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Purpose: Serum hormone profiles among different feminizing gender-affirming hormone therapies (GAHT) are poorly characterized. To address this gap, we described the serum estrogen profiles of three 17ß-estradiol preparations, taken with or without an antiandrogen, using a novel liquid chromatography-mass spectrometry (LC-MS/MS) assay in adults taking feminizing GAHT. Methods: This was a secondary analysis of 93 healthy transgender women and gender nonbinary adults taking feminizing GAHT in a prospective cross-sectional study. Eligible participants took 17ß-estradiol (sublingual tablet, transdermal patch, or intramuscular/subcutaneous injection) with or without oral spironolactone for ≥12 months before study entry. We determined serum estrone and estradiol concentrations for each hormone preparation and described the association between estrone and (1) clinically relevant estradiol concentration ranges (≤200 and >200 pg/mL) and (2) antiandrogen use. To achieve our objectives, we described our protocol for developing an LC-MS/MS assay to measure estrone and estradiol concentrations. Results: Estrone concentrations were higher among participants taking sublingual 17ß-estradiol tablets compared with transdermal or injectable preparations (p < 0.0001). Estradiol concentrations were higher for injectable versus transdermal preparations (p = 0.0201), but both were similar to sublingual tablet concentrations (p > 0.05). Estradiol >200 pg/mL (vs. ≤200 pg/mL) was associated with higher estrone concentrations among participants taking sublingual 17ß-estradiol, but not transdermal or injectable 17ß-estradiol. We observed no association between spironolactone and estrone concentrations (p > 0.5). Conclusion: Estrone concentrations were higher among transgender women and gender nonbinary adults taking sublingual 17ß-estradiol compared with transdermal or injectable preparations. The role of estrone in clinical monitoring and the influence of other antiandrogens (e.g., cyproterone acetate) on the estrogen profile remain to be determined.
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Estradiol/administração & dosagem , Terapia de Reposição de Estrogênios , Estrona/sangue , Minorias Sexuais e de Gênero/estatística & dados numéricos , Pessoas Transgênero/estatística & dados numéricos , Administração Cutânea , Administração Sublingual , Adulto , Estudos Transversais , Feminino , Humanos , Injeções , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Transgender women and nonbinary people seeking feminizing therapy are often prescribed estrogen as a gender-affirming hormone, which will alter their reproductive hormone axis. Testosterone, estradiol, and other reproductive hormones are commonly evaluated to assess therapy, but reference intervals specific to transgender women have not been established. The objective of this study was to derive reference intervals for commonly measured analytes related to reproductive endocrinology in a cohort of healthy gender nonconforming individuals on stable feminizing hormone therapy. METHODS: Healthy transgender individuals who had been prescribed estrogen (n = 93) for at least a year were recruited from internal medicine and primary care clinics that specialize in transgender medical care. Total testosterone and estradiol were measured using immunoassay and mass spectrometry; LH, FSH, sex hormone binding globulin, prolactin, progesterone, anti-mullerian hormone (AMH), and dehydroepiandrosterone sulfate (DHEAS) were measured using immunoassay; free testosterone was calculated. Reference intervals (central 95%) were calculated according to Clinical Laboratory Standards Institute guidelines. RESULTS: The distribution of results for transgender women was different than what would be expected from cisgender men or women across all measurements. Use of spironolactone was associated with changes in the result distribution of AMH, FSH, LH, and progesterone. Compared to liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), immunoassay was sufficient for the majority of estradiol and total testosterone measurements; free testosterone added little clinical value beyond total testosterone. CONCLUSION: Reference intervals specific to transgender women should be applied when evaluating reproductive endocrine analytes. Spironolactone is a significant variable for result interpretation of some tests.
