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1.
Hum Gene Ther ; 8(5): 575-84, 1997 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-9095409

RESUMO

Nonviral gene therapy approaches use a plasmid vector to express the desired transgene. We have systematically examined several regulatory elements within plasmid vectors that govern gene expression, e.g., the promoter, enhancer, intron, and polyadenylation signal, by constructing a series of plasmids that differed only in the particular sequence element being evaluated. Of the several promoters and polyadenylation signal sequences that were tested, the human cytomegalovirus (CMV) immediate early gene promoter and the addition of polyadenylation signal sequences from the bovine growth hormone (BGH) gene or rabbit beta-globin gene produced the highest levels of expression in vitro. The inclusion of a hybrid intron 3 to the promoter further increased expression 1.6-fold. The addition of a region of the CMV enhancer 5' to several weak promoters increased expression 8- to 67-fold, and co-transfection with a second plasmid encoding a chimeric transcription factor also enhanced expression. On the basis of these results, the CMV promoter, the hybrid intron, and the BGH polyadenylation signal were selected for consistent high level expression in vitro and in the mouse lung. However, expression was transient, with greater than 60% loss of activity in the first 7 days. This transient expression was not specific to CMV promoter-containing plasmids, because plasmids containing other heterologous promoters showed a similar profile of transient expression in vivo. These comparative analyses begin to provide a basis for the development of optimized expression plasmids for gene therapy of lung diseases.


Assuntos
Citomegalovirus/genética , Elementos Facilitadores Genéticos/fisiologia , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Genes Precoces/genética , Vetores Genéticos/genética , Pulmão , Transgenes/genética , Animais , Bovinos , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Elementos Facilitadores Genéticos/genética , Genes Reporter , Humanos , Íntrons , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Poli A/metabolismo , Regiões Promotoras Genéticas , Coelhos
2.
Hum Gene Ther ; 10(10): 1667-82, 1999 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-10428212

RESUMO

Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A. Deficiency of this enzyme results in progressive deposition of the glycosphingolipid globotriaosylceramide (GL-3) in the vascular lysosomes, with resultant distension of the organelle. The demonstration of a secretory pathway for lysosomal enzymes and their subsequent recapture by distant cells through the mannose 6-phosphate receptor pathway has provided a rationale for somatic gene therapy of lysosomal storage disorders. Toward this end, recombinant adenoviral vectors encoding human alpha-galactosidase A (Ad2/CEHalpha-Gal, Ad2/CMVHIalpha-Gal) were constructed and injected intravenously into Fabry knockout mice. Administration of Ad2/CEHalpha-Gal to the Fabry mice resulted in an elevation of alpha-galactosidase A activity in all tissues, including the liver, lung, kidney, heart, spleen, and muscle, to levels above those observed in normal animals. However, enzymatic expression declined rapidly such that by 12 weeks, only 10% of the activity observed on day 3 remained. Alpha-galactosidase A detected in the plasma of injected animals was in a form that was internalized by Fabry fibroblasts grown in culture. Such internalization occurred via the mannose 6-phosphate receptors. Importantly, concomitant with the increase in enzyme activity was a significant reduction in GL-3 content in all tissues to near normal levels for up to 6 months posttreatment. However, as expression of alpha-galactosidase A declined, low levels of GL-3 reaccumulated in some of the tissues at 6 months. For protracted treatment, we showed that readministration of recombinant adenovirus vectors could be facilitated by transient immunosuppression using a monoclonal antibody against CD40 ligand (MR1). Together, these data demonstrate that the defects in alpha-galactosidase A activity and lysosomal storage of GL-3 in Fabry mice can be corrected by adenovirus-mediated gene transfer. This suggests that gene replacement therapy represents a viable approach for the treatment of Fabry disease and potentially other lysosomal storage disorders.


