RESUMO
BACKGROUND: Using residual values calculated from models regressing appendicular lean mass on fat mass and height is one of several suggested strategies for adjusting appendicular lean mass for body size when measuring sarcopenia. However, special consideration is required when using this technique in different subgroups in order to capture the correct individuals as sarcopenic. OBJECTIVES: To provide guidance about how to conduct stratified analyses for the regression adjustment technique using age groups as an example. DESIGN: Cross-sectional study. SETTING: Data collected at baseline (2012-2015) for the Canadian Longitudinal Study on Aging. PARTICIPANTS: Community dwelling participants of European descent aged 45 to 85 years (n=25,399). MEASUREMENTS: Appendicular lean mass, height, and weight were measured. Sex-specific residuals were calculated in participants before and after stratifying participants by age group (45-54, 55-64, 65-74, 75-85 years). Cut offs corresponding to the sex-specific 20th percentile residual values in participants ≥65 years were determined first in the residuals calculated in all participants and residuals calculated in only those aged ≥65 years. For each set of cut offs, the percentage of age and sex-stratified participants with low appendicular lean mass were compared for the residuals calculated in all participants and the residuals calculated after stratifying by age. RESULTS: In 12,622 males and 12,737 females, regardless of the cut off used, the percentage of participants with low appendicular lean mass decreased with age when residuals were calculated after age stratification. When the residuals were calculated in all participants, the percentage of participants with sarcopenia increased from the youngest to the oldest age groups. CONCLUSIONS: Sex-specific residuals in all participants should be calculated prior to stratifying the sample by age group, or other stratification variables, for the purposes of developing appendicular lean mass cut offs or subgroup analyses.
Assuntos
Projetos de Pesquisa , Sarcopenia , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Envelhecimento , Composição Corporal , Canadá/epidemiologia , Estudos Transversais , Feminino , Humanos , Modelos Lineares , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Sarcopenia/diagnóstico , Sarcopenia/epidemiologiaRESUMO
Background: sarcopenia in ageing is a progressive decrease in muscle mass, strength and/or physical function. This review aims to summarise the definitions of sarcopenia in community-dwelling older adults and explore similarities and differences in prevalence estimates by definition. Methods: a systematic review was conducted to identify articles which estimated sarcopenia prevalence in older populations using search terms for sarcopenia and muscle mass. Overall prevalence for each sarcopenia definition was estimated stratified by sex and ethnicity. Secondary analyses explored differences between studies and within definitions, including participant age, muscle mass measurement techniques and thresholds for muscle mass and gait speed. Results: in 109 included articles, eight definitions of sarcopenia were identified. The lowest pooled prevalence estimates came from the European Working Group on Sarcopenia/Asian Working Group on Sarcopenia (12.9%, 95% confidence interval: 9.9-15.9%), International Working Group on Sarcopenia (9.9%, 3.2-16.6%) and Foundation for the National Institutes of Health (18.6%, 11.8-25.5%) definitions. The highest prevalence estimates were for the appendicular lean mass (ALM)/weight (40.4%, 19.5-61.2%), ALM/height (30.4%, 20.4-40.3%), ALM regressed on height and weight (30.4%, 20.4-40.3%) and ALM / body mass index (24.2%, 18.3-30.1%) definitions. Within definitions, the age of study participants and the muscle mass cut points used were substantive sources of between-study differences. Conclusion: estimates of sarcopenia prevalence vary from 9.9 to 40.4%, depending on the definition used. Significant differences in prevalence exist within definitions across populations. This lack of agreement between definitions needs to be better understood before sarcopenia can be appropriately used in a clinical context.
