RESUMO
Two types of interventions to remyelinate the adult demyelinated central nervous system were investigated in heterozygous transgenic mice overexpressing the proteolipid protein gene. 1) A cocktail of trophic factors, "TS1," was directed toward the activation of the endogenous pool of neural progenitors to increase the number of myelinating oligodendrocytes (OL) in the brain. 2) A combinatorial approach in which OL progenitors were coinjected with TS1 into the corpus callosum of wild-type and He4e transgenic mice that displayed hindlimb paralysis. The levels of locomotor ability in these mice were evaluated after a single treatment. The data showed that a single administration of either one of the interventions had similar therapeutic effects, alleviating the symptoms of demyelination and leading to the recovery of hindlimb function. Histological and immunofluorescent examination of brain sections showed extensive remyelination that was sufficient to reverse hindlimb paralysis in transgenic mice. When the interventions were administered prior to hindlimb paralysis, He4e mice were able to walk up to 1 year of age without paralysis.
Assuntos
Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Proteína Proteolipídica de Mielina/metabolismo , Recuperação de Função Fisiológica/fisiologia , Amidinas/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Corpo Caloso/metabolismo , Corpo Caloso/transplante , Meios de Cultivo Condicionados/farmacologia , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/cirurgia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Comportamento Exploratório/fisiologia , Gangliosídeos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiopatologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Proteínas de Filamentos Intermediários/genética , Camundongos , Camundongos Transgênicos , Microscopia Confocal/métodos , Proteína Básica da Mielina/metabolismo , Proteína Proteolipídica de Mielina/genética , Proteínas do Tecido Nervoso/genética , Nestina , Proteínas de Neurofilamentos/metabolismo , Neuroglia/química , Recuperação de Função Fisiológica/efeitos dos fármacos , Recuperação de Função Fisiológica/genética , Fatores de TempoRESUMO
Myelin proteolipid protein (PLP), the major myelin protein in the CNS, has been thought to function in myelin assembly. Thus, mutations within the gene coding for PLP (Plp) cause hypomyelination, such as the jimpy phenotype in mice and Pelizaeus-Merzbacher disease in humans. However, these mutants often exhibit premature death of oligodendrocytes, which form CNS myelin. To elucidate the functional roles of Plp gene products in the maturation and/or survival of oligodendrocytes, we produced transgenic mice overexpressing the Plp gene by introducing extra wild-type mouse Plp genes. Surprisingly, transgenic mice bearing 4 more Plp genes exhibited dysmyelination in the CNS, whereas those with 2 more Plp genes showed normal myelination at an early age (3 weeks after birth), but later developed demyelination. Overexpression of the Plp gene resulted in arrested maturation of oligodendrocytes, and the severity of arrest was dependent on the extent of overexpression. Overexpression also led to oligodendrocyte cell death, apparently caused by abnormal swelling of the Golgi apparatus. Thus, tight regulation of Plp gene expression is necessary for normal oligodendrocyte differentiation and survival, and its overexpression can be the cause of both dys- and demyelination.
Assuntos
Doenças Desmielinizantes/genética , Proteínas da Mielina/genética , Fibras Nervosas Mielinizadas/patologia , Neuroglia/patologia , Animais , Sequência de Bases , Encéfalo/fisiopatologia , Clonagem Molecular , Primers do DNA , Doenças Desmielinizantes/patologia , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Dados de Sequência Molecular , Proteína Proteolipídica de Mielina , Oligodendroglia/patologia , Linhagem , RNA Mensageiro/genética , Mapeamento por RestriçãoRESUMO
In the neurological mutant mouse reeler, the histological organization of the neocortex develops abnormally and essentially results in an inversion of the relative positions of the cortical layers. The reeler mutation, therefore, provides an insight into the molecular mechanisms underlying the formation of the cortical layers. We have generated a monoclonal antibody (CR-50) that probes a distinct allelic antigen present in wild-type but not in reeler mutant mice. CR-50 reacted specifically with Cajal-Retzius neurons, one of the first cortical neurons to differentiate in the neocortex, but whose functional role is not known. When dissociated cerebral cortical cells were incubated with CR-50 in reaggregation culture, the genotype-dependent histogenetic assembly of wild-type cortical cells resembled that of reeler mutants. These findings revealed that the selective expression of a distinct molecule on Cajal-Retzius neurons is critical for the normal lamination of cortical neurons in the mammalian neocortex.
