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1.
Transplant Proc ; 45(6): 2469-75, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23953564

RESUMO

The outcomes of organ transplantation have improved due to better immunosuppressive drugs, surgical techniques, and management of complications. However, ischemia-reperfusion injury remains a challenge affecting graft survival. In this study, we employed injection of a protein transduction domain (PTD) to inhibit the c-Jun NH2-terminal kinase (JNK) pathway thereby attenuating ischemia-reperfusion injury in a porcine model. The PTD-JNK inhibitor (JNKI) was administered into the renal artery, allowing it to be taken into various elements including vascular endothelial cells by endocytosis via the PTD. Serum creatinine and blood urea nitrogen concentrations were lower among PTD-JNKI than controls. In addition, renal tissue blood flow was maintained in the PTD-JNKI group, resulting in less tissue injury and fewer apoptotic cells. These results suggested that the PTD technique improved renal transplantation outcomes.


Assuntos
Peptídeos Penetradores de Células/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/sangue , Nitrogênio da Ureia Sanguínea , Permeabilidade da Membrana Celular , Peptídeos Penetradores de Células/administração & dosagem , Peptídeos Penetradores de Células/metabolismo , Isquemia Fria/efeitos adversos , Creatinina/sangue , Citoproteção , Modelos Animais de Doenças , Endocitose , Feminino , Injeções Intra-Arteriais , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Rim/irrigação sanguínea , Rim/enzimologia , Rim/patologia , Nefropatias/enzimologia , Nefropatias/etiologia , Nefropatias/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/metabolismo , Artéria Renal , Circulação Renal/efeitos dos fármacos , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Suínos , Fatores de Tempo , Fator de Necrose Tumoral alfa/sangue , Resistência Vascular/efeitos dos fármacos
2.
Neuroscience ; 200: 13-8, 2012 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-22088430

RESUMO

Oxytocin (OT) levels in plasma increase during sexual response and are significantly lower in patients with depression. A drug for the treatment of sexual dysfunction, sildenafil, enhances the electrically evoked release of OT from the posterior pituitary. In this study, we showed that sildenafil had an antidepressant-like effect through activation of an OT signaling pathway. Application of sildenafil reduced depression-related behavior in male mice. The antidepressant-like effect was blocked by an OT receptor (OTR) antagonist and was absent in OTR knockout (KO) mice. Sildenafil increased the phosphorylation of cAMP response element-binding protein (CREB) in the hippocampus. The OTR antagonist inhibited sildenafil-induced CREB phosphorylation and sildenafil had no effect on CREB phosphorylation in OTR KO mice. These results suggest sildenafil to have an antidepressant-like effect through the activation of OT signaling and to be a promising drug for the treatment of depression.


Assuntos
Antidepressivos/uso terapêutico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Depressão/tratamento farmacológico , Ocitocina/metabolismo , Piperazinas/uso terapêutico , Sulfonas/uso terapêutico , Compostos de Anilina/farmacologia , Animais , Benzamidas/farmacologia , Depressão/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Comportamento Exploratório/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Resposta de Imobilidade Tônica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação/efeitos dos fármacos , Purinas/uso terapêutico , Receptores de Ocitocina/deficiência , Fatores Sexuais , Comportamento Sexual Animal/efeitos dos fármacos , Citrato de Sildenafila , Natação/psicologia
3.
Transplant Proc ; 43(5): 1489-94, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21693223

