scholarly journals Quinolone Resistance Due to Reduced Target Enzyme Expression

2003 ◽  
Vol 185 (23) ◽  
pp. 6883-6892 ◽  
Author(s):  
Dilek Ince ◽  
David C. Hooper
Keyword(s):  
Steady State ◽  
Quinolone Resistance ◽  
Start Codon ◽  
Cell Multiplication ◽  
Compensatory Mechanism ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Resistance Phenotype ◽  
Allelic Exchange ◽  
Steady State Levels

ABSTRACT We report for the first time low-level quinolone resistance mediated by decreased expression of topoisomerase IV in Staphylococcus aureus. A single-step mutant of wild-type S. aureus strain ISP794, P18 selected by using twice the MIC of premafloxacin, had four- and four- to eightfold greater MICs of premafloxacin and ciprofloxacin, respectively, than the wild type. Sequencing of parEC and gyrBA with their promoter regions revealed a point mutation (G→A) 13 bp upstream of the start codon of parE. Genetic linkage studies showed that there was a high level of correlation between the mutation and the resistance phenotype, and allelic exchange confirmed the contribution of the mutation to resistance. Decreased expression of ParE and decreased steady-state levels of parEC transcripts in P18 and in resistant allelic exchange mutants were observed. The steady-state levels of gyrBA and topB transcripts were increased in P18 but not in two resistant allelic exchange mutants, and sequencing upstream of either gene did not reveal a difference between ISP794 and P18. The steady-state levels of topA transcripts were similar in the various strains. Growth competition experiments performed at 30, 37, and 41°C with a susceptible allelic exchange strain and a resistant allelic exchange strain suggested that loss of fitness was associated with reduced levels of ParE at 41°C. However, P18 had a growth advantage over ISP794 at all temperatures, suggesting that a compensatory mechanism was associated with the increased levels of gyrBA and topB transcripts. Thus, reduced levels of ParE appear to be compatible with cell survival, although there may be a fitness cost during rapid cell multiplication, which might be overcome by compensatory mechanisms without reversion of the resistance phenotype.

scholarly journals The critical role of T cells in glucocorticoid-induced osteoporosis

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Lin Song ◽  
Lijuan Cao ◽  
Rui Liu ◽  
Hui Ma ◽  
Yanan Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Steady State ◽  
Side Effects ◽  
Critical Role ◽  
Wild Type ◽  
Receptor Activator ◽  
Steady State Levels ◽  
Induced Apoptosis

AbstractGlucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

scholarly journals An AMP nucleosidase gene knockout in Escherichia coli elevates intracellular ATP levels and increases cold tolerance

2007 ◽  
Vol 4 (1) ◽  
pp. 53-56 ◽  
Author(s):  
Brittany A Morrison ◽  
Daniel H Shain
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Atp Hydrolysis ◽  
Gene Knockout ◽  
Compensatory Mechanism ◽  
Wild Type ◽  
Intracellular Atp ◽  
Atp Levels ◽  
Glacier Ice ◽  
Mesophilic Bacterium

Disparate psychrophiles (e.g. glacier ice worms, bacteria, algae and fungi) elevate steady-state intracellular ATP levels as temperatures decline, which has been interpreted as a compensatory mechanism to offset reductions in molecular motion and Gibb's free energy of ATP hydrolysis. In this study, we sought to manipulate steady-state ATP levels in the mesophilic bacterium, Escherichia coli , to investigate the relationship between cold temperature survivability and elevated intracellular ATP. Based on known energetic pathways and feedback loops, we targeted the AMP nucleotidase ( amn ) gene, which is thought to encode the primary AMP degradative enzyme in prokaryotes. By knocking out amn in wild-type E. coli DY330 cells using recombineering methodology, we generated a mutant (AMNk) that elevated intracellular ATP levels by more than 30% across its viable temperature range. As temperature was lowered, the relative ATP disparity between AMNk and DY330 cells increased to approximately 66% at 10°C, and was approximately 100% after storage at 0°C for 5–7 days. AMNk cells stored at 0°C for 7 days displayed approximately fivefold higher cell viability than wild-type DY330 cells treated in the same manner.

scholarly journals Alterations in the DNA topoisomerase IV grlA gene responsible for quinolone resistance in Staphylococcus aureus.

