RESUMO
Protein phosphatase 1D (PPM1D, Wip1) is induced by the tumor suppressor p53 during DNA damage response signaling and acts as an oncoprotein in several human cancers. Although PPM1D is a potential therapeutic target, insights into its atomic structure were challenging due to flexible regions unique to this family member. Here, we report the first crystal structure of the PPM1D catalytic domain to 1.8 Å resolution. The structure reveals the active site with two Mg2+ ions bound, similar to other structures. The flap subdomain and B-loop, which are crucial for substrate recognition and catalysis, were also resolved, with the flap forming two short helices and three short ß-strands that are followed by an irregular loop. Unexpectedly, a nitrogen-oxygen-sulfur bridge was identified in the catalytic domain. Molecular dynamics simulations and kinetic studies provided further mechanistic insights into the regulation of PPM1D catalytic activity. In particular, the kinetic experiments demonstrated a magnesium concentration-dependent lag in PPM1D attaining steady-state velocity, a feature of hysteretic enzymes that show slow transitions compared with catalytic turnover. All combined, these results advance the understanding of PPM1D function and will support the development of PPM1D-targeted therapeutics.
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DNA helicase RECQ1 (also known as RECQL or RECQL1) is a candidate breast cancer susceptibility gene significantly correlated with clinical outcomes of sporadic breast cancer patients. Prior studies have suggested that RECQ1 maintains genomic stability by regulating a wide variety of core cellular functions including DNA replication, DNA damage response, and transcription. However, it is unclear which, if any, of these are the primary functions of RECQ1 as related to its role in suppressing breast cancer. We describe here an unbiased integrative genomics approach that enabled us to discover a previously unknown regulatory role of RECQ1 in promoting Estrogen Receptor alpha (ERα) expression and the expression of specific ERα target genes in ER positive breast cancer cells. We discuss potential future applications of similar experimental strategies in advancing the mechanistic understanding and elucidating specific new details of genome-wide functions of RECQ1 and other RecQ helicases in maintaining genomic stability and preventing cancer.
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Neoplasias da Mama , RecQ Helicases , Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Feminino , Predisposição Genética para Doença , Instabilidade Genômica , Humanos , RecQ Helicases/genéticaRESUMO
Nuclear factor of activated T cells (NFAT) transcription factors are required for induction of T-cell cytokine production and effector function. Although it is known that activation via the T-cell antigen receptor (TCR) results in 2 critical steps, calcineurin-mediated NFAT1 dephosphorylation and NFAT2 up-regulation, the molecular mechanisms underlying each are poorly understood. Here we find that T cell p38, which is activated by an alternative pathway independent of the mitogen-activated protein (MAP) kinase cascade and with different substrate specificities, directly controls these events. First, alternatively (but not classically) activated p38 was required to induce the expression of the AP-1 component c-Fos, which was necessary for NFAT2 expression and cytokine production. Second, alternatively (but not classically) activated p38 phosphorylated NFAT1 on a heretofore unidentified site, S79, and in its absence NFAT1 was unable to interact with calcineurin or migrate to the nucleus. These results demonstrate that the acquisition of unique specificities by TCR-activated p38 orchestrates NFAT-dependent T-cell functions.
Assuntos
Fatores de Transcrição NFATC/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Calcineurina , Comunicação Celular , Humanos , Imunidade Celular/genética , Imunidade Celular/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Fosforilação , Proteólise , Proteínas Proto-Oncogênicas c-fos , Receptores de Antígenos de Linfócitos T/fisiologia , Especificidade por Substrato , Linfócitos T , Fatores de TranscriçãoRESUMO
BACKGROUND: Fatigue is prevalent in hemodialysis patients who for survival follow a strict dialysis treatment regimen - dialysis and non-dialysis days. As a result, the daily activities, symptom burden, and clinical outcomes of hemodialysis patients vary significantly between dialysis and non-dialysis days. Fatigue is one of the most reported debilitating symptoms by hemodialysis patients with profound negative impact on their quality of life. Prior studies assessed fatigue during the preceding 7 or 30 days and did not discriminate fatigue characteristics between dialysis and non-dialysis days. We aimed to characterize and compare fatigue severity and fatigue interference with daily activities between dialysis and non-dialysis days. METHODS: Hemodialysis patients self-reported fatigue on consecutive dialysis and non-dialysis days using the 9-item Brief Fatigue Inventory. The differences in fatigue characteristics between dialysis and non-dialysis days were analyzed using one-way ANCOVA. RESULTS: Global fatigue burden was worse on a dialysis day compared to a non-dialysis day (P for all < 0.001). Age and education were associated with fatigue, but hemodialysis-related variables were not. A significant inverse association of physical activity with fatigue severity observed on non-dialysis day; there was also a negative association between the normalized protein catabolic rate and fatigue severity on both dialysis and non-dialysis days. The positive association of depression with fatigue severity and fatigue interference were consistent on both dialysis and non-dialysis days. None of these factors, however, explained differences in fatigue characteristics between dialysis and non-dialysis days. CONCLUSIONS: Fatigue, measured in severity and interference, was more pronounced on a dialysis day relative to a non-dialysis day. These differences were not explained by age, sex, education, hemodialysis-related variables, habitual exercise, nutritional status, and or depression. The quantitative measures of fatigue characteristics may facilitate future interventional trials design and better fatigue management for hemodialysis patients.