Assuntos
Moléculas de Adesão Celular Neuronais/análise , Córtex Cerebral/citologia , Proteínas da Matriz Extracelular/análise , Mutação , Proteínas do Tecido Nervoso/análise , Neurônios/citologia , Animais , Anticorpos Monoclonais , Adesão Celular , Comunicação Celular , Espaço Extracelular/metabolismo , Hibridomas/imunologia , Imuno-Histoquímica , Camundongos , Camundongos Mutantes Neurológicos , Neurônios/química , Proteína Reelina , Serina Endopeptidases , Distribuição TecidualRESUMO
The distribution of protein arginine N-methyltransferase 3 (PRMT3) was investigated in the mouse brain using indirect immunofluorescence. PRMT3 was observed to be localized in the cell bodies and dendrites of neurons but not in the axons and glial cells, indicating that PRMT3 is involved in neuronal function. The distribution of the immunoreactive neurons in the brain was uneven, indicating that PRMT3 plays a role in specific neuronal systems such as the motor and limbic systems, as well as functions related to the cerebellum. The present ontogenetic analysis of PRMT1 and PRMT3 using Western blot methodology clearly revealed that PRMT3 develops during the perinatal stage and its expression is maintained even in adulthood. PRMT1, on the other hand, is expressed transiently during the early embryonic stage. These findings indicate that PRMT3 is related with neuronal function in both young and adult brains, while PRMT1 has roles in the immature brain, such as the formation of neural circuits.
Assuntos
Encéfalo/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Western Blotting/métodos , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Células Cultivadas , Embrião de Mamíferos , Expressão Gênica/fisiologia , Hipocampo/citologia , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Ratos , Ratos WistarRESUMO
To unearth glioma-specific genes in human glioblastoma, the serial analysis of gene expression technique was applied to a primary glioblastoma, using cultured human astrocytes as a normal control. Among the top 147 most-expressed tags in glioblastoma, we found a tag, TTTTGGGTAT, that originated from an unidentified gene and which was not detected in human astrocyte cultures. Real-time quantitative reverse transcription-PCR showed that MAGE-E1 expression was 2.6-15-fold enriched in glioblastoma relative to human astrocytes. Expressed sequence tags containing this tag were homologous to the melanoma-associated antigen gene (MAGE) family, and this new cDNA, named MAGE-E1, was cloned by the 5'-rapid amplification of cDNA ends technique. Three alternatively spliced variants (MAGE-E1a-c) were found, and deduced amino acid sequence showed that MAGE-E1a and -E1b shared the MAGE-conserved region, whereas -E1c did not. This suggests that although MAGE-E1c is expressed from one of the MAGE family, it has distinct functions from other members. Tissue distribution analysis showed that MAGE-E1 was distinct from other MAGEs. MAGE-E1 expression was detected only in brain and ovary among normal tissues. Interestingly, MAGE-E1a and/or -E1b were specifically expressed in glioma cells among cancer cells. These results indicate that MAGE-E1 is a novel and glioma-specific member of MAGE family.
