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1.
Molecules ; 24(15)2019 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-31362457

RESUMO

: It has long been established that mitochondrial dysfunction in Alzheimer's disease (AD) patients can trigger pathological changes in cell metabolism by altering metabolic enzymes such as the mitochondrial 17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10), also known as amyloid-binding alcohol dehydrogenase (ABAD). We and others have shown that frentizole and riluzole derivatives can inhibit 17ß-HSD10 and that this inhibition is beneficial and holds therapeutic merit for the treatment of AD. Here we evaluate several novel series based on benzothiazolylurea scaffold evaluating key structural and activity relationships required for the inhibition of 17ß-HSD10. Results show that the most promising of these compounds have markedly increased potency on our previously published inhibitors, with the most promising exhibiting advantageous features like low cytotoxicity and target engagement in living cells.


Assuntos
17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 17-Hidroxiesteroide Desidrogenases/química , Benzotiazóis/química , Ureia/química , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Desenho de Drogas , Humanos , Mitocôndrias/metabolismo , Estrutura Molecular , Relação Estrutura-Atividade
2.
Sci Immunol ; 3(30)2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30578351

RESUMO

Hundreds of patients with autosomal recessive, complete IL-12p40 or IL-12Rß1 deficiency have been diagnosed over the last 20 years. They typically suffer from invasive mycobacteriosis and, occasionally, from mucocutaneous candidiasis. Susceptibility to these infections is thought to be due to impairments of IL-12-dependent IFN-γ immunity and IL-23-dependent IL-17A/IL-17F immunity, respectively. We report here patients with autosomal recessive, complete IL-12Rß2 or IL-23R deficiency, lacking responses to IL-12 or IL-23 only, all of whom, unexpectedly, display mycobacteriosis without candidiasis. We show that αß T, γδ T, B, NK, ILC1, and ILC2 cells from healthy donors preferentially produce IFN-γ in response to IL-12, whereas NKT cells and MAIT cells preferentially produce IFN-γ in response to IL-23. We also show that the development of IFN-γ-producing CD4+ T cells, including, in particular, mycobacterium-specific TH1* cells (CD45RA-CCR6+), is dependent on both IL-12 and IL-23. Last, we show that IL12RB1, IL12RB2, and IL23R have similar frequencies of deleterious variants in the general population. The comparative rarity of symptomatic patients with IL-12Rß2 or IL-23R deficiency, relative to IL-12Rß1 deficiency, is, therefore, due to lower clinical penetrance. There are fewer symptomatic IL-23R- and IL-12Rß2-deficient than IL-12Rß1-deficient patients, not because these genetic disorders are rarer, but because the isolated absence of IL-12 or IL-23 is, in part, compensated by the other cytokine for the production of IFN-γ, thereby providing some protection against mycobacteria. These experiments of nature show that human IL-12 and IL-23 are both required for optimal IFN-γ-dependent immunity to mycobacteria, both individually and much more so cooperatively.


Assuntos
Imunidade Inata/imunologia , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-23/imunologia , Infecções por Micobactéria não Tuberculosa/imunologia , Mycobacterium/imunologia , Humanos , Interleucina-12/deficiência , Interleucina-12/genética , Interleucina-23/deficiência , Interleucina-23/genética , Linhagem
3.
Patient Prefer Adherence ; 12: 1907-1916, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30288031