Assuntos
Vida Independente/estatística & dados numéricos , Sarcopenia/epidemiologia , Fatores Etários , Idoso/estatística & dados numéricos , Feminino , Humanos , Masculino , Força Muscular , Prevalência , Sarcopenia/diagnóstico , Fatores SexuaisRESUMO
As Newfoundland has the highest rate of familial colorectal cancer (CRC) in the world, we started a population-based clinic to provide colonoscopic and Lynch syndrome (LS) screening recommendations to families of CRC patients based on family risk. Of 1091 incident patients 51% provided a family history. Seventy-two percent of families were at low or intermediate-low risk of CRC and colonoscopic screening recommendations were provided by letter. Twenty-eight percent were at high and intermediate-high risk and were referred to the genetic counsellor, but only 30% (N = 48) were interviewed by study end. Colonoscopy was recommended more frequently than every 5 years in 35% of families. Lower family risk was associated with older age of proband but the frequency of screening colonoscopy recommendations varied across all age groups, driven by variability in family history. Twenty-four percent had a high MMR predict score for a Lynch syndrome mutation, and 23% fulfilled the Provincial Program criteria for LS screening. A population-based approach in the provision of colonoscopic screening recommendations to families at risk of CRC was limited by the relatively low response rate. A family history first approach to the identification of LS families was inefficient.
Assuntos
Neoplasias Colorretais/genética , Programas de Rastreamento , Idoso , Colonoscopia , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais Hereditárias sem Polipose/genética , Feminino , Aconselhamento Genético/estatística & dados numéricos , Predisposição Genética para Doença , Humanos , Masculino , Programas de Rastreamento/estatística & dados numéricos , Terra Nova e Labrador/epidemiologia , Recusa do Paciente ao TratamentoRESUMO
BACKGROUND: Stearoyl-CoA desaturase-1 (SCD1) is rate limiting for the conversion of saturated fatty acids palmitate (16:0) and stearate (18:0) to monounsaturated fatty acids palmitoleate (16:1n7) and oleate (18:1n9), respectively. Given that reduced SCD1 activity is associated with improved insulin sensitivity and decreased body weight, there is considerable interest to elucidate the role of this enzyme in adipocytes. During adipogenesis, SCD1 levels increase concomitantly with the accumulation of triacylglycerol (TG); however, the extent to which reduced SCD1 activity can influence TG synthesis and metabolic pathways in differentiating adipocytes remains relatively unexplored. OBJECTIVE: The aim of this work was to delineate how reduced SCD1 activity affects gene expression, protein content and cellular fatty acids in differentiating murine preadipocytes. METHODS: 3T3-L1 preadipocytes were treated with an SCD1 inhibitor (10 nM) throughout differentiation. After 7 days, global gene expression, protein content and fatty acid profiles were examined using microarrays, western blotting and gas chromatography, respectively. RESULTS: SCD1 inhibition increased the abundance of 16:0 and 18:0 (45% and 194%, respectively) and decreased 16:1n7 and 18:1n7 (61% and 35%, respectively) in differentiated preadipocytes. Interestingly, 18:1n9 levels increased by 61%. The augmented 18:0 suggested a possible increase in elongase activity. Elongase 6 (Elovl6) gene expression was increased 2.8-fold (P = 0.04); however, changes were not detected for ELOVL6 protein content. Microarray analysis revealed that genes affecting TG synthesis were downregulated with SCD1 inhibition, which coincided with a 33% decrease in cellular TG content. CONCLUSION: These results provide new mechanistic insight into the role of SCD1 as a regulator of fatty acid profiles and TG synthesis in adipocytes, and reinforce that modulating SCD1 activity may help reduce the risk of obesity-related complications.
Assuntos
Células 3T3-L1/enzimologia , Acetiltransferases/metabolismo , Adipócitos/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Estearoil-CoA Dessaturase/metabolismo , Triglicerídeos/biossíntese , Animais , Western Blotting , Diferenciação Celular , Células Cultivadas , Cromatografia Gasosa , Regulação para Baixo , Elongases de Ácidos Graxos , Ácidos Graxos/metabolismo , Regulação Enzimológica da Expressão Gênica , Camundongos , Análise Serial de Tecidos , Regulação para CimaRESUMO
This study investigated the effects of temperature (22, 35, and 45 degrees C), storage time (48, 30, and 15 d), and packaging type on the quality of white wine in bag-in-box (BIB) during the secondary shelf life. Several enological parameters (color and contents of free and total SO2, total aldehyde, and total phenol) were monitored and correlated with oxygen transmission rate (OTR) and Fourier transform infrared (FTIR) spectral data. Time and temperature had significant effects on color development and SO2 depletion during storage. The increased absorbance at 420 nm was correlated with decreases of free SO2 and total SO2. Overall, total phenol content correlated negatively with total aldehyde content. The variance of the enological parameters can be correlated with the OTR data, indicating the barrier properties for the tested packages were different. FTIR-ATR spectra of the wine were analyzed chemometrically using PLS algorithm. The resulting models were able to predict the A(420), free SO2, total SO2, total phenol, total aldehyde, and storage time of the wines. This technique can potentially be used as an efficient tool to evaluate the quality of wine.
