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Biomedical subjects

J Capobianco

Publications and source records attributed to J Capobianco.

5 recordsLinked to original sources
PubMed1999

Induction of ribosome methylation in MLS-resistant Streptococcus pneumoniae by macrolides and ketolides.

One major mechanism for resistance to macrolide antibiotics in Streptococcus pneumoniae is MLS (macrolide, lincosamide, and streptogramin B) resistance, manifested when the 23S rRNA is methylated by the product of an erm gene. This modification results in the decreased binding of all known macrolide, lincosamide, and streptogramin B antibiotics to the ribosome. More than 30 ermAM-containing clinical isolates of S. pneumoniae were examined in our lab and showed high-level resistance (MIC > or =128 microg/ml) to erythromycin, azithromycin, tylosin, clindamycin, and ketolide (macrolides that lack the cladinose sugar) TE-802. We found that the new generation of ketolides A965 and A088 displayed variable activity against the same group of resistant S. pneumoniae strains. To understand the basis of variability of the minimal inhibitory concentration (MIC) values of A965 and A088, we examined the effects of a series of macrolides and ketolides on the level of 23S rRNA methylation in five ermAM-containing resistant S. pneumoniae isolates. We show here that the basal levels of ribosomal methylation vary from strain to strain. The level of rRNA methylation can be strongly induced by erythromycin, azithromycin, and TE-802, resulting in high-level of resistance to these compounds. Ketolide A965 and A088, however, are weak inducers at sub-MIC drug concentrations, therefore showing variable activities in strains with differential methylation levels.

Anti-Bacterial Agents↗
PubMed1994

Characterization of (1,3)-beta-glucan synthase in Candida albicans: microsomal assay from the yeast or mycelial morphological forms and a permeabilized whole-cell assay.

A systematic evaluation of the in vitro (1,3)-beta-glucan synthase assay parameters was performed using microsomes prepared from Candida albicans from either yeast or mycelial phase cells. Enzyme activities of both yeast and mycelial phase microsomes depended on the presence of guanosine-5'-O-(3-thiophosphate) and either bovine serum albumin or a detergent [W-1 (polyoxyethylene ether detergent) or Brij-35 (polyoxyethylene ether, 23 lauryl ether)]. Brij-35 was included in standard assays as it was compatible with the permeabilized whole-cell assay. Microsomes derived from both the yeast and mycelial phases generally yielded similar glucan synthase activities under a range of different assay conditions. Brij-35 significantly stabilized the enzyme, yielding a half-life of 5.6 d at 4 degrees C, compared with 0.9 d without detergent. The addition of detergent during mechanical breakage of yeast cells dramatically improved glucan synthase stability and activity. Enzyme catalysis was linear for at least 75 min with 100 micrograms protein from microsomes of yeast cells grown to mid-exponential phase, with an apparent Km for UDP-glucose of 1.1 mM. The pH and temperature optima were 7.75 and 30 degrees C, respectively. Glucan synthase activity was highest in cells derived from early mid-exponential phase and declined to a basal level by stationary phase. A permeabilization-based in situ assay for glucan synthase was developed. Cells were permeabilized with 2% (v/v) solution of toluene/methanol (1:1) and assayed for glucan synthase activity using standard reaction mixtures. Reactions were linear for 30 min and were inhibited by known inhibitors of glucan synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoglycosides↗
PubMed1994

Tight binding of clarithromycin, its 14-(R)-hydroxy metabolite, and erythromycin to Helicobacter pylori ribosomes.

Clarithromycin is a recently approved macrolide with improved pharmacokinetics, antibacterial activity, and efficacy in treating bacterial infections including those caused by Helicobacter pylori, an agent implicated in various forms of gastric disease. We successfully isolated ribosomes from H. pylori and present the results of a study of their interaction with macrolides. Kinetic data were obtained by using 14C-labeled macrolides to probe the ribosomal binding site. Clarithromycin, its parent compound erythromycin, and its 14-(R)-hydroxy metabolite all bound tightly to H. pylori ribosomes. Kd values were in the range of 2 x 10(-10) M, which is the tightest binding interaction observed to date for a macrolide-ribosome complex. This tight binding was due to very slow dissociation rate constants of 7.07 x 10(-4), 6.83 x 10(-4), and 16.6 x 10(-4) min-1 for clarithromycin, erythromycin, and 14-hydroxyclarithromycin, respectively, giving half-times of dissociation ranging from 7 to 16 h, the slowest yet measured for a macrolide-ribosome complex. These dissociation rate constants are 2 orders of magnitude slower than the dissociation rate constants of macrolides from other gram-negative ribosomes. [14C]clarithromycin was bound stoichiometrically to 50S ribosomal subunits following incubation with 70S ribosomes and subsequent separation of the 30S and 50S subunits by sucrose density gradient centrifugation. These data predict that the lower MIC of clarithromycin compared with that of erythromycin for H. pylori is likely due to a faster rate of intracellular accumulation, possibly because of increased hydrophobicity.

Centrifugation, Density Gradient↗
PubMed1984

Isolation and analysis of inhibitors of transposon Tn3 site-specific recombination.

We have constructed a genetic bioassay for inhibitors of site-specific recombination by transposon Tn3 resolvase. Of 6,000 compounds tested, 26 inhibited in vivo, and 5 of these 26 inhibited in vitro. At least two inhibitors also inhibit the topoisomerase of resolvase. We have also identified analogs of A1062 which inhibit.

DNA Transposable Elements↗