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PubMed1990

Hepatic changes produced by 30-day administration of a novel aminocyclitol antibiotic, trospectomycin sulfate, to laboratory animals.

The studies described here were done to characterize the hepatic response to a new aminocyclitol antibiotic, trospectomycin sulfate, administered intravenously (beagle dog) or subcutaneously (Sprague-Dawley rat) at a variety of dose levels, to investigate reversibility of observed changes, and to document any untoward effects of subchronic trospectomycin sulfate administration. Both species showed significant elevations in serum levels of alanine and aspartate transaminases in higher dose groups. In the dog only, a transient neuromuscular blockade was also observed within higher dose groups. No other functional, morphological, or serum chemical changes were observed. Examination of liver by electron microscopy revealed the presence of cytoplasmic lamellar inclusion bodies, concentrated in the bile canalicular region of the hepatocytes. Occurrence of the lamellar bodies and coincident transaminase increases were found to be reversible upon discontinuance of treatment (studied in the dog). Electron microscopy of acid phosphatase cytochemistry in the rat indicated that most, but not all, of the lamellar bodies contained this enzyme. This observation suggests that they may be derived from the lysosome, or once formed become lysosomal.

Acid Phosphatase↗

PubMed1991

Mutants lacking individual ribosomal proteins as a tool to investigate ribosomal properties.

We have isolated and characterized mutants which lack one or two of sixteen of the proteins of the Escherichia coli ribosome. The mutation responsible in each case mapped close to, and probably in, the corresponding gene. A conditional lethal phenotype and a variable degree of impairment in growth was observed. The missing protein was readily restored to the organelle if E coli or other eubacterial ribosomal proteins were added to a suspension of the mutant particles. The mutants have been used to investigate the role of individual proteins in ribosome function and assembly. They have also aided in the topographic pinpointing of proteins on the surface of the organelle.

Aminoglycosides↗

PubMed1982

Isolation of the origin of replication of the IncW-group plasmid pSa.

The origin of replication of the IncW plasmid pSa has been cloned and the function of this origin in Escherichia coli examined. A 1.9-kb region of DNA is required for efficient autonomous replication, and a 0.47-kb fragment within this region can initiate replication only in the presence of an autonomously replicating derivative of pSa. An Mr 35,000 protein (repA) is encoded adjacent to the origin and is required for efficient initiation of replication. The derivatives examined provide information suggesting a direct role of partition factors in plasmid replication and incompatibility.

Bacterial Proteins↗

PubMed1982

Construction and characterization of new cloning vehicles. VI. Plasmid pBR329, a new derivative of pBR328 lacking the 482-base-pair inverted duplication.

The 4150-bp plasmid pBR329 was constructed by the the insertion into pBR327 of an 877-bp DNA fragment carrying the Cmr gene from pBR328. This new cloning vector does not contain the 482-bp inverted duplication that has been reported to be present in pBR325 and pBR328 (Prentki et al., 1981). In pBR329 the Cmr gene lacks its original promoter but is transcribed counterclockwise toward the Apr gene by a promoter located to the right of the HindIII site in the Tcr gene.

Base Sequence↗

PubMed1988

Two new tools: multi-purpose cloning vectors that carry kanamycin or spectinomycin/streptomycin resistance markers.

We have constructed two new multi-purpose cloning vectors, pJKKmf(-) and pJKSp/Smf(-), that carry resistance to kanamycin (Km) and spectinomycin/streptomycin (Sp/Sm), respectively. These plasmids are based on pGEM3Zf(-) and therefore contain a pUC-vector-derived multiple cloning site for 13 restriction sites flanked by T7 and SP6 RNA polymerase promoters, the alpha-peptide coding region of beta-galactosidase for blue/white colony screening and the bacteriophage f1 origin of replication for production of single-stranded DNA in the presence of a helper phage. We have used these vectors to reclone sequences from a maize genomic library, to produce radiolabeled RNA probes and to make single-stranded DNA.

Cloning, Molecular↗

PubMed1992

Novel streptococcal-integration shuttle vectors for gene cloning and inactivation.

