Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 38
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Rev Neurol (Paris) ; 172(10): 594-606, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27633507

RESUMO

Myofibrillar myopathies (MFM) are mostly adult-onset diseases characterized by progressive morphological alterations of the muscle fibers beginning in the Z-disk and the presence of protein aggregates in the sarcoplasm. They are mostly caused by mutations in different genes that encode Z-disk proteins, including DES, CRYAB, LDB3, MYOT, FLNC and BAG3. A large family of French origin, presenting an autosomal dominant pattern, characterized by cardiac arrhythmia associated to late-onset muscle weakness, was evaluated to clarify clinical, morphological and genetic diagnosis. Muscle weakness began during adult life (over 30 years of age), and had a proximal distribution. Histology showed clear signs of a myofibrillar myopathy, but with unusual, large inclusions. Subsequently, genetic testing was performed in MFM genes available for screening at the time of clinical/histological diagnosis, and desmin (DES), αB-crystallin (CRYAB), myotilin (MYOT) and ZASP (LDB3), were excluded. LMNA gene screening found the p.R296C variant which did not co-segregate with the disease. Genome wide scan revealed linkage to 7q.32, containing the FLNC gene. FLNC direct sequencing revealed a heterozygous c.3646T>A p.Tyr1216Asn change, co-segregating with the disease, in a highly conserved amino acid of the protein. Normal filamin C levels were detected by Western-blot analysis in patient muscle biopsies and expression of the mutant protein in NIH3T3 showed filamin C aggregates. This is an original FLNC mutation in a MFM family with an atypical clinical and histopathological presentation, given the presence of significantly focal lesions and prominent sarcoplasmic masses in muscle biopsies and the constant heart involvement preceding significantly the onset of the myopathy. Though a rare etiology, FLNC gene should not be excluded in early-onset arrhythmia, even in the absence of myopathy, which occurs later in the disease course.


Assuntos
Arritmias Cardíacas/etiologia , Filaminas/genética , Debilidade Muscular/etiologia , Doenças Musculares/complicações , Doenças Musculares/genética , Mutação de Sentido Incorreto/genética , Adolescente , Adulto , Idade de Início , Idoso , Sequência de Aminoácidos , Análise Mutacional de DNA , Família , Feminino , Genoma Humano , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Miofibrilas/patologia , Linhagem , Adulto Jovem
2.
Rev Neurol (Paris) ; 171(10): 715-29, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26342832

RESUMO

Myofibrillar myopathies (MFM) have been described in the mid-1990s as a group of diseases sharing common histological features, including an abnormal accumulation of intrasarcoplasmic proteins, the presence of vacuoles and a disorganization of the intermyofibrillar network beginning at the Z-disk. The boundaries of this concept are still uncertain, and whereas six genes (DES, CRYAB, LDB3/ZASP, MYOT, FLNC and BAG3) are now classically considered as responsible for MFM, other entities such as FHL1 myopathy or Hereditary Myopathy with Early Respiratory Failure linked to mutations of titin can now as well be included in this group. The diagnosis of MFM is not always easy; as histological lesions can be focal, and muscle biopsy may be disappointing; this has led to a growing importance of muscle imaging, and the selectivity of muscle involvement has now been described in several disorders. Due to the rarity of these myopathies, if some clinical patterns (such as distal myopathy associated with cardiomyopathy due to desmin mutations) are now well known, surprises remain possible and should lead to systematic testing of the known genes in case of a typical histological presentation. In this paper, we aim at reviewing the data acquired on the six main genes listed above as well as presenting the experience from two French reference centres, Paris and Marseilles.


Assuntos
Miofibrilas/patologia , Miopatias Congênitas Estruturais/patologia , Adolescente , Adulto , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/genética , Músculo Esquelético/patologia , Miofibrilas/genética , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/terapia , Adulto Jovem
3.
Phys Biol ; 10(1): 016001, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23234811

RESUMO

Mechanics is now recognized as crucial in cell function. To date, the mechanical properties of cells have been inferred from experiments which investigate the roles of actin and microtubules ignoring the intermediate filaments (IFs) contribution. Here, we analyse myoblasts behaviour in the context of myofibrillar myopathy resulting from p.D399Y desmin mutation which disorganizes the desmin IF network in muscle cells. We compare the response of myoblasts expressing either mutated or wild-type desmin to cyclic stretch. Cells are cultivated on supports submitted to periodic uniaxial stretch of 20% elongation amplitude and 0.3 Hz frequency. We show that during stretching cycles, cells expressing mutated desmin reduce their mean amplitude both for the elongation and spreading area compared to those expressing wild-type desmin. Even more unexpected, the reorientation angles are altered in the presence of p.D399Y desmin. Yet, at rest, the whole set of those parameters are similar for the two cell populations. Thus, we demonstrate that IFs affect the mechanical properties and the dynamics of cell reorientation. Since these processes are known due to actin cytoskeleton, these results suggest the IFs implication in mechanics signal transduction. Further studies may lead to better understanding of their contribution to this process.


