Presence Of Rifamycin Research Articles

Marinomonas epiphytica sp. nov., isolated from a marine intertidal macroalga.

A novel Gram-stain-negative, aerobic marine bacterial strain, SAB-3T, was isolated from brown macroalgae (Dictyota sp.) growing in the Arabian sea, Goa, India. The strain grew optimally at 30 °C, with 2.0-4.0 % (w/v) NaCl and at pH 7.0 on marine agar medium. Strain SAB-3T was unable to hydrolyse aesculin and did not grow in the presence of rifamycin but showed resistance to antibiotics such as cefadroxil and co-trimoxazole. The major fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and C16 : 0, and Q-8 was the major ubiquinone. The major polar lipids were phosphatidylglycerol and phosphatidylethanolamine. The DNA G+C content was 41.0 mol%. 16S rRNA gene sequencing and phylogenetic analysis indicated that the strain was a member of the genus Marinomonas with Marinomonas aquiplantarum IVIA-Po-159T (97.6 % similarity), Marinomonas posidonica IVIA-Po-181T (97.5 %) and Marinomonas dokdonensis DSM 17202T (97.4 %) as the closest relatives. Whole genome relatedness determined through DNA-DNA hybridization revealed values of 40-50 % (below the 70 % threshold recommended for species delineation) with the above three species, thus confirming it as representing a distinct and novel species of the genus Marinomonas for which the name Marinomonas epiphytica sp. nov. is proposed. The type strain is SAB-3T (=JCM 31365T=KCTC 52293T=MTCC 12569T).

Understanding the interplay of drug transporters involved in the disposition of rosuvastatin in the isolated perfused rat liver using a physiologically-based pharmacokinetic model

The role of hepatic uptake (Oatp1a1 and Oatp1b4) and efflux (Bcrp and Mrp2) transporters in the disposition of rosuvastatin were investigated using the isolated perfused rat liver (IPRL). A simple physiologically-based pharmacokinetic model was developed to quantitatively determine the interplay between the individual transporters.Uptake and elimination of rosuvastatin in the IPRL was rapid and extensive. In the presence of rifamycin (an equipotent inhibitor of both Oatp1a1 and Oatp1a4) the perfusate clearance of rosuvastatin was reduced, but rifampicin (a potent inhibitor of Oatp1a4) had no effect upon the perfusate clearance. This might indicate a limited role for Oatp1a4, but it is possible that Oatp1a1 (or other uptake transporters) may have redundancy in their affinity for rosuvastatin.In the presence of GF120918 (a potent inhibitor of Bcrp) and in the Wistar TR- rat (a naturally occurring mutant not expressing Mrp2) the biliary clearance was reduced and virtually abolished in the TR- pre-incubated GF120918. Bcrp and Mrp2 appear to represent the primary efflux mechanisms for rosuvastatin in the rat.Rosuvastatin disposition in the IPRL is mediated in part by Oatp1a1 and efflux is almost entirely by Mrp2 and Bcrp. Other uptake processes may be involved.

Screening of rifamycin producing marine sponge bacteria by LC–MS–MS

HPLC–MS–MS has been used for the identification and characterisation of rifamycin B and rifamycin SV in various strains of the marine sponge-derived bacterium Salinispora. Gradient elution using acetonitrile/water/ammonium acetate was used to separate the rifamycins from the matrix and negative ion-electrospray mass spectrometry was used for detection and confirmation. The presence of rifamycin in bacterial extracts was confirmed by matching retention times, parent ion spectra and the fragmentated parent ion spectra of the standard compounds and the bacterial extracts. All strains of the marine sponge bacterium Salinispora tested were found to contain rifamycin thus an alternate actinobacterial source of rifamycin was established.

Monitoring the ingestion of anti-tuberculosis drugs by simple non-invasive methods

This investigation retrospectively assessed inexpensive non-invasive qualitative methods to monitor the ingestion of anti-tuberculosis drugs isoniazid, rifampicin and rifapentine. Results showed that commercial test strips detected the isoniazid metabolites isonicotinic acid and isonicotinylglycine as efficiently as the isonicotinic acid method in 150 urine samples. The presence of rifamycins in urine samples ( n = 1085) was detected by microbiological assay techniques and the sensitivity compared to the n-butanol extraction colour test in 91 of these specimens. The proportions detected by the two methods were significantly different and the sensitivity of the n-butanol procedure was only 63.8% (95% CL 51.2–76.4%) as compared to that of the superior microbiological method. Final validation ( n = 691) showed that qualitative assays measure isoniazid and rifamycin ingestion with an efficiency similar to high-performance liquid chromatography. The qualitative procedures may therefore be valuable in clinical trials and in tuberculosis clinics to confirm drug ingestion.

