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1.
Am J Pathol ; 155(3): 995-1004, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10487857

RESUMO

In this study we describe the generation of a transgenic mouse model with neuronal overexpression of the human cyclooxygenase-2, h(COX)-2, to explore its role in excitotoxicity. We report that overexpression of neuronal hCOX-2 potentiates the intensity and lethality of kainic acid excitotoxicity in coincidence with potentiation of expression of the immediate early genes c-fos and zif-268. In vitro studies extended the in vivo findings and revealed that glutamate excitotoxicity is potentiated in primary cortico-hippocampal neurons derived from hCOX-2 transgenic mice, possibly through potentiation of mitochondrial impairment. This study is the first to demonstrate a cause-effect relationship between neuronal COX-2 expression and excitotoxicity. This model system will allow the systematic examination of the role of COX-2 in mechanisms of neurodegeneration that involve excitatory amino acid pathways.


Assuntos
Agonistas de Aminoácidos Excitatórios/toxicidade , Proteínas Imediatamente Precoces , Isoenzimas/biossíntese , Ácido Caínico/toxicidade , Camundongos Transgênicos/genética , Neurônios/enzimologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Animais , Northern Blotting , Encéfalo/enzimologia , Encéfalo/metabolismo , Células Cultivadas , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Proteínas de Ligação a DNA/biossíntese , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce , Embrião de Mamíferos , Expressão Gênica , Ácido Glutâmico/toxicidade , Humanos , Isoenzimas/genética , Proteínas de Membrana , Camundongos , Especificidade de Órgãos/genética , Prostaglandina-Endoperóxido Sintases/genética , Proteínas Proto-Oncogênicas c-fos/biossíntese , RNA Mensageiro/biossíntese , Convulsões/induzido quimicamente , Convulsões/genética , Fatores de Transcrição/biossíntese
2.
Eur J Immunol ; 29(2): 466-76, 1999 02.
Artigo em Inglês | MEDLINE | ID: mdl-10064062

RESUMO

Taking the antisense approach to inhibit the expression of specific protein kinase C (PKC) isoforms, we investigated the function of PKC alpha in T cell activation by transfecting Jurkat cells with an episomal vector (pREP3) containing a copy of the corresponding gene in the antisense orientation. Transfected Jurkat cells were selected with hygromycin and cloned by limiting dilution. Two (as1/as2) stably transfected antisense PKC alpha-pREP3 clones (as PKC alpha-pREP3) exhibited consistently reductions (76% and 85%, respectively) of PKC alpha levels when analyzed by immunoblotting and immunoprecipitation and also of PKC alpha mRNA (75%, as determined by Northern blotting) when compared to control clones (C1/C2) containing the pREP3 vector alone. The ability of the as-PKC alpha-pREP3 construct to specifically reduce PKC alpha levels in both clones was demonstrated by Western blots probed with antibodies against the PKC beta isozyme (the form structurally more similar to PKC alpha) and other representative isoenzymes expressed in Jurkat cells (PKC delta, epsilon, theta, and mu). Stimulation of transfected Jurkat clones with phorbol-12-myristate-13 alone or in the presence of ionomycin resulted in significant reduction of IL-2R alpha expression, TNF-alpha production, and the induction of transcriptional activity of a pIL-2/Luc construct in both as PKC alpha-reduced clones. The magnitude of these decrements paralleled the reductions of PKC alpha expression. The loss of the effects in clone as1 after a high number of passages correlated with the recovery of normal levels of PKC alpha protein, suggesting a link between these processes. Thus, the findings of this study demonstrate the essential role that PKC alpha plays in major events of the T lymphocyte activation process.