Assuntos
Desmina/química , Desmina/genética , Doenças Musculares/fisiopatologia , Mioblastos/metabolismo , Estresse Mecânico , Adesão Celular , Células Cultivadas , Humanos , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Doenças Musculares/genética , Doenças Musculares/metabolismo , Mutação
4.
Acta Myol ; 30(2): 121-6, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22106715

RESUMO

The term myofibrillar myopathies (MFM) refers to uncommon neuromuscular disorders that pathologically are characterized by myofibrillar degeneration and ectopic expression of several proteins. MFM are partly caused by mutations in genes that encode mainly Z-disk-related proteins (desmin, alphaB-crystallin, myotilin, ZASP, filamin C and BAG3). We reviewed clinical, light and electron microscopy, immunohistochemistry, immunoblotting and genetic findings of 21 patients with MFM (15 unrelated patients and three pairs of brothers) investigated at our neuromuscular center. MFM patients begin to show symptoms at any age, from juvenile to late adult life and present a different distribution of muscle weakness. Cardiac involvement and peripheral neuropathy are common. Typical histological features include focal areas with reduction/loss of ATPase and oxidative enzyme activity, and amorphous material (eosinophilic on hematoxylin and eosin and dark blue on Engel-Gomori trichrome) in these abnormal fiber areas. Electron microscopy shows disintegration of myofibrils starting from the Z-disk and accumulation of granular and filamentous material among the myofilaments. Immunohistochemical studies demonstrate focal accumulation of desmin, alphaB-crystallin and myotilin in abnormal muscle fibers while immunoblot analysis does not highlight differences in the expression of these proteins also including ZASP protein. Therefore, unlike immunoblot, immunohistochemistry together with light and electron microscopy is a useful diagnostic tool in MFM. Finally three of our 21 patients have missense mutations in the desmin gene, two brothers carry missense mutations in the gene encoding myotilin, one has a missense mutation in alphaB-crystallin, and none harbour pathogenic variations in the genes encoding ZASP and BAG3.


Assuntos
Proteínas Contráteis/genética , Proteínas do Citoesqueleto/genética , Debilidade Muscular/etiologia , Distrofias Musculares/etiologia , Miofibrilas , Idade de Início , Estudos de Coortes , Proteínas Contráteis/metabolismo , Proteínas do Citoesqueleto/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Cardiopatias/etiologia , Cardiopatias/fisiopatologia , Humanos , Imuno-Histoquímica , Padrões de Herança , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Debilidade Muscular/patologia , Debilidade Muscular/fisiopatologia , Distrofias Musculares/epidemiologia , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Mutação , Miofibrilas/metabolismo , Miofibrilas/patologia , Doenças do Sistema Nervoso Periférico/etiologia , Doenças do Sistema Nervoso Periférico/fisiopatologia
5.
Neuromuscul Disord ; 18(8): 656-66, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18653338

RESUMO

We studied the ultrastructural characteristics in patients with myofibrillar myopathy (MFM) and differentiated between MFM-subtypes using electron microscopic (EM) findings. The ultrastructural findings in 19 patients with different genetically proven MFMs (9 desmin, 5 alphaB-crystallin, 3 ZASP, 2 myotilin) were analyzed. In one ZASPopathy, we additionally performed an immunoEM study, using antibodies against desmin, alphaB-crystallin, ZASP and myotilin. The ultrastructural findings in desminopathies and alphaB-crystallinopathies were very similar and consisted of electrondense granulofilamentous accumulations and sandwich formations. They differed in the obvious presence of early apoptotic nuclear changes in alphaB-crystallinopathies. ZASPopathies were characterized by filamentous bundles (labeled with the myotilin antibody on immunoEM), and floccular accumulations of thin filamentous material. Tubulofilamentous inclusions in sarcoplasm and myonuclei in combination with filamentous bundles were characteristic for myotilinopathies. We conclude that MFMs ultrastructural findings can direct diagnostic efforts towards the causal gene mutated, and that EM should be included in the diagnostic workup of MFMs.