Assuntos
Manipulação de Alimentos/métodos , Embalagem de Alimentos/instrumentação , Temperatura Alta , Oxigênio/química , Vinho/análise , Aldeídos/análise , Reação de Maillard , Modelos Químicos , Oxirredução , Permeabilidade , Fenóis/análise , Pigmentação , Controle de Qualidade , Espectroscopia de Infravermelho com Transformada de Fourier , Dióxido de Enxofre/análise , Fatores de TempoRESUMO
OBJECTIVES: We evaluated the in vitro activity of posaconazole against nine Candida species using minimum fungicidal concentration (MFC) measurements and time-kill methods. METHODS: MFCs of posaconazole were determined for 209 clinical isolates (32 Candida albicans, 30 Candida glabrata, 21 Candida tropicalis, 29 Candida krusei, 28 Candida parapsilosis sensu stricto, 50 Candida inconspicua, 13 Candida kefyr, 3 Candida lusitaniae and 3 Candida guilliermondii) and 7 ATCC Candida strains. The following strains were tested in time-kill studies: 3 strains each of C. glabrata, C. kefyr, C. guilliermondii and C. lusitaniae; 2 C. tropicalis; 4 C. albicans; 4 C. inconspicua; 9 C. krusei; 12 C. parapsilosis; and 7 ATCC strains. RESULTS: Posaconazole was fungicidal in both MFC and time-kill experiments (at 2 mg/L within 48 h in time-kill assays) against each C. krusei, C. inconspicua and C. lusitaniae strain and was fungistatic against each C. albicans, C. glabrata, C. tropicalis and C. guilliermondii strain. For the C. parapsilosis strains, posaconazole MFCs were Assuntos
Antifúngicos/farmacologia
, Candida/efeitos dos fármacos
, Triazóis/farmacologia
, Relação Dose-Resposta a Droga
, Farmacorresistência Fúngica
, Testes de Sensibilidade Microbiana
, Fatores de Tempo
RESUMO
Posaconazole (POS) is a newer triazole with activity against yeasts and moulds. POS and fluconazole were tested in vitro against 32 Candida albicans, 30 C. glabrata, 21 C. tropicalis, 29 C. krusei, 28 C. parapsilosis, 50 C. inconspicua, 13 C. kefyr and 5 C. famata isolates using CLSI broth microdilution method (BMD). We compared E-test and a modified BMD using polyethylene-glycol (PEG) as solvent to the CLSI method. BMDs and E-test were performed according to CLSI and the manufacturer's instructions respectively. Geometric means of POS MICs using BMD were 0.71, 0.22 and 0.21 microg ml(-1) against C. glabrata, C. krusei and C. inconspicua, respectively, and remained below 0.1 microg ml(-1) against all other species tested. One of two C. albicans and two of three C. glabrata isolates resistant to fluconazole showed MICs above 8 microg ml(-1) to POS. The impact of using PEG instead of DMSO had only a minor effect (agreements above 95% with the exception of C. parapsilosis). E-tests read after 24 h showed good agreement with the BMD. POS exhibited excellent in vitro activity against Hungarian Candida strains. E-test showed good correlation with the CLSI method, but to facilitate the comparability of results we believe that DMSO should be used as solvent in the BMD.
