beta-lactamase production by Haemophilus influenzae. (7/158)

Production of beta-lactamase by 15 strains of Haemophilus influenzae has been investigated. All the strains produce a constitutive beta-lactamase, which readily hydrolyses penicillin G, ampicillin, and cephaloridine. The beta-lactamase produced by these strains is indistinguishable from the type-IIIa enzyme commonly found in strains of Escherichia coli. The beta-lactamase gene has been transferred from the enzyme-producing strains of Haemophilus to strains of H. parainfluenzae and a strain of E. coli.  (+info)

Current status of the treatment of syphilis. (8/158)

Penicillin remains the treatment of choice for syphilis, with sustained low blood levels curing virtually all patients having early syphilis and halting disease progression in most patients with symptomatic syphilis. Tetracycline, erythromycin or cephalothin yields similar cure rates for patients with early syphilis who are allergic to penicillin. The efficacy of non-penicillin regimens for the treatment of late syphilis is uncertain. Results of Venereal Disease Research Laboratory (VDRL) or other reagin tests should become negative or remain at very low titer following adequate therapy, although results of Treponema pallidum immobilization (TPI) and fluorescent treponemal antibody-absorbed (FTA-ABS) tests often remain positive.  (+info)

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