Assuntos
Doenças Musculares/genética , Doenças Musculares/patologia , Miofibrilas/genética , Miofibrilas/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Idoso , Conectina , Cristalinas/genética , Proteínas do Citoesqueleto/genética , Desmina/genética , Feminino , Humanos , Proteínas com Domínio LIM , Masculino , Proteínas dos Microfilamentos , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Mitocôndrias Musculares/ultraestrutura , Proteínas Musculares/genética , Doenças Musculares/diagnóstico , Mutação/genética , Mutação/fisiologia , Retículo Sarcoplasmático/ultraestrutura
6.
Brain ; 127(Pt 4): 723-34, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14724127

RESUMO

Desmin myopathy is a recently identified disease associated with mutations in desmin or alphaB-crystallin. Typically, the illness presents with lower limb muscle weakness slowly spreading to involve truncal, neck-flexor, facial, bulbar and respiratory muscles. Skeletal myopathy is often combined with cardiomyopathy manifested by conduction blocks and arrhythmias resulting in premature sudden death. Sections of the affected skeletal and cardiac muscles show abnormal fibre areas containing amorphous eosinophilic deposits seen as granular or granulofilamentous material on electron microscopic examination. Immuno-staining for desmin is positive in each region containing abnormal structures. The inheritance pattern in familial desmin myopathy is autosomal dominant or autosomal recessive, but many cases have no family history. At least some, and probably most, non-familial desmin myopathy cases are associated with de novo desmin mutations. Age of disease onset and rate of progression may vary depending on the type of inheritance and location of the causative mutation. Multiple mutations have been identified in the desmin gene: point substitutions, insertion, small in-frame deletions and a larger exon-skipping deletion. The majority of these mutations are located in conserved alpha-helical segments of desmin. Many of the missense mutations result in changing the original amino acid into proline, which is known as a helix breaker. Studies of transfected cell cultures indicate that mutant desmin is assembly-incompetent and able to disrupt a pre-existing filamentous network in dominant-negative fashion. Disease-associated desmin mutations in humans or transgenic mice cause accumulation of chimeric intracellular aggregates containing desmin and other cytoskeletal proteins. alphaB-crystallin serves in the muscle as a chaperone preventing desmin aggregation under various forms of stress. If mutated, alphaB-crystallin may cause a myopathy similar to those resulting from desmin mutations. Routine genetic testing of patients for mutations in desmin and alphaB- crystallin genes is now available and necessary for establishing an accurate diagnosis and providing appropriate genetic counselling. Better understanding of disease pathogenesis would stimulate research focused on developing specific treatments for these conditions.


Assuntos
Desmina/genética , Doenças Musculares/genética , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Mutação , Fenótipo , Cadeia B de alfa-Cristalina/genética
7.
Clin Neuropathol ; 21(5): 220-31, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12365725

RESUMO

Myofibrillar or desmin-related myopathies encompass neuromuscular disorders with abnormal deposits of desmin and myofibrillar alterations. We report 3 unrelated patients presenting with proximal and distal myopathy, and, as a unique congenital syndrome, diffusely distributed myopathy, osteoporosis and myopia. Muscle biopsies shared cytoplasmic inclusions, rimmed vacuoles, and ragged-red-like fibers. Sarcoplasmic inclusions, either plaque-like or amorphous, strongly immunoreacted on dystrophin and variably for desmin, alphaB crystallin and ubiquitin. Cyclin-dependent kinases CDK1, CDK2 and CDK5 were overexpressed in affected fibers. Ultrastructurally, focal myofibrillar disruption was accompanied by tubulo-filamentous inclusions in one case and abundant glycogen and enlarged mitochondria displaying respiratory chain dysfunction at biochemistry in another case. Molecular analysis of the alphaB crystallin gene coding sequence and exons 4, 5 and 6 of the desmin gene did not reveal any mutation. The morphologic denominator of hyaline structures and areas of myofibrillar destruction occurs in heterogeneous conditions and may overlap with features of inclusion body myopathy and mitochondrial myopathy.