A Comparative Study of the Redox-Cycling of a Quinone (Rifamycin S) and A Quinonimine (Rifabutin) Antibiotic by Rat Liver Microsomes

Rifamycin S and rifabutin are clinical drugs used to treat tuberculosis and leprosy. The formation of reactive oxygen species during the redox-cycling of rifamycin S (quinone) and rifabutin (quinonimine) was evaluated. The semiquinone (or semiquinonimine) and hydroquinone (or hydroquinonimine) formed during the reduction of the parent molecules by microsomal electron transfer in the presence of nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) or nicotinamide-adenine dinucleotide, reduced (NADH) reoxidizes in air to generate superoxide radical and hydrogen peroxide. In the presence of added iron, hydroxyl radicals, formed by the Fenton reaction, were detected using 5,5′-dimethyl-1-pyrroline-N-oxide as the spin-trap. Rifamycin S, a quinone, redox cycles more efficiently than rifabutin, a quinonimine, as approximately five times the concentration of hydroxyl radical adduct of 5,5′-dimethyl-1-pyroline-N-oxide (DMPO) was detected, when compared with rifabutin. The NADPH-dependent microsomal production of hydroxyl radical in the presence of rifamycin S was somewhat higher than the NADH-rifamycin S system with most iron chelators. However, with rifabutin, NADH-dependent microsomal production of hydroxyl radical was higher than that found with the NADPH-rifabutin system. An exception was the iron chelator, diethylene-triamine-pentacetic acid (DTPA), in which NADPH-dependent rates exceeded the rates with NADH with both antibiotics. Rat liver sub-mitochondrial particles also generated hydroxyl radical in the presence of NADH and either rifamycin S or rifabutin. The electron transport chain inhibitors such as rotenone and antimycin A enhanced the signal intensity of DMPO-OH, suggesting NADH dehydrogenase (complex I) as the major component involved in the reduction of rifamycin S. Rifamycin S was shown to be readily reduced to rifamycin SV, the corresponding hydroquinone by Fe(II); under similar conditions Fe(II) did not reduce rifabutin. Using optical spectroscopy, we determined that rifamycin S forms a complex with Fe(II). The stoichiometry of the complex was Fe(rifamycin S) 3 in phosphate buffer at pH 7.4. Rifabutin did not form a detectable complex with Fe(II). The redox cycling of rifamycin S and rifabutin did not cause microsomal lipid peroxidation. In fact, the Fe:ATP induced lipid peroxidation was completely inhibited by these two molecules. These results indicate that rifamycin S and rifabutin can interact with rat liver microsomes to undergo redox-cycling, with the subsequent production of hydroxyl radicals when iron complexes are present. Compared to NADPH, NADH is almost as effective (rifamycin S) or even more effective (rifabutin) in promoting these interactions. These interactions may play a role in the hepatotoxicity associated with the use of these antibiotics. Copyright © 1996 Elsevier Science Inc.

Transformation of rifamycin B with growing and resting cells of Curvularia lunata

Growing and resting cell systems of Curvularia lunata were used for the transformation of rifamycin B to rifamycin S. In the case of growing cells, rifamycin B was added at the time of inoculation and at the different phases of growth. Interestingly, it was found that C. lunata could grow in the presence of rifamycin B and could convert rifamycin B to rifamycin S. Growing cells 24 and 48 h of age were capable of transforming rifamycin B. Resting cells, cultivated at the exponential or early stationary phase, were found to be very active, and the resting cells of different ages were repeatedly used for the transformation reaction. Growing cells of 72 and 96 h were not capable of transforming rifamycin B, whereas resting cells of similar ages were very active. Due to the adsorption of rifamycins by the growing and resting cells of C. lunata, the stoichiometric yield of rifamycin S was not obtained.