RESUMO

Background: Higher levels of patient activation for self-managing health are associated with positive clinical and health care utilization outcomes. Identifying a patient's activation level can guide clinicians to tailor interventions to improve their health. Effective self-management of atrial fibrillation (AF) requires patient activation to participate in treatment decisions, prevent complications, and manage risk factors. Yet, little is known about activation in patients with AF. The purpose of this descriptive study was to identify patient activation levels and factors associated with activation in patients with AF. Methods: Patients (N=123), 66% male, with a mean (SD) age of 59.9 (11.3) years seeking treatment for AF at an arrhythmia clinic completed the Patient Activation Measure (PAM), Atrial Fibrillation Severity Scale, Knowledge about Atrial Fibrillation test, Hospital Anxiety Depression Scale, Godin Leisure-Time Exercise Questionnaire, and Patient Assessment of Chronic Illness Care. Sociodemographic and clinical data were obtained from medical records. PAM scores were categorized into Levels 1-4. Associations among patient-reported outcomes, sociodemographic, and clinical variables were analyzed using Fisher's exact tests and Kruskal-Wallis procedures. Results: The PAM scores of nearly half (45.5%) of the patients were at Level 3, while the scores of 38% were at Level 4. Male sex (P=0.02), higher education (P=0.004), being employed (P=0.005), lower body mass index (P=0.03), tobacco abstinence (P=0.02), less AF symptom burden (P=0.006), less depression (P≤0.0001) and anxiety (P=0.006), greater knowledge of AF (P=0.01), and higher levels of physical activity (P=0.02) were associated with higher activation levels. Conclusion: Higher levels of patient activation in those with AF were associated with a more positive health status and educational attainment. Additional research to describe activation in patients with AF is warranted to identify patients at risk for low activation and to tailor interventions to activation level.

4.
Mayo Clin Proc Innov Qual Outcomes ; 2(3): 218-225, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30225453

RESUMO

Atrial fibrillation (AF) is the most common cardiac arrhythmia in adults and is associated with an increased risk of stroke, heart failure, and death. Therapy for this pervasive arrhythmia is complex, involving multiple options that chiefly manage symptoms and prevent stroke. Current therapeutic strategies are also of limited efficacy, and can present potentially life-threatening side effects and/or complications. Emerging research suggests that the burden of AF can be reduced by improving patient understanding of the arrhythmia and teaching patients to adopt and maintain lifestyle and behavior changes. Shared medical appointments (SMAs) have been successfully used to deliver education and develop patient coping and disease management skills for patients with complex needs, but there is a paucity of studies examining the use of SMAs for managing AF. Moreover, few studies have examined strategies for implementing SMAs into routine clinical care. We detail our approach for (1) adapting a patient-centered SMA curriculum; (2) designing an evaluation comparing SMAs to routine care on patient outcomes; and (3) implementing SMAs into routine clinical practice. We conclude that evaluation and implementation of SMAs into routine clinical practice requires considerable planning and continuous engagement from committed key stakeholders, including patients, family members, schedulers, clinical staff, nurse educators, administrators, and billing specialists.

5.
Biomed Chromatogr ; 32(12): e4349, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30051494

RESUMO

Although reactive oxygen/nitrogen species (ROS/RNS) have a fundamental role in physiological processes, enhanced ROS/RNS production induced by exogenous sources, including drugs and other xenobiotics, may result in serious damage to biomolecules. Oxidative/nitrosative stress is being intensively investigated and might be responsible for a variety of health side effects. The present liquid chromatography-tandem mass spectrometry (LC-MS/MS) method provides reliable and accurate simultaneous measurement of malondialdehyde (MDA) and 3-nitrotyrosine (3-NT) in cultured human hepatoma (HepG2) cells. Sample preparation process involving ultrasonic homogenization, alkaline hydrolysis of protein-bound MDA and 3-NT, deproteination, derivatization of MDA by 2,4-dinitrophenylhydrazine and solid-phase extraction was optimized, ensuring the isolation and purification of desired analytes. Additionally, nonprotein thiols and nonprotein disulfides were measured using HPLC-UV. The established lower limit of quantification (0.025 nmol/mL for MDA; 0.0125 nmol/mL for 3-NT) allowed their LC-MS/MS determination in HepG2 cells exposed to model oxidizing agent, tert-butyl hydroperoxide (t-BOOH). The results show significant changes in MDA and 3-NT concentrations and alterations in thiol redox-state in dependence on the t-BOOH concentration and duration of its incubation in HepG2 cells. Concurrent evaluation of oxidative/nitrosative stress biomarkers in the in vitro model may significantly facilitate assessment of toxicity of newly developed drugs in preclinical trials and thus improve their safety profile.