Assuntos
Desmina/genética , Desmina/ultraestrutura , Doenças Musculares/genética , Doenças Musculares/patologia , Miofibrilas/patologia , Miofibrilas/ultraestrutura , Adulto , Idoso , Criança , Feminino , Humanos , Masculino , Miofibrilas/genética
8.
Hum Mutat ; 18(5): 388-96, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11668632

RESUMO

Desmin-related myopathy is a familial or sporadic disease characterized by skeletal muscle weakness and cardiomyopathy as well as the presence of intracytoplasmic aggregates of desmin-reactive material in the muscle cells. Previously, two kinds of deletions and eight missense mutations have been identified in the desmin gene and proven to be responsible for the disorder. The present study was conducted to determine structural and functional defects in a pathogenic desmin variant that caused a disabling disorder in an isolated case presenting with distal and proximal limb muscle weakness and cardiomyopathy. We identified a novel heterozygous Q389P desmin mutation located at the C-terminal part of the rod domain as the causative mutation in this case. Transfection of desmin cDNA containing the patient's mutation into C2.7, MCF7, and SW13 cells demonstrated that the Q389P mutant is incapable of constructing a functional intermediate filament network and has a dominant negative effect on filament formation. We conclude that Q389P mutation is the molecular event leading to the development of desmin-related myopathy.


Assuntos
Desmina/genética , Desmina/metabolismo , Variação Genética/genética , Mutação de Sentido Incorreto/genética , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/fisiopatologia , Adulto , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Cardiomiopatias/complicações , Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Linhagem Celular , Cristalinas/genética , Análise Mutacional de DNA , Desmina/química , Genes Dominantes/genética , Humanos , Filamentos Intermediários/metabolismo , Filamentos Intermediários/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Debilidade Muscular/complicações , Debilidade Muscular/genética , Debilidade Muscular/fisiopatologia , Miopatias Congênitas Estruturais/complicações , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade , Células Tumorais Cultivadas
9.
Rev Neurol (Paris) ; 156(5): 497-504, 2000 May.
Artigo em Francês | MEDLINE | ID: mdl-10844369

RESUMO

Two familial cases of a myopathy remarkable by the presence of a granulo-filamentar, electron dense material were reported in 1978. In a second step, in 1988, it was demonstrated that this material contained an abnormally-phosphorylated desmin. During the last twenty years, the occurrence of new cases in this family confirmed the autosomal dominant inheritance of the disease, and made it potentially informative for molecular genetics studies. This allowed first to map the disease on chromosome11q21-23, and afterwards to identify a mutation within a gene coding for a chaperone protein, alphaBcrystallin. An extensive clinical, pathological and genetic study of this princeps family is herein reported in detail. First, it showed the possible detection of histopathological changes in presymptomatic patients. Second, it allowed to demonstrate the simultaneous occurrence of both alphaBcrystallin and desmin in the granulo-filamentar aggregates. Third, this study provided a precise knowledge of the evolution rate of the disease. The analysis of similar observations reported in the literature clearly shows the clinical, pathological and genetic heterogeneity of this new neuro-muscular disorder.


Assuntos
Citoesqueleto de Actina/genética , Cristalinas/genética , Grânulos Citoplasmáticos/genética , Desmina/genética , Miopatias Congênitas Estruturais/genética , Citoesqueleto de Actina/patologia , Adulto , Idoso , Biópsia , Grânulos Citoplasmáticos/patologia , Feminino , Seguimentos , Humanos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Miopatias Congênitas Estruturais/diagnóstico , Miopatias Congênitas Estruturais/patologia , Linhagem
10.
Cell Biol Toxicol ; 15(3): 153-61, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10580548

RESUMO

A human endothelial cell line is a convenient tool for exploring cell physiology and testing drugs and toxics. Several attempts have been made using SV40 to immortalize endothelial cells. We used human umbilical vein endothelial cells (HUVEC) transformed with a construct made of promoter of the vimentin gene and SV40 Tag. The proliferation of immortalized vascular endothelial cells (IVEC), as measured by [methyl-3H]thymidine incorporation, was compared to that of HUVEC in the presence of endothelial cell growth factor and cytokines: tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma). Inhibition of [methyl-3H]thymidine incorporation by IL-1beta was lower than that observed with HUVEC, while TNF-alpha reduced the proliferation of IVEC and HUVEC to similar extents. Induction of intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and E-selectin by TNF-alpha, measured by a radiometric technique, was similar in IVEC and HUVEC, while the induction of E-selectin by IL-1beta on IVEC was limited and significantly different from that observed on HUVEC (p<0.001). The number of 125I-IL-1beta binding sites on IVEC is 3-fold less than on HUVEC and the IL-1beta receptor number was reduced. Dexamethasone treatment of IVEC restored their reactivity to IL-1beta and corrected the IL-1beta binding and the receptor number. These results showed that the introduction of SV40 gene not only immortalized the cell but also altered IL-1 receptor expression. This alteration may be improved by addition of corticosteroids to the cell culture, which extends the possibility of using IVEC as a model of endothelial cells.