RESUMO

BACKGROUND: Milrinone (MIL), a phosphodiesterase (PDE) 3 inhibitor, exhibits cardiotonic and angioectatic effects. Various PDE inhibitors have been shown to suppress inflammatory cytokines. In this study, we evaluated the angioectatic and anti-inflammatory cytokine effects of MIL on renal function after warm ischemia in a rat ischemia-reperfusion (I-R) injury model. MATERIALS AND METHODS: MIL or control solution was perfused from the left renal artery to the right kidney, and the left kidney was excised. The right renal artery, vein, and ureter were clamped and then released after 50 minutes to produce warm ischemia. We evaluated control (n = 7), MIL (n = 7), and sham operation (n = 7) groups for serum creatinine, blood urea nitrogen (BUN), blood flow, expression of tumor necrosis factor (TNF)-α mRNA, apoptosis index, and histological evidence of acute tubular necrosis. RESULTS: Serum creatinine and BUN concentrations peaked at 24 hours after reperfusion. MIL treatment significantly reduced serum creatinine (control group 1.27 ± 0.45 mg/dL vs MIL group 0.77 ± 0.19 mg/dL, P < .05; sham 0.35 ± 0.2 mg/dL) and BUN (control 67.6 ± 13.6 mg/dL vs MIL 51.0 ± 8.8 mg/dL, P < .05; sham 23.0 ± 4.2 mg/dL) levels at 24 hours. Thereafter, serum creatinine and BUN concentrations in the MIL group remained significantly lower compared with the control group for 120 hours (P < .05). MIL group exhibited significantly higher tissue blood flow, less acute tubular necrosis, lower expression of TNF-α mRNA in renal tissue, and lower apoptotic index (P < .05). CONCLUSIONS: MIL maintained renal tissue blood flow by its vasodilatory effect, suppressed expression of TNF-α mRNA by increasing intracellular cyclic adenosine monophosphate, and ultimately decreased tubular cell apoptosis, thus protecting renal function after warm I-R injury.


Assuntos
Rim/efeitos dos fármacos , Milrinona/uso terapêutico , Traumatismo por Reperfusão/prevenção & controle , Animais , Sequência de Bases , Nitrogênio da Ureia Sanguínea , Creatinina/sangue , Primers do DNA , Rim/irrigação sanguínea , Ratos
4.
Transplant Proc ; 42(7): 2427-9, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20832520

RESUMO

OBJECTIVE: Kidney grafts with multiple renal arteries (MRAs) are not uncommon, but they do make transplantation more difficult. Laparoscopic graft nephrectomy has become the standard; however, the safety and reliability must be maintained for both a donor and a recipient even in case of MRAs. This study evaluated the short-term outcomes of living donor renal transplant using grafts with MRAs procured by laparoscopic nephrectomy. PATIENTS AND METHODS: This study reviewed all living donor kidney transplantations performed from January 2008 to June 2009, which were divided into 3 groups according to the number of renal graft arteries. The serum creatinine level, warm ischemic time (WIT), rewarming time, total ischemic time (TIT), operative time, acute rejection episodes, and complications in each group were evaluated. RESULTS: The serum creatinine level showed no difference among the groups. Longer TIT was observed in the MRAs group, but WIT and rewarming time did not differ. The acute rejection rate was not different. There were no vessel complications in any donors and recipients. CONCLUSION: Harvesting kidney grafts with MRAs by laparoscopic nephrectomy requires a longer TIT; however, transplantation can be performed safely and reliably for both donors and recipients.


Assuntos
Transplante de Rim/métodos , Laparoscopia/métodos , Doadores Vivos , Nefrectomia/métodos , Artéria Renal/cirurgia , Adolescente , Adulto , Idoso , Anastomose Cirúrgica/métodos , Criança , Pré-Escolar , Creatinina/sangue , Rejeição de Enxerto/epidemiologia , Humanos , Artéria Ilíaca/cirurgia , Imunossupressores/uso terapêutico , Transplante de Rim/imunologia , Laparoscopia/normas , Pessoa de Meia-Idade , Nefrectomia/normas , Estudos Retrospectivos , Segurança , Coleta de Tecidos e Órgãos/métodos
5.
Transplant Proc ; 41(10): 4274-6, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20005383

RESUMO

OBJECTIVE: The aim of this study was to evaluate the availability of a pancreatic allograft biopsy via a laparotpmy. PATIENTS AND METHODS: From September 2004 to November 2007, 17 pancreas transplantations were performed: 15 simultaneous pancreas and kidney transplantations (SPK), 1 pancreas transplant alone (PTA), and one pancreas after kidney transplantation (PAK). Thirteen pancreatic allograft biopsies were obtained via an open laparotomy. This study evaluated the complications associated with this procedure, the rate of obtaining an adequate sample, and the relationship between biopsy-proven rejections and laboratory markers. In SPK cases we evaluated the synchronization between pancreas and kidney rejection. The pancreatic samples were diagnosed according to the Drachenberg classification. RESULTS: No complications resulted from the procedure. The rate of obtaining adequate samples was 84.6%. Pancreas rejection correlated with elevation of the laboratory markers in 71.4%. Simultaneous pancreas and kidney rejection occurred in 62.5%, only kidney in 25%, and only pancreas in 12.5%. CONCLUSION: A pancreas graft biopsy was absolutely imperative to improve the outcome in PTA, and even in SPK cases. A pancreatic allograft biopsy via a laparotomy was a safe, necessary and easy procedure to obtain an accurate diagnosis of rejection among pancreas transplantation patients.