RNA and ribosomal protein patterns during aerial spore germination in Streptomyces granaticolor.

Disruption of the external sheath of Streptomyces granaticolor aerial spores and subsequent cultivation in a rich medium result in a synchronous germination. This method was used to analyze RNA and protein patterns during the germination. The germination process took place through a sequence of time-ordered events. RNA and protein synthesis started during the first 5 min and net DNA synthesis at 60-70 min of germination. Within the first 10 min of germination, synthesis of RNA was not sensitive to the inhibitory effect of rifamycin. During this period rRNA and other species including 4-5-S RNA were synthesized. Dormant spores contained populations of ribosomes or ribosomal precursors that were structurally and functionally defective. The ribosomal particles bound a sporulation pigment(s) of the melanine type. The ribosomal proteins complexed to the pigments formed insoluble aggregates which were easily removed from the ribosomes by one wash with 1 M NH4Cl. During the first 10 min of germination, pigment(s) were liberated from the complexes with the ribosomes and protein extracts of the washed ribosomes had essentially the same pattern as the extracts of ribosomes of vegetative cells. These structural alterations were accompanied by enhancement of the ribosome activities in polypeptide synthesis in vivo and in vitro. When the spores were incubated with a 14C-labelled amino acid mixture in the presence of rifamycin, only three proteins (GS1, GL1 and GS9) were identified to be radiolabelled in the extracts from the washed ribosomes. These experiments indicate that liberation of the sporulation pigment(s) from the complexes with ribosomal proteins and assembly of de novo synthesized proteins and proteins from a preexisting pool in the spore are involved in the reactivation of the ribosomes of dormant spores of S. granaticolor.

Glucocorticoids modify the rate of ribosomal RNA synthesis in rat thymus cells by regulating the polymerase elongation rate

The mechanism by which glucocorticoids inhibit RNA polymerase A activity, and hence rRNA synthesis, in rat thymus cells has been investigated. Studies of the intranuclear distribution of RNA polymerase A between chromatin bound (“engaged”) and unbound (“free”) forms revealed that the steroid-mediated inhibition of the activity of the “engaged” form of the enzyme was not accompanied by significant changes in “free” pool activity. In the presence of rifamycin AF/0-13, an inhibitor of re-initiation of RNA polymerase A, the rate of [ 3H]UMP incorporation into RNA was slower in nuclei from steroid-treated cells than in those from control cells, although in both conditions similar plateau levels of UMP incorporation were attained. Direct measurements of the numbers of transcribing RNA polymerase A molecules and of elongation rates showed that the inhibition of pre-rRNA synthesis was the result of a decrease in enzyme elongation rate; no significant change was observed in the number of transcribing enzymes. The steroid-induced inhibition of pre-rRNA synthesis was selectively abolished by mild proteolysis of nuclei, suggesting the involvement of a labile, regulatory glucocorticoid-induced protein. It is concluded that glucocorticoid treatment of rat thymus cells decreases 45S rRNA synthesis primarily by decreasing the polyribonucleotide elongation rate of RNA polymerase A, possibly by modification of the enzyme.

In vitro transcription by mouse plasmocytoma RNA polymerase II supplemented with nuclear protein fractions

Mouse plasmocytoma (MOPC 31C) nuclear proteins were mildly extracted and fractionated into four fractions. Among them, a nonhistone (CP-I) or a saline-wash (NS-I) fraction enhanced the activity of RNA polymerase II (enzyme II) purified from MOPC 31C when each of these fractions was preincubated with enzyme II and native DNA before incubation for RNA synthesis in the presence of rifamycin AF/013. The enhancing activity of these fractions depended on the preincubation time, and was heat and trypsin sensitive. These results suggest the presence of chromatin-bound and unbound protein factors capable of accelerating some preinitiation reaction of transcription catalyzed by RNA polymerase II. The kinetics profile of the rifamycin AF/013-resistant transcription enhanced by fraction NS-I was evidently different from those observed when DNase I (or II)-treated or sonicated DNA was used as template. This fact as well as inefficiency of any kind of DNA other than native eucaryotic DNA for the enhancement by fraction NS-I indicate that the enhanced transcription takes place at certain specific sites of DNA. Evidence for a direct interaction between enzyme II and a factor involved in fraction NS-I was obtained by the following three kinds of analysis: kinetics employing preincubation of enzyme II and fraction NS-I without DNA; sucrose gradient centrifugation and gel permeation chromatography; and electrophoresis under nondenaturing conditions. The results of these analyses suggest that the enhancement by this factor correlates with some structural alteration of enzyme II accompanied by a change in its surface charge.