Assuntos
Regulação Enzimológica da Expressão Gênica/imunologia , Isoenzimas/genética , Ativação Linfocitária/genética , Proteína Quinase C/genética , RNA Antissenso/genética , Linfócitos T/imunologia , Humanos , Células Jurkat , Ativação Linfocitária/imunologia , Proteína Quinase C-alfa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Transfecção
3.
Exp Neurol ; 144(2): 339-49, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9168834

RESUMO

We explored the constitutive expression, maturational regulation, and relation to kainic-acid-induced apoptosis of cyclooxygenase (COX)-2 mRNA in rat brain. In adult rats, COX-2 mRNA was expressed primarily in limbic structures. Constitutive COX-2 mRNA expression increased markedly between Postnatal Day 7 (P7) and P14, reaching adult levels by P21. Despite intense KA-induced seizures, no COX-2 mRNA induction was found before P14 in any brain region examined. During response to KA-induced seizures in adult brain, COX-2 mRNA induction paralleled temporally and overlapped anatomically the appearance of cellular morphological features of apoptosis in subsets of cells of the pyramidal neuron layer of the hippocampal formation, amygdaloid complex, and pyriform cortex. While COX-2 mRNA showed KA-induced elevation in the granule cell layer of the dentate gyrus, no detectable morphological features of apoptosis were found in this region. Finally, monotypic culture of rat corticohippocampal neurons confirmed the neuronal expression of COX-2 in vitro and revealed that COX-2 is induced during response to glutamate treatment, leading to neuron death. These studies may provide novel insights into the role of COX-2 and mechanisms of action of nonsteroidal anti-inflammatory drugs in Alzheimer's disease.


Assuntos
Doença de Alzheimer/enzimologia , Encéfalo/enzimologia , Isoenzimas/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Prostaglandina-Endoperóxido Sintases/biossíntese , Doença de Alzheimer/tratamento farmacológico , Tonsila do Cerebelo/enzimologia , Tonsila do Cerebelo/patologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Apoptose/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Células Cultivadas , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Indução Enzimática/efeitos dos fármacos , Agonistas de Aminoácidos Excitatórios/farmacologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hipocampo/enzimologia , Hipocampo/patologia , Isoenzimas/genética , Ácido Caínico/farmacologia , Ácido Caínico/toxicidade , Degeneração Neural/efeitos dos fármacos , Proteínas do Tecido Nervoso/genética , Especificidade de Órgãos , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Convulsões/induzido quimicamente , Convulsões/enzimologia , Convulsões/patologia
4.
Br J Rheumatol ; 35(8): 711-8, 1996 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-8761181

RESUMO

An inducible form of cyclooxygenase-2 (COX-2) has been shown to be upregulated in vitro by various pro-inflammatory agents, such as lipopolysaccharide, IL-1 and TNF, COX-2 appears to be responsible for the increase in prostaglandin synthesis at the site of inflammation. To examine the involvement of COX-2 in inflammation, we analysed the expression of this gene in human rheumatoid arthritis (RA) and in rat adjuvant-induced arthritis. Immunocytochemical studies of synovial membrane biopsies from human RA, osteoarthritic (OA) and normal joints using a COX-2 specific antibody showed positive staining in RA, but not in normal synovial membranes. Specifically, expression of COX-2 was detected in synovial lining cells, lymphoid aggregates and endothelial cells of blood vessels. Although some positive staining was observed in the OA joints, the number of stained cells was dramatically lower and the staining of the cells was less intense than in the rheumatoid tissue. By reverse transcription and polymerase chain reaction analysis, COX-2 mRNA was detected in the rat adjuvant arthritic limb, whereas no COX-2 mRNA was detectable in the normal limb. These observations indicate that COX-2 expression is upregulated in inflammatory joint disease and that COX-2 is a potential therapeutic target for specific inhibition.