Assuntos
Biópsia/métodos , Transplante de Pâncreas/patologia , Rejeição de Enxerto/patologia , Humanos , Transplante de Rim/imunologia , Transplante de Rim/patologia , Laparotomia/métodos , Cavidade Peritoneal/diagnóstico por imagem , Cavidade Peritoneal/patologia , Estudos Retrospectivos , Transplante Homólogo/patologia , Ultrassonografia
6.
Neuroscience ; 107(3): 363-71, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11718992

RESUMO

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) plays an essential role in vesicular transport and the release of neurotransmitters and hormones through associations with NSF and SNAP receptors (SNAREs). Three isoforms (alpha, beta and gamma) of SNAP are expressed in mammals. We have generated isoform-specific antibodies and studied the expression and distribution of these SNAP isoforms in the rat nervous system. Each antibody specifically recognized alpha-, beta- or gamma-SNAP in an isoform-specific manner in immunoblots of brain homogenate. Alpha- and gamma-SNAP were ubiquitously expressed in various tissues, whereas beta-SNAP was expressed only in brain. After subcellular fractionation of brain homogenates, all three isoforms were recovered in both soluble and particulate fractions. Immunohistochemistry revealed that alpha- and beta-SNAP were generally differentially distributed both in synaptic and non-synaptic regions, including brain white matter. The presynaptic location of both alpha- and beta-SNAP was confirmed by immunoelectron microscopy. At the neuromuscular junction, immunoreactive alpha-SNAP was identified in synaptic vesicles, while in the cerebellum, beta-SNAP was present in the presynaptic membranes of basket neuron and mossy fiber terminals. From these results we suggest that both alpha- and beta-SNAP may play an important role in neurotransmitter release as well as in constitutive vesicular transport.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Sistema Nervoso/metabolismo , Proteínas de Transporte Vesicular , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Encéfalo/metabolismo , Proteínas de Transporte/genética , Immunoblotting , Imuno-Histoquímica , Proteínas de Membrana/genética , Sistema Nervoso/ultraestrutura , Ratos , Solubilidade , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida , Frações Subcelulares/metabolismo , Distribuição Tecidual
8.
J Biosci Bioeng ; 89(3): 247-51, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-16232737

RESUMO

The anaerobic oxidation of dissolved H2S to elemental sulfur was studied at 23 degrees C and pH 6.5+/-0.3 in continuous culture of the phototrophic green sulfur bacterium Prosthecochloris aestuarii. The number of cells formed in the cultures was proportional to the amount of H2S oxidized, and the growth yield was independent of light intensity. The specific growth rate was significantly dependent on the dissolved H2S concentration and light intensity. The kinetic data were analyzed with a rate expression as a function of each rate-limiting factor. Under illumination by white fluorescent lamps, the specific oxidation rate of P. aestuarii reached a maximum of 2.02 x 10(-14) mol-H2S.h(-1).cell(-1) when the dissolved H2S concentration was 2.1 mM at 5000 lx. Simultaneous use of near infrared LED (light-emitting diode) and white fluorescent lamps provided a 35% increase in the maximum specific H2S oxidation rate.

10.
Infect Immun ; 66(10): 4811-6, 1998 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-9746583

RESUMO

The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra. The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT. The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT. Binding experiments with rat brain synaptosomes revealed that 125I-labeled 111/NT bound to a single binding site with a dissociation constant (Kd) of 2.5 nM; the value was rather lower than that (0.42 nM) of 125I-Okra/NT for the high-affinity binding site. In the lipid vesicles reconstituted with ganglioside GT1b, 125I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the Kd values accorded with those of the low- and high-affinity binding sites in synaptosomes. However, 125I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b. Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner. These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity.