Seven new streptococcal integration shuttle vectors have been constructed which contain different antibiotic-resistance-encoding genes capable of expression in both Streptococcus sp. and Escherichia coli. These plasmids can replicate in E. coli, but not in streptococci because of the absence of a streptococcal origin of replication. The size, antibiotic resistance, and number of unique restriction sites available for cloning for each plasmid are as follows: pSF141 (7.6 kb, CmR and KmR, 7 sites), pSF143 (5.7 kb, TcR, 6 sites), pSF148 (7.3 kb, CmR and SpR, 7 sites), pDL285 (3.4 kb, KmR, 3 sites), pDL286 (3.1 kb, SpR, 4 sites), pSF151 (3.5 kb, KmR, 10 sites), pSF152 (3.2 kb, SpR, 9 sites). If these plasmids carry a fragment of streptococcal DNA they can specifically integrate into the chromosome via Campbell-like, homologous recombination. Therefore, they should be useful for gene inactivation, cloning, chromosomal walking, or linkage analysis in streptococci. The availability of these integration plasmids resistant to different antibiotics, along with the previously described plasmid, pVA891 (ErR), should also allow the construction of mutants possessing multiple insertionally inactivated genes useful for a variety of genetic studies.

Cloning, Molecular↗

PubMed1995

Direct linkage of str-, S10- and spc-related gene clusters in Thermus thermophilus HB8, and sequences of ribosomal proteins L4 and S10.

We have analyzed the genomic region harboring str-S10-spc-related gene clusters in the thermophilic bacterium, Thermus thermophilus (Tt) HB8. This study was initiated for the purpose of isolating the gene encoding ribosomal (r-) protein S10 which is assumed to be involved in the antitermination of transcription at the rRNA-encoding genes in Bacteria. The S10-related gene cluster encodes the same set of r-proteins as in Escherichia coli. However, the gene coding for elongation factor Tu (the last gene of the str operon in E. coli) is separated by only eight nucleotides (nt) from the gene encoding r-protein S10 (the first gene of the S10 operon in E. coli), and the genes encoding r-protein S17 (the last gene of the S10 operon in E. coli) and L14 (the first gene of the spc operon in E. coli) overlap. This suggests that all three gene clusters are cotranscribed from a single promoter preceding the str-related operon. In addition, we determined the complete nt sequences of the Tt genes encoding r-proteins L4 and S10. Tt L4 shows the lowest degree of conservation among the known L4 r-proteins from Bacteria. Tt S10 has the highest proportion of basic amino acids (aa) and the lowest number of acidic aa, as compared with its homologues from Bacteria and Archaea, which might be related to its possible role in binding to the boxA RNA of nascent rRNA transcripts at high temperatures.

Amino Acid Sequence↗

PubMed1996

Genetic and transcriptional organization of the Bacillus subtilis spc-alpha region.

We used chromosomal walking methods to isolate a 10.8-kb region from the major ribosomal protein (r-protein) gene cluster of Bacillus subtilis (Bs). The gene order in this region, given by gene product, was r-proteins L16-L29-S17-L14-L24-L5-S14-S8-L6-L18-S5-L30-L15-SecY-adenylate kinase (Adk)-methionine aminopeptidase (Map)-initiation factor 1 (IF1)-L36-S13-S11-alpha subunit of RNA polymerase-L17. The region cloned, therefore, contains the homologues for the last three genes of the Escherichia coli (Ec) S10 operon, together with entire spc and alpha operons. This Bs organization differs from the corresponding region in Ec by the inclusion of the genes encoding Adk, Map and IF1 between the genes encoding SecY and L36. Plasmid integration experiments indicated that all 22 genes comprise a single large transcriptional unit controlled from a major promoter which lies upstream from the gene encoding r-protein L16. Promoter probe experiments located lesser activities internal to this large transcriptional unit, the secY and map promoters. The secY promoter region (psecY) contained two activities, each principally functioning in the stationary growth phase when high protein export is required. Thus, the Bs S10-spc-alpha region differs from its Ec counterpart in both genetic and transcriptional organization. Given this difference in transcriptional organization, the mechanisms coordinating expression of the translational apparatus are also likely to differ between Ec and Bs.

Amino Acid Sequence↗

PubMed1995

Cultured hepatocytes as investigational models for hepatic toxicity: practical applications in drug discovery and development.