Assuntos
Artrite Experimental/enzimologia , Artrite Reumatoide/enzimologia , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , RNA Mensageiro/metabolismo , Membrana Sinovial/enzimologia , Adulto , Idoso , Animais , Especificidade de Anticorpos , Sequência de Bases , Ciclo-Oxigenase 2 , Modelos Animais de Doenças , Feminino , Humanos , Isoenzimas/imunologia , Masculino , Proteínas de Membrana , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Prostaglandina-Endoperóxido Sintases/imunologia , Coelhos , Ratos , Ratos Sprague-Dawley
5.
Biochim Biophys Acta ; 1254(1): 112-6, 1995 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-7811740

RESUMO

We have isolated a murine macrophage cDNA encoding a 12-lipoxygenase, that represents the homolog of the human 15-lipoxygenase. The predicted amino acid sequence of this lipoxygenase is highly similar to the rat 12-lipoxygenase isolated from brain and human 15-lipoxgenase. The recombinant enzyme expressed in Cos-7 cells oxidizes arachidonic acid to 12- and 15-HETE with a profile similar to that obtained from peritoneal macrophages. A polyclonal antibody generated against a putative peptide recognizes a 75 kDa protein in cell extracts from mouse peritoneal macrophages and transfected Cos-7 cells. The lipoxygenase cDNA hybridizes to a 2.5 kb mRNA present in peritoneal macrophages, lung, spleen, heart and liver. RT-PCR analysis indicates that the same lipoxygenase is expressed in mouse reticulocytes. A partial genomic clone for this lipoxygenase has also been characterized. Southern blot analysis of mouse genomic DNA indicates that this is a single copy gene.


Assuntos
Lipoxigenase/genética , Macrófagos/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Western Blotting , Clonagem Molecular , DNA Complementar , Humanos , Lipoxigenase/química , Camundongos , Dados de Sequência Molecular , Ratos , Alinhamento de Sequência
6.
Cell Immunol ; 153(1): 28-38, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8287492

RESUMO

PKC pseudosubstrate consensus sequences can be found in the amino terminal region of all the different PKC isoenzymes characterized to date. Here we have used four peptides corresponding to the putative pseudosubstrate sequences from the PKC isoenzymes alpha, gamma, delta, and epsilon. These peptides showed PKC inhibitory activity when tested in a PKC-specific enzyme assay at concentrations of 25 to 100 microM, similar to what has been reported for the myristylated peptide KRTLR. Although the presence of a myristyl group at the amino terminal end of any of these peptides is not essential for their inhibitory activity, myristylation increased the inhibitory activity significantly. By contrast, the myristylated control peptide (GALRQQKNVHEVKN) was not active even at a 100 microM concentration. All of the PKC inhibitory peptides were also able to block PKC activity in a cell assay as demonstrated by their ability to inhibit the induction of IL-2R and TNF-beta expression in Jurkat cells. Finally, we confirmed a previous report of the inhibitory activity of the myristylated peptide KRTLR and showed that other related peptides (N-m-RLTRK, N-m-RRLKT) are also active in these assays.


Assuntos
Ativação Linfocitária , Peptídeos/farmacologia , Proteína Quinase C/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Complexo CD3/fisiologia , Linhagem Celular , Primers do DNA/química , Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Linfotoxina-alfa/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Proteína Quinase C/antagonistas & inibidores , RNA Mensageiro/genética , Receptores de Interleucina-2/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
7.
J Immunol ; 150(5): 1746-54, 1993 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-8436813

RESUMO

Activation of protein kinase C (PKC) in T cells leads to a variety of responses including IL-2 production and IL-2 receptor expression. PKC consists of several isoforms that exhibit some different in vitro properties. We have set up a Western blotting system to explore the regulation of PKC isoforms during T cell activation. In Jurkat T lymphoma cells, PKC alpha, beta, delta, epsilon, and zeta were detected. PKC alpha and beta existed primarily in the cytosol, translocated to the membrane fraction after 10 minutes of treatment with PMA, and almost completely disappeared within 16 h. A larger fraction of PKC delta and epsilon existed in the membrane fraction compared to PKC alpha or beta, and PKC epsilon translocated to the membrane fraction rapidly. Translocation of PKC delta was not apparent after 1 h treatment with PMA, but total PKC delta protein was reduced within 4 to 6 h of treatment. Consistent with this, overnight treatment with PMA caused down-regulation of both PKC delta and epsilon, but to a lesser degree than was observed with PKC beta. Anti-PKC zeta antibody detected two bands at 82 and 75 kDa. The 75-kDa band existed mostly in the cytosol fraction and showed no translocation or down regulation after PMA. We present evidence that this 75-kDa band represents PKC zeta. Similar PMA-induced translocation responses were observed in murine thymocytes showing that the responses are not unique to PKC isoforms in Jurkat. These results demonstrate that it is possible for the PKC isoforms to be differentially regulated during T cell activation.