Assuntos
Toxinas Botulínicas/toxicidade , Botulismo/microbiologia , Proteínas de Ligação ao Cálcio , Clostridium botulinum/patogenicidade , Metaloendopeptidases/toxicidade , Neurotoxinas/toxicidade , Animais , Toxinas Botulínicas/imunologia , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas Tipo A , Botulismo/epidemiologia , Humanos , Lactente , Japão/epidemiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/imunologia , Metaloendopeptidases/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurotoxinas/imunologia , Neurotoxinas/metabolismo , Ligação Proteica , Proteínas R-SNARE , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sinaptossomos/metabolismo , Sinaptotagmina I , Sinaptotagminas
11.
Microb Pathog ; 25(2): 91-9, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9712688

RESUMO

Clostridium botulinum type B neurotoxin (BoNT/B) recognizes a complex of synaptotagmin II and ganglioside GT1b or GD1a as the high-affinity toxin binding site. Recombinant deletion mutants of synaptotagmin II allowed us to demonstrate that the N-terminal domain including the transmembrane region retains BoNT/B binding activity while the C-terminal domain is not involved in constituting the BoNT/B receptor. BoNT/B binding to reconstituted lipid vesicles containing synaptotagmin II and gangliosides showed that GT1b and GD1a confer the difference in the maximum binding capacity but not in the dissociation constant. The direct binding of GT1b to the deletion mutants revealed that the transmembrane region is required to bind GT1b, suggesting that synaptotagmin II binds to the ceramide portion of gangliosides within the plasma membrane. A monoclonal antibody against GT1b effectively inhibited not only BoNT/B binding to the reconstituted lipid vesicles and brain synaptosomes but also type A BoNT (BoNT/A) binding to brain synaptosomes. In addition, the monoclonal antibody antagonized the action of both BoNT/A and BoNT/B on synaptic transmission of rat superior cervical ganglion neurons. These results suggest that GT1b functions as a component of the receptor complex.


Assuntos
Toxinas Botulínicas/metabolismo , Encéfalo/microbiologia , Clostridium botulinum/fisiologia , Gangliosídeos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sinaptossomos/microbiologia , Animais , Anticorpos Monoclonais , Sítios de Ligação , Toxinas Botulínicas Tipo A , Sequência de Carboidratos , Membrana Celular/microbiologia , Gangliosídeos/análise , Gangliosídeos/química , Gangliosídeos/imunologia , Cinética , Dados de Sequência Molecular , Mutagênese , Proteínas do Tecido Nervoso/química , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Sinaptotagmina II
12.
J Int Med Res ; 25(5): 247-54, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9364287

RESUMO

Tenascin is a component of the extracellular matrix that responds rapidly to inflammation or injury. The activity index and chronicity index of immunoglobulin A nephropathy are mainly used to decide whether or not steroid therapy is indicated, but are sometimes difficult to evaluate histologically. We investigated whether tenascin staining of the glomeruli was an indicator of the activity or chronicity indices in patients with immunoglobulin A nephropathy. Tenascin staining was evaluated immunohistochemically in 38 renal specimens, including 32 from patients with immunoglobulin A nephropathy and six from control kidneys, and the extent of staining was scored. Tenascin staining was correlated with the chronicity index (r = 0.643, P < 0.0003) but not with the activity index.


Assuntos
Glomerulonefrite por IGA/fisiopatologia , Tenascina/fisiologia , Adolescente , Adulto , Idoso , Criança , Creatinina/sangue , Feminino , Glomerulonefrite por IGA/sangue , Humanos , Imuno-Histoquímica , Glomérulos Renais/química , Glomérulos Renais/patologia , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Coloração e Rotulagem
13.
Hiroshima J Med Sci ; 46(3): 93-7, 1997 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-9353969

RESUMO

In July 1994, a 70-year-old woman was diagnosed as having nephrotic syndrome with proteinuria of 8 to 10 g/day and a serum albumin level of 1.8 g/dl. She was hospitalized in August 1994 for investigation. The urinary findings then normalized, with urinary protein and occult blood both negative and total urinary protein excretion at 0 g/day. A renal biopsy was performed, and spontaneous remission of minimal change nephrotic syndrome was diagnosed. This is an interesting case involving rapid remission of minimal change nephrotic syndrome in an elderly patient.