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Pessoas Transgênero , Feminino , Humanos , Masculino , Valores de Referência , Espectrometria de Massas em Tandem , TestosteronaRESUMO
BACKGROUND: Gender-affirming therapy with testosterone is commonly prescribed to aid in the masculinization of transgender men. Sex-hormone concentrations are routinely measured, but interpretation of results can be difficult due to the lack of published reference intervals. METHODS: Healthy transgender individuals who had been prescribed testosterone (n = 82) for at least a year were recruited from internal medicine and primary care clinics that specialize in transgender medical care. Total testosterone and estradiol were measured using immunoassay and mass spectrometry; LH, FSH, SHBG, prolactin, progesterone, anti-Müllerian hormone (AMH), and dehydroepiandrosterone sulfate (DHEAS) were measured using immunoassay; free testosterone was calculated. Reference intervals (central 95%) were calculated according to Clinical Laboratory Standards Institute guidelines. RESULTS: When evaluating general endocrine laboratory tests in people using masculinizing hormones, reference intervals for cisgender men can be applied for total and free testosterone and SHBG and reference intervals for cisgender women can be applied for prolactin. Reference intervals for estradiol, LH, FSH, AMH, and DHEAS differ from those used for cisgender men and cisgender women, and therefore should be interpreted using intervals specific to the transmasculine population. For testosterone and estradiol, results from immunoassays were clinically equivalent to mass spectrometry. CONCLUSION: Masculinizing hormones will alter the concentrations of commonly evaluated endocrine hormones. Providers and laboratories should use appropriate reference intervals to interpret the results of these tests.
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Pessoas Transgênero , Estrogênios , Feminino , Humanos , Imunoensaio , Masculino , Valores de Referência , TestosteronaRESUMO
Increasing laboratory automation and efficiency requires quality assurance (QA) approaches to ensure that reported results are precise and accurate. Prerequisites for designing optimal QA strategies include an in-depth understanding of the laboratory processes, the expected results, and of the mechanisms that can cause erroneous results. Oftentimes, a laboratory's own data, extracted from the laboratory information system, electronic medical record, and/or clinical data warehouse are necessary to master the aforementioned requirements. Data-driven QA utilizes retrospective and/or prospective laboratory results to minimize errors in the clinical laboratory due to pre-analytical or analytical vulnerabilities. Additionally, exploitation of this data may improve result interpretation. The objective of this review is to illustrate specific examples of data-driven QA approaches for several areas of the clinical laboratory and for different phases of the testing cycle.
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Tireotropina/sangue , Adulto , Humanos , Hipotireoidismo/diagnóstico , Hipotireoidismo/terapia , MasculinoRESUMO
BACKGROUND: Serum thyroid-stimulating hormone (TSH) reference intervals are dependent on population characteristics, including prevalent thyroid disease and iodine status. Studies in the US have demonstrated increasing TSH levels with age, and the American Thyroid Association recommends higher TSH goals for older patients taking thyroid supplementation, but few laboratories offer age-specific reference intervals for TSH. Our objective was to establish TSH reference ranges in our racially diverse population in northern California. METHODS: Data mining of electronic medical records was used with the a posteriori approach to select a euthyroid reference population for TSH reference intervals. A report gathered all TSH results from 2 weeks from >1 year in the past, excluding results from patients with thyroid-related disease or medication use at any time before or after the TSH test. RESULTS: The reference population numbered 33038 and consisted of approximately 44% of the total TSH results reported in the selected time periods. The population identified as 46.5% white, 18.3% Asian, 17.0% Hispanic/Latino, 8.0% black/African American, and 10.3% other or unknown. These data demonstrate an increase in the median and 97.5 percentile of TSH levels with increasing age in adults. No clinically significant difference was seen between female and male individuals or between the self-identified races, except for lower TSH levels in the black/African American population. CONCLUSIONS: The a posteriori approach using data mining for disease-specific criteria proved to be an efficient method for obtaining a large healthy reference population. Age-specific TSH reference ranges could prevent inappropriate diagnoses of subclinical hypothyroidism in older patients.
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INTRODUCTION: Beckman Coulter recently reformulated their commercial TSH assay with primary calibration to the World Health Organization 3rd TSH international standard. An extensive evaluation of the performance characteristics for this assay was completed. METHODS: Intra-day and inter-day precision was evaluated using 3 concentrations of commercial quality control material. Linearity, reportable range, stability, sensitivity and susceptibility to common inferences were determined using pooled patient specimens. Inter-assay variability was assessed across 5 different platforms (n=47 patient specimens). RESULTS: Intra-day and inter-day CVs were <10% at all concentrations evaluated. The LOQ, LOD and LOB were 0.0047µIU/ml (10% CV), 0.0012µIU/ml and 0.0005µIU/ml, respectively. Variable bias was observed for the TSH3 assay when evaluated against the previous generation assay and other platforms, but overall TSH3 gave comparable results. CONCLUSIONS: The TSH3 assay for UniCel DxI 800, is precise, highly sensitive and comparable to the previous generation assay. The assay is acceptable for clinical testing.