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Fadiga/etiologia , Diálise Renal/efeitos adversos , Atividades Cotidianas , Adulto , Análise de Variância , Exercício Físico , Feminino , Humanos , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Qualidade de Vida , Autorrelato , Índice de Gravidade de DoençaRESUMO
ZAP-70 is a tyrosine kinase that is essential for initiation of T cell antigen receptor (TCR) signaling. We have found that T cell p38 MAP kinase (MAPK), which is directly phosphorylated and activated by ZAP-70 downstream of the TCR, in turn phosphorylates Thr-293 in the interdomain B region of ZAP-70. Mutant T cells expressing ZAP-70 with an alanine substitution at this residue (ZAP-70T293A) had enhanced TCR proximal signaling and increased effector responses. Lack of ZAP-70T293 phosphorylation increased association of ZAP-70 with the TCR and prolonged the existence of TCR signaling microclusters. These results identify a tight negative feedback loop in which ZAP-70-activated p38 reciprocally phosphorylates ZAP-70 and destabilizes the signaling complex.
Assuntos
Genes Codificadores dos Receptores de Linfócitos T/fisiologia , Proteína-Tirosina Quinase ZAP-70/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Aminoácidos , Regulação da Expressão Gênica , Humanos , Células Jurkat , Fosforilação , Transdução de Sinais , Proteína-Tirosina Quinase ZAP-70/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.
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Ativadores de Enzimas/química , Fosfopeptídeos/química , Proteína Fosfatase 2C/química , Bibliotecas de Moléculas Pequenas/química , Ativadores de Enzimas/isolamento & purificação , Ativadores de Enzimas/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Proteína Fosfatase 2C/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/isolamento & purificação , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato , Proteína Supressora de Tumor p53/químicaRESUMO
BACKGROUND: Fatigue is one of the most debilitating symptoms reported by maintenance hemodialysis (MHD) patients. Hemodialysis causes marked depletion in plasma essential amino acids. We studied the cross-sectional relationship of pre- and post-hemodialysis branched-chain amino acids (BCAAs) concentrations with fatigue in MHD patients. METHODS: MHD patients self-reported fatigue during a dialysis session using the Brief Fatigue Inventory. Pre- and post-dialysis plasma levels of BCAAs (valine, leucine, and isoleucine) were measured using HPLC-mass spectrometry. RESULTS: The mean age of study participants (n = 114) was 54.8 ± 12.8 years. Plasma levels of BCAAs decreased significantly post-dialysis compared to pre-dialysis (303.8 ± 9.4 vs. 392.1 ± 9.4 µM/L, p < 0.0001). Fatigue score increased as a function of age (p = 0.015). There was no association between pre-dialysis plasma levels of BCAAs and fatigue. A significant negative correlation was found between post-dialysis plasma levels of BCAAs and fatigue (p < 0.05). CONCLUSIONS: These preliminary findings suggest that disruption in BCAAs homeostasis may play a role in precipitating fatigue.
Assuntos
Aminoácidos de Cadeia Ramificada/sangue , Fadiga/epidemiologia , Falência Renal Crônica/terapia , Diálise Renal/efeitos adversos , Adulto , Idoso , Aminoácidos de Cadeia Ramificada/metabolismo , Estudos de Coortes , Estudos Transversais , Fadiga/sangue , Fadiga/diagnóstico , Fadiga/etiologia , Feminino , Humanos , Falência Renal Crônica/sangue , Masculino , Pessoa de Meia-Idade , Autorrelato/estatística & dados numéricosRESUMO
Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E-c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.