Assuntos
Antígenos de Neoplasias/genética , Neoplasias Encefálicas/genética , Glioblastoma/genética , Antígenos de Neoplasias/biossíntese , Astrócitos/imunologia , Sequência de Bases , Neoplasias Encefálicas/imunologia , Clonagem Molecular , DNA Complementar/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/imunologia , Humanos , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Regulação para CimaRESUMO
Polysialic acid (PSA) is a carbohydrate composed of a linear homopolymer of alpha-2-8-linked sialic acid residues and is mainly attached to the neural cell adhesion molecule (NCAM). Because of the large negative charge of PSA, presence of PSA attenuates the adhesive property of NCAM and increases the cellular motility. PSA expression on NCAM is developmentally regulated, and PSA plays important roles in formation and remodeling of the neural system through regulation of the adhesive property of NCAM. Expression of the polysialated form of NCAM has been also demonstrated in some malignant tumors, such as Wilms' tumor and small cell lung cancer. Despite the possible importance as an onco-developmental antigen, however, significance of PSA expression in most malignant tumors has not been revealed. Therefore, PSA expression in non-small cell lung cancer was assessed in the present study. PSA was expressed only in 5 (20.8%) of 24 pathological stage I cases, whereas it was expressed in most stage IV cases (76.8%, 11 of 14 cases). PSA expression was correlated with nodal metastasis and distant metastasis, but not with local extent of the primary tumor. Next, expression of polysialyltransferase genes (PST and STX genes) which controlled formation of PSA, was examined. The PST gene was constantly expressed in both normal lung tissue and tumor tissue of all cases. In contrast, the STX gene was not expressed in normal lung tissue of any case, and STX gene expression in tumor tissue was closely correlated with tumor progression. The STX gene was expressed only in 1 (4.2%) of 24 stage I cases, whereas it was expressed in most stage IV cases (85.7%, 12 of 14 cases). These results suggested that the PSA and STX genes could be new targets of cancer therapy as well as important clinical markers.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Ácidos Siálicos/biossíntese , Sialiltransferases/biossíntese , Adulto , Idoso , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Moléculas de Adesão de Célula Nervosa/biossíntese , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácidos Siálicos/genética , Sialiltransferases/genéticaRESUMO
Polysialic acid (PSA) is a carbohydrate attached mainly to the neural cell adhesion molecule. Because PSA is composed of a linear homopolymer of alpha-2-8-linked sialic acid residues and has a large negative charge, the presence of PSA attenuates the adhesive property of neural cell adhesion molecule and increases cellular motility. In an earlier study, we demonstrated that PSA and STX, a polysialyltransferase, were associated with tumor progression in non-small cell lung cancer (NSCLC) (F. Tanaka et al., Cancer Res., 60: 3072-3080, 2000). Therefore, in the present study, to assess the prognostic significance of PSA in resected NSCLC, a total of 236 patients who underwent complete resection for pathological (p)-stage I-IIIa disease were reviewed retrospectively. PSA was expressed in 44 of 236 (18.6%) patients, and the expression was correlated with p-stage disease. For all p-stage patients, 5-year survival rates for those with PSA-positive and PSA-negative tumors were 52.1% and 71.3%, respectively, demonstrating a significantly worse prognosis for the PSA-positive patients (P = 0.012). Analysis for only p-stage I patients also demonstrated a significantly worse prognosis for the PSA-positive patients; 5-year survival rates of the PSA-positive and the PSA-negative patients were 45.1% and 83.5%, respectively, (P < 0.001). In addition, there proved to be no difference in the postoperative survival among p-stage I, II, and IIIa patients when PSA expression was positive. Multivariate analysis confirmed that PSA expression was an independent factor to predict poor prognosis in resected NSCLC. These results suggested that PSA could be an important clinical marker and that preoperative induction and/or postoperative adjuvant therapies should be performed for PSA-positive NSCLC, even if the disease is classified as p-stage I.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Ácidos Siálicos/biossíntese , Biomarcadores Tumorais/biossíntese , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Moléculas de Adesão de Célula Nervosa/biossíntese , Prognóstico , Taxa de SobrevidaRESUMO
During neuronal differentiation and maturation, electrical excitability is essential for proper gene expression and the formation of synapses. The expression of ion channels is crucial for this process; in particular, voltage-gated K(+) channels function as the key determinants of membrane excitability. Previously, we reported that the A-type K(+) current (I(A)) and Kv4.2 K(+) channel subunit expression increased in cultured cerebellar granule cells with time. To examine the correlation between ion currents and the action potential, in the present study, we measured developmental changes of action potentials in cultured granule cells using the whole-cell patch-clamp method. In addition to an observed increment of I(A), we found that the Na(+) current also increased during development. The increase in both currents was accompanied by a change in the membrane excitability from the nonspiking type to the repetitive firing type. Next, to elucidate whether Kv4.2 is responsible for the I(A) and to assess the effect of Kv4 subunits on action potential waveform, we transfected a cDNA encoding a dominant-negative mutant Kv4.2 (Kv4.2dn) into cultured cells. Expression of Kv4.2dn resulted in the elimination of I(A) in the granule cells. This result demonstrates that members of the Kv4 subfamily are responsible for the I(A) in developing granule cells. Moreover, elimination of I(A) resulted in shortening of latency before the first spike generation. In contrast, expression of wild-type Kv4.2 resulted in a delay in latency. This indicates that appearance of I(A) is critically required for suppression of the excitability of granule cells during their maturation.