Assuntos
Cromatografia Líquida/métodos , Malondialdeído/análise , Espectrometria de Massas em Tandem/métodos , Tirosina/análogos & derivados , Células Hep G2 , Humanos , Limite de Detecção , Neoplasias Hepáticas/metabolismo , Estresse Oxidativo , Reprodutibilidade dos Testes , Tirosina/análise
6.
Chem Res Toxicol ; 31(7): 534-547, 2018 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-29847927

RESUMO

Mosquito-borne diseases (including malaria) belong among the leading causes of death in humans. Vector control is a crucial part of the global strategy for management of mosquito-associated diseases, when insecticide use is the most important component in this effort. However, drug and insecticide resistance threaten the successes made with existing methods. Reduction or elimination of malaria is not possible without effective mosquito control. This article reviews current strategies of intervention in vector control to decrease transmission of disease and covers current relevant knowledge in molecular biology, biochemistry, and medicinal chemistry.


Assuntos
Anopheles/parasitologia , Resistência a Inseticidas , Malária/patologia , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Desenho de Drogas , Humanos , Inseticidas/química , Inseticidas/farmacologia , Malária/parasitologia , Mosquitos Vetores/efeitos dos fármacos
7.
J Am Chem Soc ; 139(40): 13981-13984, 2017 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-28933848

RESUMO

The cothermolysis of As4 and [Cp″2Zr(CO)2] (Cp″ = η5-C5H3tBu2) results in the formation of [Cp″2Zr(η1:1-As4)] (1) in high yields and the arsenic-rich complex [(Cp″2Zr)(Cp″Zr)(µ,η2:2:1-As5)] (2) as a minor product. In contrast to yellow arsenic, 1 is a light-stable, weighable and storable arsenic source for subsequent reactions. The transfer reaction of 1 with [Cp‴Fe(µ-Br)]2 (Cp‴ = η5-C5H2tBu3) yields the unprecedented bond isomeric complexes [(Cp‴Fe)2(µ,η4:4-As4)] (3a) and [(Cp‴Fe)2(µ,η4:4-cyclo-As4)] (3b). In contrast, the analogous reaction with the CpBn derivative [CpBnFe(µ-Br)]2 (CpBn = η5-C5(CH2(C6H5)5) leads exclusively to the triple decker complex [(CpBnFe)2(µ,η4:4-As4)] (4) possessing the tetraarsabutadiene-type ligand analogous to 3a. To elucidate the stability of the bonding isomers 3a and 3b, DFT calculations were performed. The oxidation of 4 with AgBF4 affords [(CpBnFe)2(µ,η5:5-As5)][BF4] (5), which is a product expanded by one arsenic atom, instead of the expected complex [(CpBnFe)2(µ,η4:4-cyclo-As4)]+.

8.
Angew Chem Int Ed Engl ; 56(25): 7307-7311, 2017 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-28508464

RESUMO

The redox chemistry of [Cp*Fe(η5 -As5 )] (1, Cp*=η5 -C5 Me5 ) has been investigated by cyclic voltammetry, revealing a redox behavior similar to that of its lighter congener [Cp*Fe(η5 -P5 )]. However, the subsequent chemical reduction of 1 by KH led to the formation of a mixture of novel Asn scaffolds with n up to 18 that are stabilized only by [Cp*Fe] fragments. These include the arsenic-poor triple-decker complex [K(dme)2 ][{Cp*Fe(µ,η2:2 -As2 )}2 ] (2) and the arsenic-rich complexes [K(dme)3 ]2 [(Cp*Fe)2 (µ,η4:4 -As10 )] (3), [K(dme)2 ]2 [(Cp*Fe)2 (µ,η2:2:2:2 -As14 )] (4), and [K(dme)3 ]2 [(Cp*Fe)4 (µ4 ,η4:3:3:2:2:1:1 -As18 )] (5). Compound 4 and the polyarsenide complex 5 are the largest anionic Asn ligand complexes reported thus far. Complexes 2-5 were characterized by single-crystal X-ray diffraction, 1 H NMR spectroscopy, EPR spectroscopy (2), and mass spectrometry. Furthermore, DFT calculations showed that the intermediate [Cp*Fe(η5 -As5 )]- , which is presumably formed first, undergoes fast dimerization to the dianion [(Cp*Fe)2 (µ,η4:4 -As10 )]2- .