Studies on sex-organ development. Changes in the oestrogenic response of the chick Müllerian duct as measured by chromatin template and ribonucleic acid initiation capacity

Assays of transcription in vitro, with Escherichia coli RNA polymerase or wheat-germ RNA polymerase II, were used to characterize chromatin templates isolated from the left Müllerian duct of the chick embryo during normal development, and during development in the presence of diethylstilboestrol. Control Müllerian-duct template capacity with E. coli RNA polymerase decreased from 6.42% on day 10 to 4.34% by day 15 of development. Similar results were found with wheat-germ RNA polymerase II. In the presence of rifampicin and heparin, the prokaryotic enzyme transcribed a number-average RNA chain of 670 nucleotide residues, at an average rate of 110 nucleotide residues/min, from Müllerian-duct chromatin of all developmental stages. From day 10 to day 15 there was a 44% decrease in the number of initiation sites for E. coli RNA polymerase on Müllerian-duct chromatin. A 47% decline was observed when these chromatins were transcribed with excess RNA polymerase II in the presence of rifamycin Af/013. Signs of increasing responsiveness to oestrogen developed between days 10 and 16. Embryos exposed to maximally responsive doses of diethylstilboestrol for 2 days showed increases in Müllerian-duct chromatin template capacity, RNA-chain initiation sites, wet weight, protein and RNA. The changes seen in the oviduct of the 1-week-old chick injected for 2 days with diethylstilboestrol were defined as 100% responses. By comparison, the Müllerian duct, after exposure to diethylstilboestrol from day 10 to day 12, from day 13 to day 15 or from day 16 to day 18, showed a 15%, 39% and 72% template response respectively, and a 42%, 56% and 85% initiation-site change respectively. A similar developmental trend was observed in all parameters. It is concluded that oestrogenic responsiveness in the developing Müllerian duct increases from day 10 to nearly maximal values by day 16 of development, and that this transition is paralleled by a progressive restriction of genomic activity.

Nuclear transcription in vitro. Sensitivity to inhibition by ribosyldichlorobenzimidazole and rifamycin AF/013

L cell nuclear preparations were shown to transcribe RNA for periods up to 1 h at 37 degrees C. Nearly 70% of the transcription products were sensitive to inhibition by 1 microgram/mL of alpha-amanitin, indicating that they were transcribed by RNA polymerase II. Analysis of polyphosphorylated termini of in vitro synthesized RNA showed the presence of a phosphatase activity which prevents quantitative recovery of these termini. The finding of in vitro labeled polyphosphorylated termini in RNA greater than 12 S after short periods of incubation shows initiation in vitro for this size class. The labeling of these polyphosphorylated termini is decreased in the presence of rifamycin AF/013. The use of two apparent inhibitors of initiation, rifamycin AF/013 and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), has allowed detection of in vitro initiated transcripts of heterogeneous nuclear RNA. Both of these inhibitors act primarily at later times of incubation, in contrast to alpha-amanitin which acts on elongation and inhibits in vitro RNA synthesis immediately. The selective pattern of DRB inhibition on hnRNA is retained in vitro and some accumulation of large-size molecules is observed. It can be estimated that about 30% of the greater than 12S hnRNA sequences transcribed in vitro are sensitive to DRB and 48% of greater than 12S RNA are sensitive to rifamycin AF/013 inhibition.

Replication of RNA bacteriophages in the presence of rifamycin

Replication of RNA bacteriophages in the presence of rifamycin was studied in different Escherichia coli strains that vary in RNase content but are not isogenic: AB259 RNase+, Q13 RNase I- PNPase-, AB105 RNase I- RNase III-. It was found that rifamycin did not affect characteristics of phage replication such as the general pattern of viral RNA synthesis and intracellular development of the phage. These characteristics are strain specific and independent of the cell growth rate, which defines only phage release. The inhibition of cell division by rifamycin interfered with the release of the phage and thus produced an apparent effect of rifamycin on phage replication.