Assuntos
Dexametasona/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Glucocorticoides/farmacologia , Interleucina-1/farmacologia , Receptores de Interleucina-1/biossíntese , Linhagem Celular Transformada , Selectina E/biossíntese , Endotélio Vascular/efeitos dos fármacos , Inibidores do Crescimento/farmacologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Interferon gama/farmacologia , Receptores de Adesão de Leucócito/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais , Molécula 1 de Adesão de Célula Vascular/biossíntese
11.
J Virol ; 72(12): 9553-60, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9811688

RESUMO

Theiler's murine encephalomyelitis virus is a neurotropic murine picornavirus which replicates permissively and causes a cytopathic effect in the BHK-21 cell line. We examined the interactions between the GDVII and DA strains of Theiler's virus and BHK-21 host cell proteins in a virus overlay assay. We observed binding of the virions to two proteins of approximately 60 kDa. These proteins were microsequenced and identified as desmin and vimentin, two main components of the intermediate filament network. The association between desmin or vimentin and virions was demonstrated by immunoprecipitation. Anti-desmin and anti-vimentin monoclonal antibodies precipitated GDVII or DA virions from extracts of infected BHK-21 cells. The intracellular distributions of virions and of the desmin and vimentin intermediate filaments of BHK-21 cells were investigated by two-color immunofluorescence confocal microscopy. Following infection, the intermediate filament network was rearranged into a shell-like structure which surrounded a viral inclusion. Finally, close contact between GDVII virus particles and 10-nm intermediate filaments was observed by electron microscopy.


Assuntos
Filamentos Intermediários/virologia , Theilovirus/patogenicidade , Animais , Linhagem Celular , Cricetinae , Desmina/metabolismo , Corpos de Inclusão Viral/ultraestrutura , Filamentos Intermediários/metabolismo , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Ligação Proteica , Theilovirus/crescimento & desenvolvimento , Theilovirus/fisiologia , Vimentina/metabolismo
12.
Pathol Biol (Paris) ; 46(1): 39-45, 1998 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9769935

RESUMO

Vimentin is an intermediate filament protein mainly specific of the mesoderm in vivo. In vitro vimentin synthesis is characteristic of proliferating cells, regardless of their embryonal origin, and is switched off upon differentiation of certain precursor cells. Vimentin gene expression is upregulated in some metastatic tumour cells, appearing as a marker of oncogenic progression. The vimentin network has been suggested to participate in several steps of viral infections. The promoter of the vimentin gene is comprised of multiple elements responsible for its complex transcriptional regulation. Among them, an NF-kappa B- and two AP1-binding sites mediate growth factor responsiveness. Two negative elements are present, one of which is deregulated by the HTLV-1 activator protein Tax. Transcription factor PEA3, encoded by a member of the ets oncogene family, activates the vimentin promoter in mammary tumour cells. In vitro, 878 base pairs of the vimentin 5'-regulatory region are sufficient to give high levels of transcription. These sequences were coupled to the SV40 large T antigen-encoding gene to achieve immortalization of new cell lines, either by transfection of primary cultures, or by derivation of cell explants from transgenic mice expressing the vimentin-SV40 construct. This allowed us, for instance, to immortalize endothelial, myogenic or renal epithelial cells, otherwise difficult to maintain in culture without loss of their differentiated phenotypes.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Vimentina/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Sobrevivência Celular/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Substâncias de Crescimento/farmacologia , Humanos , Regiões Promotoras Genéticas , Replicação Viral
13.
Nat Genet ; 20(1): 92-5, 1998 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9731540

RESUMO

Desmin-related myopathies (DRM) are inherited neuromuscular disorders characterized by adult onset and delayed accumulation of aggregates of desmin, a protein belonging to the type III intermediate filament family, in the sarcoplasma of skeletal and cardiac muscles. In this paper, we have mapped the locus for DRM in a large French pedigree to a 26-cM interval in chromosome 11q21-23. This region contains the alphaB-crystallin gene (CRYAB), a candidate gene encoding a 20-kD protein that is abundant in lens and is also present in a number of non-ocular tissues, including cardiac and skeletal muscle. AlphaB-crystallin is a member of the small heat shock protein (shsp) family and possesses molecular chaperone activity. We identified an R120G missense mutation in CRYAB that co-segregates with the disease phenotype in this family. Muscle cell lines transfected with the mutant CRYAB cDNA showed intracellular aggregates that contain both desmin and alphaB-crystallin as observed in muscle fibers from DRM patients. These results are the first to identify a defect in a molecular chaperone as a cause for an inherited human muscle disorder.