Assuntos
Adenovírus Humanos , Doença de Fabry/terapia , Técnicas de Transferência de Genes , Vetores Genéticos , alfa-Galactosidase/genética , Animais , Linhagem Celular , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Terapia de Imunossupressão , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Tempo , Triexosilceramidas/metabolismo , alfa-Galactosidase/metabolismo
3.
Hum Gene Ther ; 10(11): 1833-43, 1999 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-10446923

RESUMO

For gene therapy to be effective in the treatment of chronic diseases, plasmid DNA (pDNA) vectors that provide persistent expression of therapeutic levels of the transgene product are desirable. Studies in the lung with adenovirus vectors showed that products of the adenovirus E4 region can act both in cis and in trans to increase the duration of expression when transcription of the transgene was under the control of the human cytomegalovirus (CMV) promoter. To determine if these E4-encoded proteins could also effect greater persistence of expression from a nonviral vector, a complex composed of cationic lipid GL-67, a CMV promoter plasmid (pCF1-CAT), and an E4-containing adenovirus vector (Ad2/betagal-4) was instilled into the lungs of BALB/c nu/nu mice. Significant increases in the duration of transgene expression were observed for up to 10 weeks postinstillation compared with expression from mice instilled with control complexes containing an adenovirus vector deleted of most of E4 (Ad2/betagal-2). This effect could also be observed in immunodeficient NIH-rnu rats as well as in immunocompetent BALB/c mice. Studies with CMV promoter mutants indicated that a region proximal to the promoter was necessary for the E4-mediated increase in longevity of expression. In addition to the CMV promoter, a CMV enhancer-human mucin I (MUC-I) hybrid promoter also responded to these E4-encoded proteins with increased persistence of transgene expression, but a human interleukin 8 (IL-8) promoter did not. Ad2/betagal-4 could be replaced by a pDNA vector expressing only the E4 region, indicating that products of the E4 region alone were sufficient in the absence of expression from the rest of the adenovirus genome. Further analysis indicated that the protein encoded by open reading frame 3 (ORF3) alone was sufficient for conferring the increase in persistence of expression. These data indicate that expression of a single protein from the adenovirus genome can significantly improve the duration of transgene expression from pDNA vectors, and increases the feasibility of using nonviral vectors for the treatment of chronic diseases.


Assuntos
Adenoviridae/genética , Proteínas E4 de Adenovirus/genética , Expressão Gênica , Vetores Genéticos , Pulmão/metabolismo , Plasmídeos , Transgenes , Proteínas E4 de Adenovirus/metabolismo , Animais , Resinas de Troca de Cátion/metabolismo , Cloranfenicol O-Acetiltransferase/metabolismo , Fibrose Cística/terapia , Citomegalovirus/genética , Técnicas de Transferência de Genes , Terapia Genética , Humanos , Imunocompetência , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas , Ratos , alfa-Galactosidase/metabolismo
4.
Hum Gene Ther ; 7(14): 1701-17, 1996 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-8886841

RESUMO

Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Técnicas de Transferência de Genes , Lipídeos , Pulmão , Adenovírus Humanos/genética , Animais , Transporte Biológico , Cátions , Células Cultivadas , DNA Recombinante/administração & dosagem , Portadores de Fármacos , Eletrólitos/metabolismo , Epitélio/fisiologia , Expressão Gênica , Vetores Genéticos/genética , Humanos , Lipídeos/síntese química , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ratos , Ratos Endogâmicos F344 , Relação Estrutura-Atividade , Transfecção , Transgenes/genética
5.
J Neuropathol Exp Neurol ; 38(2): 165-76, 1979 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-233531

RESUMO

An in vitro model system was developed to study the effects that immune cells might have on herpes simplex virus (HSV) infection of rat dorsal root ganglia in culture. Our results demonstrate that rat splenic lymphocytes and peritoneal exudate cells are incapable of replicating HSV even if these cells come from sensitized animals and are stimulated in vitro. Both cell preparations can offer some protection to rat dorsal root ganglia from the effects of HSV infection. The data suggest that an immunologically non-specific (not mediated by sensitized cells) type of protection is important to neurons, while an immunologically specific (mediated by sensitized cells) protection is most beneficial to fibroblasts. This system can be utilized to study the mechanism of latency since the neurons of sensory ganglia are the natural site of latent herpes virus.