Assuntos
Metais/metabolismo , Fosfopeptídeos/metabolismo , Proteína Fosfatase 2C/química , Proteína Fosfatase 2C/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica , Proteína Fosfatase 2C/genética , Homologia de Sequência , Especificidade por SubstratoRESUMO
Localized separation of strands of duplex DNA is a necessary step in many DNA-dependent processes, including transcription and replication. Little is known about how these strand separations occur. The strand-separated E.coli RNA polymerase-promoter open-complex structure showed four bases of the non-template strand, the master base -11A, -7, -6 and +2, in a flipped state and inserted into protein pockets. To explore whether any property of these bases in the duplex state pre-disposes them to flipping, NMR studies were performed on a wild-type promoter in the duplex state. Measurement of relaxation times indicates that a limited number of base pairs, including the flipped ones, have faster opening rates than the rest. Molecular dynamics studies also show an inherently high dynamic character of the -11A:T base pair in the wild-type strand-paired state. In order to explore the role of the RNA polymerase in the flipping process, we have used 2-aminopurine as a fluorescent probe. Slower kinetics of the increase of 2-aminopurine fluorescence was observed with RNA polymerases containing several mutant σ70s. This may be interpreted as the protein playing an important role in enhancing the flipping rate. These results suggest that flipping of -11A, and perhaps other flipped bases observed in the open-complex, is facilitated by its inherent proclivity to open-up with further assistance from the protein, thus leading to a strand-open state. Other DNA-based processes that require strand-separation may use similar pathways for strand separation. We conclude that not only basepair stability, but also dynamics may play an important role in the strand-separation.
Assuntos
DNA/química , Iniciação da Transcrição Genética , 2-Aminopurina/química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Escherichia coli/genética , Corantes Fluorescentes/química , Cinética , Simulação de Dinâmica Molecular , Mutação , Regiões Promotoras GenéticasRESUMO
PPM serine/threonine protein phosphatases function in signaling pathways and require millimolar concentrations of Mn2+ or Mg2+ ions for activity. Whereas the crystal structure of human PP2Cα displayed two tightly bound Mn2+ ions in the active site, recent investigations of PPM phosphatases have characterized the binding of a third, catalytically essential metal ion. The binding of the third Mg2+ to PP2Cα was reported to have millimolar affinity and to be entropically driven, suggesting it may be structurally and catalytically important. Here, we report the use of hydrogen/deuterium exchange-mass spectrometry and molecular dynamics to characterize conformational changes in PP2Cα between the active and inactive states. In the presence of millimolar concentrations of Mg2+, metal-coordinating residues in the PP2Cα active site are maintained in a more rigid state over the catalytically relevant time scale of 30-300 s. Submillimolar Mg2+ concentrations or introduction of the D146A mutation increased the conformational mobility in the Flap subdomain and in buttressing helices α1 and α2. Residues 192-200, located in the Flap subdomain, exhibited the greatest interplay between effects of Mg2+ concentration and the D146A mutation. Molecular dynamics simulations suggest that the presence of the third metal ion and the D146A mutation each produce distinct conformational realignments in the Flap subdomain. These observations suggest that the binding of Mg2+ to the D146/D239 binding site stabilizes the conformation of the active site and the Flap subdomain.
Assuntos
Medição da Troca de Deutério , Proteína Fosfatase 2C/química , Proteína Fosfatase 2C/metabolismo , Sítios de Ligação , Humanos , Espectrometria de Massas , Conformação ProteicaRESUMO
Transcription is initiated when RNA polymerase recognizes the duplex promoter DNA in the closed complex. Due to its transient nature, the closed complex has not been well characterized. How the initial promoter recognition occurs may offer important clues to regulation of transcription initiation. In this article, we have carried out single-base pair substitution experiments on two Escherichia coli promoters belonging to two different classes, the -35 and the extended -10, under conditions which stabilize the closed complex. Single-base pair substitution experiments indicate modest base-specific effects on the stability of the closed complex of both promoters. Mutations of base pairs in the -10 region affect the closed complexes of two promoters differently, suggesting different modes of interaction of the RNA polymerase and the promoter in the two closed complexes. Two residues on σ(70) which have been suggested to play important role in promoter recognition, Q437 and R436, were mutated and found to have different effects on the closed-complex stability. DNA circular dichroism (CD) and FRET suggest that the promoter DNA in the closed complex is distorted. Modeling suggests two different orientations of the recognition helix of the RNA polymerase in the closed complex. We propose that the RNA polymerase recognizes the sequence dependent conformation of the promoter DNA in the closed complex.