Assuntos
Isoenzimas/metabolismo , Ativação Linfocitária , Proteína Quinase C/metabolismo , Linfócitos T/enzimologia , Acetato de Tetradecanoilforbol/farmacologia , Animais , Transporte Biológico , Regulação para Baixo , Feminino , Humanos , Linfoma de Células T/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase C/imunologia , Linfócitos T/imunologia , Células Tumorais Cultivadas
9.
Cell Immunol ; 142(2): 303-12, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1623554

RESUMO

Regulation of protein kinase C (PKC) isoform mRNAs has been studied in the immature, murine B lymphoma WEHI-231 by the MAPPing protocol and by slot blot analysis of unamplified mRNA. This membrane IgM (mIgM)-positive cell line has been previously used as a model to study signal transduction by mIgM in immature B lymphocytes and the role of those signals in the induction of immune tolerance in the B cell compartment. Stimulation of the cells by anti-mu antibodies, phorbol ester, or Ca2+ ionophore caused growth arrest and death of the cells. IL 4 and IL 5 slowed the growth of the cells. Of these stimuli, only anti-mu stimulation affected PKC mRNA levels. Anti-mu treatment caused a transient decrease in the amount of PKC-zeta isoform mRNA within 3 hr. Within 24 hr levels returned toward normal. Anti-mu had little or no effect on the expression of mRNA for the alpha, beta, delta, or epsilon isoforms of PKC. WEHI-231 cells do not express PKC-gamma. Although anti-mu treatment blocked progression of the cells from the G0/G1 stage into the S phase of cell cycle, viable sort selected cells in either the G0/G1 or the S/G2/M phases showed no clear difference in the expression of PKC-zeta message. Thus, there is not preferential regulation of expression of PKC-zeta during stages of the cell cycle. The results show that mIgM on WEHI-231 cells can transduce a signal that is not mediated by PKC or Ca2+ mobilization alone. The signal causes transient, selective down-regulation of mRNA encoding the zeta PKC isoform.


Assuntos
Anticorpos Anti-Idiotípicos/química , Imunoglobulina M/química , Isoenzimas/genética , Proteína Quinase C/genética , RNA Mensageiro/análise , Animais , Morte Celular , Membrana Celular/química , Regulação para Baixo , Camundongos , Transdução de Sinais , Células Tumorais Cultivadas
10.
Cell Immunol ; 142(1): 197-206, 1992 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-1586956

RESUMO

Protein kinase C (PKC), which plays a pivotal role in lymphocyte activation, represents a homologous family of at least nine proteins. Seven genes that encode PKC proteins have been identified. Since the regulatory properties and substrate specificities of the isoforms are not identical in vitro, it is possible that each isoform plays a unique role in cell activation. Toward an understanding of the role of PKC isoforms in lymphocyte activation we have studied the expression of mRNA encoding six of the isoforms (alpha, beta, gamma, delta, epsilon, and zeta) in T cell clones and B cell lines. PKC isoform phenotyping was done by MAPPing using isoform-specific primers and slot-blot analyses of mRNA were performed using specific probes. T cell clones and B cell lines were determined to express levels of the delta, epsilon, and zeta isoforms of PKC that were detectable by MAPPing. Plasmacytomas did not express PKC-beta message detectable by MAPPing. Slot blot analyses and Western blot analyses with peptide-specific antibody confirmed that B cell plasmacytomas did not express PKC-beta mRNA or protein. T cell clones and B cell lines were similar in that none expressed PKC-gamma. In cells that expressed PKC isoforms that were detectable by the MAPPing protocol, there was heterogeneity in the relative abundance of isoform mRNA (PKC-delta and -beta) and protein (PKC-beta and -epsilon). Such diversity of isoform expression could be responsible for the differential responsiveness of lymphocyte clones to activating stimuli.


