RESUMO
In 2015 HMS DUNCAN was chosen as the intervention ship for an Institute of Naval Medicine (INM)-led project entitled Second Sea Lord (2SL)'s Feeding the Fleet Initiative. During her nine-month maiden deployment on Operation KIPION, a healthy lifestyle intervention was initiated, encompassing executive health, catering services, medical services and physical training. The key enabler of the success of the intervention was the formation of an effective Unit Health Committee. This article presents the lessons identified and provides useful examples from the intervention, which aimed to create a healthy lifestyle culture on board a deployed ship.
Assuntos
Comportamentos Relacionados com a Saúde , Promoção da Saúde/métodos , Militares/psicologia , Navios , Humanos , Medicina Naval , Reino UnidoRESUMO
African swine fever virus is a large DNA virus which can cause an acute haemorrhagic fever in pigs resulting in high mortality. No vaccine is available, limiting options for control. The virus encodes up to 165 genes and virus particles are multi-layered and contain more than 50 proteins. Pigs immunised with natural low virulence isolates or attenuated viruses produced by passage in tissue culture and by targeted gene deletions can be protected against challenge with virulent viruses. CD8+ cells are required for protection induced by attenuated strain OURT88/3. Passive transfer of antibodies from immune to naïve pigs can also induce protection. Knowledge of the genome sequences of attenuated and virulent strains and targeted gene deletions from virulent strains have identified a number of virus genes involved in virulence and immune evasion. This information can be used to produce rationally attenuated vaccine strains. Virus antigens that are targets for neutralising antibodies have been identified and immunisation with these recombinant proteins has been shown to induce partial protection. However knowledge of antigens which encode the dominant protective epitopes recognised by CD8+ T cells is lacking.
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Vírus da Febre Suína Africana/imunologia , Febre Suína Africana/prevenção & controle , Vacinas Virais/imunologia , África/epidemiologia , Febre Suína Africana/epidemiologia , Febre Suína Africana/virologia , Vírus da Febre Suína Africana/fisiologia , Animais , Anticorpos Antivirais , Genoma Viral , Genótipo , Epidemiologia Molecular , Filogeografia , Pesquisa , Suínos , Replicação ViralRESUMO
UNLABELLED: Summary. BACKGROUND: Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F-actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin's function in platelets is unknown. OBJECTIVE: To determine whether vinculin is required for the functions of platelets and their major integrin, α(IIb) ß(3) . METHODS: The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4-Cre mice. Platelet and integrin functions were studied in vivo and ex vivo. RESULTS: Vinculin was undetectable in platelets from Vcl fl/fl Cre(+) mice, as determined by immunoblotting and fluorescence microscopy. Vinculin-deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on α(IIb) ß(3) with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin-deficient platelets displayed normal agonist-induced fibrinogen binding to α(IIb) ß(3) , aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin-deficient platelets adhered to immobilized fibrinogen or collagen normally, under both static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin-deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to those in wild-type littermates in response to carotid artery injury with FeCl(3) . CONCLUSION: Despite promoting membrane cytoskeleton integrity when mechanical force is applied to α(IIb) ß(3) , vinculin is not required for the traditional functions of α(IIb) ß(3) or the platelet actin cytoskeleton.
Assuntos
Actinas/química , Plaquetas/metabolismo , Citoesqueleto/metabolismo , Vinculina/fisiologia , Animais , Linhagem da Célula , Colágeno/química , Fibrinogênio/química , Deleção de Genes , Megacariócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência/métodos , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Vinculina/metabolismoRESUMO
We study the properties of a polycarbonate melt near a nickel surface as a model system for the interaction of polymers with metal surfaces by employing a multiscale modeling approach. For bulk properties, a suitably coarse-grained bead-spring model is simulated by molecular dynamics methods with model parameters directly derived from quantum chemical calculations. The surface interactions are parametrized and incorporated by extensive quantum mechanical density functional calculations using the Car-Parrinello method. We find strong chemisorption of chain ends, resulting in significant modifications of the melt composition when compared to an inert wall.