Assuntos
DNA Bacteriano/química , RNA Polimerases Dirigidas por DNA/química , Regiões Promotoras Genéticas , Fator sigma/química , Pareamento de Bases , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Modelos Moleculares , Mutação , Ligação Proteica , Fator sigma/metabolismoRESUMO
Photoreceptor death causes blinding inheritable retinal diseases, such as retinitis pigmentosa (RP). As disease progression often outpaces therapeutic advances, finding effective treatments is urgent. This study focuses on developing a targeted approach by evaluating the efficacy of small peptides derived from pigment epithelium-derived factor (PEDF), known to restrict common cell death pathways associated with retinal diseases. Peptides with affinity for the PEDF receptor, PEDF-R, (17-mer and H105A) delivered via eye drops reached the retina, efficiently promoted photoreceptor survival, and improved retinal function in RP mouse models based on both the rd10 mutation and the rhodopsin P23H mutation. Additionally, intravitreal delivery of AAV-H105A vectors delayed photoreceptor degeneration in the latter RP mouse model. Furthermore, peptide H105A specifically prevented photoreceptor death induced by oxidative stress, a contributing factor to RP progression, in human retinal organoids. This promising approach for peptide eye drop delivery holds significant potential as a therapeutic for preventing photoreceptor death in retinal disorders, offering a high safety profile, low invasiveness and multiple delivery options.
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Optimizing energy use in the kidney is critical for normal kidney function. Here, we investigate the effect of hyperglycemia and sodium-glucose cotransporter 2 (SGLT2) inhibition on urinary amino acid excretion in individuals with type 1 diabetes (T1D). The open-label ATIRMA trial assessed the impact of 8 weeks of 25 mg empagliflozin orally once per day in 40 normotensive normoalbuminuric young adults with T1D. A consecutive 2-day assessment of clamped euglycemia and hyperglycemia was evaluated at baseline and posttreatment visits. Principal component analysis was performed on urinary amino acids grouped into representative metabolic pathways using MetaboAnalyst. At baseline, acute hyperglycemia was associated with changes in 25 of the 33 urinary amino acids or their metabolites. The most significant amino acid metabolites affected by acute hyperglycemia were 3-hydroxykynurenine, serotonin, glycyl-histidine, and nicotinic acid. The changes in amino acid metabolites were reflected by the induction of four biosynthetic pathways: aminoacyl-tRNA; valine, leucine, and isoleucine; arginine; and phenylalanine, tyrosine, and tryptophan. In acute hyperglycemia, empagliflozin significantly attenuated the increases in aminoacyl-tRNA biosynthesis and valine, leucine, and isoleucine biosynthesis. Our findings using amino acid metabolomics indicate that hyperglycemia stimulates biosynthetic pathways in T1D. SGLT2 inhibition may attenuate the increase in biosynthetic pathways to optimize kidney energy metabolism.
Assuntos
Compostos Benzidrílicos , Diabetes Mellitus Tipo 1 , Glucosídeos , Hiperglicemia , Adulto Jovem , Humanos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Transportador 2 de Glucose-Sódio , Leucina , Isoleucina , Aminoácidos/metabolismo , Hiperglicemia/tratamento farmacológico , Valina , RNA de TransferênciaRESUMO
Kidney dysfunction often leads to neurological impairment, yet the complex kidney-brain relationship remains elusive. We employed spatial and bulk metabolomics to investigate a mouse model of rapid kidney failure induced by mouse double minute 2 ( Mdm2) conditional deletion in the kidney tubules to interrogate kidney and brain metabolism. Pathway enrichment analysis of focused plasma metabolomics panel pinpointed tryptophan metabolism as the most altered pathway with kidney failure. Spatial metabolomics showed toxic tryptophan metabolites in the kidneys and brains, revealing a novel connection between advanced kidney disease and accelerated kynurenine degradation. In particular, the excitotoxic metabolite quinolinic acid was localized in ependymal cells adjacent to the ventricle in the setting of kidney failure. These findings were associated with brain inflammation and cell death. A separate mouse model of acute kidney injury also had an increase in circulating toxic tryptophan metabolites along with altered brain inflammation. Patients with advanced CKD similarly demonstrated elevated plasma kynurenine metabolites and quinolinic acid was uniquely correlated with fatigue and reduced quality of life in humans. Overall, our study identifies the kynurenine pathway as a bridge between kidney decline, systemic inflammation, and brain toxicity, offering potential avenues for diagnosis and treatment of neurological issues in kidney disease.