Assuntos
Antifúngicos/farmacologia , Candida/classificação , Candida/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Triazóis/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/microbiologia , Dimetil Sulfóxido/farmacologia , Farmacorresistência Fúngica , Fluconazol/farmacologia , Humanos , Testes de Sensibilidade Microbiana/normas , Polietilenoglicóis/farmacologia , Solventes/farmacologiaRESUMO
The in vitro activity of the novel triazole antifungal agent posaconazole (Noxafil; SCH 56592) was assessed in 45 laboratories against approximately 19,000 clinically important strains of yeasts and molds. The activity of posaconazole was compared with those of itraconazole, fluconazole, voriconazole, and amphotericin B against subsets of the isolates. Strains were tested utilizing Clinical and Laboratory Standards Institute broth microdilution methods using RPMI 1640 medium (except for amphotericin B, which was frequently tested in antibiotic medium 3). MICs were determined at the recommended endpoints and time intervals. Against all fungi in the database (22,850 MICs), the MIC(50) and MIC(90) values for posaconazole were 0.063 microg/ml and 1 mug/ml, respectively. MIC(90) values against all yeasts (18,351 MICs) and molds (4,499 MICs) were both 1 mug/ml. In comparative studies against subsets of the isolates, posaconazole was more active than, or within 1 dilution of, the comparator drugs itraconazole, fluconazole, voriconazole, and amphotericin B against approximately 7,000 isolates of Candida and Cryptococcus spp. Against all molds (1,702 MICs, including 1,423 MICs for Aspergillus isolates), posaconazole was more active than or equal to the comparator drugs in almost every category. Posaconazole was active against isolates of Candida and Aspergillus spp. that exhibit resistance to fluconazole, voriconazole, and amphotericin B and was much more active than the other triazoles against zygomycetes. Posaconazole exhibited potent antifungal activity against a wide variety of clinically important fungal pathogens and was frequently more active than other azoles and amphotericin B.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Aspergillus/efeitos dos fármacos , Candida/efeitos dos fármacos , Cryptococcus/efeitos dos fármacos , Aspergilose/microbiologia , Aspergillus/genética , Aspergillus/isolamento & purificação , Candida/genética , Candida/isolamento & purificação , Candidíase/microbiologia , Criptococose/microbiologia , Cryptococcus/genética , Cryptococcus/isolamento & purificação , Farmacorresistência Fúngica , Fluconazol/farmacologia , Fungos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Itraconazol/farmacologia , Testes de Sensibilidade Microbiana , Micoses/microbiologia , Pirimidinas/farmacologia , Triazóis/farmacologia , VoriconazolRESUMO
Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.
Assuntos
Aminoglicosídeos , Antibacterianos/farmacologia , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/enzimologia , Metiltransferases/metabolismo , Animais , Antibacterianos/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Farmacorresistência Bacteriana/fisiologia , Enterococcus faecium/genética , Genes Bacterianos , Humanos , Metiltransferases/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos/genética , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Ribossomos/metabolismoRESUMO
Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.
Assuntos
Aminoglicosídeos , Antibacterianos/farmacologia , RNA Arqueal/efeitos dos fármacos , RNA Bacteriano/efeitos dos fármacos , RNA Ribossômico 23S/efeitos dos fármacos , Sítios de Ligação , Resistência Microbiana a Medicamentos/genética , Halobacterium salinarum/química , Halobacterium salinarum/genética , Halobacterium salinarum/isolamento & purificação , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , RNA Arqueal/química , RNA Bacteriano/química , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genéticaRESUMO
Chemical mutagenesis of Staphylococcus aureus RN450 generated two strains that displayed a stable reduction (30- to 60-fold) in susceptibility to evernimicin. Cell-free translation reactions demonstrated that the resistance determinant was located in the ribosomal fraction. Compared to ribosomes isolated from a wild-type strain, ribosomes from the mutant strains displayed an 8- to 10-fold reduction in affinity for [(14)C]evernimicin. In contrast, the mutants displayed no alteration in either binding affinity or in vitro susceptibility to erythromycin. Exponential cultures of the mutant strains accumulated significantly less [(14)C]evernimicin than the wild-type strain, suggesting that accumulation is dependent on the high affinity that evernimicin displays for its binding site. Sequencing rplP (encodes ribosomal protein L16) in the mutant strains revealed a single base change in each strain, which resulted in a substitution of either cysteine or histidine for arginine at residue 51. Introduction of a multicopy plasmid carrying wild-type rplP into the mutant strains restored sensitivity to evernimicin, confirming that the alterations in rplP were responsible for the change in susceptibility. Overexpression of the mutant alleles in S. aureus RN450 had no effect on susceptibility to evernimicin, demonstrating that susceptibility is dominant over resistance.