Assuntos
Gânglios Espinais/microbiologia , Linfócitos/imunologia , Simplexvirus/crescimento & desenvolvimento , Animais , Líquido Ascítico/citologia , Células Cultivadas , Fibroblastos/patologia , Gânglios Espinais/patologia , Herpes Simples/imunologia , Imunização , Neurônios/patologia , Ratos , Simplexvirus/imunologia , Baço/citologia , Replicação Viral
6.
J Neuropathol Exp Neurol ; 36(4): 680-92, 1977 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-196052

RESUMO

The ultrastructural consequences of herpes simplex virus type 2 (strain R-2) infection of organotypic cultures of embryonic rat dorsal root ganglia were studied. The intial consequences (dilation of endoplasmic reticulum and/or Golgi apparatus and distortion of mitochondrial structure) occurred in the cytoplasm. These effects were followed by several nuclear changes including loss of nucleoplasm, and margination of chromatin. Extensive nuclear membrane proliferation was accompanied by the viral maturation process. Two previously unreported observations were made. First, productive virus replications occurred in glial cells, as well as in neurons. Mature, enveloped virus was produced by nuclear budding and envelopment in the cytoplasm in both cell types. Second, neurons were observed to participate in polykaryocyte formation with other neurons and with glial cells. These polykaryocytes were usually composed of only three or four cells. Neuronal-glial polykaryocytes were more prevalent than neuronal-neuronal polykaryocytes. In general, however, the ultrastructural changes in neurons and glial cells in culture were consistent with previously reported changes occurring in nervous tissue of experimental animals suffering from acute herpes simplex virus infections. Therefore, the utilization of this in vitro system to further study the pathogenesis of acute herpetic infections of sensory ganglia appears to be reasonable.


Assuntos
Gânglios Espinais/ultraestrutura , Herpes Simples/patologia , Simplexvirus , Animais , Corpos de Inclusão Viral , Microscopia Eletrônica , Bainha de Mielina/ultraestrutura , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Técnicas de Cultura de Órgãos , Especificidade de Órgãos , Ratos , Replicação Viral
7.
J Neuropathol Exp Neurol ; 40(4): 380-9, 1981 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-6265604

RESUMO

Dissociated rat dorsal root ganglion cultures were infected with herpes simplex virus type 1 and studied electrophysiologically throughout the course of infection. Alterations in the electrical parameters of the action potentials of these neurons occurred as early as 4 hours post infection. The maximum rate of rise, spike amplitude, and overshoot were decreased, and the full width at half maximum and resting membrane potential were increased. At 6 hours post infection there is a movement back to normal values for these parameters. This reversal is transient, however, and the types of alterations seen at 4 hours post infection are more pronounced at 12 hours post infection. The first morphological alterations occur at 16 hours post infection, at which time less than 50% of the neurons impaled produced action potentials. Application of cyclohexamide to inhibit protein synthesis 1 hour post infection prevented electrophysiological changes normally seen at 4 hours and 6 hours post infection. It is concluded that the sodium conductance is specifically reduced by acute HSV infection, and that this reduction is attributed to viral specific or modified host cell protein synthesis.


Assuntos
Gânglios Espinais/fisiopatologia , Herpes Simples/fisiopatologia , Potenciais de Ação , Animais , Permeabilidade da Membrana Celular , Células Cultivadas , Potenciais da Membrana , Proteínas de Membrana/biossíntese , Neurônios/patologia , Ratos , Sódio/fisiologia , Transmissão Sináptica
8.
Talanta ; 33(8): 657-60, 1986 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18964162

RESUMO

Analytical methods for the assay of anthranilic acid and for determination of the impurities methyl anthranilate, anthranoylanthranilic acid and 3- and 4-aminobenzoic acid are described. A Microbondapak C18 column is used for both the assay and the impurity determination. The assay is based on isocratic development with a mobile phase of 35:65 v v methanol/pH-3 phosphate buffer, with benzoic acid as internal standard. The impurities are separated by gradient elution. The standard deviation of the assay method is about 1% and the limit of detection for the impurities is about 0.01%.

