Assuntos
Anticorpos Facilitadores/imunologia , Macrófagos/virologia , Síndrome Respiratória Aguda Grave/virologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Animais , Anticorpos Antivirais/imunologia , Células Cultivadas , Chlorocebus aethiops , Desenho de Fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/patogenicidade , Síndrome Respiratória Aguda Grave/imunologia , Células Vero , Vacinas Virais/imunologia , Replicação ViralRESUMO
1. A SARS vaccine was produced based on recombinant native full-length Spike-protein trimers (triSpike) and efficient establishment of a vaccination procedure in rodents. 2. Antibody-mediated enhancement of SARS-CoV infection with anti-SARS-CoV Spike immune-serum was observed in vitro. 3. Antibody-mediated infection of SARS-CoV triggers entry into human haematopoietic cells via an FcγR-dependent and ACE2-, pH-, cysteine-protease-independent pathways. 4. The antibody-mediated enhancement phenomenon is not a mandatory component of the humoral immune response elicited by SARS vaccines, as pure neutralising antibody only could be obtained. 5. Occurrence of immune-mediated enhancement of SARS-CoV infection raises safety concerns regarding the use of SARS-CoV vaccine in humans and enables new ways to investigate SARS pathogenesis (tropism and immune response deregulation).
Assuntos
Anticorpos Neutralizantes/metabolismo , Anticorpos Facilitadores , Glicoproteínas de Membrana/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/imunologia , Proteínas do Envelope Viral/imunologia , Internalização do Vírus , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Antivirais/metabolismo , Linhagem Celular Tumoral , Cisteína Proteases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Endogâmicos BALB C , Monócitos , Peptidil Dipeptidase A/metabolismo , Receptores Fc/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Glicoproteína da Espícula de Coronavírus , VacinasRESUMO
The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.
Assuntos
Proteínas do Nucleocapsídeo/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/metabolismo , Montagem de Vírus , Animais , Chlorocebus aethiops , Proteínas M de Coronavírus , Proteínas do Nucleocapsídeo de Coronavírus , Humanos , Glicoproteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas do Nucleocapsídeo/genética , Glicoproteína da Espícula de Coronavírus , Células Vero , Proteínas do Envelope Viral/genética , Proteínas da Matriz Viral/genética , Proteínas Viroporinas , Virossomos/metabolismo , Virossomos/ultraestruturaRESUMO
Imatinib mesylate determines a favorable clinical course in most Ph positive Chronic Myeloid Leukemia (CML) patients in the chronic phase. Cytogenetic response is usually evaluated by analyzing 20-25 bone marrow metaphases using standard banding techniques. Since this methodology has very low sensitivity, we compared the results obtained by standard banding techniques to the ones obtained by fluorescent in situ hybridization (FISH). This was also done to identify any possible discrepancies between the two techniques. We analyzed 40 Ph+ CML patients in the chronic phase who had previously been treated with interferon alpha (IFNalpha) and who were receiving imatinib. The studies were performed by utilizing the same BM cell samples fixed in acetic acid/methanol, before imatinib therapy and then quarterly. Comparison of cytogenetic results to FISH results at 3 and 6 months of imatinib treatment showed that some patients who had achieved major cytogenetic response (i.e.<35% of examined metaphases showing Ph), showed retention of a higher number of persisting Ph+ cells when examined by FISH, and they did not achieve major FISH response (i.e. <35% of examined interphase cells show the BCR-ABL fusion signal). The discrepancy we found between the results that were obtained by analyzing metaphases and interphase cells disappeared in the subsequent examinations. Moreover, we found that 4 patients (10%) were still Ph+ in all the metaphases we examined even though they achieved excellent clinical response. On the basis of this small series of patients, we suggest that cytogenetic evaluation of patients on imatinib therapy should be performed by utilizing the classic banding technique (metaphase examination), but also by using the FISH technique (interphase examination), since the two methodologies may provide different results.