9.
Artigo em Inglês | MEDLINE | ID: mdl-29322032

RESUMO

The DsbA homolog of Francisella tularensis was previously demonstrated to be required for intracellular replication and animal death. Disruption of the dsbA gene leads to a pleiotropic phenotype that could indirectly affect a number of different cellular pathways. To reveal the broad effects of DsbA, we compared fractions enriched in membrane proteins of the wild-type FSC200 strain with the dsbA deletion strain using a SILAC-based quantitative proteomic analysis. This analysis enabled identification of 63 proteins with significantly altered amounts in the dsbA mutant strain compared to the wild-type strain. These proteins comprise a quite heterogeneous group including hypothetical proteins, proteins associated with membrane structures, and potential secreted proteins. Many of them are known to be associated with F. tularensis virulence. Several proteins were selected for further studies focused on their potential role in tularemia's pathogenesis. Of them, only the gene encoding glyceraldehyde-3-phosphate dehydrogenase, an enzyme of glycolytic pathway, was found to be important for full virulence manifestations both in vivo and in vitro. We next created a viable mutant strain with deleted gapA gene and analyzed its phenotype. The gapA mutant is characterized by reduced virulence in mice, defective replication inside macrophages, and its ability to induce a protective immune response against systemic challenge with parental wild-type strain. We also demonstrate the multiple localization sites of this protein: In addition to within the cytosol, it was found on the cell surface, outside the cells, and in the culture medium. Recombinant GapA was successfully obtained, and it was shown that it binds host extracellular serum proteins like plasminogen, fibrinogen, and fibronectin.


Assuntos
Francisella tularensis/enzimologia , Francisella tularensis/patogenicidade , Deleção de Genes , Gliceraldeído-3-Fosfato Desidrogenases/deficiência , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Isomerases de Dissulfetos de Proteínas/deficiência , Animais , Proteínas Sanguíneas/metabolismo , Modelos Animais de Doenças , Francisella tularensis/imunologia , Camundongos , Viabilidade Microbiana , Ligação Proteica , Proteoma/análise , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Virulência , Fatores de Virulência/análise
10.
Chem Commun (Camb) ; 52(90): 13217-13220, 2016 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-27738672

RESUMO

Reduction of [Cp*Fe(η5-E5)] (E = P, As) with divalent lanthanide reagents usually leads to reduction of [Cp*Fe(η5-E5)] followed by a Ln-E bond formation. In contrast, by using the sterically encumbered reagent [(DippForm)2Sm(thf)2] (DippForm = {(2,6-iPr2C6H3)NC(H)[double bond, length as m-dash]N(2,6-iPr2C6H3)}-), ring-opening of thf and reduction of the polypnictide is observed. This leads to two new 3d/4f polyphosphide or polyarsenide complexes [(DippForm)2Sm(Cp*Fe)E5{(CH2)4O}{(DippForm)2Sm(thf)}], in which [(DippForm)2Sm(thf)2] and [Cp*Fe(η5-E5)] are linked by a ring-opened thf molecule and no Ln-E bond formation is observed.