9.
Exp Neurol ; 225(2): 436-44, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20673762

RESUMO

Gaucher disease is caused by a deficit in the enzyme glucocerebrosidase. As a consequence, degradation of the glycolipids glucosylceramide (GluCer) and glucosylsphingosine (GluSph) is impaired, and their subsequent buildup can lead to significant pathology and early death. Type 1 Gaucher patients can be treated successfully with intravenous replacement enzyme, but this enzyme does not reach the CNS and thus does not ameliorate the neurological involvement in types 2 and 3 Gaucher disease. As one potential approach to treating these latter patients, we have evaluated intracerebroventricular (ICV) administration of recombinant human glucocerebrosidase (rhGC) in a mouse model of neuronopathic Gaucher disease. ICV administration resulted in enzyme distribution throughout the brain and alleviated neuropathology in multiple brain regions of this mouse model. Treatment also resulted in dose-dependent decreases in GluCer and GluSph and significantly extended survival. To evaluate the potential of continuous enzyme delivery, a group of animals was treated ICV with an adeno-associated viral vector encoding hGC and resulted in a further extension of survival. These data suggest that ICV administration of rhGC may represent a potential therapeutic approach for type 2/3 Gaucher patients. Preclinical evaluation in larger animals will be needed to ascertain the translatability of this approach to the clinic.


Assuntos
Doença de Gaucher/enzimologia , Glucosilceramidase/administração & dosagem , Longevidade/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Doença de Gaucher/genética , Doença de Gaucher/patologia , Vetores Genéticos , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Imuno-Histoquímica , Injeções Intraventriculares , Estimativa de Kaplan-Meier , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo
10.
J NeuroAIDS ; 1(3): 51-71, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-16873171

RESUMO

Varying degrees of neurological dysfunction are observed in AIDS patients who develop AIDS dementia complex (ADC). Data from a large number of in vivo and in vitro rodent studies have suggested a role for the HIV envelope glycoprotein gp 120 in this process. These studies were initiated to clarify possible effects of recombinant gp120 on signal transduction systems and the synthesis of specific ADC-related cytokines in human neuroblastoma cells. Out results indicate that gp120 on signal transduction systems and the synthesis of specific ADC-related cytokines in human neuroblastoma cells. Our results indicate that gp120 did not induce the synthesis of cAMP, IPs or NO, nor did it alter agonist-induced synthesis of these molecules. In addition, it did not induce the synthesis of IL-6 and TNFα. However, it did activate a src-family protein tyrosine kinase which phosphorylates several substrates, including prominent proteins in the 115 and 60 kDa range. This gp120-induced tyrosine phosphorylation may contribute to neurological dysfunction since protein tyrosine kinases are known to be involved in processes important for pre- and post-synaptic neuronal function.


Assuntos
Citocinas , HIV-1 , Complexo AIDS Demência , Citocinas/farmacologia , Proteína gp120 do Envelope de HIV/química , HIV-1/metabolismo , Humanos , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo
11.
J Gen Virol ; 68 ( Pt 9): 2501-7, 1987 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-3655745

RESUMO

Persistent mumps virus infections were established in rat pheochromocytoma (PC-12) and human medulloblastoma (TE-671) continuous cell lines. Significant amounts of infectious virus were produced by the PC-12 cells; infectious virus production by the TE-671 cells was limited. This restricted replication may be due to decreased production of viral envelope glycoproteins by TE-671 cells. The presence of virus changed the distribution of stimulus-evoked electrical responsiveness of both cell lines from responsiveness composed primarily of normal, rapidly rising, all-or-nothing action potentials to one dominated by abnormal, slowly rising, graded responses or by no response at all. Such changes have the potential to disrupt neural integration within the nervous system, and suggest a new mechanism by which persistent virus infections might play a role in chronic neurological and/or mental disease.