Assuntos
Aminoglicosídeos , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Mutação/fisiologia , Proteínas Ribossômicas/genética , Staphylococcus aureus/metabolismo , Alelos , Sequência de Aminoácidos , Substituição de Aminoácidos , Clonagem Molecular , Resistência Microbiana a Medicamentos , Dados de Sequência Molecular , Mutação/genética , Plasmídeos/genética , Biossíntese de Proteínas/genética , Ribossomos/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
Spontaneous mutants of susceptible clinical and laboratory isolates of Streptococcus pneumoniae exhibiting reduced susceptibility to evernimicin (SCH27899; MIC, 0.5 to 4.0 mg/liter) were selected on plates containing evernimicin. Four isolates that did not harbor mutations in rplP (which encodes ribosomal protein L16) were further analyzed. Whole chromosomal DNA or PCR products of the 23S ribosomal DNA (rDNA) operons from these mutants could be used to transform the susceptible S. pneumoniae strain R6 to resistance at frequencies of 10(-5) and 10(-4), respectively, rates 10- to 100-fold lower than that for a single-allele chromosomal marker. The transformants appeared slowly (48 to 72 h) on selective medium, and primary transformants passaged on nonselective medium produced single colonies that displayed heterogeneous susceptibilities to evernimicin. A single passage on selective medium of colonies derived from a single primary transformant homogenized the resistance phenotype. Sequence analysis of the 23S rDNA and rRNA from the resistant mutants revealed single, unique mutations in each isolate at the equivalent Escherichia coli positions 2469 (A --> C), 2480 (C --> T), 2535 (G --> A), and 2536 (G --> C). The mutations map within two different stems of the peptidyltransferase region of domain V. Because multiple copies of rDNA are present in the chromosome, gene conversion between mutant and wild-type 23S rDNA alleles may be necessary for stable resistance. Additionally, none of the characterized mutants showed cross-resistance to any of a spectrum of protein synthesis inhibitors, suggesting that the target site of evernimicin may be unique.
Assuntos
Aminoglicosídeos , Antibacterianos/farmacologia , RNA Ribossômico 23S/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Alelos , Proteínas de Bactérias/genética , Sequência de Bases , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , RNA Bacteriano/análise , RNA Bacteriano/genética , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , Streptococcus pneumoniae/genética , Transformação BacterianaRESUMO
Evernimicin (SCH 27899) is a new antibiotic with activity against a wide spectrum of gram-positive bacteria and activity against some gram-negative bacteria. Previous metabolic labeling studies indicated that evernimicin specifically inhibited protein synthesis in Staphylococcus aureus. Using a susceptible Escherichia coli strain, we demonstrated that evernimicin also inhibited protein synthesis in E. coli. In cell-free translation assays with extracts from either E. coli or S. aureus, evernimicin had a 50% inhibitory concentration of approximately 125 nM. In contrast, cell-free systems derived from wheat germ and rabbit reticulocytes were inhibited only by very high levels of evernimicin. Evernimicin did not promote transcript misreading. [(14)C]evernimicin specifically bound to the 50S subunit from E. coli. Nonlinear regression analysis of binding data generated with 70S ribosomes from E. coli and S. aureus and 50S subunits from E. coli returned dissociation constants of 84, 86, and 160 nM, respectively. In binding experiments, performed in the presence of excess quantities of a selection of antibiotics known to bind to the 50S subunit, only the structurally similar drug avilamycin blocked binding of [(14)C]evernimicin to ribosomes.