11.
DNA Repair (Amst) ; 38: 117-126, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26774442

RESUMO

Expanded tandem repeat sequences in DNA are associated with at least 40 human genetic neurological, neurodegenerative, and neuromuscular diseases. Repeat expansion can occur during parent-to-offspring transmission, and arise at variable rates in specific tissues throughout the life of an affected individual. Since the ongoing somatic repeat expansions can affect disease age-of-onset, severity, and progression, targeting somatic expansion holds potential as a therapeutic target. Thus, understanding the factors that regulate this mutation is crucial. DNA repair, in particular mismatch repair (MMR), is the major driving force of disease-associated repeat expansions. In contrast to its anti-mutagenic roles, mammalian MMR curiously drives the expansion mutations of disease-associated (CAG)·(CTG) repeats. Recent advances have broadened our knowledge of both the MMR proteins involved in disease repeat expansions, including: MSH2, MSH3, MSH6, MLH1, PMS2, and MLH3, as well as the types of repeats affected by MMR, now including: (CAG)·(CTG), (CGG)·(CCG), and (GAA)·(TTC) repeats. Mutagenic slipped-DNA structures have been detected in patient tissues, and the size of the slip-out and their junction conformation can determine the involvement of MMR. Furthermore, the formation of other unusual DNA and R-loop structures is proposed to play a key role in MMR-mediated instability. A complex correlation is emerging between tissues showing varying amounts of repeat instability and MMR expression levels. Notably, naturally occurring polymorphic variants of DNA repair genes can have dramatic effects upon the levels of repeat instability, which may explain the variation in disease age-of-onset, progression and severity. An increasing grasp of these factors holds prognostic and therapeutic potential.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Doença/genética , Instabilidade Genômica , Expansão das Repetições de Trinucleotídeos/genética , Animais , Sequência de Bases , Humanos , Dados de Sequência Molecular , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/metabolismo , Polimorfismo Genético
12.
Nucleic Acids Res ; 42(16): 10473-87, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25147206

RESUMO

R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats.


Assuntos
Expansão das Repetições de DNA , Instabilidade Genômica , Proteínas/genética , Expansão das Repetições de Trinucleotídeos , Proteína C9orf72 , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Células HeLa , Humanos , Neurônios/metabolismo , RNA/química , RNA/metabolismo , Ribonuclease H/metabolismo
13.
Chemistry ; 20(13): 3759-68, 2014 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-24615817

RESUMO

Reactions of the sandwich complexes [Cp*Fe(η(5)-E5)] (Cp*=η(5)-C5Me5; E=P (1), As (2)) with the monovalent Group 13 metals Tl(+), In(+), and Ga(+) containing the weakly coordinating anion [TEF] ([TEF]=[Al{OC(CF3)3}4](-)) are described. Here, the one-dimensional coordination polymers [M(µ,η(5):η(1 -E5 FeCp*)3]n [TEF]n (E=P, M=Tl (3 a), In (3 b), Ga (3 c); E=As, M=Tl (4 a), In (4 b)) are obtained as sole products in good yields. All products were analyzed by single-crystal X-ray diffraction, revealing a similar assembly of the products with η(5)-bound E5 ligands and very weak σ-interactions between one P or As atom of the ring to the neighbored Group 13 metal cation. By exchanging the [TEF] anion of 4 a for the larger [FAl] anion ([FAl]=[FAl{OC6F10(C6F5)}3](-)), the coordination compound [Tl{(η(5)-As5)FeCp*}3][FAl] (5) without any σ-interactions of the As5-ring is obtained. All products are readily soluble in CH2 Cl2 and exhibit a dynamic coordination behavior in solution, which is supported by NMR spectroscopy and ESI-MS spectrometry as well as by osmometric molecular-weight determination. For a better understanding of the proceeding equilibrium DFT calculations of the cationic complexes were performed for the gas phase and in solution. Furthermore, the (31)P{(1)H} magic-angle spinning (MAS) NMR spectra of 3 a-c are presented and the first crystal structure of the starting material 2 was determined.


Assuntos
Compostos Ferrosos/química , Compostos Organometálicos/química , Compostos Organofosforados/química , Arsênico/química , Cátions/química , Cristalografia por Raios X , Gálio/química , Índio/química , Espectroscopia de Ressonância Magnética , Modelos Químicos , Conformação Molecular , Estrutura Molecular , Tálio/química
14.
Appl Microbiol Biotechnol ; 97(23): 10103-15, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24162084

RESUMO

The intracellular pathogens have the unique capacity to sense the host cell environment and to respond to it by alteration in gene expression and protein synthesis. Proteomic analysis of bacteria exposed directly to the host cell milieu might thus greatly contribute to the elucidation of processes leading to bacterial adaptation and proliferation inside the host cell. Here we have performed a global proteome analysis of a virulent Francisella tularensis subsp. holarctica strain during its intracellular cycle within the macrophage-like murine cell line J774.2 using the metabolic pulse-labeling of bacterial proteins with (35)S-methionine and (35)S-cysteine in various periods of infection. The two-dimensional gel analysis revealed macrophage-induced bacterial proteome changes in which 64 identified proteins were differentially expressed in comparison to controls grown in tissue culture medium. Nevertheless, activation of macrophages with interferon gamma before in vitro infection decreased the number of detected alterations in protein levels. Thus, these proteomic data indicate the F. tularensis ability to adapt to the intracellular hostile environment that is, however, diminished by prior interferon gamma treatment of host cells.