Assuntos
Transformação Celular Viral , Vírus da Caxumba/genética , Neoplasias das Glândulas Suprarrenais , Animais , Antígenos Virais/análise , Linhagem Celular , Neoplasias Cerebelares , Replicação do DNA , Humanos , Meduloblastoma , Potenciais da Membrana , Vírus da Caxumba/crescimento & desenvolvimento , Feocromocitoma , Ratos , Replicação Viral
12.
Arch Virol ; 79(1-2): 123-30, 1984.
Artigo em Inglês | MEDLINE | ID: mdl-6320769

RESUMO

Various immune mechanisms were tested for their ability to alter a normally productive infection of herpes simplex virus in cultures of dissociated rat sensory neurons. These mechanisms included anti-HSV antibody with and without the aid of complement, HSV-sensitized "T" cells, natural killer cells, and "K" cells plus anti-HSV antibody (ADCC reactions). Although both anti-HSV antibody plus complement and the ADCC mechanism significantly limited infectious HSV production, no mechanism was capable of preventing the eventual infection of the majority of neurons. It appears, therefore, that none of these mechanisms by themselves can convert a normally productive infection of sensory neurons in culture to a non-productive or latent infection.


Assuntos
Herpes Simples/imunologia , Neurônios Aferentes/microbiologia , Simplexvirus/imunologia , Animais , Anticorpos Antivirais/imunologia , Efeito Citopatogênico Viral , Células Matadoras Naturais/imunologia , Ratos , Simplexvirus/crescimento & desenvolvimento , Linfócitos T/imunologia
13.
J Gen Virol ; 70 ( Pt 3): 749-54, 1989 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-2543758

RESUMO

Rat pheochromocytoma (PC12) cells, persistently infected with mumps virus (MV), failed to generate full-sized stimulus-evoked action potentials (SEAPs) when examined by intracellular electrophysiological recording techniques. Application of tetrodotoxin (TTX) had little or no effect on MV-reduced SEAPs, indicating that the number of functional voltage-gated Na+ channels was decreased or their operation was blocked by the virus. In contrast, MV-infected cells generated normal Ca2+ spikes when bathed in a solution containing TTX, tetraethylammonium ions and a high concentration (20 mM) of Ca2+. In addition, when infected cells bathed in TTX were superfused with Co2+ the SEAP profile reverted to that typical of PC12 cells with functional voltagegated K+ channels only. These observations indicate that MV affects voltage-gated Na+ channels, but spares voltage-gated Ca2+ and K+ channels of persistently infected cells.


Assuntos
Caxumba/fisiopatologia , Canais de Sódio/fisiologia , Neoplasias das Glândulas Suprarrenais/complicações , Neoplasias das Glândulas Suprarrenais/fisiopatologia , Animais , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/fisiologia , Linhagem Celular , Potenciais Evocados/efeitos dos fármacos , Feocromocitoma/complicações , Feocromocitoma/fisiopatologia , Ratos , Canais de Sódio/efeitos dos fármacos , Tetrodotoxina/farmacologia , Células Tumorais Cultivadas
14.
J Bacteriol ; 105(1): 190-9, 1971 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-4925120

RESUMO

The 50S ribosomal subunits obtained from exponential-phase and valine-limited cultures of Streptococcus faecalis 9790 differed in protein content. Approximately 35% of the valine-limited subunits showed protein losses when analyzed by CsCl density centrifugation after formaldehyde fixation. Results of density gradient analysis and of acrylamide gel electrophoresis localized the protein to a "split" subunit fraction. No differences could be detected in the protein contents of 30S subunits from exponential-phase and valine-limited cultures.


Assuntos
Acrilatos , Arginina/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/biossíntese , Técnicas Bacteriológicas , Isótopos de Carbono , Centrifugação com Gradiente de Concentração , Césio , Cloretos , Meios de Cultura
15.
Infect Immun ; 28(2): 620-3, 1980 May.
Artigo em Inglês | MEDLINE | ID: mdl-6249745

RESUMO

The peripheral infection of neurons from dissociated rat sensory ganglia by herpes simplex virus was studied with a newly developed tissue culture system. The results indicated that the virus arrives at the neuronal soma by an axoplasmic route and that at least some of these neurons are productively infected since eventually all cells in the culture appeared to be destroyed by the virus. This system showed promise in elucidating the possible roles of various effector cells, antiviral antibody, or antiviral compounds in the mechanism of herpes simplex virus latency.