Assuntos
Síndrome Nefrótica/fisiopatologia , Idoso , Feminino , Humanos , Rim/fisiopatologia , Remissão Espontânea
14.
Biochem Biophys Res Commun ; 239(1): 57-62, 1997 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-9345269

RESUMO

The molecular mechanisms of exocytosis from two types of secretory organelles, synaptic-like microvesicles and secretory vesicles, were compared by measuring acetylcholine (ACh) and catecholamine (CA) release from a newly isolated PC12 subclone, PC12-C3 which contains a high level of Ach. Digitonin-permeabilized PC12-C3 cells released both transmitters with similar Ca(2+)-dependency. Ca(2+)-evoked Ach and CA release from permeabilized cells were increased in the presence of MgATP, suggesting the existence of a MgATP-dependent priming step prior to the Ca(2+)-triggered fusion step in both ACh release and CA release. The non-hydrolyzable analogue of GTP guanosine 5'-(gamma-thio)triphosphate (GTP gamma S), produced both ACh and CA release from permeabilized cells in the absence of Ca2+. Pretreatment with a phorbol ester which activates protein kinase C, potentiated depolarization-induced ACh and CA release from unpermeabilized cells. These results indicated that exocytosis from two distinct vesicle populations are mediated by the same basic molecular mechanisms.


Assuntos
Acetilcolina/metabolismo , Catecolaminas/metabolismo , Exocitose , Vesículas Sinápticas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Cromatografia Líquida de Alta Pressão , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Células PC12 , Potássio/metabolismo , Ratos , Vesículas Sinápticas/efeitos dos fármacos
15.
Am J Nephrol ; 17(5): 413-6, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9382157

RESUMO

To investigate the relationship between localization of hepatocyte growth factor (HGF) and tubulointerstitial lesions (TILs) in the cortical area of renal biopsy specimens, a clinicopathological study was performed in 35 patients with IgA nephropathy. HGF was detected by an enzyme-antibody method and TILs were assessed semiquantitatively by light microscopy. HGF was observed mainly on epithelial cells in the tubules, but not in the glomeruli. Fourteen patients had biopsies that were positive for HGF. There was a correlation between HGF positivity and histological damage, the TIL grade, and several clinical parameters determined at biopsy. Thus, HGF is related to TILs in IgA nephropathy, and may be a factor in the exacerbation of this disease.


Assuntos
Glomerulonefrite por IGA/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Córtex Renal/metabolismo , Túbulos Renais/metabolismo , Adolescente , Adulto , Idoso , Biópsia , Feminino , Fibrose , Glomerulonefrite por IGA/complicações , Glomerulonefrite por IGA/patologia , Humanos , Imuno-Histoquímica , Córtex Renal/ultraestrutura , Túbulos Renais/ultraestrutura , Masculino , Pessoa de Meia-Idade , Fotomicrografia
16.
Brain Res ; 737(1-2): 351-5, 1996 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-8930391

RESUMO

In digitonin-permeabilized adrenal chromaffin cells, Ca(2+)-induced catecholamine release can be resolved into at least two sequential steps: a MgATP-dependent priming step and a MgATP-independent Ca(2+)-triggered step. Botulinum neurotoxins types A and E cleaved SNAP-25, and blocked MgATP-independent Ca(2+)-induced catecholamine release from the permeabilized chromaffin cells. When the permeabilized cells were primed by pretreatment with MgATP, the amount of SNAP-25 associated with VAMP-2 decreased, and the fraction of SNAP-25 proteolyzed by the neurotoxins increased. These results suggest that dissociation of SNAP-25 and VAMP-2 occurs during the MgATP-dependent priming step, and SNAP-25 plays some important roles in the subsequent MgATP-independent step.


Assuntos
Trifosfato de Adenosina/farmacologia , Células Cromafins/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Cálcio/farmacologia , Catecolaminas/metabolismo , Bovinos , Permeabilidade da Membrana Celular , Células Cromafins/química , Digitonina , Eletroforese em Gel de Poliacrilamida , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Proteínas de Membrana/isolamento & purificação , Proteínas do Tecido Nervoso/isolamento & purificação , Testes de Precipitina , Proteínas Qa-SNARE , Proteínas R-SNARE , Proteína 25 Associada a Sinaptossoma
17.
J Biol Chem ; 271(24): 14548-53, 1996 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-8662851