Assuntos
Proteínas de Bactérias/química , Francisella tularensis/fisiologia , Interações Hospedeiro-Patógeno , Tularemia/microbiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eletroforese em Gel Bidimensional , Francisella tularensis/química , Francisella tularensis/genética , Humanos , Macrófagos/microbiologia , Camundongos , Dados de Sequência Molecular , Proteômica
15.
Microbiology ; 159(Pt 11): 2364-2374, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24014665

RESUMO

Francisella tularensis is a highly infectious facultative intracellular bacterium and aetiological agent of tularaemia. The conserved hypothetical lipoprotein with homology to thiol/disulphide oxidoreductase proteins (FtDsbA) is an essential virulence factor in F. tularensis. Its protein sequence has two different domains: the DsbA_Com1_like domain (DSBA), with the highly conserved catalytically active site CXXC and cis-proline residue; and the domain amino-terminal to FKBP-type peptidyl-prolyl isomerases (FKBP_N). To establish the role of both domains in tularaemia infection models, site-directed and deletion mutagenesis affecting the active site (AXXA), the cis-proline (P286T) and the FKBP_N domain (ΔFKBP_N) were performed. The generated mutations led to high attenuation with the ability to induce full or partial host protective immunity. Recombinant protein analysis revealed that the active site CXXC as well as the cis-proline residue and the FKBP_N domain are necessary for correct thiol/disulphide oxidoreductase activity. By contrast, only the DSBA domain (and not the FKBP_N domain) seems to be responsible for the in vitro chaperone activity of the FtDsbA protein.


Assuntos
Francisella tularensis/enzimologia , Francisella tularensis/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Análise Mutacional de DNA , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Deleção de Sequência , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
16.
J Exp Med ; 210(9): 1743-59, 2013 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-23897980

RESUMO

Kaposi sarcoma (KS), a human herpes virus 8 (HHV-8; also called KSHV)-induced endothelial tumor, develops only in a small fraction of individuals infected with HHV-8. We hypothesized that inborn errors of immunity to HHV-8 might underlie the exceedingly rare development of classic KS in childhood. We report here autosomal recessive OX40 deficiency in an otherwise healthy adult with childhood-onset classic KS. OX40 is a co-stimulatory receptor expressed on activated T cells. Its ligand, OX40L, is expressed on various cell types, including endothelial cells. We found OX40L was abundantly expressed in KS lesions. The mutant OX40 protein was poorly expressed on the cell surface and failed to bind OX40L, resulting in complete functional OX40 deficiency. The patient had a low proportion of effector memory CD4(+) T cells in the peripheral blood, consistent with impaired CD4(+) T cell responses to recall antigens in vitro. The proportion of effector memory CD8(+) T cells was less diminished. The proportion of circulating memory B cells was low, but the antibody response in vivo was intact, including the response to a vaccine boost. Together, these findings suggest that human OX40 is necessary for robust CD4(+) T cell memory and confers apparently selective protective immunity against HHV-8 infection in endothelial cells.


Assuntos
Padrões de Herança/genética , Receptores OX40/deficiência , Receptores OX40/genética , Sarcoma de Kaposi/genética , Adulto , Idade de Início , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Formação de Anticorpos/imunologia , Sequência de Bases , Membrana Celular/metabolismo , Criança , Cisteína/genética , Feminino , Células HEK293 , Homozigoto , Humanos , Memória Imunológica/imunologia , Espaço Intracelular/metabolismo , Contagem de Linfócitos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Receptores OX40/química , Receptores OX40/metabolismo , Sarcoma de Kaposi/sangue , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/patologia , Subpopulações de Linfócitos T/metabolismo , Adulto Jovem
17.
J Prosthet Dent ; 108(3): 165-72, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22944312