Assuntos
Neurônios/microbiologia , Simplexvirus , Animais , Técnicas de Cultura , Ratos
16.
Infect Immun ; 34(2): 588-95, 1981 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-6273322

RESUMO

Concanavalin A and wheat germ agglutinin are capable of preventing a productive peripheral infection of dissociated rat sensory neurons in culture by herpes simplex virus type 1. Concanavalin A binds to the herpes simplex virion, rendering it inactive, whereas wheat germ agglutinin binds to the peripheral neuritic extensions of the sensory neurons, rendering them incapable of initiating a productive viral infection. This latter effect (i) seems to be specific for wheat germ agglutinin since other lectins have no effect, (ii) is not the result of cellular cytotoxicity, (iii) is dependent on an N-acetylneuraminic acid moiety, and (iv) may be due either to viral receptor site masking or to binding of wheat germ agglutinin to the neuritic receptor molecule for herpes simplex virus.


Assuntos
Lectinas/farmacologia , Neurônios Aferentes/microbiologia , Simplexvirus/crescimento & desenvolvimento , Animais , Células Cultivadas , Concanavalina A/metabolismo , Concanavalina A/farmacologia , Gânglios Espinais/microbiologia , Lectinas/metabolismo , Metabolismo , Ratos , Receptores Virais/metabolismo , Simplexvirus/metabolismo , Aglutininas do Germe de Trigo
17.
J Pharmacol Exp Ther ; 255(2): 497-503, 1990 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1978729

RESUMO

This study tests the hypothesis that atrial natriuretic factor (ANF) and C-ANF(4-23)-NH2 (C-ANF) augment cGMP generation and inhibit both cAMP generation and depolarization-induced catecholamine release in nerve growth factor treated pheochromocytoma cells by a pertussis toxin (PTX)-sensitive mechanism. Synthetic rat ANF(99-126) and the clearance receptor antagonist C-ANF (10(-12)-10(-9) M) inhibited basal and 5 microM vasoactive intestinal peptide (VIP)-induced cAMP generation in a concentration-dependent manner. These actions of ANF and C-ANF were blocked by 12-18 h pretreatment with PTX (100 ng/ml), suggesting ANF receptor coupling to adenylate cyclase via an inhibitory guanine nucleotide-binding protein. Both ANF (10(-11)-10(-9) M) and C-ANF (10(-11)-10(-8) M) also inhibited K(+)-induced catecholamine release in a concentration-dependent manner. ANF (10(-11)-10(-8) M) increased cGMP generation in a concentration-dependent manner but C-ANF did not. The accumulation of cGMP in response to ANF was not altered by treatment with PTX. Therefore, PTX dissociated the increased concentrations of cGMP from the ANF-mediated depression of evoked catecholamine release. C-ANF also dissociated elevations in cGMP concentrations from an ANF-mediated attenuation of evoked catecholamine release. The results of this study indicate that ANF inhibits adrenergic neurotransmission independent of guanylate cyclase.


Assuntos
Fator Natriurético Atrial/farmacologia , Dopamina/metabolismo , Guanilato Ciclase/fisiologia , Norepinefrina/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Toxina Adenilato Ciclase , Neoplasias das Glândulas Suprarrenais/patologia , Animais , AMP Cíclico/biossíntese , GMP Cíclico/biossíntese , Toxina Pertussis , Feocromocitoma/patologia , Potássio/farmacologia , Ratos , Células Tumorais Cultivadas , Fatores de Virulência de Bordetella/farmacologia
18.
J Bacteriol ; 168(2): 785-90, 1986 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-3782025

RESUMO

We constructed a genetic map of the fla-che region of the Rhizobium meliloti chromosome using cotransduction with bacteriophage phi M12. Several other chromosomal markers located in the general area are included in the map. We isolated plasmids carrying wild-type DNA inserts that complement the mapped mutations from a genomic library carried in the broad-host-range vector pLAFR1. The complementation data obtained from the clones confirmed the contransduction map and clarified the exact order of several of the behavioral genes. A restriction map of this area was developed by using the cloned DNA. One of the five individual EcoRI fragments subcloned from the original clones complemented two of the behavioral mutations.