RESUMO
The X-linked form of Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy that arises in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32), which is expressed by Schwann cells. We recently showed that Cx32 containing the CMTX-associated mutation, Ser-85-Cys (S85C), forms functional cell-cell channels in paired Xenopus oocytes. Here, we describe that this mutant connexin also shows increased opening of hemichannels in nonjunctional surface membrane. Open hemichannels may damage the cells through loss of ionic gradients and small metabolites and increased influx of Ca(2+), and provide a mechanism by which this and other mutant forms of Cx32 may damage cells in which they are expressed. Evidence for open hemichannels includes: (i) oocytes expressing the Cx32(S85C) mutant show greatly increased conductance at inside positive potentials, significantly larger than in oocytes expressing wild-type Cx32 (Cx32WT); and (ii) the induced currents are similar to those previously described for several other connexin hemichannels, and exhibit slowly developing increases with increasing levels of positivity and reversible reduction when intracellular pH is decreased or extracellular Ca(2+) concentration is increased. Although increased currents are seen, oocytes expressing Cx32(S85C) have lower levels of the protein in the surface and in total homogenates than do oocytes expressing Cx32WT; thus, under the conditions examined here, hemichannels in the surface membrane formed of the Cx32(S85C) mutant have a higher open probability than hemichannels formed of Cx32WT. This increase in functional hemichannels may damage Schwann cells and ultimately lead to loss of function in peripheral nerves of patients harboring this mutation.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Conexinas/genética , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Ligação Genética/genética , Mutação/genética , Cromossomo X/genética , Animais , Cálcio/farmacologia , Condutividade Elétrica , Junções Comunicantes/efeitos dos fármacos , Humanos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Xenopus laevis , Proteína beta-1 de Junções ComunicantesRESUMO
The balance between acceptable plasma-product safety and the demand for those products involves a finite (but very low) level of risk for transmitting infectious diseases. The Food and Drug Administration (FDA) has focused on the use of a triple safety net to provide safe plasma products. This safety net includes donor selection, plasma testing, and inventory hold (look back and retrieval). Although each part of this safety net contributes to reducing the overall viral load in plasma products, each part may not contribute equally to a safe product in all cases. Donor selection is not universally reliable, and plasma antibody testing can be negative for a disease during its seroconversion window period. However, inventory hold (especially for first-time donors) has added to the usefulness of the other safety-net components. This review article provides a brief overview of each safety component and subsequent related improvements, including advances in antibody testing for donor screening and the addition of PCR testing for donor minipools and viral inactivation procedures during plasma-product manufacturing. Dilemmas created by this safety approach are also discussed, including the debate over zero risk of product versus cost and the impact on donors when they test positive for the various diagnostic tests used. Other dilemmas include the decision to include or exclude currently used plasma tests with the advent of polymerase chain reaction (PCR) testing, expanded use of PCR to test for other plasma-transmitted diseases that impart limited risk to recipients, and the role of antigen/antibody interactions in plasma products, especially as the latter relate to plasma-pool and final-product testing.
Assuntos
Qualidade de Produtos para o Consumidor , Plasma/virologia , United States Food and Drug Administration , Humanos , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/normas , Reação em Cadeia da Polimerase/estatística & dados numéricos , Testes Sorológicos/economia , Testes Sorológicos/normas , Estados UnidosRESUMO
Viral reduction techniques help assure the highest level of plasma-product safety that is possible using today's technology. However, worldwide travel and changing demographics could bring new pathogens into focus and thus require plasma-product manufacturers to be continually vigilant in their efforts to refine current reduction techniques and develop and apply new methods. This review focuses on all the currently used viral reduction techniques, why viral reduction continues to be important (even with the use of polymerase chain reaction techniques to improve plasma screening), and possible future directions for viral reduction. When exploring current approaches, including heat treatment (especially pasteurization), solvent/detergent, pH changes, and ultrafiltration, this review also includes (where applicable) the negative impact of these techniques on product integrity and/or viruses not destroyed. Other techniques (eg, chromatography) and the fractionation steps relevant in the production of selected plasma products may also impart viral reduction and are briefly assessed. Relying on an extensive review of current online and printed literature, this article also discusses the current US and European guidelines and regulations that impact viral reduction techniques. This information includes emphasis on the recommendation to use at least 2 reduction techniques (each with a different mode of action) for each plasma product so as to substantially reduce both enveloped and nonenveloped viruses. The approach to validation studies and results from specific studies are reviewed, and future approaches for viral reduction are described.