Assuntos
Cerebelo/fisiologia , Neurônios/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/fisiologia , Potenciais de Ação/fisiologia , Algoritmos , Animais , Linhagem Celular , Cerebelo/citologia , Grânulos Citoplasmáticos/fisiologia , Eletrofisiologia , Proteínas de Fluorescência Verde , Humanos , Cinética , Proteínas Luminescentes , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Mutação/genética , Mutação/fisiologia , Oligonucleotídeos/genética , Técnicas de Patch-Clamp , Canais de Potássio/genética , Canais de Potássio ShalRESUMO
Rats on a protein-free diet synthesized less DNA after partial hepatectomy than rats on a normal diet. In regenerating livers of animals on the protein-free diet, induction of several enzymes involved in the DNA precursor synthetic pathway, and especially ribonucleotide reductase, were depressed. When young rats were maintained solely by parenteral nutrition after partial hepatectomy, exogenous amino acids were more important than the exogenous energy source for induction of enzymes involved in synthesis of DNA and its pyrimidine nucleotide precursors. In particular, induction of ribonucleotide reductase appeared to be controlled by exogenous amino acids. Tryptophan, methionine, phenylalanine, leucine, valine, isoleucine and threonine seemed to stimulate the induction of this enzyme most after partial hepatectomy.
Assuntos
Aminoácidos/farmacologia , DNA/biossíntese , Regeneração Hepática , Nutrição Parenteral Total , Nutrição Parenteral , Deficiência de Proteína/metabolismo , Animais , Dieta , Enzimas/metabolismo , Hepatectomia , Masculino , Ratos , Ratos EndogâmicosRESUMO
Jimpy is one of many related mutations affecting the myelin proteolipid protein gene that causes severe hypomyelination in the central nervous system (CNS). Underlying the hypomyelination is a failure of oligodendrocytes (OLs) to differentiate, and the premature death of large numbers of OLs during the developmental period. Previous light and electron microscopic evidence suggested that jimpy OLs die in a manner consistent with programmed cell death. We have used TUNEL staining as a biochemical marker for apoptosis in conjunction with immunostaining for OL and myelin markers. At 13 - 14 days postnatal, a time when the number of dying OLs in jimpy CNS is increased more than five times normal, there are only modest increases (70% in spinal cord; 20% in cerebral cortex) in TUNEL labeled cells in mutant CNS tissues. The results in vitro are similar, and only a small per cent of TUNEL labeled cells have the antigenic phenotype of OLs. The discrepancy between numbers of dying and TUNEL labeled cells suggests either that most jimpy OLs do not undergo programmed cell death or that the biochemical pathways leading to their death do not involve DNA fragmentation which is detected by the TUNEL method. We also present evidence that jimpy OLs show increased survival and enhanced differentiation when they are grown in vitro in medium conditioned by cells lines which express products of the proteolipid protein gene. Cell lines expressing proteolipid protein and the alternatively spliced DM20 protein have differential effects on cell numbers and production of myelin-like membranes.
Assuntos
Apoptose/efeitos dos fármacos , Sistema Nervoso Central/fisiologia , Proteínas do Tecido Nervoso , Oligodendroglia/metabolismo , Animais , Apoptose/genética , Astrócitos/metabolismo , Diferenciação Celular/genética , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Fragmentação do DNA/genética , Camundongos , Camundongos Jimpy , Proteína Proteolipídica de Mielina/farmacologia , Bainha de Mielina/metabolismo , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
The mouse proteolipid protein (PLP) gene was cloned into the lambda bacteriophage Charon 4A. The organization and the nucleotide sequence of the exons of the mouse PLP gene were quite similar to those of their human counterparts, consisting of seven exons. The transcription of the PLP gene started from multiple sites. There was a unique sequence tandemly repeated four times, sharing homology with the herpes simplex virus DR2 sequence, upstream from the transcribed region. Expression of the myelin basic protein (MBP) is also restricted to the oligodendrocytes in the central nervous system as is the PLP expression. Homology search against the mouse MBP gene revealed that several boxes in the 5'-flanking region of PLP show a high degree of homology with the sequence present in the MBP 5'-flanking region, possibly of importance in the concomitant expression of both genes in the central nervous system. PLP-mRNA in jimpy mutant mice does not contain exon 5 and its content is greatly reduced. We analyzed the jimpy PLP-mRNA and showed that the transcription initiated from the same sites as those in normal mice. Cloning and sequencing of the 5'-flanking region of the jimpy PLP gene revealed that there were no mutations in the promoter region of the jimpy PLP gene. Therefore, it is likely that a mutation, presumably existing within the jimpy PLP gene, caused the skipping of exon 5 and directly affected the mRNA level.