Assuntos
Linfócitos B/enzimologia , Proteína Quinase C/análise , RNA Mensageiro/análise , Linfócitos T/enzimologia , Animais , Linhagem Celular , Linfoma/enzimologia , Mieloma Múltiplo/enzimologia , Proteína Quinase C/química
11.
Biotechniques ; 12(1): 30, 32, 34-5, 1992 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1310411

RESUMO

The PCR technique can use protein-derived oligonucleotide sequences as primers to develop probes for screening recombinant libraries. Here we report a method with highly degenerate mixtures of oligonucleotides as primers for the PCR that eliminates the need to identify or isolate the DNA sequences derived by PCR. The method uses the pool of PCR-generated DNA sequences radiolabeled during the extension reaction as a probe, combined with highly stringent hybridization and wash conditions that permit only homologous sequences to hybridize and therefore target desired clones. This technique was used successfully to clone the receptor for tumor necrosis factor.


Assuntos
Sondas de DNA/síntese química , DNA/análise , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Sequência de Bases , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Radioisótopos de Fósforo , Receptores de Superfície Celular/química , Receptores do Fator de Necrose Tumoral
12.
Cell Immunol ; 138(2): 265-79, 1991 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-1934072

RESUMO

Interferon gamma (IFN-gamma) is the most potent known lymphokine for activating macrophages and has been shown to induce expression of HLA-DR in THP-1 cells, a monocytic tumor cell line which expresses many of the properties of monocytes, in a dose- and time-dependent manner. Experiments were designed to examine, by FACS analysis and by measurement of messenger RNA levels, the molecular mechanism regulating the expression of HLA-DR molecules. The expression of HLA-DR molecules induced by IFN-gamma was blocked by the protein kinase C (PKC) inhibitors sphingosine, staurosporine, and H7. H7 when added up to 20 hr after the initial stimulation with IFN-gamma prevented the further expression of HLA-DR. The general kinase inhibitors H8, H9, and HA1004, all less potent PKC inhibitors than H7, did not block the IFN-gamma-induced expression of HLA-DR at the concentrations employed. W7, a calmodulin antagonist, but not a PKC inhibitor, was also unable to prevent the IFN-gamma-induced expression of HLA-DR. Treatment of THP-1 with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, alone or with Ca2+ ionophore A23187, was unable to induce HLA-DR expression. However, pretreatment with PMA for 24 hr prior to IFN-gamma stimulation decreased the IFN-gamma-induced expression of HLA-DR without decreasing IFN-gamma receptor levels. These results suggest that PKC plays a significant role in the IFN-gamma-induced signal transduction pathway leading to the expression of HLA-DR in cells of the mononuclear phagocytic lineage, and that PKC activity is required throughout the course of events leading to the actual expression of HLA-DR.


Assuntos
Antígenos HLA-DR/biossíntese , Interferon gama/farmacologia , Proteína Quinase C/fisiologia , Transdução de Sinais , Ativação Enzimática/efeitos dos fármacos , Antígenos HLA-DR/genética , Humanos , Leucemia Monocítica Aguda/imunologia , Monócitos/imunologia , Sistemas do Segundo Mensageiro/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas
13.
J Immunol ; 147(2): 405-9, 1991 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-2071891