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Plasma/virologia , Esterilização/métodos , Guias como Assunto , Humanos , Controle de Qualidade , Esterilização/normasRESUMO
Although the true risk of transmitting (classical) Creutzfeld-Jakob Disease (CJD) and variant CJD (vCJD) via transfusion is likely very minimal, a review of prions and the impact of these associated prion diseases is timely because of their current effect on safety policies in the blood-plasma industry. Various types of human and animal prion diseases are outlined and reviewed, with emphasis on the importance of cross-species transmission as is relevant for vCJD. Review of the prion theory focuses on the relationship of prions to disease pathophysiology, prion resistance to protein modification, and potential prion transmission. Causes (with emphasis on iatrogenic CJD) and diagnosis of clinical CJD are described and contrasted with the same for vCJD. The origin of vCJD, the pathophysiologic questions surrounding this condition, and the latest diagnostic tests and research are also reviewed. Comparison of transmission feasibility versus actual transmission of CJD or vCJD by blood or blood products is then explored. Reasons for the discrepancy between theoretic and actual transmission for CJD and the body of evidence provided by look-back studies are examined. When compared with CJD, reasons are provided for the higher theoretic risk of transmitting vCJD by blood products. Studies evaluating transmission by blood products in animals are considered. Transfusion practices that include European and US criteria to prevent vCJD through blood products are reviewed. This includes the debate surrounding product leukoreduction, deferral of donors at high risk either for exposure to vCJD or for contracting CJD, targeted elimination of donor plasma, and how some policies may have contributed to product shortages.
Assuntos
Síndrome de Creutzfeldt-Jakob/transmissão , Plasma , Animais , Qualidade de Produtos para o Consumidor , Síndrome de Creutzfeldt-Jakob/epidemiologia , Síndrome de Creutzfeldt-Jakob/genética , Guias como Assunto , Humanos , Estados Unidos , United States Food and Drug AdministrationRESUMO
Phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) mediates cell motility and changes in cell shape in response to extracellular stimuli. In platelets, it is synthesized from PI4P by PIP5K in response to stimulation of a G-protein-coupled receptor by an agonist, such as the thrombin. In the present study, we have addressed the pathway that induces PIP5K I alpha activation following the addition of thrombin. Under resting condition expressed PIP5K I alpha was predominantly localized in a perinuclear distribution. After stimulation of the thrombin receptor, PAR1, or overexpression of a constitutively active variant of G alpha(q), PIP5K I alpha translocated to the plasma membrane. Movement of PIP5K I alpha to the cell membrane was dependent on both GTP-bound Rac and Rho, but not Arf, because: 1) inactive GDP-bound variants of either Rac or Rho blocked the translocation induced by constitutively active G alpha(q), 2) constitutively GTP-bound active variants of Rac or Rho induced PIP5K I alpha translocation in the absence of other stimuli, and 3) constitutively active variants of Arf1 or Arf6 failed to induce membrane translocation of PIP5K I alpha. In addition, a dominant negative variant of Rho blocked the PIP5K I alpha membrane translocation induced by constitutively active Rac, whereas dominant negative variants of either Rac or Arf6 failed to block PIP5K I alpha membrane translocation induced by constitutively active Rho. This implies that the effect on PIP5K I alpha by Rac is indirect, and requires the activation of Rho. In contrast to the findings with PIP5K I alpha, the related lipid kinase PIP4K failed to undergo translocation after stimulation by small GTP-binding proteins Rac or Rho. We also tested whether membrane localization of PIP5K I alpha correlated with an increase in its lipid kinase activity and found that co-expressing of PIP5K I alpha with either constitutively active G alpha(q), Rac, or Rho led to a 5- to 7-fold increase in PIP5K I alpha activity. Thus, these findings suggest that stimulation of a G-protein-coupled receptor (PAR1) leads to the sequential activation of G alpha(q), Rac, Rho, and PIP5K I alpha. Once activated and translocated to the cell membrane, PIP5K I alpha becomes available to phosphorylate PI4P to generate PI4,5P(2) on the plasma membrane.