1996 ◽  
Vol 40 (5) ◽  
pp. 1157-1163 ◽  
Author(s):  
J Yamagishi ◽  
T Kojima ◽  
Y Oyamada ◽  
K Fujimoto ◽  
H Hattori ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Gene Dosage ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Wild Type Allele ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Dna Topoisomerase ◽  
Transformation Experiment ◽  
Dna Fragment

A 4.2-kb DNA fragment conferring quinolone resistance was cloned from a quinolone-resistant clinical isolate of Staphylococcus aureus and was shown to possess a part of the grlB gene and a mutated grlA gene. S-80-->F and E-84-->K mutations in the grlA gene product were responsible for the quinolone resistance. The mutated grlA genes responsible for quinolone resistance were dominant over the wild-type allele, irrespective of gene dosage in a transformation experiment with the grlA gene alone. However, dominance by mutated grlA genes depended on gene dosage when bacteria were transformed with the grlA and grlB genes in combination. Quinolone-resistant gyrA mutants were easily isolated from a strain, S. aureus RN4220, carrying a plasmid with the mutated grlA gene, though this was not the case for other S. aureus strains lacking the plasmid. The elimination of this plasmid from such quinolone-resistant gyrA mutants resulted in marked increases in quinolone susceptibility. These results suggest that both DNA gyrase and DNA topoisomerase IV may be targets of quinolones and that the quinolone susceptibility of organisms may be determined by which of these enzymes is most quinolone sensitive.

scholarly journals In Vitro Activities of Novel Nonfluorinated Quinolones PGE 9262932 and PGE 9509924 against Clinical Isolates of Staphylococcus aureus and Streptococcus pneumoniae with Defined Mutations in DNA Gyrase and Topoisomerase IV

2002 ◽  
Vol 46 (6) ◽  
pp. 1651-1657 ◽  
Author(s):  
Mark E. Jones ◽  
Ian A. Critchley ◽  
James A. Karlowsky ◽  
Renée S. Blosser-Middleton ◽  
Franz-Josef Schmitz ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Streptococcus Pneumoniae ◽  
Dna Gyrase ◽  
Quinolone Resistance ◽  
Clinical Isolates ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Selection Of

ABSTRACT Two 8-methoxy nonfluorinated quinolones (NFQs), PGE 9262932 and PGE 9509924, were tested against contemporary clinical isolates of Staphylococcus aureus (n = 122) and Streptococcus pneumoniae (n = 69) with genetically defined quinolone resistance-determining regions (QRDRs). For S. aureus isolates with wild-type (WT) sequences at the QRDRs, the NFQs demonstrated activities 4- to 32-fold more potent (MICs at which 90% of isolates are inhibited [MIC90s], 0.03 μg/ml) than those of moxifloxacin (MIC90, 0.12 μg/ml), gatifloxacin (MIC90, 0.25 μg/ml), levofloxacin (MIC90, 0.25 μg/ml), and ciprofloxacin (MIC90, 1 μg/ml). Against S. pneumoniae isolates with WT sequences at gyrA and parC, the NFQs PGE 9262932 (MIC90, 0.03 μg/ml) and PGE 9509924 (MIC90, 0.12 μg/ml) were 8- to 64-fold and 2- to 16-fold more potent, respectively, than moxifloxacin (MIC90, 0.25 μg/ml), gatifloxacin (MIC90, 0.5 μg/ml), levofloxacin (MIC90, 2 μg/ml), and ciprofloxacin (MIC90, 2 μg/ml). The MICs of all agents were elevated for S. aureus isolates with alterations in GyrA (Glu88Lys or Ser84Leu) and GrlA (Ser80Phe) and S. pneumoniae isolates with alterations in GyrA (Ser81Phe or Ser81Tyr) and ParC (Ser79Phe or Lys137Asn). Fluoroquinolone MICs for S. aureus strains with double alterations in GyrA combined with double alterations in GrlA were ≥32 μg/ml, whereas the MICs of the NFQs for strains with these double alterations were 4 to 8 μg/ml. The PGE 9262932 and PGE 9509924 MICs for the S. pneumoniae isolates did not exceed 0.5 and 1 μg/ml, respectively, even for isolates with GyrA (Ser81Phe) and ParC (Ser79Phe) alterations, for which levofloxacin MICs were >16 μg/ml. No difference in the frequency of selection of mutations (<10−8 at four times the MIC) in wild-type or first-step mutant isolates of S. aureus or S. pneumoniae was detected for the two NFQs. On the basis of their in vitro activities, these NFQ agents show potential for the treatment of infections caused by isolates resistant to currently available fluoroquinolones.

scholarly journals Effects of nonsense mutations on nuclear and cytoplasmic adenine phosphoribosyltransferase RNA.