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Técnicas de Laboratório Clínico/instrumentação , Tireotropina/análise , Viés , Calibragem , Técnicas de Laboratório Clínico/normas , Humanos , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Efficient tools are needed to stage liver disease before treatment of patients infected with hepatitis C virus (HCV). Compared to biopsy, several studies demonstrated favorable performance of noninvasive multianalyte serum fibrosis marker panels [fibrosis-4 (FIB-4) index] and aspartate aminotransferase (AST)-to-platelet ratio index (APRI), but suggested cutoffs vary widely. Our objective was to evaluate FIB-4 index and APRI and their component tests for staging fibrosis in our HCV-infected population and to determine practical cutoffs to help triage an influx of patients requiring treatment. METHODS: Transient elastography (TE) results from 1731 HCV-infected patients were mapped to an F0-F4 equivalent scale. Each patient's APRI and FIB-4 index were calculated. Areas under the receiver operator curve (AUROCs) and false-positive and false-negative rates were calculated to retrospectively compare the performance of the indices and their component tests. RESULTS: The highest AUROCs for distinguishing severe (F3-F4) from mild-to-moderate (F0-F2) fibrosis had overlapping 95% CIs: APRI (0.77; 0.74-0.79), FIB-4 index (0.76; 0.73-0.78), and AST (0.74; 0.72-0.77). Cutoffs had false-negative rates of 2.7%-2.8% and false-positive rates of 6.4%-7.4% for all 3 markers. CONCLUSIONS: AST was as effective as FIB-4 index and APRI at predicting fibrosis. Published cutoffs for APRI and FIB-4 index would have been inappropriate in our population, with false-negative rates as high as 11%. For our purposes, no serum fibrosis marker was sufficiently sensitive to rule-out significant fibrosis, but cutoffs developed for AST, FIB-4 index, and APRI all had specificities of 79.2%-80.3% for ruling-in severe fibrosis and could be used to triage 1/3 of our population for treatment without waiting for TE or liver biopsy.
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BACKGROUND: Measurement of tacrolimus using the ARCHITECT immunoassay analyzer requires a manual extraction step that puts clinical laboratory workers at risk for ergonomic injury. Therefore, we developed 2 batched extraction systems for tacrolimus measurement on the ARCHITECT analyzer and describe their features herein. METHODS: Two batched extraction methods were developed at 2 different laboratories. The batched extraction methods allow processing of at least 20 specimens at a time. We evaluated the analytical performance of those methods and compared them with the United States Food and Drug Administration (FDA)-cleared process for manually extracting individual specimens. RESULTS: Comparing the performance of batched- and individual-extraction methods revealed that both methods had comparable between-day imprecision, high patient-results correlation (R2 values ≥0.9869), equivalent functional sensitivity (0.48 ng/mL), and good linearity between 1 ng per mL and 25 ng per mL. Further, we observed decreased delta check-identified errors using the batched method. CONCLUSION: The 2 developed batched extraction methods for tacrolimus measurement that we describe herein demonstrate excellent performance and can replace individual specimen extraction.