Assuntos
Regulação da Expressão Gênica , Genes , Proteínas da Mielina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA , Camundongos , Camundongos Jimpy , Dados de Sequência Molecular , Mutação , Proteína Proteolipídica de Mielina , RNA Mensageiro , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
To obtain high-titer recombinant retroviruses, we constructed plasmid pDL+, which carries the extended Psi region and the polyomavirus early region, including the replication origin and the early gene. Although pDL+ is useful for obtaining high-titer recombinant retroviruses, this vector plasmid is difficult to modify further for tissue-specific expression of foreign genes. To overcome this problem, the coding region of the polyomavirus early gene was expressed in the packaging cell lines. We modified the packaging cell lines, psi2 and PA317, by stably introducing the polyomavirus early gene, and established psiMP34 and psiMP37 from psi2, and PAMP51 from PA317. In the transient expression system using plasmids with and without the polyomavirus replication origin, the titers of recombinant retrovirus produced by these cell lines were 10-100 times higher than produced by the parent cell line and reached levels of 0.5-1.5 x 106 cfu/ml. Expression of the polyomavirus early gene in the packaging cell lines did not stimulate the production of replication-competent retrovirus. We also routinely established stable clones producing retrovirus titers over 1 x 10(7) cfu/ml from psiMP34 and PAMP51. We found that the activity of the long terminal repeat (LTR) promoter is stimulated by the polyomavirus early region protein(s) in these cell lines. Therefore, increases titer can be expected to occur in all the retroviral vectors in which LTR promoter is used to transcribe the retroviral genome.
Assuntos
Polyomavirus/genética , Regiões Promotoras Genéticas/genética , Montagem de Vírus/genética , Células 3T3 , Animais , Linhagem Celular , Células Clonais , Regulação Viral da Expressão Gênica , Proteínas Imediatamente Precoces/genética , Camundongos , Plasmídeos , Polyomavirus/metabolismo , Recombinação Genética , Retroviridae/genética , Retroviridae/crescimento & desenvolvimento , Transdução Genética , Transfecção , Replicação Viral/genéticaRESUMO
The "bystander effect" describes the killing of nearby unmodified cells and herpes simplex thymidine kinase (HTK)-transduced cells by ganciclovir (GCV) treatment. This effect is thought to be produced by contact between these cells. In this study, we showed that injected glioma cells migrated rapidly to a place distant from the injection point whereas injected virus-producing fibroblast cells did not migrate in a murine brain model. Moreover, the initially injected glioma cells and glioma cells injected at a later time mix very well, even at a place distant from the injection point. This suggested that glioma cells migrating after injection could be targeted by HTK-modified glioma cells introduced in a second injection and be killed together by GCV treatment. Therefore, we injected HTK-modified glioma cells 3 days after injection of wild glioma cells to investigate whether wild-type glioma cells that migrated to a place distant from the injection point could also be killed by GCV treatment. Tumor growth was suppressed after the GCV treatment. Suppression of tumor growth of wild glioma cells is not solely mediated by the immune response, which may be triggered by the killing of HTK-modified glioma cells with GCV, because inoculation of HTK-modified glioma to the contralateral side followed by GCV treatment did not cure the initial wild glioma. Moreover, the migration of the second inoculum of glioma cells is necessary for effective killing, because early administration of GCV resulted in insufficient killing.