1996 ◽  
Vol 16 (8) ◽  
pp. 4426-4435 ◽  
Author(s):  
O Kessler ◽  
L A Chasin
Keyword(s):  
Steady State ◽  
Actinomycin D ◽  
Chinese Hamster ◽  
Nuclear Pore ◽  
Mrna Levels ◽  
Wild Type ◽  
Adenine Phosphoribosyltransferase ◽  
Aprt Gene ◽  
Steady State Levels ◽  
Nonsense Codons

We have analyzed Chinese hamster ovary (CHO) cell mutants bearing nonsense codons in four of the five exons of the adenine phosphoribosyltransferase (aprt) gene and have found a pattern of mRNA reduction similar to that seen in systems studied previously: a decrease in steady-state mRNA levels of 5- to 10-fold for mutations in exons 1, 2, and 4 but little effect for mutations in the 3'-most exon (exon 5). Nuclear aprt mRNA levels showed a similar decrease. Nonsense-containing aprt mRNA decayed at the same rate as wild-type mRNA in these cell lines after inhibition of transcription with actinomycin D. Nonsense-containing aprt mRNA is associated with polysomes, ruling out a model in which stable residual mRNA escapes degradation by avoiding translation initiation. A tetracycline-responsive form of the aprt gene was used to compare the stability of nonsense-containing and wild-type aprt mRNAs without globally inhibiting transcription. In contrast to measurements made in the presence of actinomycin D, after inhibition of aprt transcription with tetracycline, a nonsense-mediated destabilization of aprt mRNA was indeed demonstrable. The increased rate of decay of cytoplasmic aprt mRNA seen here could account for the nonsense-mediated reduction in steady-state levels of aprt mRNA. However, the low levels of nonsense-bearing aprt mRNA in the nucleus suggest a sensibility of mRNA to translation or translatability before it exits that compartment. Quantitation of the steady-state levels of transcripts containing introns revealed no accumulation of partially spliced aprt RNA and hence no indication of nonsense-mediated aberrancies in splicing. Our results are consistent with a model in which translation facilitates the export of mRNA through a nuclear pore. However, the mechanism of this intriguing nucleocytoplasmic communication remains to be determined.

Multiple mechanisms are responsible for altered expression of ornithine decarboxylase in overproducing variant cells

1986 ◽  
Vol 6 (8) ◽  
pp. 2865-2871
Author(s):  
L McConlogue ◽  
S L Dana ◽  
P Coffino
Keyword(s):  
Steady State ◽  
Gene Amplification ◽  
Ornithine Decarboxylase ◽  
Mrna Levels ◽  
Wild Type ◽  
Altered Expression ◽  
Cells Of Origin ◽  
Multistep Process ◽  
Steady State Levels ◽  
Synthesis And Degradation

We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.

HSP70.1 and -70.3 are required for late-phase protection induced by ischemic preconditioning of mouse hearts

2003 ◽  
Vol 285 (2) ◽  
pp. H866-H874 ◽  
Author(s):  
Craig R. Hampton ◽  
Akira Shimamoto ◽  
Christine L. Rothnie ◽  
Jeaneatte Griscavage-Ennis ◽  
Albert Chong ◽  
...  
Keyword(s):  
Steady State ◽  
Infarct Size ◽  
Ischemic Preconditioning ◽  
Tissue Injury ◽  
Late Phase ◽  
Wild Type ◽  
Hsp70 Protein ◽  
Steady State Levels ◽  
Shock Proteins

We investigated the role of inducible heat shock proteins 70.1 and 70.3 (HSP70.1 and HSP70.3, respectively) in myocardial ischemic preconditioning (IP) in mice. Wild-type (WT) mice and HSP70.1- and HSP70.3-null [HSP70.1/3(–/–)] mice were subjected to IP and examined 24 h later during the late phase of protection. IP significantly increased steady-state levels of HSP70.1 and HSP70.3 mRNA and expression of inducible HSP70 protein in WT myocardium. To assess protection against tissue injury, mice were subjected to 30 min of regional ischemia and 3 h of reperfusion. In WT mice, IP reduced infarct size by 43% compared with sham IP-treated mice. In contrast, IP did not reduce infarct size in HSP70.1/3(–/–) mice. Absence of inducible HSP70.1 and HSP70.3 had no effect, however, on classical or early-phase protection produced by IP, which significantly reduced infarct size in HSP70.1/3(–/–) mice. We conclude that inducible HSP70.1 and HSP70.3 are required for late-phase protection against infarction following IP in mice.

scholarly journals Contribution of the ATP Binding Site of ParE to Susceptibility to Novobiocin and Quinolones in Streptococcus pneumoniae