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Imunoensaio/métodos , Imunossupressores/sangue , Manejo de Espécimes/métodos , Tacrolimo/sangue , Humanos , Sensibilidade e Especificidade , Estados UnidosRESUMO
CONTEXT: An index case of a clinically euthyroid woman of South Asian descent was identified with discordant TSH results: undetectable TSH on our routine assay and normal TSH on an alternate assay. Low TSH concentrations due to functionally compromising TSH mutations have been reported. Here we describe a new phenomenon of functional TSH that is undetectable by 4 widely used US Food and Drug Administration (FDA)-approved TSH immunoassays marketed by a single vendor. OBJECTIVE: The purpose of this study was to identify additional cases and investigate the cause of the falsely undetectable TSH. DESIGN: All samples with TSH results of <0.01 µIU/mL were retested with a second TSH assay. Discordant samples were evaluated on up to 8 FDA-approved TSH immunoassays and the TSHß gene was sequenced. Retrospectively, thyroid function tests, diagnoses, and medications from 1.6 million individuals were analyzed. RESULTS: Out of approximately 2 million individuals, we have identified a cohort of 20 hypothyroid and euthyroid patients of shared ethnicity with falsely undetectable TSH (<0.01 µIU/mL) in 4 of 8 commercially available TSH assays. Half of these individuals were initially treated based on repeated falsely undetectable TSH values (7 euthyroid patients were treated with methimazole and 2 hypothyroid patients had doses of levothyroxine decreased). In all cases, a retrospective chart review revealed that clinical assessments and free T4 and total T3 results were inconsistent with the undetectable TSH results. Specific antibodies failing to detect TSH in these cases were identified in the 4 affected assays. A novel TSHß point mutation was identified. CONCLUSIONS: Our data suggest that these individuals have a previously unrecognized, functionally normal, TSH variant to which some monoclonal antibodies fail to bind. To assure appropriate patient management, clinicians and laboratorians need to be aware that certain TSH variants may be undetectable in some hyperselective TSH assays.
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Erros de Diagnóstico , Hipotireoidismo/diagnóstico , Tireotropina/sangue , Adulto , Idoso , Povo Asiático/estatística & dados numéricos , California/epidemiologia , Estudos de Coortes , Erros de Diagnóstico/estatística & dados numéricos , Reações Falso-Negativas , Feminino , Humanos , Hipotireoidismo/sangue , Hipotireoidismo/epidemiologia , Imunoensaio/normas , Imunoensaio/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Testes de Função Tireóidea/normas , Testes de Função Tireóidea/estatística & dados numéricos , Adulto JovemRESUMO
BACKGROUND: Metastatic brain cancers are the most common intracranial tumor and occur in about 15% of all cancer patients. In up to 10% of these patients, the primary tumor tissue remains unknown, even after a time consuming and costly workup. The Pathwork Tissue of Origin Test (Pathwork Diagnostics, Redwood City, CA, USA) is a gene expression test to aid in the diagnosis of metastatic, poorly differentiated and undifferentiated tumors. It measures the expression pattern of 1,550 genes in these tumors and compares it to the expression pattern of a panel of 15 known tumor types. The purpose of this study was to evaluate the performance of the Tissue of Origin Test in the diagnosis of primary sites for metastatic brain cancer patients. METHODS: Fifteen fresh-frozen metastatic brain tumor specimens of known origins met specimen requirements. These specimens were entered into the study and processed using the Tissue of Origin Test. Results were compared to the known primary site and the agreement between the two results was assessed. RESULTS: Fourteen of the fifteen specimens produced microarray data files that passed all quality metrics. One originated from a tissue type that was off-panel. Among the remaining 13 cases, the Tissue of Origin Test accurately predicted the available diagnosis in 12/13 (92.3%) cases. DISCUSSION: This study demonstrates the accuracy of the Tissue of Origin Test when applied to predict the tissue of origin of metastatic brain tumors. This test could be a very useful tool for pathologists as they classify metastatic brain cancers.
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Neoplasias Encefálicas/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Primárias Desconhecidas/genética , Adulto , Biópsia , Neoplasias Encefálicas/secundário , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Primárias Desconhecidas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Adulto JovemRESUMO
A case is presented of a 39-year-old woman who suffered severe debilitation because of a hemorrhagic stroke in the context of substance abuse. The patient presented to the emergency room with rapidly diminishing mental status, hypertension, and vasoconstriction; her friends provided a history of ingestion of cocaine, 3,4-methylenedioxymethamphetamine (MDMA), and 2C-I, a novel designer amine. A multi-targeted LC-MS/MS method for sympathomimetic amines and related drugs in urine detected and quantified 2C-I and MDA, while ruling out MDMA. The cause of the stroke was determined to be an underlying cerebrovascular abnormality called Moyamoya, secondary to substance abuse. In clinical laboratories, gas chromatography-mass spectrometry or liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation of a positive amphetamine immunoassay is usually directed only towards amphetamine, methamphetamine, MDMA and MDA. This report demonstrates the utility of testing for a wider menu of compounds using LC-MS/MS in order to better characterize the prevalence and toxicities of novel amines such as 2C-I.