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The PPM phosphatases require millimolar concentrations of Mg²âº or Mn²âº to activate phosphatase activity in vitro. The human phosphatases PP2Cα (PPM1A) and Wip1 (PPM1D) differ in their physiological function, substrate specificity, and apparent metal affinity. A crystallographic structure of PP2Cα shows only two metal ions in the active site. However, recent structural studies of several bacterial PP2C phosphatases have indicated three metal ions in the active site. Two residues that coordinate the third metal ion are highly conserved, suggesting that human PP2C phosphatases may also bind a third ion. Here, isothermal titration calorimetry analysis of Mg²âº binding to PP2Cα distinguished binding of two ions to high affinity sites from the binding of a third ion with a millimolar affinity, similar to the apparent metal affinity required for catalytic activity. Mutational analysis indicated that Asp239 and either Asp146 or Asp243 was required for low-affinity binding of Mg²âº, but that both Asp146 and Asp239 were required for catalysis. Phosphatase activity assays in the presence of MgCl2, MnCl2, or mixtures of the two, demonstrate high phosphatase activity toward a phosphopeptide substrate when Mg²âº was bound to the low-affinity site, whether Mg²âº or Mn²âº ions were bound to the high affinity sites. Mutation of the corresponding putative third metal ion-coordinating residues of Wip1 affected catalytic activity similarly both in vitro and in human cells. These results suggest that phosphatase activity toward phosphopeptide substrates by PP2Cα and Wip1 requires the binding of a Mg²âº ion to the low-affinity site.
Assuntos
Domínio Catalítico , Magnésio/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Alanina/química , Sequência de Aminoácidos , Ácido Aspártico/química , Catálise , Células HEK293 , Humanos , Manganês/metabolismo , Fosfopeptídeos/metabolismo , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2C , Alinhamento de SequênciaRESUMO
The aim of this study was to evaluate the psychometric properties of the Brief Fatigue Inventory (BFI) in hemodialysis patients. During a dialysis day, patients completed both 9-item BFI and 21-item Beck Depression Inventory (BDI)-II questionnaires. The psychometric properties of the BFI were assessed in terms of reliability and validity. The BFI had an overall Cronbach's coefficient alpha of .92. Inter-item correlation coefficients between BFI items ranged from .38 to. 81 (all p < .0001). Exploratory factor analysis revealed bidimensional factor structure of the BFI-fatigue "severity" and fatigue "interference" explaining 11.0% and 62.0% of the total variance in the data set, respectively. In criterion validity analysis, BFI composite score correlated significantly with the total BDI-II score-Pearson correlation coefficient .40 (p < .0001). These preliminary results support the satisfactory psychometric properties of the BFI in assessing fatigue among hemodialysis patients during a dialysis day in a clinic setting.
Assuntos
Neoplasias , Humanos , Psicometria , Reprodutibilidade dos Testes , Diálise Renal , Inquéritos e Questionários , FadigaRESUMO
Our study aims to review the role of neoadjuvant chemotherapy (NACT) followed by interval debulking surgery (IDS) in patients with advanced endometrial cancer. Patients with advanced endometrial cancer treated with NACT followed by IDS at our institute from January 2010 to January 2020 were recruited. Data pertaining to baseline patient characteristics, surgical details, histopathology/imaging reports, treatment and follow up details including the development of recurrence and death were collected from institutional database. Disease free survival (DFS) and overall survival (OS) were calculated using Kaplan Meier survival curves. We recruited 31 patients for our study. About 83.9% patients showed partial response and 6.4% patients responded completely to NACT with none of the patients developing disease progression. Complete cytoreduction was achieved in 90.3% patients, optimal cytoreduction in 3.2% patients while 6.5% patients had suboptimal surgery. On completion of primary treatment, complete remission was achieved by 80.6% patients while 16.1% patients had progressive disease. Median follow up period was 21 months (range 1- 61 months). During follow up period, 51.6% patients developed recurrent disease after achieving complete remission and 61.3% patients died of disease progression/recurrence. The median DFS and median OS of the cohort was 15 months and 21 months respectively. The 2 year DFS for the cohort was 34.1% and the 3 year OS was 30.5%. NACT followed by IDS is a reasonably good option for advanced stage endometrial cancer not amenable to primary surgery. Innovative treatments are warranted in this cluster of patients.