Assuntos
Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Células COS , Núcleo Celular/metabolismo , Ativação Enzimática , Genes Dominantes , Guanosina Trifosfato/metabolismo , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Transporte Proteico , Trombina/metabolismo , TransfecçãoRESUMO
CMTX, the X-linked form of Charcot-Marie-Tooth disease, is an inherited peripheral neuropathy arising in patients with mutations in the gene encoding the gap junction protein connexin 32 (Cx32). In this communication, we describe the expression levels and biophysical parameters of seven mutant forms of Cx32 associated with CMTX, when expressed in paired Xenopus oocytes. Paired oocytes expressing the R15Q and H94Q mutants show junctional conductances not statistically different from that determined for Cx32WT, though both show a trend toward reduced levels. The S85C and G12S mutants induce reduced levels of junctional conductance. Three other mutants (R15W, H94Y and V139M) induce no conductance above baseline when expressed in paired oocytes. Analysis of the conductance voltage relations for these mutants shows that the reduced levels of conductance are entirely (H94Y and V139M) or partly (S85C and R15W) explicable by a reduced open probability of the mutant hemichannels. The R15Q and H94Q mutations also show alterations in the conductance voltage relations that would be expected to minimally (H94Q) or moderately (R15Q) reduce the available gap junction communication pathway. The reduction in G12S induced conductance cannot be explained by alterations in hemichannel open probability and are more likely due to reduced junction formation. These results demonstrate that many CMTX mutations lead to loss of function of Cx32. For these mutations, the loss of function model is likely to explain the pathogenesis of CMTX.
Assuntos
Substituição de Aminoácidos , Doença de Charcot-Marie-Tooth/genética , Conexinas/genética , Junções Comunicantes/fisiologia , Mutação de Sentido Incorreto , Cromossomo X/genética , Animais , Doença de Charcot-Marie-Tooth/metabolismo , Doença de Charcot-Marie-Tooth/patologia , Conexinas/química , Conexinas/fisiologia , Feminino , Junções Comunicantes/química , Humanos , Potenciais da Membrana , Oócitos , Técnicas de Patch-Clamp , Conformação Proteica , RNA Mensageiro/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Xenopus laevis , Proteína beta-1 de Junções ComunicantesRESUMO
Our recent study indicates that lysophosphatidylcholine (LPC) enhances Sp1 binding and Sp1-dependent endothelial nitric oxide synthase (eNOS) promoter activity via the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK-1) signaling pathway (Cieslik, K., Lee, C.-M., Tang, J.-L., and Wu, K. K. (1999) J. Biol. Chem. 274, 34669-34675). To identify upstream signaling molecules, we transfected human endothelial cells with dominant negative and active mutants of Ras and evaluated their effects on eNOS promoter activity. Neither mutant altered the basal or LPC-induced eNOS promoter function. By contrast, a dominant negative mutant of phosphatidylinositol 3-kinase gamma (PI-3Kgamma) blocked the promoter activity induced by LPC. Wortmannin and LY 294002 had a similar effect. AG-490, a selective inhibitor of Janus kinase 2 (Jak2), also reduced the LPC-induced Sp1 binding and eNOS promoter activity to the basal level. LPC induced Jak2 phosphorylation, which was abolished by LY 294002 and the dominant negative mutant of PI-3Kgamma. LY 294002 and AG-490 abrogated MEK-1 phosphorylation induced by LPC but had no effect on Raf-1. These results indicate that PI-3Kgamma and Jak2 are essential for LPC-induced eNOS promoter activity. This signaling pathway was sensitive to pertussis toxin, suggesting the involvement of a G(i) protein in PI-3Kgamma activation. These results indicate that LPC enhances Sp1-dependent eNOS promoter activity by a pertussis toxin-sensitive, Ras-independent novel pathway, PI-3Kgamma/Jak2/MEK-1/ERK1/2.