Drugs can fail at any phase during discovery, preclinical or clinical development due to unacceptable levels of toxicity, and liver is commonly the principle target organ. Investigational toxicology methods, using appropriate models and hypotheses, can often resolve problems, identify toxic chemical substituents and salvage therapeutic discovery programs. While in vivo models are used to investigate hepatic drug effects in the context of toxicokinetics and systemic influences, cell culture models provide in vitro systems for investigating specific mechanisms in a precisely controlled environment. Using primary hepatocytes isolated from laboratory animals, we have explored several drug-induced hepatic disorders that surfaced during different phases of drug discovery and development. Additionally, the use of human hepatocytes has allowed us to address concerns for human exposure, examine human relevance of animal data, and provide perspective on problems encountered in clinical trials.

Animals↗

PubMedDate not supplied

Haemophilus test medium interpretive criteria for disk diffusion susceptibility tests with cefdinir, cefetamet, cefmetazole, cefpodoxime, cefdaloxime (RU29246, HR-916 metabolite), and trospectomycin.

The disk diffusion zones and the MICs of six newer antimicrobials with significant activity against Haemophilus influenzae were compared using the Haemophilus test medium (HTM) and National Committee for Clinical Laboratory Standards methods. The rank order of potency was cefpodoxime (MIC50, < or = 0.03 micrograms/ml) > cefetamet > cefdinir > cefdaloxime = trospectomycin > cefmetazole (MIC50, 2 micrograms/ml). Susceptible breakpoint interpretive criteria for HTM tests were established that conformed to prior recommendations for each drug when tested against other species. Absolute agreement between methods ranged from 89% to 100% with < or = 1% false-susceptible interpretive errors. The recommended, tentative disk diffusion susceptible interpretive criteria were for 5-micrograms cefdinir disks > or = 20 mm (MIC correlate, < or = 1 micrograms/ml); for 10-micrograms cefetamet disks > or = 18 mm (MIC correlate, < or = 4 micrograms/ml); for 30-micrograms cefetamet disks > or = 21 mm (MIC correlate, < or = 4 micrograms/ml); for 30-micrograms cefmetazole disks > or = 16 mm (MIC correlate, < or = 16 micrograms/ml); for 10-micrograms cefpodoxime disks > or = 21 mm (MIC correlate, < or = 2 micrograms/ml); for 30-micrograms cefdaloxime disks > or = 23 mm (MIC correlate, < or = 2 micrograms/ml) and for 30-micrograms trospectomycin disks > or = 17 mm (MIC correlate, < or = 16 micrograms/ml). beta-Lactamase-negative, ampicillin-resistant (BLNAR) H. influenzae isolates consistently had the highest MICs for each cephalosporin tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Ampicillin Resistance↗

PubMed1992

The bactericidal activity and postantibiotic effect of trospectomycin.

Trospectomycin sulfate (trospectomycin, TRS) is a novel, broad-spectrum, aminocyclitol antibiotic that is being developed clinically for the treatment of upper respiratory tract infections, bacterial vaginosis, pelvic inflammatory disease, and gonorrhea. This study investigated the bactericidal activity (by time-kill kinetics) and the postantibiotic effect (PAE) of TRS. Species-dependent bacteriostatic/bactericidal activity was observed for TRS; the antibiotic was bacteriostatic for Staphylococcus epidermidis, Enterococcus faecalis, and Escherichia coli, and bactericidal for Haemophilus influenzae, Neisseria gonorrhoeae, Moraxella catarrhalis, and Bacteroides fragilis (one of two test strains). When TRS was tested at four times its minimum inhibitory concentration or at a maximum test concentration of 32 micrograms/ml, with a 1-hr exposure period, the following PAE values were recorded: S. epidermidis 30032, 1.8 hr, En. faecalis ATCC 29212, 1.6 hr, E. coli UC 311, 1.5 hr, E. coli UC 9451, 1.5 hr, H. influenzae 30063, greater than 4.0 hr, B. fragilis ATCC 25285, 5.2 hr, and B. fragilis UC 12199, 6.7 hr. The broad-spectrum PAE that was observed for TRS is somewhat unique compared with other antibiotics.

Anti-Bacterial Agents↗

PubMed1971