Assuntos
Antineoplásicos/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Neoplasia Residual/diagnóstico , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Adulto , Idoso , Benzamidas , Medula Óssea/metabolismo , Medula Óssea/patologia , Bandeamento Cromossômico , Análise Citogenética , Resistencia a Medicamentos Antineoplásicos , Humanos , Mesilato de Imatinib , Hibridização in Situ Fluorescente , Interferon-alfa/efeitos adversos , Interfase , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Metáfase , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Terapia de Salvação , Taxa de SobrevidaRESUMO
Connexins are the protein subunits of gap junction channels that allow a direct signaling pathway between networks of cells. The specific role of connexin channels in the homeostasis of different organs has been validated by the association of mutations in several human connexins with a variety of genetic diseases. Several connexins are present in the mammalian cochlea and at least four of them have been proposed as genes causing sensorineural hearing loss. We have started our functional analysis by selecting nine mutations in Cx26 that are associated with non-syndromic recessive deafness (DFNB1). We have observed that both human Cx26 wild-type (HCx26wt) and the F83L polymorphism, found in unaffected controls, generated electrical conductance between paired Xenopus oocytes, which was several orders of magnitude greater than that measured in water-injected controls. In contrast, most recessive Cx26 mutations (identified in DFNB1 patients) resulted in a simple loss of channel activity. In addition, the V37I mutation, originally identified as a polymorphism in heterozygous unaffected individuals, was devoid of function and thus may be pathologically significant. Unexpectedly, we have found that the recessive mutation V84L retained functional activity in both paired Xenopus oocytes and transfected HeLa cells. Furthermore, both the magnitude of macroscopic junctional conductance and its voltage-gating properties were indistinguishable from those of HCx26wt. The identification of functional differences of disease causing mutations may lead to define which permeation or gating properties of Cx26 are necessary for normal auditory function in humans and will be instrumental in identifying the molecular steps leading to DFNB1.
Assuntos
Conexinas/genética , Conexinas/metabolismo , Surdez/genética , Surdez/metabolismo , Mutação , Animais , Conexina 26 , Conexinas/química , Feminino , Junções Comunicantes/metabolismo , Genes Recessivos , Células HeLa , Humanos , Técnicas In Vitro , Ativação do Canal Iônico , Oócitos/metabolismo , Polimorfismo Genético , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Transfecção , XenopusRESUMO
A report on the Ninth International Gap Junction Conference, Honolulu, USA, 4-9 August 2001.
Assuntos
Comunicação Celular/fisiologia , Conexinas/fisiologia , Doença de Charcot-Marie-Tooth/metabolismo , Conexinas/química , Conexinas/genética , Conexinas/metabolismo , Junções Comunicantes/fisiologia , Conformação Proteica , Transdução de Sinais , Sinapses , Terminologia como AssuntoRESUMO
The discovery that the dominant X-linked form of Charcot-Marie-Tooth disease (CMTX), a genetic disease of the peripheral nervous system (PNS), is associated with mutations in connexin32 (Cx32) has brought attention to the importance of connexins in glial cell biology. To gain further insight into the consequences of Cx32 deficiency, we have undertaken a detailed characterization of the gene expression profile of Schwann cells isolated from the sciatic nerve of wild-type and Cx32-null mice. Schwann cells exhibit two distinct phenotypes, myelinating and nonmyelinating, which are defined by their different morphology with respect to axons and by their unique profile of gene expression. Our findings show that, regardless of the mouse genotype, cultured Schwann cells express similar levels of messages for a number of connexins and for genes characteristic of both the myelinating and the nonmyelinating phenotypes. Furthermore, we have identified Cx36, a member of the gamma subclass of connexins, which are preferentially expressed in neuronal cells of mouse brain and retina, as an additional connexin present in Schwann cells. Mice lacking Cx32, however, exhibited a marked up-regulation of glial fibrillary acidic protein (GFAP), a cytoskeletal protein usually synthesized only by nonmyelinating Schwann cells. This observation was extended to the PNS in vivo and did not reflect a general perturbation of the expression of other nonmyelinating Schwann cell genes. These findings demonstrate that the absence of Cx32 results in a distinct pattern of gene dysregulation in Schwann cells and that Schwann cell homeostasis is critically dependent on the correct expression of Cx32 and not just any connexin. Identifying the relationship between increased GFAP expression and the absence of Cx32 could lead to the definition of specific roles for Cx32 in the control of myelin homeostasis and in the development of CMTX.