Assuntos
Endotélio Vascular/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Isoenzimas/metabolismo , Óxido Nítrico Sintase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Anticorpos/farmacologia , Linhagem Celular , Cromonas/farmacologia , Classe Ib de Fosfatidilinositol 3-Quinase , Endotélio Vascular/citologia , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes ras , Humanos , Isoenzimas/genética , Janus Quinase 2 , Lisofosfatidilcolinas/farmacologia , MAP Quinase Quinase 1 , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Morfolinas/farmacologia , Óxido Nítrico Sintase Tipo III , Toxina Pertussis , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Fator de Transcrição Sp1/metabolismo , Transfecção , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since HOXB4 plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of HOXB4 expression in human hematopoietic stem cells. HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5' promoter. Culture of highly purified human CD34(+) bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3-10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20-50%. Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro. Cotransfection assays in both K562 and CD34(+) cells showed that USF-1 and USF-2, but not MITF, induce the HOXB4 promoter in response to signals stimulating stem cell self-renewal, through activation of the mitogen-activated protein kinase pathway. Thus hematopoietic expression of the human HOXB4 gene is regulated by the binding of USF-1 and USF-2, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.
Assuntos
Proteínas de Ligação a DNA , Células-Tronco Hematopoéticas , Proteínas de Homeodomínio/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Células da Medula Óssea , Biblioteca Genômica , Humanos , Células K562 , Proteínas Quinases Ativadas por Mitógeno , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transdução de Sinais , Ativação Transcricional , Fatores Estimuladores Upstream , Proteínas rasRESUMO
AIMS: To adapt the Diabetes Health Profile (DHP-1) for use with English speaking patients with Type 2 diabetes mellitus and to evaluate the psychometric properties of the adapted measure in a UK and Danish sample of insulin, tablet and diet-treated patients with Type 2 diabetes. METHODS: Following linguistic adaptation using the forward-backward translation procedure, the 32-item DHP-1 was sent to 650 and 800 consecutively selected UK and Danish patients with Type 2 diabetes. Construct validity was assessed using principal axis factoring. Factor stability was assessed across language groups using the coefficient of congruence. Reliability was evaluated using Cronbach's alpha and multi-trait analysis, including item convergent/discriminant validity. Subscale discriminant validity was assessed through known groups with one-way ANOVA and post hoc Scheffe tests for multiple comparisons. RESULTS: Eighteen items (56.25%) were retained following initial item analysis. A three-factor solution accounting for 45.6% and 40.3% of the total explained variance was identified in the UK and Danish samples, respectively. Factors were interpreted as psychological distress (PD), barriers to activity (BA) and disinhibited eating (DE). Factor congruence between language groups ranged from 0.98 to 0.99 and Cronbach's alpha ranged between 0.70 and 0.88. Item scaling success for both language versions was 88.9%. BA scores discriminated between treatment groups in both language groups (F = 24.24, P < 0.001; F = 7.68, P < 0.001) and PD scores in the UK sample (F = 20.97, P < 0.001). CONCLUSIONS: The DHP-18 developed for use with patients with Type 2 diabetes has been shown to have satisfactory internal reliability and validity and measurement equivalence across language groups.