RESUMO

The expression of the different protein kinase C (PKC) isozymes in mouse thymocytes was studied to determine if there is a correlation between isozyme expression and thymocyte phenotype. Expression of PKC isozymes in thymocyte subsets (distinguished by the CD4 or CD8 Ag) was determined by message amplification phenotyping. The expression of mRNA for PKC-alpha, -beta, -epsilon, and -zeta, but not -gamma or -delta isozymes, was detected in all of the unstimulated thymocyte subpopulations analyzed. Thus no differences in the pattern of PKC isozyme expression were found that could be correlated with thymocyte phenotype. However, it was noted that the levels of PKC mRNA expression were affected by different stimuli in unfractionated thymocytes. Whereas mRNA levels of PKC-alpha and -beta were down-regulated by PMA and ionomycin treatment, no significant changes were seen in the levels of PKC-epsilon mRNA with these agents. PKC-epsilon mRNA decreased in thymocytes exposed to Con A similar to what has been reported for PKC-epsilon protein. PKC-zeta mRNA was also down-regulated by PMA or ionomycin, and the combination of both compounds caused a more rapid and drastic effect. Finally, PKC-delta mRNA expression was induced transiently in thymocytes only after exposure to PMA or Con A, and this induction was inhibited by ionomycin treatment. These results indicate that message levels of specific isoforms of PKC are uniquely regulated and suggest an additional level of control of PKC activity in activated lymphocytes.


Assuntos
Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Subpopulações de Linfócitos T/enzimologia , Animais , Sequência de Bases , Concanavalina A/farmacologia , Expressão Gênica , Ionomicina/farmacologia , Isoenzimas/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Oligonucleotídeos/química , Reação em Cadeia da Polimerase , Proteína Quinase C/genética , RNA Mensageiro/genética , Acetato de Tetradecanoilforbol/farmacologia
14.
Int Immunol ; 2(8): 765-74, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2083234

RESUMO

Soluble immune response suppressor (SIRS) is a low-molecular-weight protein (approximately 10,000 daltons) produced by mitogen- or interferon-activated T lymphocytes that can block development of humoral or cell-mediated immune responses in vivo or in vitro. As previously reported, murine SIRS is heterogeneous, eluting in two broad peaks on high performance reverse phase chromatography as well as displaying several broad isoelectric point forms. A putative N-terminal 21 amino acid sequence has been obtained for one of the less hydrophobic isoforms, SIRS-alpha 7. The sequence of SIRS-alpha 7 is unique in mammals but shows a remarkable homology to the family of short neurotoxins (short neurotoxin 1) found in sea snake, adder, and cobra species. A degenerate oligonucleotide probe based on the protein sequence was synthesized and was shown to hybridize to SIRS messenger RNA as measured by SIRS synthesis in a rabbit reticulocyte lysate system. A synthetic polypeptide based on the 21-residue sequence was also prepared and coupled to thyroglobulin or keyhole limpet hemocyanin. These were used to prepare rabbit antisera that neutralize SIRS bioactivity and precipitate authentic SIRS.


Assuntos
Venenos Elapídicos/genética , Neurotoxinas/genética , Fatores Supressores Imunológicos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sondas de DNA , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico
15.
Lymphokine Res ; 8(1): 9-18, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2469910

RESUMO

Suppressor T-cell hybridomas specific for the synthetic polypeptide antigen L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) release TsF spontaneously and are not dependent on exogenous sources of lymphokines for their growth. IL-2 has no effect on the cell growth of these hybridomas and little overall effect is observed on protein biosynthesis. Nevertheless, the addition of IL-2 to one of these hybridomas (762 B3.7), leads to a substantial increase in suppressor factor (TsF2) production as measured by both bioactivity and direct analysis of 35S-methionine incorporation into TsF2. Treatment of the TsF2 producing hybridoma with phorbol myristate acetate (PMA) causes an increase in the level of IL-2 receptor expression in this hybridoma and enhances the effects of IL-2 on the biosynthesis of TsF2. These data suggest that in addition to its growth promoting properties, IL-2 may provide a signal that triggers suppressor cells to produce antigen specific suppressor factors.