Assuntos
Antineoplásicos/uso terapêutico , Antivirais/uso terapêutico , Neoplasias Encefálicas/terapia , Ganciclovir/uso terapêutico , Terapia Genética/métodos , Glioma/terapia , Timidina Quinase/genética , Proteínas Virais/genética , Animais , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/fisiopatologia , Comunicação Celular , Terapia Combinada , Feminino , Vetores Genéticos , Glioma/enzimologia , Glioma/fisiopatologia , Glioma/secundário , Camundongos , Camundongos Endogâmicos C3H , Metástase Neoplásica , Células Tumorais CultivadasRESUMO
Serum-induced inactivation of retroviruses is the most critical limitation for in vivo gene transfer therapy. To solve this problem, we searched for reagents that protect retroviruses from inactivation. The effects of the protease inhibitors FOY-007 and FOY-305 and of an inhibitor of the complement pathway FUT-175, all of which have been used clinically, were investigated. All of these agents protected against the inactivation of retroviruses by human serum, with 1 microM FUT-175 providing the most effective protection. Thus, the co-administration of FUT-175 with retroviruses may make retrovirus-mediated in vivo gene transfer feasible for the treatment of patients. FUT-175 dose-dependently inhibited the classical pathway of complement in a hemolysis protection assay of sensitized sheep erythrocytes with guinea pig serum or by cell-lysis assay of mouse fibroblasts with human serum. However, increasing the FUT-175 concentration by 10-fold (10 microM) did not produce further protection against retroviral inactivation in most human sera. There was also no correlation between the serum-induced inactivation of retroviruses and either the amount of anti-alpha-galactosyl (anti-alpha-Gal) antibody or the complement activity in human serum. These results suggest that retroviruses are not inactivated by utilizing the same pathway leading to cell lysis by the classical complement system.
Assuntos
Sangue , Proteínas Inativadoras do Complemento/farmacologia , Guanidinas/farmacologia , Retroviridae/efeitos dos fármacos , Ativação Viral/efeitos dos fármacos , Células 3T3 , Animais , Anticorpos/sangue , Benzamidinas , Relação Dose-Resposta a Droga , Cobaias , Humanos , Camundongos , OvinosRESUMO
The jimpy mouse, an X-linked recessive dysmyelinating and demyelinating mutant, has been shown to contain abnormal myelin proteolipid protein (PLP) mRNA. To understand the molecular basis of the mutation, we analyzed the structure of PLP mRNA by an RNase-mapping procedure, using the probes specific to each exon of the mouse PLP gene. We found that the fifth exon of the PLP gene is not utilized in the jimpy.
Assuntos
Química Encefálica , Doenças Desmielinizantes/veterinária , Éxons , Camundongos Jimpy/genética , Camundongos Mutantes Neurológicos/genética , Proteínas da Mielina/genética , Splicing de RNA , RNA Mensageiro/genética , Doenças dos Roedores/genética , Animais , Sequência de Bases , DNA/genética , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/metabolismo , Esclerose Cerebral Difusa de Schilder , Modelos Animais de Doenças , Camundongos , Dados de Sequência Molecular , Proteínas da Mielina/análise , Proteína Proteolipídica de Mielina , Doenças dos Roedores/metabolismoRESUMO
Genes of the melanoma-associated antigen (MAGE) family are characterized by the expression of tumor antigens on a malignant melanoma recognized by autologous cytolytic T lymphocytes. We have previously identified novel members of the MAGE gene family expressed in human glioma and named them MAGE-E1a-c. In the present study, we have revealed the genomic structure of MAGE-E1 by sequence analysis of a human chromosome bacterial artificial chromosome clone containing the MAGE-E1 gene. The MAGE-E1 gene is composed of 13 exons, and three of these (exon 2, exon 3 and exon 12) are alternatively spliced in each variant (E1a-c). The open reading frame encoding the MAGE-E1 peptides initiates in exon 2 and ends in exon 13. We have also demonstrated that the MAGE-E1 gene is located in Xp11 through the analysis of radiation hybrid panels. The genomic structure of MAGE-E1 is markedly similar to that of MAGE-D and its chromosomal locus is also identical to that of MAGE-D, but these features contrast with those of other MAGEs. These results suggest that MAGE-D and -E1 may be evolutionarily distant from other members of the MAGE family, and the two may be ancestral genes for the others.
Assuntos
Antígenos de Neoplasias/genética , Genes/genética , Processamento Alternativo , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos , Clonagem Molecular , DNA/química , DNA/genética , Éxons , Humanos , Íntrons , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Cromossomo X/genéticaRESUMO
We recently developed a novel promoter assay system using a retroviral vector (pIP200 series). Transcription from the internal promoter, which had been inserted for the promoter assay, was shown to be interfered with by transcription from the upstream long terminal repeat (LTR). Here we report a new high-titer 'self-inactivating' vector, in which transcription interference was virtually eliminated. This new vector was constructed by introducing only a very minor mutation into the 'TATA box' in the 3'-LTR. This mutation was successfully transferred to the 5'-LTR after reverse transcription, yielding a provirus incapable of transcribing viral RNA. The viral titer was not reduced by the mutation, permitting general application of this virus.