Assuntos
Comparação Transcultural , Diabetes Mellitus Tipo 2/psicologia , Diabetes Mellitus Tipo 2/terapia , Psicometria , Qualidade de Vida , Afeto , Dinamarca , Diabetes Mellitus Tipo 2/fisiopatologia , Dieta para Diabéticos , Feminino , Nível de Saúde , Humanos , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Idioma , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Comportamento Social , Inquéritos e Questionários , Reino UnidoRESUMO
Intercellular channels formed by connexins (gap junctions) are sensitive to the application of transjunctional voltage (V(j)), to which they gate by the separate actions of their serially arranged hemichannels (Harris, A. L., D. C. Spray, and M. V. L. Bennett. 1981. J. Gen. Physiol. 77:95-117). Single channel studies of both intercellular and conductive hemichannels have demonstrated the existence of two separate gating mechanisms, termed "V(j)-gating" and "loop gating" (Trexler, E. B., M. V. L. Bennett, T. A. Bargiello, and V. K. Verselis. 1996. Proc. Natl. Acad. Sci. U.S.A. 93:5836-5841). In Cx32 hemichannels, V(j)-gating occurs at negative V(j) (Oh, S., J. B. Rubin, M. V. L. Bennett, V. K. Verselis, and T. A. Bargiello. 1999. J. Gen. Physiol. 114:339-364; Oh, S., C. K. Abrams, V. K. Verselis, and T. A. Bargiello. 2000. J. Gen. Physiol. 116:13-31). A negative charge substitution at the second amino acid position in the N-terminus reverses the polarity of V(j)-gating of Cx32 hemichannels (Verselis, V. K., C. S. Ginter, and T. A. Bargiello. 1994. Nature. 368:348-351;. J. Gen. Physiol. 116:13-31). We report that placement of a negative charge at the 5th, 8th, 9th, or 10th position can reverse the polarity of Cx32 hemichannel V(j)-gating. We conclude that the 1st through 10th amino acid residues lie within the transjunctional electric field and within the channel pore, as in this position they could sense changes in V(j) and be largely insensitive to changes in absolute membrane potential (V(m)). Conductive hemichannels formed by Cx32*Cx43E1 containing a negatively charged residue at either the 8th or 10th position display bi-polar V(j)-gating; that is, the open probability of hemichannels formed by these connexins is reduced at both positive and negative potentials and is maximal at intermediate voltages. In contrast, Cx32*Cx43E1 hemichannels with negative charges at either the 2nd or 5th positions are uni-polar, closing only at positive V(j). The simplest interpretation of these data is that the Cx32 hemichannel can adopt at least two different open conformations. The 1st-5th residues are located within the electric field in all open channel conformations, while the 8th and 10th residues lie within the electric field in one conformation and outside the electric field in the other conformation.
Assuntos
Conexinas/química , Conexinas/metabolismo , Junções Comunicantes/metabolismo , Ativação do Canal Iônico , Substituição de Aminoácidos , Animais , Sequência de Bases , Fenômenos Biofísicos , Biofísica , Conexinas/genética , Eletroquímica , Feminino , Técnicas In Vitro , Potenciais da Membrana , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos Antissenso/genética , Oócitos/metabolismo , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus , Proteína beta-1 de Junções ComunicantesRESUMO
The African swine fever virus protein A238L inhibits activation of NFAT transcription factor by binding calcineurin and inhibiting its phosphatase activity. NFAT controls the expression of many immunomodulatory proteins. Here we describe a 14-amino-acid region of A238L that is needed and sufficient for binding to calcineurin. By introducing mutations within this region, we have identified a motif (PxIxITxC/S) required for A238L binding to calcineurin; a similar motif is found in NFAT proteins. Peptides corresponding to this domain of A238L bind calcineurin but do not inhibit its phosphatase activity. Binding of A238L to calcineurin stabilizes the A238L protein in cells. Although A238L-mediated suppression of NF-kappaB-dependent gene expression occurs by a different mechanism, the A238L-calcineurin interaction may be required to stabilize A238L.
Assuntos
Calcineurina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Chlorocebus aethiops , Dados de Sequência Molecular , Fatores de Transcrição NFATC , Fragmentos de Peptídeos/metabolismo , Suínos , Células Vero , Proteínas Virais/metabolismoRESUMO
Pleckstrin is a 40-kD phosphoprotein containing NH(2)- and COOH-terminal pleckstrin homology (PH) domains separated by a disheveled-egl 10-pleckstrin (DEP) domain. After platelet activation, pleckstrin is rapidly phosphorylated by protein kinase C. We reported previously that expressed phosphorylated pleckstrin induces cytoskeletal reorganization and localizes in microvilli along with glycoproteins, such as integrins. Given the role of integrins in cytoskeletal organization and cell spreading, we investigated whether signaling from pleckstrin cooperated with signaling pathways involving the platelet integrin, alphaIIbbeta3. Pleckstrin induced cell spreading in both transformed (COS-1 & CHO) and nontransformed (REF52) cell lines, and this spreading was regulated by pleckstrin phosphorylation. In REF52 cells, pleckstrin-induced spreading was matrix dependent, as evidenced by spreading of these cells on fibrinogen but not on fibronectin. Coexpression with alphaIIbbeta3 did not enhance pleckstrin-mediated cell spreading in either REF52 or CHO cells. However, coexpression of the inactive variant alphaIIbbeta3 Ser753Pro, or beta3 Ser753Pro alone, completely blocked pleckstrin-induced spreading. This implies that alphaIIbbeta3 Ser753Pro functions as a competitive inhibitor by blocking the effects of an endogenous receptor that is used in the signaling pathway involved in pleckstrin-induced cell spreading. Expression of a chimeric protein composed of the extracellular and transmembrane portion of Tac fused to the cytoplasmic tail of beta3 completely blocked pleckstrin-mediated spreading, whereas chimeras containing the cytoplasmic tail of beta3 Ser753Pro or alphaIIb had no effect. This suggests that the association of an unknown signaling protein with the cytoplasmic tail of an endogenous integrin beta-chain is also required for pleckstrin-induced spreading. Thus, expressed phosphorylated pleckstrin promotes cell spreading that is both matrix and integrin dependent. To our knowledge, this is the first example of a mutated integrin functioning as a dominant negative inhibitor.