RESUMO

STATEMENT OF PROBLEM: The accuracy of impressions has been described in 1 or 2 dimensions, whereas it is most desirable to evaluate the accuracy of impressions spatially, in 3 dimensions. PURPOSE: The purpose of this study was to demonstrate the accuracy and reproducibility of a 3-dimensional (3-D) approach to assessing impression preciseness and to quantitatively comparing the occlusal correctness of gypsum dies made with different impression materials. MATERIAL AND METHODS: By using an aluminum replica of a maxillary molar, single-step dual viscosity impressions were made with 1 polyether/vinyl polysiloxane hybrid material (Identium), 1 vinyl polysiloxane (Panasil), and 1 polyether (Impregum) (n=5). Corresponding dies were made of Type IV gypsum and were optically digitized and aligned to the virtual reference of the aluminum tooth. Accuracy was analyzed by computing mean quadratic deviations between the virtual reference and the gypsum dies, while deviations of the dies among one another determined the reproducibility of the method. The virtual reference was adapted to create 15 occlusal contact points. The percentage of contact points deviating within a ±10 µm tolerance limit (PDP(10) = Percentage of Deviating Points within ±10 µm Tolerance) was set as the index for assessing occlusal accuracy. Visual results for the difference from the reference tooth were displayed with colors, whereas mean deviation values as well as mean PDP(10) differences were analyzed with a 1-way ANOVA and Scheffé post hoc comparisons (α=.05). RESULTS: Objective characterization of accuracy showed smooth axial surfaces to be undersized, whereas occlusal surfaces were accurate or enlarged when compared to the original tooth. The accuracy of the gypsum replicas ranged between 3 and 6 µm, while reproducibility results varied from 2 to 4 µm. Mean (SD) PDP(10)-values were: Panasil 91% (±11), Identium 77% (±4) and Impregum 29% (±3). One-way ANOVA detected significant differences among the subjected impression materials (P<.001). CONCLUSIONS: The accuracy and reproducibility of impressions were determined by 3-D analysis. Results were presented as color images and the newly developed PDP(10)-index was successfully used to quantify spatial dimensions for complex occlusal anatomy. Impression materials with high PDP(10)-values were shown to reproduce occlusal dimensions the most accurately.


Assuntos
Materiais para Moldagem Odontológica , Técnica de Moldagem Odontológica , Imagem Tridimensional , Análise de Variância , Materiais para Moldagem Odontológica/normas , Técnica de Moldagem Odontológica/normas , Modelos Dentários , Precisão da Medição Dimensional , Humanos , Dente Molar/anatomia & histologia , Polivinil , Reprodutibilidade dos Testes , Resinas Sintéticas , Siloxanas , Estatísticas não Paramétricas
18.
J Clin Invest ; 122(9): 3239-47, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22850876

RESUMO

Epidermodysplasia verruciformis (EV) is a rare genetic disorder characterized by increased susceptibility to specific human papillomaviruses, the betapapillomaviruses. These EV-HPVs cause warts and increase the risk of skin carcinomas in otherwise healthy individuals. Inactivating mutations in epidermodysplasia verruciformis 1 (EVER1) or EVER2 have been identified in most, but not all, patients with autosomal recessive EV. We found that 2 young adult siblings presenting with T cell deficiency and various infectious diseases, including persistent EV-HPV infections, were homozygous for a mutation creating a stop codon in the ras homolog gene family member H (RHOH) gene. RHOH encodes an atypical Rho GTPase expressed predominantly in hematopoietic cells. Patients' circulating T cells contained predominantly effector memory T cells, which displayed impaired TCR signaling. Additionally, very few circulating T cells expressed the ß7 integrin subunit, which homes T cells to specific tissues. Similarly, Rhoh-null mice exhibited a severe overall T cell defect and abnormally small numbers of circulating ß7-positive cells. Expression of the WT, but not of the mutated RHOH, allele in Rhoh-/- hematopoietic stem cells corrected the T cell lymphopenia in mice after bone marrow transplantation. We conclude that RHOH deficiency leads to T cell defects and persistent EV-HPV infections, suggesting that T cells play a role in the pathogenesis of chronic EV-HPV infections.