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3,4-Metilenodioxianfetamina/efeitos adversos , Drogas Desenhadas/efeitos adversos , Dimetoxifeniletilamina/análogos & derivados , Alucinógenos/efeitos adversos , Hemorragias Intracranianas/etiologia , Acidente Vascular Cerebral/etiologia , 3,4-Metilenodioxianfetamina/análise , Adulto , Cromatografia Gasosa , Drogas Desenhadas/análise , Dimetoxifeniletilamina/efeitos adversos , Dimetoxifeniletilamina/análise , Feminino , Toxicologia Forense , Cromatografia Gasosa-Espectrometria de Massas , Alucinógenos/análise , Humanos , Doença de Moyamoya/complicações , Doença de Moyamoya/diagnóstico , Quadriplegia/etiologia , Detecção do Abuso de Substâncias/métodos , Transtornos Relacionados ao Uso de Substâncias/complicaçõesRESUMO
The process of bacterial conjugation involves the transfer of a conjugative plasmid as a single strand. The potentially deleterious SOS response, which is normally triggered by the appearance of single-stranded DNA, is suppressed in the recipient cell by a conjugative plasmid system centered on the product of the psiB gene. The F plasmid PsiB protein inhibits all activities of the RecA protein, including DNA binding, DNA strand exchange, and LexA protein cleavage. The proteins known to negatively regulate recombinases, such as RecA or Rad51, generally work at the level of dismantling the nucleoprotein filament. However, PsiB binds to RecA protein that is free in solution. The RecA-PsiB complex impedes formation of RecA nucleoprotein filaments on DNA.
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Proteínas de Bactérias/metabolismo , Recombinases Rec A/metabolismo , Resposta SOS em Genética/fisiologia , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Conjugação Genética/fisiologia , Troca Genética/genética , DNA/genética , DNA/metabolismo , DNA Circular/genética , DNA Circular/metabolismo , DNA Circular/ultraestrutura , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Polarização de Fluorescência , Modelos Genéticos , Poli T/metabolismo , Ligação Proteica/fisiologia , Recombinases Rec A/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/metabolismoRESUMO
BACKGROUND: Approximately 6% of new-onset seizures are drug-related, but there is currently no reliable way to determine if a seizure is drug-induced. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool that allows simultaneous detection of numerous analytes of diverse chemical nature in patient samples. This allows a single analysis to incorporate many compounds relevant to a particular clinical presentation, such as suspected drug-induced seizures. We investigated whether results from a seizure panel using LC-MS/MS could affect patient care. METHODS: We developed a semiquantitative LC-MS/MS assay to detect 12 chemically diverse drugs implicated in drug-related seizures. We collected leftover serum and plasma samples from patients who had seized, performed solid-phase extraction, and analyzed the samples using a hybrid triple quadrupole/linear ion trap mass spectrometer. After assembling a team of medical and toxicology experts, we developed and used a scoring system to determine whether the results of the seizure panel would have affected patient treatment in each case where a drug was detected. RESULTS: In an analysis of 157 samples from patients who seized, 17 (11%) were found to be positive for a drug on the seizure panel. The team of experts determined that the test results probably or definitely would have affected treatment in 7 (41%) of these cases. CONCLUSIONS: A test that detects the presence of drugs implicated in drug-induced seizures can help physicians determine if an unexplained seizure is drug-related and thus potentially better direct patient care. Additionally, LC-MS/MS is an effective tool for answering clinically driven questions.
