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 JovemRESUMO
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 JovemRESUMO
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 , MutaçãoRESUMO
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/metabolismoRESUMO
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/fisiopatologiaRESUMO
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/ultraestruturaRESUMO
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 CultivadasRESUMO
The promoter of the human vimentin-encoding gene (VIM) contains two enhancers separated by a negative region. The distal and proximal enhancers bind the transcription factors, AP-1 and NK-kappaB, respectively, which contribute to serum induction of Vim synthesis. We were interested in looking for particular regulatory elements that might be responsible for tissue-specific extinction and culture-dependent activation of human VIM. We have identified a 48-bp sequence in the distal enhancer which had not been reported before. This sequence includes a negative element, NE2, which confers transcriptional repression in transfection experiments and binds at least two factors in vitro. NE2 may participate in the differentiation-stage-specific control of VIM expression which involves multiple regulatory sequences and several positive and negative trans-acting factors.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Vimentina/biossíntese , Vimentina/genética , Composição de Bases , Sequência de Bases , Sítios de Ligação , Cloranfenicol O-Acetiltransferase/biossíntese , Primers do DNA , Desoxirribonuclease I , Células HeLa , Humanos , Immunoblotting , Dados de Sequência Molecular , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência do Ácido Nucleico , Fator de Transcrição AP-1/metabolismo , TransfecçãoRESUMO
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/farmacologiaRESUMO
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/fisiologiaRESUMO
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/genéticaRESUMO
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 , LinhagemAssuntos
Enterocolite/diagnóstico por imagem , Índio , Neutrófilos , Radioisótopos , Humanos , CintilografiaAssuntos
Citoesqueleto/genética , Regulação da Expressão Gênica , Filamentos Intermediários/genética , Fusos Musculares/ultraestrutura , Animais , Diferenciação Celular/genética , Desmina/genética , Proteína Glial Fibrilar Ácida/genética , Humanos , Proteínas de Neurofilamentos/genética , Vimentina/genéticaRESUMO
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 ViralRESUMO
Lipophilicity of the injectable form of some antibiotics was measured with a micromethod. The antibiotic was dissolved in a small volume (1 ml) of phosphate buffer at a physiological pH (pH = 7.4) and was extracted by a small volume of octanol (1 ml). HPLC determinations of the antibiotic were performed in the two phases. log P ranged from +1.3 for chloramphenicol to -4.3 for ceftriaxone. A linear relationship was established for a few antibiotics between the log P values found in our experiments and the log permeability calculated from data in the literature for human and rat brain. This linear relationship enabled the brain concentrations of antibiotics to be predicted in man and rats.
Assuntos
Antibacterianos/farmacocinética , Barreira Hematoencefálica/efeitos dos fármacos , Metabolismo dos Lipídeos , Animais , Cromatografia Líquida de Alta Pressão , Humanos , Permeabilidade , Ratos , Especificidade da EspécieRESUMO
The external counting method was used to simultaneously measure the kinetics of intravenously injected polymorphonuclear cells labelled with Technetium 99m, in the lungs, liver and blood of rats. When radiolabelled polymorphonuclear leukocytes (PMN) are injected into animals, a large part is taken up by the various organs. This uptake may result from intravascular aggregation, capillary blocking (in which case the transit of the PMN is slowed down), vascular margination or, finally, from tissue infiltration. A rapid increase in PMN was observed initially in the lungs (first seconds), followed by a progressive decrease (t1/2 = 50 min). At the same time, PMN in the liver increased and that in the blood slowly decreased. The kinetics of distribution in the lungs, liver and blood were not modified when the PMN were injected through the aorta, via a carotid catheter, instead of intravenously. Since there was no shunt, vascular resistance was not the cause of the slow transit of PMN through the organs, and we favour the hypothesis that vascular margination of PMN is responsible for their kinetics. When heat damaged PMN were injected, the kinetics of their distribution in the lung, liver and in the blood were clearly altered.
Assuntos
Circulação Hepática , Fígado/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Neutrófilos/diagnóstico por imagem , Circulação Pulmonar , Animais , Circulação Sanguínea , Cinética , Masculino , Neutrófilos/fisiologia , Cintilografia , Ratos , Ratos Endogâmicos , TecnécioRESUMO
The aim of this study was to investigate a new method to enhance the efficiency to create mammalian cell lines. Cell immortalization was achieved by intranuclear microinjection of a recombinant DNA construct composed of a constitutive promoter controlling the genes encoding immortalizing proteins; the sequences coding for the large T and small t antigens were fused downstream of regulatory elements from the vimentin gene, the activation of which characterizes the vast majority of cells growing in vitro. Data show that the efficiency of the immortalizing procedures using the SV40 early genes could be enhanced by the control elements derived from the human vimentin (HuVim) 5' sequences that contained nucleotides -878 to +93 from the CAP site. This HuVim 830-T/t recombinant was used to create cell lines from numerous primary cultures of different origins: rabbit, porcine and human endothelial cells, rabbit and bovine epithelial cells. A set of large T-expressing cells was derived, and these cells retained characteristics of differential cells: binding of Ulex europaeus lectin and synthesis of Factor VIII for human endothelial cells; network of cytokeratin for bovine oviductal cells and rabbit mammary cells.
Assuntos
Técnicas de Cultura/métodos , Transformação Genética , Animais , Antígenos Virais de Tumores/genética , Diferenciação Celular , Divisão Celular , Linhagem Celular , Endotélio/citologia , Células Epiteliais , Genes Virais/genética , Humanos , Regiões Promotoras Genéticas/genética , Coelhos , Vírus 40 dos Símios/genética , Vimentina/genéticaRESUMO
A method for determining drug concentration relationships between plasma and cerebrospinal fluid (CSF) in rats is described. Continuous CSF samples were collected directly from the third anterior ventricle with an indwelling cannula inserted through the bregma point, and drug concentrations were determined by high-pressure liquid chromatography and radioimmunoassay micromethods. Three antibiotics with different abilities to cross the blood-CSF barrier (chloramphenicol, piperacillin, and gentamicin) were tested. This method was found to be reproducible for each drug even if the antibiotic levels were low and the sample volumes very small. Peak CSF concentrations occurred between 0.75 and 1.25 h after injection for all three antibiotics. Percent penetration values at 1 h were 50, 1.2, and 5.4% for chloramphenicol, piperacillin, and gentamicin, respectively.
Assuntos
Cloranfenicol/líquido cefalorraquidiano , Gentamicinas/líquido cefalorraquidiano , Piperacilina/líquido cefalorraquidiano , Animais , Cateterismo/veterinária , Cloranfenicol/metabolismo , Cromatografia Líquida de Alta Pressão , Gentamicinas/metabolismo , Cinética , Masculino , Piperacilina/metabolismo , Radioimunoensaio , Ratos , Ratos EndogâmicosRESUMO
Disposition of cefsulodin (125 mg X kg-1) was studied in rat frontal cortex after intravenous injection of a bolus by two methods. In vivo voltametry and high performance liquid chromatography of cefsulodin in brain extracts gave different concentration values. The ratio of concentrations determined by the two methods was similar to the ratio of the extravascular volume to total volume. Thus, these findings evidenced the distribution of cefsulodin in the extravascular fluid of the rat frontal cortex.