Assuntos
Conexinas/genética , Células de Schwann/fisiologia , Animais , Células Cultivadas , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/fisiopatologia , Primers do DNA , Expressão Gênica/fisiologia , Proteína Glial Fibrilar Ácida/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Bainha de Mielina/fisiologia , Fenótipo , Células de Schwann/citologia , Nervo Isquiático/citologia , Proteína beta-1 de Junções ComunicantesRESUMO
Major advances in the identification of genes implicated in idiopathic epilepsy have been made. Generalized epilepsy with febrile seizures plus (GEFS+), benign familial neonatal convulsions and nocturnal frontal lobe epilepsy, three autosomal dominant idiopathic epilepsies, result from mutations affecting voltage-gated sodium and potassium channels, and nicotinic acetylcholine receptors, respectively. Disruption of GABAergic neurotransmission mediated by gamma-aminobutyric acid (GABA) has been implicated in epilepsy for many decades. We now report a K289M mutation in the GABA(A) receptor gamma2-subunit gene (GABRG2) that segregates in a family with a phenotype closely related to GEFS+ (ref. 8), an autosomal dominant disorder associating febrile seizures and generalized epilepsy previously linked to mutations in sodium channel genes. The K289M mutation affects a highly conserved residue located in the extracellular loop between transmembrane segments M2 and M3. Analysis of the mutated and wild-type alleles in Xenopus laevis oocytes confirmed the predicted effect of the mutation, a decrease in the amplitude of GABA-activated currents. We thus provide the first genetic evidence that a GABA(A) receptor is directly involved in human idiopathic epilepsy.
Assuntos
Epilepsia/genética , Mutação , Receptores de GABA-A/genética , Sequência de Aminoácidos , Segregação de Cromossomos , Sequência Conservada , Condutividade Elétrica , Epilepsia Neonatal Benigna/genética , Epilepsia do Lobo Frontal/genética , Epilepsia Generalizada/genética , Feminino , Humanos , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem , Subunidades Proteicas , Convulsões Febris/genética , Homologia de Sequência de AminoácidosRESUMO
By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.
Assuntos
Conexinas/genética , Junções Comunicantes/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Conexinas/química , Conexinas/classificação , Conexinas/metabolismo , Junções Comunicantes/química , Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Oócitos/fisiologia , Técnicas de Patch-Clamp , Filogenia , Alinhamento de Sequência , Distribuição Tecidual , Xenopus laevisRESUMO
Cx26 has been implicated in dominant (DFNA3) and recessive (DFNB1) forms of nonsyndromic sensorineural deafness. While most homozygous DFNB1 Cx26 mutations result in a simple loss of channel activity, it is less clear how heterozygous mutations in Cx26 linked to DFNA3 cause hearing loss. We have tested the ability of one dominant mutation (W44C) to interfere with wild-type human Cx26 (HCx26wt). HCx26wt induced robust electrical conductance between paired oocytes, and facilitated dye transfer between transfected HeLa cells. In contrast, oocyte pairs injected with only W44C were not electrically coupled above background levels, and W44C failed to dye couple transfected HeLa cells. Moreover, W44C dramatically inhibited intercellular conductance of HCx26wt when co-expressed in an equal ratio, and the low levels of residual conductance displayed altered gating properties. A nonfunctional recessive mutation (W77R) did not inhibit the ability of HCx26wt to form functional channels when co-injected in the same oocyte pairs, nor did it alter HCx26wt gating. These results provide evidence for a functional dominant negative effect of the W44C mutant on HCx26wt and explain how heterozygous Cx26 mutations could contribute to autosomal dominant deafness, by resulting in a net loss, and/or alteration, of Cx26 function.
Assuntos
Conexinas/genética , Surdez/genética , Mutação , Animais , Comunicação Celular/fisiologia , Conexina 26 , Conexinas/metabolismo , Eletrofisiologia , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Ativação do Canal Iônico/fisiologia , Isoquinolinas/metabolismo , Oócitos/fisiologia , Xenopus laevisAssuntos
Conexinas/metabolismo , Proteínas do Olho/metabolismo , Canais Iônicos/metabolismo , Retina/metabolismo , Rajidae/metabolismo , Animais , Conexinas/genética , Proteínas do Olho/genética , Feminino , Ativação do Canal Iônico , Oócitos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rajidae/genética , XenopusRESUMO
Electrical synapses (gap junctions) in neuronal circuits have become a major focus in the study of network properties such as synchronization and oscillation (Galarreta and Hestrin, 1999; Gibson et al., 1999). Despite the recent progress made in unraveling the contribution of gap junctions to network behavior, little is known about the molecular composition of the junctional constituents. By cloning gap junction proteins [connexins (Cxs)] from zebrafish retina and through functional expression, we demonstrate that the retina possesses a high degree of connexin diversity, which may account for differential functional properties of electrical synapses. Three new Cxs, designated as zebrafish Cx27.5 (zfCx27.5), zfCx44.1, and zfCx55.5, and the carp ortholog of mammalian Cx43 were cloned. By in situ hybridization and in situ RT-PCR, we demonstrate that the four fish connexin mRNAs show differential localization in the retina. Transient functional expression in paired Xenopus oocytes and in the neuroblastoma N2A cell line indicate an extreme range of electrophysiological properties of these connexins in terms of voltage dependence and unitary conductance. For instance, the new zfCx44.1 exhibited high sensitivity to voltage-induced closure with currents decaying rapidly for transjunctional potentials >10 mV, whereas zfCx55.5 channels showed an opposite voltage dependence in response to voltage steps of either polarity. Moreover, although zfCx44.1 channels showed unitary conductance as high as any previously reported for junctional channels (nearly 300 pS), zfCx55. 5 and zfCx27.5 exhibited much lower unitary conductances (<60 pS).