Assuntos
Cristalinas/genética , Cristalinas/metabolismo , Desmina/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Doenças Musculares/genética , Mutação , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Cricetinae , Cristalinas/ultraestrutura , Desmina/ultraestrutura , Feminino , Marcadores Genéticos , Proteínas de Choque Térmico/ultraestrutura , Humanos , Escore Lod , Masculino , Microscopia Imunoeletrônica , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/ultraestrutura , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Doenças Musculares/metabolismo , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
14.
Blood Coagul Fibrinolysis ; 9(2): 153-65, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9622213

RESUMO

A stable immortalized venous endothelial cell (IVEC) line, obtained by transfection of human umbilical vein endothelial cells (HUVEC), retains many normal differentiated endothelial characteristics. We compared the fibrinolytic activities of IVEC and HUVEC, and observed that IVEC express a more profibrinolytic phenotype than HUVEC, since they bind and activate plasminogen more efficiently, produce more tissue plasminogen activator and urokinase-type plasminogen activator antigens, and secrete less plasminogen activator inhibitor-1 antigen both under basal conditions and after stimulation with lipopolysaccharide, phorbol ester and tumor necrosis factor. Moreover, immunostaining and Western blotting of IVEC for the plasminogen/tissue plasminogen activator receptor annexin II, as well as Northern blotting of annexin II mRNA, revealed similar patterns of surface expression in IVEC and HUVEC. Plasminogen activator inhibitor-2 is expressed similarly in both cell types. IVEC may be a useful human model for functional and pharmacological explorations and modulations of fibrinolytic system components.


Assuntos
Endotélio Vascular/fisiologia , Fibrinólise/fisiologia , Anexina A2/metabolismo , Linhagem Celular Transformada , Endotélio Vascular/citologia , Humanos , Receptores de Superfície Celular/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tecidual/biossíntese , Ativador de Plasminogênio Tecidual/metabolismo , Veias Umbilicais/citologia , Veias Umbilicais/fisiologia
15.
Biol Cell ; 89(2): 85-97, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9351189

RESUMO

Research over the past few years on the function of intermediate filaments in cells in culture has not produced convincing results, because the key role of intermediate filaments is within tissues and at certain periods of development. Only recently the technique of gene knockout has been used to examine intermediate filaments in mice and has provided the first evidence that intermediate filaments are directly involved in cell resilience and the maintenance of tissue integrity. Knockout of the gene encoding keratin K8 is lethal in the embryo, and results in hepatic or intestinal lesions, while knockout of the K14 or K10 genes leads to rupture of stratified epithelia. Knockout of the gene encoding desmin causes the rupture of skeletal and cardiac muscle, and collapse of blood vessel walls. Knockout of the gene coding for GFAP leads to a loss of cerebral white matter, and knockout of the gene coding for vimentin causes degeneration of the cerebellar Purkinje cells. The results reveal the lack of compensation by another intermediate filament. Tissues without intermediate filaments fall apart; they are mechanically unstable, unable to resist physical stress, and this leads to cell degeneration. By maintaining the shape and plasticity of the cell, the intermediate filament network acts as an integrator within the cell space. The state of mechanical force imposed on a tissue or a cell can alter the shape of certain elements of the cytoskeleton and thus participate to the control of cell functions.


Assuntos
Filamentos Intermediários/fisiologia , Adaptação Fisiológica , Animais , Fenômenos Biomecânicos , Epitélio/fisiologia , Proteína Glial Fibrilar Ácida/química , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/fisiologia , Proteínas de Filamentos Intermediários/química , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/fisiologia , Queratinas/química , Queratinas/genética , Queratinas/fisiologia , Camundongos , Camundongos Knockout , Músculos/fisiologia , Fenômenos Fisiológicos do Sistema Nervoso , Estresse Mecânico , Vimentina/química , Vimentina/genética , Vimentina/fisiologia
16.
Lab Invest ; 76(1): 25-36, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9010447

