ABSTRACT
A comprehensive set of bicyclomycin-resistant mutants of transcription termination protein Rho has been characterized in Escherichia coli by in vivo and in vitro assays. Several of the mutant Rho proteins have functional defects. Strains with either the L208R or the S266A mutation in the bacterial chromosome have a higher intracellular concentration of the Rho protein than strains containing a wild-type copy of the rho gene. Strains carrying the L187R, L208R or S266A mutations in the chromosome also have a mutant phenotype; a plasmid-located arabinose promoter is constitutively de-repressed in these strains. The L208R and S266A mutant strains also have a rate of growth defect. When the S266A mutation is located on a high-copy plasmid, the mutant grows more slowly than a wild-type strain. In contrast to the majority of the bicyclomycin-resistant mutants, these two mutants show clear phenotypic differences from wild-type cells. These differences are also seen in vitro. In vitro transcription termination by RhoL208R and RhoS266A is defective at the lambda tR1 terminator, but can be enhanced by NusG. These functionally defective Rho mutations have been located near the putative catalytic site on a model of Rho based on the F1-ATPase. This indicates that this region of the Rho molecule is crucial for Rho function. The crucial region overlaps the putative bicyclomycin-binding site, suggesting an explanation for the efficacy of bicyclomycin as an antibiotic.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Escherichia coli/genetics , Rho Factor/genetics , Drug Resistance, Microbial , Escherichia coli/growth & development , Escherichia coli/metabolism , Genes, Reporter , Models, Molecular , Mutation , Phenotype , Plasmids , Rho Factor/analysisABSTRACT
A total of 38 bicyclomycin-resistant mutants of Escherichia coli transcription termination protein Rho have been isolated. The locations of their mutations identify the ATP-binding region as the functional domain inhibited by bicyclomycin. Strains containing the S266C, S266A and L208R Rho mutations are very resistant to bicyclomycin in vivo. In a similar way, the mutant Rho proteins containing these mutations are very resistant to bicyclomycin in vitro. These data suggest that Ser266 and Leu208 might make direct contact with the antibiotic. These two residues are close to each other in the tertiary structure of a model of Rho based on the alpha and beta subunits of the F(1) ATPase, supporting the validity of the model. The strain containing the G337S Rho mutation also has high bicyclomycin resistance, and the proximity of L208, S266 and G337 in the quaternary structure of the Rho model has enabled a candidate bicyclomycin-binding pocket to be delineated. As a whole, the bicyclomycin sensitivities of the mutants are consistent with the locations of their respective mutations in the model of Rho based on the F(1) ATPase, therefore supporting the emerging consensus model of Rho structure.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Escherichia coli/drug effects , Mutation/genetics , Proton-Translocating ATPases/chemistry , Rho Factor/chemistry , Rho Factor/genetics , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Amino Acid Substitution/drug effects , Amino Acid Substitution/genetics , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/metabolism , DNA Mutational Analysis , Drug Resistance, Microbial/genetics , Escherichia coli/genetics , Hydrolysis/drug effects , Hydroxylamine/pharmacology , Microbial Sensitivity Tests , Models, Molecular , Molecular Sequence Data , Mutagenesis/drug effects , Mutagenesis/genetics , Mutagens/pharmacology , Mutation/drug effects , Phenotype , Protein Binding , Protein Conformation , Proton-Translocating ATPases/metabolism , Rho Factor/metabolismSubject(s)
Health Policy , Health Promotion/methods , Parenting , Parents/education , Pediatric Nursing/methods , Child , Humans , Job Description , State Medicine , United KingdomABSTRACT
This study shows that blood from young children (< 3 years) with end stage liver disease, contains less IGF-I, IGF-II and IGFBPs than blood from normally growing children without liver disease, despite the provision of adequate calories. These low levels are likely to contribute to growth failure in children with end stage liver disease.
Subject(s)
Growth Disorders/blood , Insulin-Like Growth Factor II/metabolism , Insulin-Like Growth Factor I/metabolism , Liver Diseases/blood , Carrier Proteins/blood , Child, Preschool , Chronic Disease , Female , Growth Disorders/etiology , Humans , Infant , Insulin-Like Growth Factor Binding Proteins , Liver Diseases/complications , MaleABSTRACT
We examined the effects of estradiol and progesterone agonist, promegestone (R5020), on secretion of insulin-like growth factors (IGF) and binding proteins (IGFBPs) by T-47D human breast cancer cells cultured in a serum-free defined medium. Estrogen, IGF-I and IGF-II promoted and R5020 inhibited growth under these conditions. Cells secreted IGFs and four IGFBPs. The amounts of all IGFBPs in cell-conditioned media were increased by estradiol and reduced by R5020, which also abolished the stimulatory effect of estradiol on IGFBPs without inducing an IGFBP protease. Therefore estrogen and progesterone may alter growth of breast cancers by regulating tumour secretion of IGFBPs and hence change carcinoma responsiveness to IGFs.
Subject(s)
Carrier Proteins/metabolism , Estradiol/pharmacology , Insulin-Like Growth Factor II/metabolism , Promegestone/pharmacology , Somatomedins/metabolism , Breast Neoplasms , Carrier Proteins/analysis , Cell Division/drug effects , Culture Media, Serum-Free , Female , Humans , Insulin-Like Growth Factor Binding Proteins , Insulin-Like Growth Factor II/analysis , Insulin-Like Growth Factor II/pharmacology , Radioimmunoassay , Tumor Cells, CulturedABSTRACT
Insulin-like growth factors (IGF) and binding proteins were measured in serum from pregnant and nonpregnant women. IGF-I determined by immunoassay after acid-ethanol extraction was increased by pregnancy (p less than 0.005) and was highest in the third trimester (p less than 0.01). Size exclusion chromatography of serum in acid before assay (i) gave a very similar IGF-I pattern, (ii) showed that IGF-II was much higher than IGF-I and (iii) revealed less serum IGF-binding protein activity in pregnancy and lactation. All IGF-binding proteins except binding protein-1 were markedly reduced by pregnancy. This indicates a major change in the main carrier protein for IGFs in the circulation and suggests that tissue targetting of IGFs may be altered during pregnancy.