The influence of steroid-receptor complexes on the stages of transcription of target-tissue chromatin

Earlier investigations established that complexes formed between 5α-dihydrotestosterone and receptor macromolecules derived from rat ventral prostate subcellular fractions cause changes in the nature and extent of transcription of prostate chromatin by homologous RNA polymerases. The present report describes an investigation into relationships between concentrations of cellular receptor sites and initiation and elongation of RNA chains on prostatic chromatin from rats after castration or after androgen administration to castrated animals. Occupied receptor sites were measured by precipitation with protamine sulphate and replacement of endogenously bound steroid with exogenous [ 3H]-steroid. Initiation of RNA chains was measured by incorporation of [γ- 32P]-purine nucleotides and chain elongation by incorporation of [ 3H]-UMP under conditions which precluded reinitiation i.e. in the presence of rifamycin AF/013 or under high salt conditions. Castration resulted in a marked depletion of cellular receptors, particularly nuclear sites, and a decrease in initiation sites on chromatin. Testosterone propionate, depending on the time of administration, partially or completely maintained the nuclear content of receptor, but not cytoplasmic receptor. Androgen increased initiation sites above levels observed on prostate chromatin of castrated rats. RNA chain length appeared dependent on androgenic status, but other reasons for this effect are discussed.

The synthesis of nucleic acids in Bacillus subtilis infected with phage PBS 1.

Unlike other phage systems, the development of PBS 1 was found to be insensitive to rifamycin SV. The incorporation of 3H-uridine into trichloroacetic acid precipitable and alkali-labile material (RNA), in PBS-1-infected cells, was greatly reduced by rifamycin. Observations that RNA synthesized in the presence of rifamycin was hybridizable exclusively with the phage DNA and that actinomycin D inhibited the phage growth indicated that the synthesis of a new species of RNA was required for the development of PBS 1. The host DNA synthesis was reduced to a very low level 5 min after infection. The phage DNA synthesis was also apparently reduced markedly by rifamycin when determined with 3H-uridine as labelling material. On the other hand, rifamycin did not affect the incorporation of 3H-deoxycytidine into the phage DNA, suggesting that phage DNA synthesis was in fact insensitive to rifamycin. It is not clear how rifamycin inhibits the incorporation of 3H-uridine into nucleic acids in PBS-1-infected cells.

Rifamycin-resistant mutation in E. coli: effect of rifamycin on DNA-dependent RNA polymerase and on R17 phage growth.

Rifamycin-resistant mutants in E. coli, K12, Hfr strain, were isolated without mutagenic treatment. The frequency of natural mutation of this type appeared to be approximately 10−9. Selection of resistant strains was effected by plating 109 cells on agar plates containing 50 μg of rifamycin per milliliter. A total of 11 strains resistant to rifamycin thus isolated remained Hfr and, in the presence of rifamycin, permitted the growth of the RNA-containing, male specific coliphage R17, which is sensitive to rifamycin in a wild-type E. coli. DNA-dependent RNA polymerase was prepared from these resistant mutants and examined for sensitivity toward rifamycin and other antibiotics. It was found that the RNA polymerase of the resistant strain is indeed resistant to rifamycin. However, both rifamycin-resistant and -sensitive enzymes are inhibited by the other three antibiotics (acridine orange, ethidium bromide, actinomycin D), which are known to interact with the template molecule.

The effect of rifamycins in the ultrastructure of Anacystis montana

The fine structure alterations produced in Anacystis montana by rifamycins were studied. The basic normal structure of this vegetative cell is a cytoplasmic matrix which extends throughout the cell. Photosynthetic lamellae and various granular inclusions are irregularly distributed throughout the cytoplasmic matrix. In these areas vesicles or vacuoles are mainly evident. When these cells are incubated in the presence of rifamycins, numerous dense bodies, which are acid phosphatase positive, appear in the cytoplasmic matrix. The origin of these dense bodies is clearly in the nucleoplasm. Also, similar dense bodies or amorphous electron dense acid phosphatase-positive substances, appear within the vacuoles. These results are discussed.