Assuntos
Hibridomas/efeitos dos fármacos , Interleucina-2/farmacologia , Fatores Supressores Imunológicos/biossíntese , Autorradiografia , Bioensaio , Divisão Celular/efeitos dos fármacos , Imunofluorescência , Humanos , RNA/biossíntese , Receptores de Interleucina-2/biossíntese , Acetato de Tetradecanoilforbol/farmacologia
16.
Int J Biochem ; 18(2): 149-53, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-2936637

RESUMO

Thymocytes, bone marrow lymphocytes, as well as lymphocytes from spleen, lymphoid nodes and peripheral blood were obtained from BALB/c mice. Subpopulations of BALB/c bone marrow T-lymphocyte precursors and immature (small) and mature (large) thymocytes, as established by the percentage of terminal deoxynucleotidyl transferase (TdT) and peanut agglutinin (PNA) positive cells, were obtained by centrifugation on discontinuous density gradients. The activities of N-acetyl-beta-glucosaminidase (NAG), beta-glucuronidase (BG), acid alpha-naphthyl acetate esterase (ANAE) and alpha-naphthyl acetate esterase (NAE) were determined by enzymatic assays of cell extracts of the diverse T-lymphocyte subpopulations, in order to follow their evolution with the maturation of the T-lymphocytes in the thymus. These activities were compared with that determined in lymphocytes from spleen, lymphoid nodes and peripheral blood. The glucidases BG and NAG and the esterases ANAE and NAE present a high decrease in their activities from bone marrow T-lymphocyte progenitors to immature thymocytes. BG, NAG and ANAE activities undergo an about 3-fold increase with the evolution of the thymocytes from small to large cells. Whereas the level of the NAE activity decreases (2-fold) with that evolution of the thymocytes. Lymphocytes from spleen and lymphoid nodes exhibit activities of the glucidases and, specially, the esterases marked by higher than those of thymocyte populations. Peripheral blood lymphocytes also present NAG, ANAE and NAE activities higher than in thymocytes, but their BG activity is lower.


Assuntos
Medula Óssea/enzimologia , Glucuronidase/metabolismo , Hexosaminidases/metabolismo , Tecido Linfoide/enzimologia , Naftol AS D Esterase/metabolismo , Linfócitos T/enzimologia , Timo/enzimologia , Animais , Cinética , Linfonodos/enzimologia , Camundongos , Baço/enzimologia , beta-N-Acetil-Hexosaminidases
17.
Int J Biochem ; 16(2): 225-9, 1984.
Artigo em Inglês | MEDLINE | ID: mdl-6323233

RESUMO

Lymphocyte populations of BALB/c mice were obtained from bone marrow, thymus, spleen, peripheral blood and lymphoid nodes. Subpopulations of thymocytes and bone marrow T-lymphocyte precursors were separated by density gradient centrifugation. The activity of adenosine deaminase (ADA) undergoes a marked increase during the evolution of bone marrow T-cell precursors to immature thymocytes, and a decrease with thymocytes maturation. The peripheral blood lymphocytes (PBL) present the lower activity of the enzyme, and lymphocytes from spleen (SL) and lymphoid nodes (LNL) show activity in the order of that in mature thymocytes. The activity of purine nucleotide phosphorylase (PNP) in the different lymphocytes populations experiments a very little variation with the T-lymphocyte differentiation. With the evolution of T-lymphocyte precursors to immature thymocytes the 5'-nucleotidase (5'-NT) activity experiment a 2-fold decrease. The thymocytes maturation is correlated with an increase in the activity of 5'-NT. The PBL present the maximal activity of the enzyme, whereas in spleen and LNL its levels of activity are in the range of that in mature thymocytes and bone marrow T-cell precursors respectively.


Assuntos
Adenosina Desaminase/isolamento & purificação , Medula Óssea/enzimologia , Tecido Linfoide/enzimologia , Nucleosídeo Desaminases/isolamento & purificação , Nucleotidases/isolamento & purificação , Pentosiltransferases/isolamento & purificação , Purina-Núcleosídeo Fosforilase/isolamento & purificação , Linfócitos T/enzimologia , 5'-Nucleotidase , Adenosina Desaminase/sangue , Animais , Centrifugação com Gradiente de Concentração , DNA Nucleotidilexotransferase/metabolismo , Precursores Enzimáticos/metabolismo , Linfonodos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Nucleotidases/sangue , Purina-Núcleosídeo Fosforilase/sangue , Baço/enzimologia , Timo/enzimologia
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