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The human transcriptional positive coactivator 4 (PC4) activates several p53-dependent genes. It has been demonstrated that this is a consequence of direct interaction with p53. Previously, we have concluded that PC4 interacts mainly with the C-terminal negative regulatory domain of p53 through its DNA binding C-terminal half. NMR chemical shift perturbation studies with peptide fragments indicated that amino acids 380-386 of p53 are crucial for interaction with PC4. This was verified by fluorescence anisotropy and sedimentation velocity studies. A peptide consisting of p53-(380-386) sequence, when attached to a cell penetration tag and nuclear localization signal, localizes to the nucleus and inhibits luciferase gene expression from a transfected plasmid carrying a Luc gene under a p53-dependent promoter. Acetylation of lysine 382/381 enhanced the binding of this peptide to PC4 by about an order of magnitude. NMR and mutagenesis studies indicated that serine 73 of PC4 is an important residue for recognition of p53. Intermolecular nuclear Overhauser effect placed aspartate 76 in the vicinity of lysine 381, indicating that the region around residues 73-76 of PC4 is important for p53 recognition. We conclude that the 380-386 region of p53 interacts with the region around residues 73-76 of PC4, and acetylation of lysine 382/381 of p53 may play an important role in modulating p53-PC4 interaction and as a consequence PC4 mediated activation of p53 target genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Lisina/química , Lisina/genética , Lisina/metabolismo , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaRESUMO
The incidence of diabetic nephropathy (DN) is growing rapidly worldwide as a consequence of the rising prevalence of Type 2 diabetes mellitus (T2DM). Among U.S. ethnic groups, Mexican Americans have a disproportionately high incidence and prevalence of DN and associated end-stage renal disease (ESRD). In communities bordering Mexico, as many as 90% of Mexican American patients with ESRD also suffer from T2DM compared to only 50% of non-Hispanic Whites (NHW). Both socio-economic factors and genetic predisposition appear to have a strong influence on this association. In addition, certain pathogenetic and clinical features of T2DM and DN are different in Mexican Americans compared to NHW, raising questions as to whether the diagnostic and treatment strategies that are standard practice in the NHW patient population may not be applicable in Mexican Americans. This article reviews the epidemiology of DN in Mexican Americans, describes the pathophysiology and associated risk factors, and identifies gaps in our knowledge and understanding that needs to be addressed by future investigations.
Assuntos
Diabetes Mellitus Tipo 2/epidemiologia , Nefropatias Diabéticas/epidemiologia , Falência Renal Crônica/epidemiologia , Americanos Mexicanos/estatística & dados numéricos , Obesidade/epidemiologia , Índice de Massa Corporal , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/complicações , Humanos , Incidência , Falência Renal Crônica/complicações , Obesidade/complicações , Pobreza , Prevalência , Fatores de Risco , Texas/epidemiologia , População Branca/estatística & dados numéricosRESUMO
BACKGROUND: Cuban Americans have a high prevalence of type 2 diabetes, placing them at risk for cardiovascular disease (CVD) and increased medical costs. Little is known regarding the lifestyle risk factors of CVD among Cuban Americans. This study investigated modifiable CVD risk factors of Cuban Americans with and without type 2 diabetes. METHODS: Sociodemographics, anthropometrics, blood pressure, physical activity, dietary intake, and biochemical parameters were collected and assessed for n=79 and n=80 Cuban Americans with and without type 2 diabetes. RESULTS: Fourteen percent with diabetes and 24 percent without diabetes engaged in the recommended level of physical activity. Over 90 percent had over the recommended intake of saturated fats. Thirty-five percent were former or current smokers. DISCUSSION: Cuban Americans had several lifestyle factors that are likely to increase the risk of CVD. Their dietary factors were associated with blood cholesterol and body weight, which has been shown to impact on medical expenses. These findings may be used for designing programs for the prevention of CVD as well as type 2 diabetes for Cuban Americans.