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Monitoramento de Medicamentos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Preparações Farmacêuticas/sangue , Convulsões/sangue , Convulsões/induzido quimicamente , Adulto , Idoso , Cromatografia Líquida , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Extração em Fase Sólida , Espectrometria de Massas em TandemRESUMO
The Escherichia coli RdgC protein is a potential negative regulator of RecA function. RdgC inhibits RecA protein-promoted DNA strand exchange, ATPase activity, and RecA-dependent LexA cleavage. The primary mechanism of RdgC inhibition appears to involve a simple competition for DNA binding sites, especially on duplex DNA. The capacity of RecA to compete with RdgC is improved by the DinI protein. RdgC protein can inhibit DNA strand exchange catalyzed by RecA nucleoprotein filaments formed on single-stranded DNA by binding to the homologous duplex DNA and thereby blocking access to that DNA by the RecA nucleoprotein filaments. RdgC protein binds to single-stranded and double-stranded DNA, and the protein can be visualized on DNA using electron microscopy. RdgC protein exists in solution as a mixture of oligomeric states in equilibrium, most likely as monomers, dimers, and tetramers. This concentration-dependent change of state appears to affect its mode of binding to DNA and its capacity to inhibit RecA. The various species differ in their capacity to inhibit RecA function.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Recombinases Rec A/metabolismo , Adenosina Trifosfatases/química , Anisotropia , Proteínas de Bactérias/química , Bacteriófagos/metabolismo , Sítios de Ligação , Ligação Competitiva , Clonagem Molecular , DNA/química , DNA de Cadeia Simples/química , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Deleção de Genes , Hidrólise , Microscopia Eletrônica , Ligação Proteica , Recombinases Rec A/química , Recombinação Genética , Serina Endopeptidases/química , Espectrofotometria , Temperatura , Fatores de TempoRESUMO
The RecX protein is a potent inhibitor of RecA activities. We identified several factors that affect RecX-RecA interaction. The interaction is enhanced by the RecA C terminus and by significant concentrations of free Mg(2+) ion. The interaction is also enhanced by an N-terminal His(6) tag on the RecX protein. We conclude that RecX protein interacts most effectively with a RecA functional state designated A(o) and that the RecA C terminus has a role in modulating the interaction. We further identified a C-terminal point mutation in RecA protein (E343K) that significantly alters the interaction between RecA and RecX proteins.
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Proteínas de Bactérias/fisiologia , Escherichia coli/fisiologia , Recombinases Rec A/antagonistas & inibidores , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Soluções Tampão , Clonagem Molecular , DNA/química , DNA de Cadeia Simples/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Regulação Bacteriana da Expressão Gênica , Hidrólise , Íons , Magnésio/química , Mutação Puntual , Estrutura Terciária de Proteína , Recombinases Rec A/química , Fatores de TempoRESUMO
The DinI and RecX proteins of Escherichia coli both modulate the function of RecA protein, but have very different effects. DinI protein stabilizes RecA filaments, preventing disassembly but permitting assembly. RecX protein blocks RecA filament extension, which can lead to net filament disassembly. We demonstrate that both proteins can interact with the RecA filament, and propose that each can replace the other. The DinI/RecX displacement reactions are slow, requiring multiple minutes even when a large excess of the challenging protein is present. The effects of RecX protein on RecA filaments are manifest at lower modulator concentrations than the effects of DinI protein. Together, the DinI and RecX proteins constitute a new regulatory network. The two proteins compete directly as mainly positive (DinI) and negative (RecX) modulators of RecA function.
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Adenosina Trifosfatases/fisiologia , Proteínas de Bactérias/fisiologia , DNA Helicases/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/metabolismo , Adenosina Trifosfatases/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , Clonagem Molecular , DNA/química , DNA Helicases/química , Relação Dose-Resposta à Radiação , Proteínas de Escherichia coli/química , Hidrólise , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , Recombinases Rec A/química , Fatores de Tempo , Raios UltravioletaRESUMO
The RecX protein is a potent inhibitor of RecA protein activities. RecX functions by specifically blocking the extension of RecA filaments. In vitro, this leads to a net disassembly of RecA protein from circular single-stranded DNA. Based on multiple observations, we propose that RecX has a RecA filament capping activity. This activity has predictable effects on the formation and disassembly of RecA filaments. In vivo, the RecX protein may limit the length of RecA filaments formed during recombinational DNA repair and other activities. RecX protein interacts directly with RecA protein, but appears to interact in a functionally significant manner only with RecA filaments bound to DNA.