RESUMO

Human bone marrow endothelial cells (HBMEC) are intimately involved in the homing of hematopoietic progenitor cells (HPC) to the bone marrow and in the regulation of proliferation and differentiation of these cells. Because availability of primary HBMEC and their capacity to be cultured in vitro are limited, we used isolated HBMEC to establish a cloned cell line by microinjection of a recombinant plasmid expressing simian virus 40 early genes under the control of a deletion mutant of the human vimentin promoter. Serum requirements for growth of a transformed HBMEC line (TrHBMEC) were markedly decreased compared with those of primary cells, and added growth factors were not required for proliferation. Cells took up acetylated low-density lipoprotein normally, bound to Ulex europaeus lectin, and stained positively for von Willebrand factor, P-selectin, CD31, CD34, CD44, very late antigen-5, and intercellular adhesion molecule-2 (ICAM-2). After treatment with TNF-alpha or lipopolysaccharide, TrHBMEC increased surface expression of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and ICAM-1 in a manner similar to primary HBMEC. In contrast, IL-1 beta elicited much less up-regulation of these adhesion molecules than in primary cells. In previous work, we reported that, in a flow adhesion model, rolling of peripheral blood CD34+ cells on primary HBMEC was E-selectin-dependent, whereas VCAM-1 and ICAM-1 contributed to firm adhesion. In the present study, we show that HPC adhere in a similar way to TrHBMEC. A less-pronounced role for VCAM-1 and ICAM-1 was found in the adhesion of HPC to human umbilical vein endothelial cells. Furthermore, significantly more CD34+ cells adhered to TNF-alpha-stimulated HBMEC and TrHBMEC than to similarly stimulated human umbilical vein endothelial cells. These data emphasize the importance of using microvessel HBMEC for studying the homing of HPC to the bone marrow, and indicate the usefulness of the above-described bone marrow endothelial cell line.


Assuntos
Antígenos CD/biossíntese , Células da Medula Óssea , Moléculas de Adesão Celular/biossíntese , Endotélio Vascular/fisiologia , Endotélio/citologia , Células-Tronco Hematopoéticas/fisiologia , Antígenos CD/análise , Antígenos CD34/análise , Antígenos Virais de Tumores/biossíntese , Medula Óssea/fisiologia , Adesão Celular , Moléculas de Adesão Celular/análise , Linhagem Celular , Células Clonais , Técnicas de Cultura/métodos , Endotélio/fisiologia , Endotélio Vascular/citologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Humanos , Imunoglobulina G , Sequências Reguladoras de Ácido Nucleico , Vírus 40 dos Símios/genética , Transfecção , Veias Umbilicais , Vimentina/biossíntese
17.
Hum Genet ; 98(4): 422-9, 1996 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8792816

RESUMO

Desmin is a muscle-specific intermediate filament that is encoded by a gene assigned to human chromosome 2q35. Desmin-related myopathies are inherited disorders characterized by an intrasarcoplasmic accumulation of desmin. Recently, the knockout of the desmin gene was shown to generate a myopathic syndrome in transgenic mice, suggesting that functional abnormality of desmin may generate similar clinical symptoms in mouse and human. To determine the potential role of the desmin gene in a well-defined desmin-related myopathy (autosomal dominant form of Fardeau), human desmin cDNAs obtained from affected and unaffected individuals were cloned, sequenced and compared. No obvious mutation was detected. A BssHII restriction fragment length polymorphism (RFLP) was identified in exon 6 of the desmin gene. This RFLP was associated with a previously identified EcoRV RFLP in exon 4 to generate a tetra-allelic system, which was tested for linkage to the desmin-related myopathy in three families. The human desmin gene was localized within an 11-cM interval on chromosome 2q using a panel of radiation hybrids. This 11-cM region was clearly excluded by linkage analysis in the three desmin-related myopathy families using a set of highly polymorphic microsatellite markers. These results suggest that the desmin gene is not primarily involved in this disease.


Assuntos
Cromossomos Humanos Par 2 , Desmina/genética , Doenças Musculares/genética , Polimorfismo de Fragmento de Restrição , Processamento Alternativo , Animais , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , DNA Complementar , Desoxirribonucleases de Sítio Específico do Tipo II , Éxons , Feminino , Ligação Genética , Genótipo , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Linhagem , Reação em Cadeia da Polimerase , Recombinação Genética
18.
Exp Cell Res ; 225(2): 268-76, 1996 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-8660914

RESUMO

The growth of muscle fibers during late development as well as in regeneration following muscle injury is the result of the proliferation and differentiation of satellite cells. However, all human cells, including satellite cells, show a limit in their proliferation. In order to define a cellular system with enhanced proliferative capacity, human satellite cells were transfected with a construct containing large T antigen from SV40 under the control of the human vimentin promoter. Vimentin is normally expressed during proliferation, and its expression is down-regulated as differentiation proceeds. In transfected cells, the construct is regulated like the endogenous vimentin gene. The effect of exogenous T antigen expression on both the proliferation and differentiation of human satellite cells was investigated. T antigen expression reduced the doubling time of human satellite cells from 36 to 20 h and increased the final proliferative capacity from 46 to 69 mean population doublings. When differentiation was triggered, although T antigen did not prevent the formation of myotubes, fusion was delayed. A similar delay was observed in the appearance of myogenin protein, one of the HLH regulatory factors, but not in the corresponding mRNA. Finally, T antigen has an effect on adult myosin isoform expression, since both adult slow and fast isoforms were only detected in myotubes negative for T antigen. These results led us to propose a model of the possible interactions between T antigen and muscle-specific factors.