Assuntos
Epidermodisplasia Verruciforme/genética , Linfócitos T/patologia , Fatores de Transcrição/deficiência , Proteínas rho de Ligação ao GTP/deficiência , Adulto , Animais , Sequência de Bases , Betapapillomavirus , Estudos de Casos e Controles , Códon sem Sentido , Consanguinidade , Suscetibilidade a Doenças , Epidermodisplasia Verruciforme/imunologia , Epidermodisplasia Verruciforme/patologia , Epidermodisplasia Verruciforme/virologia , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Integrinas/metabolismo , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Linhagem , Polimorfismo de Nucleotídeo Único , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Sequência de DNA , Transdução de Sinais , Fatores de Transcrição/genética , Proteínas rho de Ligação ao GTP/genética
19.
Virus Res ; 165(2): 179-89, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22374337

RESUMO

The potential of Herpesvirus saimiri (HVS) subgroups A, B and C and Herpesvirus ateles (HVA) to transform primary T cells to permanent growth in vitro is restricted by the primate host species and by viral variability represented by distinct viral oncoproteins. We now addressed the relation between the transforming potential of the different viruses and the signaling pathways activated by transiently expressed oncoproteins. Marmoset lymphocytes were transformed by all HVS subgroups as well as HVA, while transformation of human cells was restricted to HVS-C and, unexpectedly, HVA. NF-κB and Src-family kinase (SFK) activity was required for survival of all transformed lymphocytes. Accordingly, NF-κB was induced by oncoproteins of all viruses. In contrast, SFK-related signaling was detectable only for oncoproteins of HVS-C and HVA. Thus, the restricted transformation of human lymphocytes likely correlates with the specific SFK targeting by these oncoproteins. These results will enable further studies into novel SFK effector mechanisms relevant for T-cell proliferation.


Assuntos
Herpesvirus Saimiriíneo 2/patogenicidade , Ativação Linfocitária , Proteínas Oncogênicas/metabolismo , Rhadinovirus/patogenicidade , Transdução de Sinais , Animais , Callithrix , Células Cultivadas , Humanos , Primatas
20.
J Virol ; 86(3): 1862-73, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22130528

RESUMO

Herpesviruses establish latency in suitable cells of the host organism after a primary lytic infection. Subgroup C strains of herpesvirus saimiri (HVS), a primate gamma-2 herpesvirus, are able to transform human and other primate T lymphocytes to stable growth in vitro. The viral genomes persist as nonintegrated, circular, and histone-associated episomes in the nuclei of those latently infected T cells. Epigenetic modifications of episomes are essential to restrict the transcription during latency to selected viral genes, such as the viral oncogenes stpC/tip and the orf73/LANA. In this study, we describe a genome-wide chromatin immunoprecipitation-on-chip (ChIP-on-chip) analysis to profile the occupancy of CTCF on the latent HVS genome. We then focused on two distinct, conserved CTCF binding sites (CBS) within the orf73/LANA promoter region. Analysis of recombinant viruses harboring deletions or mutations within the CBS indicated that the lytic replication of such viruses is not substantially influenced by CTCF. However, T cells latently infected with CBS mutants were impaired in their proliferation abilities and showed a significantly reduced episomal maintenance. We detected a reduced transcription of the orf73/LANA gene in the T cells, corresponding to the reduced viral genomes; this might contribute to the loss of HVS episomes, as LANA is central in the maintenance of viral episomes in the dividing T cell populations. These data demonstrate that the episomal stability of HVS genomes in latently infected human T cells is dependent on CTCF.


Assuntos
Herpesvirus Saimiriíneo 2/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Linfócitos T/virologia , Proteínas Virais/genética , Latência Viral , Sequência de Bases , Sítios de Ligação , Fator de Ligação a CCCTC , Linhagem Celular , Imunoprecipitação da Cromatina , Cromossomos Artificiais Bacterianos , Primers do DNA , Herpesvirus Saimiriíneo 2/fisiologia , Humanos , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Proteínas Virais/metabolismo
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