Assuntos
Conexina 43/genética , Conexinas/genética , Proteínas do Olho/genética , Retina/metabolismo , Proteínas de Peixe-Zebra , Animais , Carpas , Células Cultivadas , Clonagem Molecular , Conexina 26 , Conexina 43/metabolismo , Conexinas/metabolismo , Sequência Conservada , Proteínas do Olho/metabolismo , Feminino , Junções Comunicantes/metabolismo , Expressão Gênica , Hibridização In Situ , Microinjeções , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Especificidade de Órgãos/genética , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Sinapses/metabolismo , Xenopus laevis , Peixe-ZebraRESUMO
The reasons for the molecular heterogeneity of connexin channels in vivo remain unclear. Functional replacement of one connexin gene with another has now revealed unexpected phenotypes and shows that cellular homeostasis depends not simply on cell-cell communication but also on the correct types of connexin.
Assuntos
Conexinas/genética , Junções Comunicantes/metabolismo , Animais , Comunicação Celular , Conexinas/metabolismo , Feminino , Masculino , Camundongos , Camundongos Transgênicos , MutagêneseRESUMO
In the vertebrate eye, virtually every cell type is directly coupled to its neighbors by intercellular channels present in gap junctions. Although these structures share the common property of allowing adjacent cells to directly exchange ions, second messengers and small metabolites, intercellular channels in the eye also play a specific role in distinct functions such as neuronal transmission at electrotonic synapses in the retina, and the maintenance of homeostasis in the avascular lens. The structural proteins comprising these channels, the connexins (Cx), are a multigene family of which many members are expressed in the eye, even in the same cell type. This molecular heterogeneity poses the crucial question of whether and how a diversity in gap junctional structural proteins influences intercellular communication in ocular tissues. This review will focus on two recent advances in the understanding of connexin diversity in regard to the eye. First, connexin knockouts have demonstrated that postnatal development and homeostasis in the lens requires multiple connexin proteins. Secondly, functional characterization of new connexins that are abundantly expressed in the retina has revealed biophysical properties that mimic those recorded from retinal neurons.
Assuntos
Conexinas/genética , Junções Comunicantes/fisiologia , Retina/crescimento & desenvolvimento , Retina/fisiologia , Animais , Comunicação Celular/fisiologia , Junções Comunicantes/química , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Retina/citologia , Sinapses/química , Sinapses/fisiologiaRESUMO
Charcot-Marie-Tooth disease comprises a group of genetically heterogenous disorders of the peripheral nervous system. The X-linked form of Charcot-Marie-Tooth (CMTX) is associated with mutations in the gene encoding the gap junction protein connexin32 (Cx32), which is expressed in Schwann cells. Immunocytochemical evidence suggests that Cx32 is localized to the incisures of Schmidt-Lanterman and the paranodes of myelinating Schwann cells, where it appears to form reflexive gap junctions. It is currently thought that this cytoplasmic continuity provides a much shorter diffusion pathway for the transport of ions, metabolites and second messenger molecules through intracellular channels between the adaxonal and peri-nuclear regions of Schwann cells, across the myelin sheath. This review summarizes our current understanding of the role of connexins in Schwann cells and focuses on the lessons for channel function and disease pathophysiology derived from the functional analysis of Cx32 mutations. One of the most intriguing aspects emerging from this work is that several mutations retain functional competence, although the mutated channels exhibit altered gating properties. This suggests that partial and/or selective disruption of the radial communication pathway formed by Cx32 is sufficient to cause a functional deficit and lead to the development of CMTX. The next challenge will be to define, at the molecular level, the sequence of events involved in the disease process. The presence of a group of functional mutations should help understand the cellular basis of CMTX, by allowing the identification of the specific molecules that need to be exchanged through Cx32 channels, but are excluded from the mutated ones.