Assuntos
Antígenos Transformantes de Poliomavirus/farmacologia , Músculo Esquelético/citologia , Cadeias Pesadas de Miosina/genética , Adulto , Sequência de Bases , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/fisiologia , Células Clonais/efeitos dos fármacos , Células Clonais/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Lactente , Cinética , Dados de Sequência Molecular , Fenótipo , Regiões Promotoras Genéticas/fisiologia , Transfecção , Vimentina/genética
19.
J Cell Physiol ; 167(1): 22-35, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8698837

RESUMO

Four renal cell lines were derived from glomeruli, proximal, distal, and cortical collecting tubules microdissected from the kidneys of transgenic mice carrying the temperature-sensitive mutant of the simian virus 40 large T antigen under the control of the vimentin promoter. All four cell lines contained large T antigen in their nuclei, grew rapidly, and contained vimentin filaments when grown in serum-enriched medium at the permissive temperature of 33 degrees C. The glomerular cell line formed multiple layers of cells and contained smooth muscle actin and desmin filaments, features of mesangial cells. The three tubule cell lines formed monolayers of polarized cuboid cells separated by tight junctions and having a patchy distribution of cytokeratins K8-K18. A shift from 33 degrees C to the restrictive temperature (39.5 degrees C) stopped cell growth in all cell lines and caused profound changes in the content of intermediate filaments. Vimentin was still present in mesangial-like cells, but the proximal, distal, and collecting tubule cells contained uniform networks of cytokeratins K8-K18 and desmoplakin I and II around the cell peripheries. Potassium transport, mediated by Na+-K+ ATPase pumps and specific cAMP hormonal sensitivities, significantly increased in proximal, distal, and collecting tubule cells when shifted from 33 degrees C to 39.5 degrees C. Thus, the temperature-dependent inactivation of large T antigen, responsible for the arrest of cell growth, did not affect the phenotype of mesangial-like glomerular cells but induced some changes in the expression of intermediate filaments and restored, at least partially, the main parental cell-specific functions in proximal, distal, and collecting tubule cultured cells.


Assuntos
Antígenos Virais de Tumores/biossíntese , Filamentos Intermediários/metabolismo , Rim/citologia , Animais , Antígenos Virais de Tumores/genética , Sequência de Bases , Divisão Celular/genética , Linhagem Celular , Técnicas de Transferência de Genes , Rim/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , ATPase Trocadora de Sódio-Potássio/metabolismo , Temperatura , Vimentina/biossíntese , Vimentina/genética
20.
Br J Pharmacol ; 117(5): 902-6, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8851508

RESUMO

1. The mechanism(s) responsible for injury of endothelial cells induced by human leukocyte elastase (HLE) was investigated in an immortalized venous human endothelial cell line (IVEC). 2. First, the proteinase concentrations and incubation delays necessary to trigger a significant IVEC cytotoxicity were determined by chromium assays. Thus, exposure of IVEC for 6 h to 10 micrograms ml-1 HLE resulted in 22 +/- 2.8% lysis and 36.4 +/- 5.4% detachment (mean +/- s.e. mean; n = 4; P < 0.05). 3. WEB 2086, a specific platelet-activating factor (PAF) receptor antagonist, induced a significant concentration-dependent decrease of such a lysis (39.6 +/- 7.7% protection at 100 microM; n = 4). This potential role for PAF was confirmed with two other antagonists of this lipid mediator, i.e., BN 52021 and RP 48740. 4. Finally, we demonstrated that pretreatment of IVEC with WEB 2086 protected significantly against cell lysis induced by stimulated human neutrophils, an experimental model in which HLE participates.


Assuntos
Azepinas/farmacologia , Diterpenos , Endotélio Vascular/efeitos dos fármacos , Elastase de Leucócito/farmacologia , Neutrófilos , Fator de Ativação de Plaquetas/antagonistas & inibidores , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Triazóis/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/patologia , Ginkgolídeos , Humanos , Lactonas/farmacologia , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Piridinas/farmacologia , Tiazóis/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...