Assuntos
Aminoglicosídeos , Antibacterianos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Ribossomos/metabolismo , Animais , Antibacterianos/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Ligação Competitiva/efeitos dos fármacos , Radioisótopos de Carbono , Sistema Livre de Células , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Coelhos , Ribossomos/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
The product of the Escherichia coli modE gene, ModE, is a member of a unique class of molybdate-responsive DNA binding proteins. Here we investigated the roles of the N- and C-terminal domains of ModE in mediating DNA binding and protein dimerization, respectively. Compared to the full-length protein, the N-terminal half of ModE has a greatly diminished capacity to bind the modA promoter in vitro and to repress expression from a modA-lacZ operon fusion in vivo. Fusing a protein dimerization domain, encoded by the C terminus of lambda CI repressor protein, to the truncated ModE protein generated a ModE-CI fusion protein that not only displayed a greatly increased in vivo repressor activity but could also substitute for ModE at the moaA and dmsA promoters. In the reciprocal experiment, we restored repressor activity to a truncated CI protein by addition of the C-terminal domain of ModE, which is comprised of two MopI-like subdomains. By an in vivo competition assay, we also demonstrated that the CI-ModE chimeric protein retained the ability to interact with wild-type ModE. Finally, specific deletions within the ModE portion of the CI-ModE protein chimera abolished both in vivo repression and the ability to interact with wild-type ModE. Together, these data demonstrate that the N-terminal domain of ModE is sufficient to mediate DNA binding, although efficient binding requires that ModE form a dimer, a function that is supplied by the C-terminal MopI-like subdomains.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Molibdênio/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Dimerização , Escherichia coli/genética , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/químicaRESUMO
Expression of the Escherichia coli dmsABC operon that encodes a molybdenum-containing DMSO/TMAO reductase is increased in response to anaerobiosis and repressed by nitrate. These changes are mediated by the transcription factors Fnr and NarL respectively. Interestingly, modC strains that are defective in molybdate uptake exhibit impaired anaerobic induction and no nitrate-dependent repression of the dmsABC operon. To determine if the molybdate-responsive transcription factor ModE is involved in this process, a set of dmsA-lacZ operon fusions were constructed and analysed. The pattern of dmsA-lacZ expression in response to anaerobiosis and nitrate addition was identical in both modC and modE strains, thus suggesting a regulatory role for ModE. In vitro studies confirmed that ModE bound the dmsA promoter at a high-affinity site typical of other E. coli ModE operator sites. Mutations in this site abolished ModE binding in vitro and displayed the same phenotype as a modE mutation. In contrast to previously characterized ModE operator sites, which either overlap or are located immediately upstream of the ModE-regulated promoter, the ModE site is centred 52.5 bp downstream of the major dmsA transcript start site. We identified a putative integration host factor (IHF) binding site in the intervening sequence, and in vitro studies confirmed that IHF bound this site with high affinity. Using himA mutants, we confirmed that IHF plays a role in the molybdate-dependent regulation of dmsA-lacZ expression in vivo. This study provides the first example in which ModE affects gene regulation in concert with another transcription factor.
Assuntos
Proteínas de Escherichia coli , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Molibdênio/química , Fatores de Transcrição/fisiologia , Anaerobiose , Proteínas de Bactérias/química , Sequência de Bases , Pegada de DNA , DNA Bacteriano/química , Proteínas de Ligação a DNA/química , Desoxirribonuclease I , Escherichia coli/genética , Galactosidases/análise , Fatores Hospedeiros de Integração , Dados de Sequência Molecular , Nitratos/química , Óperon/fisiologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/fisiologia , Proteínas Recombinantes de Fusão/química , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The Escherichia coli molybdate transporter, encoded by the modABCD operon, is negatively regulated by the modE gene product in response to the intracellular molybdate concentration. Utilizing an in vivo titration assay, we localized the ModE-binding site to the start of modA transcription. This localization was further characterized using in vitro gel-shift assays and DNase I footprinting. ModE bound the wild-type modA promoter with an apparent dissociation constant (Kd) of 45 nM, and addition of molybdate, in physiologically relevant amounts, significantly increased DNA binding. Consistent with these data, modA promoter fragments containing mutations that reduced ModE repression in vivo displayed proportionately higher apparent Kd values in vitro. DNase I footprinting of the modA promoter revealed a single protected region that overlapped the start site of transcription and extended from position -18 to +10, relative to the transcript start site. Gel-shifting assays, employing the promoter regions from the tor, nrf, moa and moe operons, revealed that ModE bound only the moa promoter region, with an apparent Kd of 24nM. Footprint analysis of the moaA promoter revealed a single protected region located immediately upstream of the putative -35 consensus sequence and extending from position -202 to -174, relative to the start of translation. In vivo expression of a moaA-lacZ operon fusion was stimulated twofold by ModE. However, relative to modA, binding of ModE to the moaA promoter appeared to be largely molybdate independent both in vitro and in vivo. These findings demonstrate that ModE acts both as a repressor and activator of the mod and moa operons, respectively, depending on the properties of the binding site.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Óperon/fisiologia , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição/genética , Sistema Livre de Células , Pegada de DNA , Análise Mutacional de DNA , Proteínas de Ligação a DNA/fisiologia , Eletroforese em Gel de Poliacrilamida , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Molibdênio/metabolismo , Molibdênio/farmacologia , Ligação Proteica/genética , Ligação Proteica/fisiologia , Fatores de Transcrição/fisiologia , Transcrição Gênica , Ativação TranscricionalRESUMO
Little is known about the control of latter steps of heme biosynthesis in Escherichia coli. In this study we examined the transcriptional regulation of genes that encode two intermediate heme pathway enzymes, porphobilinogen deaminase (hemC) and uroporphyrinogen III cosynthase (hemD), and the final enzyme of the pathway, ferrochelatase (hemH). We also reexamined the regulation of hemA and the gene located immediately upstream of hemA, hemM. The regulatory regions of hemC, hemH, hemA and hemM were fused to lacZ. The resultant operon fusions were inserted into the E. coli chromosome in single copy and expression monitored under conditions of oxygen and heme limitation. Expression of hemM appeared constitutive under the conditions tested here. In contrast, expression of hemCD, hemH and hemA were shown to be mildly regulated in response to heme availability. Thus, transcription of four of the nine genes of the E. coli heme pathway appears to be only mildly regulated in response to heme limitation.