RESUMO

Protein kinase C-mediated phosphorylation of a 25-kDa synaptosome-associated protein (SNAP-25) was examined in living PC12 cells. Phorbol 12-myristate 13-acetate treatment enhanced high potassium-induced [3H]-norepinephrine release, and a 28-kDa protein recognized by an anti-SNAP-25 antibody was phosphorylated on Ser residues. The molecular size of the phosphorylated band decreased slightly following treatment with Clostridium botulinum type A neurotoxin, whereas the band disappeared after treatment with botulinum type E neurotoxin, indicating that the 28-kDa protein was SNAP-25. A phosphorylation is likely to occur at Ser187, as this is the only Ser residue located between the cleavage sites of botulinum type A and E neurotoxins. SNAP-25 of PC12 cells was phosphorylated by purified protein kinase C in vitro, and the amount of syntaxin co-immunoprecipitated with SNAP-25 was decreased by phosphorylation. These results suggest that the phosphorylation of SNAP-25 may be involved in protein kinase C-mediated regulation of catecholamine release from PC12 cells.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Norepinefrina/metabolismo , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Proteínas de Transporte Vesicular , Animais , Anticorpos Monoclonais , Antígenos/química , Toxinas Botulínicas/farmacologia , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/isolamento & purificação , Neurotoxinas/farmacologia , Células PC12 , Fosforilação , Fosfosserina/análise , Potássio/farmacologia , Proteínas Qa-SNARE , Ratos , Proteínas SNARE , Proteína 25 Associada a Sinaptossoma
18.
Neurosci Lett ; 208(2): 105-8, 1996 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-8859901

RESUMO

We have previously identified synaptotagmin, a synaptic vesicle membrane protein from rat brain, as a binding protein for Clostridium botulinum type B neurotoxin. In this report, rat synaptotagmin II was expressed by transfection in Chinese hamster ovary cells and interaction with the neurotoxin was studied. In stable transfectants, the NH(2)-terminal region of synaptotagmin was exposed to the extracellular medium. Synaptotagmin-expressing cells were shown to possess an extremely low binding activity for the radiodinated toxin. However, toxin-binding was markedly increased to cells which had been treated with gangliosides G T1b or G D1a. In synapses, the intravesicular NH(2)-terminus of synaptotagmin becomes exposed at the cell surface after following exocytosis. These findings suggest that the NH(2)-terminal domain of synaptotagmin II forms the binding site for type B neurotoxin by associating with specific gangliosides in presynaptic plasma membranes.


Assuntos
Toxinas Botulínicas/metabolismo , Proteínas do Tecido Nervoso/genética , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Ligação Competitiva/fisiologia , Toxinas Botulínicas/farmacologia , Toxinas Botulínicas Tipo A , Células CHO/química , Células CHO/metabolismo , Proteínas de Transporte/genética , Cricetinae , DNA Complementar , Radioisótopos do Iodo , Camundongos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/imunologia , Ratos , Sinaptotagmina II , Transfecção
20.
J Biol Chem ; 271(12): 6567-70, 1996 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-8636067

RESUMO

P- and Q-type calcium channels, which trigger rapid neurotransmitter release at many mammalian synapses, are blocked by omega-conotoxin MVIIC. 125I-omega-Conotoxin MVIIC binding to rat cerebellar synaptosomes was not displaced by omega-conotoxins GVIA or MVIIA (Ki > 1 microM), which are selective for N-type calcium channels. Solubilized 125I-omega-conotoxin MVIIC receptors were specifically recognized by antibodies directed against alpha1A calcium channel subunits, proteins known to constitute a pore with P/Q-like channel properties. Antibodies against syntaxin 1, SNAP 25, and VAMP 2 (synaptobrevin) each immunoprecipitated a similar fraction (20-40%) of omega-conotoxin MVIIC receptors. Immunoprecipitation was not additive, suggesting that heterotrimeric (SNARE) complexes containing these three proteins interact with P/Q-type calcium channels. Immobilized monoclonal anti-syntaxin antibodies retained alpha1A calcium channel subunits of 220, 180 and 160 kDa monitored by immunoblotting with site directed antibodies. Synaptotagmin was detected in channel-associated complexes, but not synaptophysin, Rab 3A nor rat cysteine string protein. Trimeric SNARE complexes are implicated in calcium-dependent exocytosis, a process thought to be regulated by synaptotagmin. Our results indicate that these proteins interact with P/Q-type calcium channels, which may optimize their location within domains of calcium influx.


Assuntos
Canais de Cálcio/metabolismo , Cerebelo/metabolismo , Proteínas de Membrana/metabolismo , Sinaptossomos/metabolismo , Proteínas de Transporte Vesicular , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio/química , Sondas Moleculares , Venenos de Moluscos/química , Venenos de Moluscos/metabolismo , Ligação Proteica , Ratos , Proteínas SNARE , Sintaxina 1
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