Assuntos
Vetores Genéticos , Regiões Promotoras Genéticas , Retroviridae/genética , Células 3T3 , Animais , Sequência de Bases , Clonagem Molecular/métodos , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/genéticaRESUMO
The MCD peptide in bee venom induces degranulation in mast cells. The internal calcium concentration of mast cells increased and remained high following MCD stimulation. This calcium increase was blocked by pertussis toxin (Ptx) treatment, suggesting that MCD peptide activates Ptx-sensitive G-protein. Even in the absence of external calcium in the incubation medium, the calcium concentration increased by MCD treatment, but soon returned to the original level. D-MCD, the optical isomer of the MCD peptide, also increased the internal calcium concentration through a Ptx-sensitive pathway. We suggest that cationic clusters at one side of the surface are more important in activating the G-protein than the alpha-helix conformation.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Mastócitos/metabolismo , Peptídeos/farmacologia , Animais , Cálcio/metabolismo , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/fisiologia , Corantes Fluorescentes , Fura-2 , Mastócitos/efeitos dos fármacos , Mastócitos/ultraestrutura , Toxina Pertussis , Ratos , Ratos Endogâmicos , Estereoisomerismo , Fatores de Virulência de Bordetella/farmacologiaRESUMO
We have immunohistochemically investigated the expression of a reeler gene-related antigen in the mouse cerebellum by using a monoclonal antibody, CR-50. This antibody probes a distinct allelic antigen present in normal but not in reeler mutant mice, and this antigen is localized in the brain regions in which morphological abnormalities occur in reeler mice (Ogawa et al., Neuron 14: 899-912, 1995). The developing normal cerebellum showed transient immunoreactivity to CR-50 in a limited set of neurons and in the extracellular space near the pial surface. An early population of CR-50-labeled cells emerged on embryonic day (E) 13 along the dorsal cerebellar surface, comprising the nuclear transitory zone (NTZ). Bromodeoxyuridine labeling revealed the time of origin of these cells to be at E11-12. From E14 to E18, some CR-50-labeled cells were stacked in the inner border of the external granular layer (EGL), whereas others were scattered in deep areas, such as the cerebellar nuclei and the surrounding intermediate zone or white matter. In the first postnatal week, these subcortical structures became immunonegative. However, CR-50 antigen was continuously produced until the second postnatal week by another population of cells occupying i) the premigratory zone (PMZ), the inner half of the EGL, and ii) the internal granular layer (IGL). These later CR-50-positive cells were smaller than the earlier type and showed the morphology typical of granule neurons. Both types of CR-50-labeled cells were positive for a DNA-binding protein, zic. By treating living cerebellar slices with CR-50, the extracellular antigen was localized as a puncutate staining pattern in the NTZ, PMZ, and molecular layer (ML), but not in the subcortical regions and IGL. Purkinje cells were negative for CR-50 and aligned as a monolayer adjacent to the PMZ, though their dendritic trees were closely associated with the extracellular CR-50-antigen in the PMZ and ML. Staining of dissociated cells suggested that the extracellular antigen is initially present throughout the surfaces of the CR-50/anti-zic double positive neurons, and is then rearranged to concentrate on their processes contacting with Purkinje cells. The spatiotemporal expressions of the CR-50 antigen in the cerebellum are consistent with the possibility that this antigen is involved in cell-cell interactions related to the histogenetic assembly of Purkinje cells.
Assuntos
Animais Recém-Nascidos/crescimento & desenvolvimento , Antígenos/metabolismo , Cerebelo/crescimento & desenvolvimento , Neurônios/imunologia , Animais , Imuno-Histoquímica , Camundongos , Camundongos MutantesRESUMO
The complement system of the human body inactivates the infectious ability of retroviruses injected as an artificial gene transfer vector. We established new murine leukemia virus (MuLV) packaging cell lines; D2SS and D7S which express decay-accelerating factor (DAF) on their surface. Both D2SS and D7S were resistant against incubation with fresh human serum. Moreover, the retroviruses produced from these packaging cell lines were also resistant to serum treatment. This resistance can be inhibited by DAF neutralizing antibody 1C6. These data demostrate that DAF induces a partial protection of MuLV infection from the human complement system.