Assuntos
Genes Bacterianos , Rhizobium/genética , Movimento Celular , Quimiotaxia , Mapeamento Cromossômico , Cromossomos Bacterianos , Clonagem Molecular , Flagelos , Teste de Complementação Genética , Mutação , Rhizobium/fisiologia , Rhizobium/ultraestrutura , Transdução Genética
19.
J Pharmacol Exp Ther ; 260(2): 689-96, 1992 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-1346640

RESUMO

We reported previously that atrial natriuretic factor (ANF) and the ANF clearance receptor binding peptide, C-ANF(4-23)-NH2 (C-ANF), inhibit catecholamine (CA) release from rat, nerve growth factor-treated pheochromocytoma cells (PC12 cells) by a guanylate cyclase independent mechanism. This mechanism is most likely a pertussis toxin (PTX)-sensitive inhibition of adenylate cyclase. This study examines the role of the second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP), in mediating atrial natriuretic factor effects on depolarization-induced CA release from PC12 cells. The following evidence supports the hypothesis that the neuromodulatory action of atrial peptides is independent of increases in cGMP: 1) ANF does not potentiate the inhibitory effect of C-ANF on CA release or cAMP generation but still elevates cGMP concentrations in the presence of C-ANF; 2) the neuromodulatory effects of ANF and C-ANF are blocked or reversed by a membrane permeable analog of cAMP, dibutyryl cAMP; 3) ANF and C-ANF attenuate CA release in the presence of a maximally effective concentration of dibutyryl cGMP; 4) the inhibitory effect of dibutyryl cGMP is PTX-insensitive whereas the atrial peptide effect is blocked by PTX-pretreatment; and 5) dibutyryl cGMP is without effect on adenylate cyclase. These data are consistent with the hypothesis that ANF and C-ANF act via the ANF clearance (R2) receptor to suppress adenylate cyclase activity and neurotransmission.


Assuntos
Inibidores de Adenilil Ciclases , Fator Natriurético Atrial/farmacologia , Guanilato Ciclase/metabolismo , Neurotransmissores/farmacologia , Toxina Adenilato Ciclase , Animais , AMP Cíclico/fisiologia , GMP Cíclico/fisiologia , Dibutiril GMP Cíclico/farmacologia , Dopamina/metabolismo , Ativação Enzimática , Norepinefrina/metabolismo , Células PC12 , Fragmentos de Peptídeos/farmacologia , Toxina Pertussis , Fatores de Virulência de Bordetella/farmacologia
20.
J Pharmacol Exp Ther ; 250(2): 428-32, 1989 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-2547929

RESUMO

This study tests the hypothesis that atrial natriuretic factor (ANF) inhibits catecholamine release from rat pheochromocytoma cells by increasing levels of intracellular cyclic GMP (cGMP). Rat differentiated pheochromocytoma cells are a model of adrenergic nerves and allow the exploration of the effects of various hormones, autacoids, drugs and neuromodulators on adrenergic neurotransmission in cell culture. Synthetic rat ANF (99-126) inhibited K+-induced norepinephrine and dopamine release, as measured by high-performance liquid chromatography, in a concentration-dependent manner over the concentration range of 10(-11) to 10(-8) M. ANF stimulated intracellular cGMP accumulation, as measured by specific radioimmunoassay, in a concentration-dependent manner over the same concentration range. The cGMP analog, N2-2'-O-dibutyryl cGMP also inhibited K+-induced norepinephrine and dopamine release in a concentration-dependent manner. The results of this study are consistent with the hypothesis that ANF acts as an inhibitory neuromodulator in adrenergic nerves via the second messenger, cGMP.


Assuntos
Fator Natriurético Atrial/farmacologia , Catecolaminas/metabolismo , GMP Cíclico/fisiologia , Feocromocitoma/metabolismo , Animais , GMP Cíclico/análise , Dibutiril GMP Cíclico/farmacologia , Potássio/farmacologia , Ratos , Células Tumorais Cultivadas
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