Assuntos
Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Animais , Células CHO , Células COS , Linhagem Celular Transformada , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Cricetinae , Matriz Extracelular/metabolismo , Fibrinogênio/farmacologia , Expressão Gênica/fisiologia , Mutagênese/fisiologia , Fosforilação , Plasmídeos , TransfecçãoRESUMO
Long-term vitamin D and calcium supplementation is effective in reducing nonvertebral fractures in elderly people. Increased bone fragility caused by secondary hyperparathyroidism (sHPT) and impaired balance are known risk factors for hip fractures. The hypothesis is that short-term therapy with calcium and vitamin D may improve body sway as well as sHPT more effectively than calcium monotherapy. The effects of 8 weeks of supplementation with vitamin D (cholecalciferol) and calcium on body sway and biochemical measures of bone metabolism were measured. The sample consisted of 148 women (mean [+/-SD] age, 74 +/- 1 years) with a 25-hydroxycholecalciferol level below 50 nmol/liter. They received either 1200 mg of calcium plus 800 IU of vitamin D or 1200 mg of calcium per day. We measured intact parathyroid hormone (PTH), markers of bone turnover, and body sway before and after treatment. Falls and fractures among the participants were followed over a 1-year period. Compared with calcium mono, supplementation with vitamin D and calcium resulted in an increase in serum 25-hydroxyvitamin D of 72% (p < 0.0001), a decrease in the serum PTH of 18% ( p = 0.0432), and a decrease in body sway of 9% (p = 0.0435). The mean number of falls per subject during a 1-year follow-up period was 0.45 for the calcium mono group and 0.24 for the calcium and vitamin D group (p = 0.0346). Short-term supplementation with vitamin D and calcium improves sHPT and body sway and therefore may prevent falls and subsequent nonvertebral fractures in elderly women.
Assuntos
Cálcio/farmacologia , Colecalciferol/farmacologia , Hiperparatireoidismo Secundário/tratamento farmacológico , Idoso , Cálcio/sangue , Cálcio/metabolismo , Cálcio/urina , Suplementos Nutricionais , Feminino , Marcha , Humanos , Hiperparatireoidismo Secundário/metabolismo , Hiperparatireoidismo Secundário/fisiopatologia , Hormônio Paratireóideo/sangue , Fatores de Tempo , Vitamina D/análogos & derivados , Vitamina D/sangueRESUMO
Gap junctions are intercellular channels formed by the serial, head to head arrangement of two hemichannels. Each hemichannel is an oligomer of six protein subunits, which in vertebrates are encoded by the connexin gene family. All intercellular channels formed by connexins are sensitive to the relative difference in the membrane potential between coupled cells, the transjunctional voltage (Vj), and gate by the separate action of their component hemichannels (Harris, A.L., D.C. Spray, and M.V. Bennett. 1981. J. Gen. Physiol. 77:95-117). We reported previously that the polarity of Vj dependence is opposite for hemichannels formed by two closely related connexins, Cx32 and Cx26, when they are paired to form intercellular channels (Verselis, V.K., C.S. Ginter, and T.A. Bargiello. 1994. Nature. 368:348-351). The opposite gating polarity is due to a difference in the charge of the second amino acid. Negative charge substitutions of the neutral asparagine residue present in wild-type Cx32 (Cx32N2E or Cx32N2D) reverse the gating polarity of Cx32 hemichannels from closure at negative Vj to closure at positive Vj. In this paper, we further examine the mechanism of polarity reversal by determining the gating polarity of a chimeric connexin, in which the first extracellular loop (E1) of Cx32 is replaced with that of Cx43 (Cx43E1). The resulting chimera, Cx32*Cx43E1, forms conductive hemichannels when expressed in single Xenopus oocytes and intercellular