Assuntos
Proteínas da Membrana Bacteriana Externa , Proteínas de Transporte , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Heme/biossíntese , Heme/genética , Aldeído Oxirredutases/genética , Proteínas de Bactérias/genética , Carbono/metabolismo , Clonagem Molecular , Ferroquelatase/genética , Regulação Bacteriana da Expressão Gênica , Hidroximetilbilano Sintase/genética , Óperon Lac , Chaperonas Moleculares , Nitratos/metabolismo , Oxigênio/metabolismo , Uroporfirinogênio III Sintetase/genéticaRESUMO
The regulation of the Escherichia coli secA gene, whose translation is auto-repressed except when protein secretion becomes limiting, was investigated using a combination of genetic and biochemical approaches. Oligonucleotide-directed deletion and point mutagenesis was used to show that only the last quarter of the upstream gene, geneX, and the geneX-secA intergenic are essential for proper regulation. This region previously shown to contain a secretion-responsive element contains two predicted helices, helix I and II, the latter of which would occlude the secA Shine-Dalgarno sequence. Mutations that destabilized the lower portion of helix II increased secA basal expression, reduced auto-repression by SecA protein, but retained a normal pattern of derepression of secA expression during a protein export block. The introduction of compensatory mutations into helix II that were predicted to restore base-pairing restored secA regulation to wild-type levels or nearly so, suggesting that this helix does play a role in secA auto-regulation in vivo. In contrast, mutations in the lower portion of helix I decreased secA basal expression, reduced auto-repression by SecA protein, and abolished the responsiveness of secA expression to a protein export block. In this latter case introduction of compensatory mutations into helix I that were predicted to restore base-pairing did not restore proper secA regulation, indicating that specific nucleotides in this region are required for normal secA regulation. Primer-extension inhibition (toeprint) analysis with 30 S ribosoma subunits, tRNAMet, and a model segment of geneX-secA RNA carrying the relevant mutations was used to show that mutations that destabilized helix II increased ribosome binding at the secA translation initiation site, while mutations that perturbed helix I decreased ribosome binding at this site. Our results suggest strongly that there is a system of dual regulation of secA translation, whereby helix I serves as an activator element while helix II serves as a repressor element.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Mutação da Fase de Leitura , Genes Bacterianos , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Mutação Puntual , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Sequências Reguladoras de Ácido Nucleico , Ribossomos/metabolismo , Canais de Translocação SEC , Proteínas SecARESUMO
The Escherichia coli modABCD operon, which encodes a high-affinity molybdate uptake system, is transcriptionally regulated in response to molybdate availability by ModE. Here we describe a highly effective enrichment protocol, applicable to any gene with a repressor role, and establish its application in the isolation of transposon mutations in modE. In addition we show that disruption of the ModE C-terminus abolishes derepression in the absence of molybdate, implying this region of ModE controls the repressor activity. Finally, a mutational analysis of a proposed molybdate binding motif indicates that this motif does not function in regulating the repressor activity of ModE.