2005 ◽  
Vol 187 (4) ◽  
pp. 1536-1540 ◽  
Author(s):  
Philippe Dupont ◽  
Alexandra Aubry ◽  
Emmanuelle Cambau ◽  
Laurent Gutmann
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Site ◽  
Quinolone Resistance ◽  
Atp Binding ◽  
Wild Type ◽  
Topoisomerase Iv ◽  
Atp Binding Site ◽  
Open Conformation ◽  
A Subunit

ABSTRACT In Streptococcus pneumoniae, an H103Y substitution in the ATP binding site of the ParE subunit of topoisomerase IV was shown to confer quinolone resistance and hypersensitivity to novobiocin when associated with an S84F change in the A subunit of DNA gyrase. We reconstituted in vitro the wild-type topoisomerase IV and its ParE mutant. The ParE mutant enzyme showed a decreased activity for decatenation at subsaturating ATP levels and was more sensitive to inhibition by novobiocin but was as sensitive to quinolones. These results show that the ParE alteration H103Y alone is not responsible for quinolone resistance and agree with the assumption that it facilitates the open conformation of the ATP binding site that would lead to novobiocin hypersensitivity and to a higher requirement of ATP.

scholarly journals Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV.

1997 ◽  
Vol 41 (1) ◽  
pp. 175-179 ◽  
Author(s):  
D M Breines ◽  
S Ouabdesselam ◽  
E Y Ng ◽  
J Tankovic ◽  
S Shah ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mutant Allele ◽  
Quinolone Resistance ◽  
Temperature Sensitive ◽  
Resistance Locus ◽  
Topoisomerase Iv ◽  
Gene Encoding ◽  
Resistance Phenotype ◽  
E Coli ◽  
High Level

The locus nfxD, which contributes to high-level quinolone resistance in Escherichia coli KF111b (gyrAr nfxB nfxD), is only expressed in the presence of a gyrA mutation, and maps to the region of the parC and parE genes, was outcrossed into strain KF130, creating strain DH161 (gyrAr nfxD). DNA sequence analysis of DH161 revealed no changes in the topoisomerase IV parC quinolone resistance-determining region but did identify a single T-to-A mutation in parE at codon 445, leading to a change from Leu to His. Full-length cloned parE+ partially complemented the resistance phenotype in KF111b and DH161, but did not complement the resistance phenotype in strain KF130 (gyrAr). No complementation was seen with cloned, truncated parE+. To confirm these findings, gyrAr was first outcrossed from KF130 into E. coli W3110parE10 [parE temperature sensitive(Ts)] and KL16. The transduced strains KL16 and W3110parE10 were subsequently transformed with plasmids containing cloned parE from DH161 or KL16. Cloned parE from DH161 increased norfloxacin resistance in the parE(Ts) background twofold at 30 degrees C and fourfold at 42 degrees C compared to those for cloned parE from KL16. The same experiment with a non-Ts background revealed a twofold increase in the norfloxacin MIC at both 30 and 42 degrees C. These data identify the nfxD conditional resistance locus as a mutant allele of parE. This report is the first of a quinolone-resistant parE mutant and confirms the role of topoisomerase IV as a secondary target of norfloxacin in E. coli.

scholarly journals Induction and repression of the urea amidolyase gene in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (11) ◽  
pp. 3954-3964 ◽  
Author(s):  
F S Genbauffe ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Normal Control ◽  
Catabolite Repression ◽  
Gene Products ◽  
Nitrogen Catabolite Repression ◽  
Wild Type ◽  
Recombinant Plasmids ◽  
Control Pattern ◽  
Steady State Levels

The DUR1,2 gene from Saccharomyces cerevisiae has been isolated on recombinant plasmids along with all DNA between the DUR1,2 and MET8 loci. DUR1,2 was found to encode a 5.7-kilobase transcript, which is consistent with our earlier suggestion that the DUR1 and DUR2 loci are two domains of a single multifunctional gene. Steady-state levels of the DUR1,2 transcript responded to induction and nitrogen catabolite repression in the same way as urea amidolyase activity. dal81 mutants (grown with inducer) contained barely detectable amounts of DUR1,2 RNA, whereas dal80 mutants (grown without inducer) contained the same amount as a wild-type induced culture. These observations support our earlier hypothesis that DUR1,2 is transcriptionally regulated, with control being mediated by the DAL80 and DAL81 gene products. We cloned the DUR1,2-Oh mutation and found it to be a Ty insertion near sequences required for complementation of dur1,2 mutations. The ROAM phenotype of the DUR1,2-Oh mutation is sharply different from that of cis-dominant, DUR80 mutations, which enhance DUR1,2 expression but do not affect the normal control pattern of the gene. There is evidence that DUR80 mutations may also be Ty insertions, which generate phenotypes that are different from those in DUR1,2-Oh mutations.

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