channels in pairs of oocytes (Pfahnl, A., X.W. Zhou, R. Werner, and G. Dahl. 1997. Pflügers Arch. 433:733-779). We demonstrate that the polarity of Vj dependence of Cx32*Cx43E1 hemichannels in intercellular pairings is the same as that of wild-type Cx32 hemichannels and is reversed by the N2E substitution. In records of single intercellular channels, Vj dependence is characterized by gating transitions between fully open and subconductance levels. Comparable transitions are observed in Cx32*Cx43E1 conductive hemichannels at negative membrane potentials and the polarity of these transitions is reversed by the N2E substitution. We conclude that the mechanism of Vj dependence of intercellular channels is conserved in conductive hemichannels and term the process Vj gating. Heteromeric conductive hemichannels comprised of Cx32*Cx43E1 and Cx32N2E*Cx43E1 subunits display bipolar Vj gating, closing to substates at both positive and negative membrane potentials. The number of bipolar hemichannels observed in cells expressing mixtures of the two connexin subunits coincides with the number of hemichannels that are expected to contain a single oppositely charged subunit. We conclude that the movement of the voltage sensor in a single connexin subunit is sufficient to initiate Vj gating. We further suggest that Vj gating results from conformational changes in individual connexin subunits rather than by a concerted change in the conformation of all six subunits.
Assuntos
Conexinas/fisiologia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Animais , Conexinas/química , Canais Iônicos/química , Potenciais da Membrana/fisiologia , Proteínas Recombinantes de Fusão/química , Xenopus , Proteína beta-1 de Junções ComunicantesAssuntos
Diabetes Insípido , Síndrome de Secreção Inadequada de HAD , Vasopressinas/fisiologia , Diabetes Insípido/diagnóstico , Diabetes Insípido/tratamento farmacológico , Diagnóstico Diferencial , Diuréticos/uso terapêutico , Humanos , Síndrome de Secreção Inadequada de HAD/diagnóstico , Síndrome de Secreção Inadequada de HAD/terapia , Vasopressinas/sangue , Desequilíbrio HidroeletrolíticoRESUMO
The connexins are a family of homologous integral membrane proteins that form channels that provide a low resistance pathway for the transmission of electrical signals and the diffusion of small ions and non-electrolytes between coupled cells. Individuals carrying mutations in the gene encoding connexin 32 (Cx32), a gap junction protein expressed in the paranodal loops and Schmidt-Lantermann incisures of myelinating Schwann cells, develop a peripheral neuropathy - the X-linked form of Charcot-Marie-Tooth disease (CMTX). Over 160 different mutations in Cx32 associated with CMTX have been identified. Some mutations will lead to complete loss of function with no possibility of expression of functional channels. Some mutations in Cx32 lead to the abnormal accumulation of Cx32 proteins in the cytoplasm, particularly in the Golgi apparatus; CMTX may arise due to incorrect trafficking of Cx32 or to interference with trafficking of other proteins. On the other hand, many mutant forms of Cx32 can form functional channels. Some functional mutants have conductance voltage relationships that are disrupted to a degree which would lead to a substantial reduction in the available gap junction mediated communication pathway. Others have essentially normal steady-state g-V relations. In one of these cases (Ser26Leu), the only change introduced by the mutation is a reduction in the pore diameter from 7 A for the wild-type channel to less than 3 A for Ser26Leu. This reduction in pore diameter may restrict the passage of important signaling molecules. These findings suggest that in some, if not all cases of CMTX, loss of function of normal Cx32 is sufficient to cause CMTX.