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BACKGROUND: There is increasing evidence that diagnostic salivary tests measuring inflammatory biomarkers are being developed to assess inflammatory status for early detection, prevention, and progression of periodontal disease. Therefore, the aim of the present study was to investigate and identify the salivary biomarker that can predict the inflammatory status of periodontal disease. METHODS: A total of 36 patients (28 women and 8 men) with an average age of 57 years were investigated. Unstimulated saliva was collected from the recruited subjects and analyzed using SillHa, a saliva-testing device that measures bacteria count, saliva buffer capacity, acidity, leukocyte esterase, protein, and ammonia. Periodontal parameters were then obtained by clinical examination and initial periodontal therapy was performed. Data obtained with SillHa were compared with clinical periodontal parameters at baseline, re-examination (three months from baseline), and final examination (six months from re-examination). RESULTS: Leukocyte esterase activity in saliva measured by SillHa; BOP and PCR measured by clinical examination showed a significant difference between baseline and final examination and between re-examination and final examination. Patients in the lower median group (group 1) had a significant difference in leukocyte esterase activity between baseline and final examination and re-examination and final examination. In addition, patients in Group 1 had significantly lower BOP between baseline and final examination. While patients in the higher median group (group 2) showed a modest decrease in leukocyte esterase activity, which was significant only between baseline and final examination, no significant changes were observed concerning BOP. Furthermore, the associated systemic disease was observed in 30% and 81.2% of group 1 and 2 patients, respectively. CONCLUSION: The results suggest that leukocyte esterase activity in saliva measured by SillHa could serve as a reliable diagnostic marker for monitoring inflammatory status in periodontal disease.
Assuntos
Doenças Periodontais , Masculino , Humanos , Feminino , Pessoa de Meia-Idade , Doenças Periodontais/diagnóstico , Doenças Periodontais/microbiologia , Hidrolases de Éster Carboxílico , Biomarcadores/análise , Saliva/químicaRESUMO
BACKGROUND: Marfan syndrome (MFS) is a systemic disorder of connective tissues caused by insufficient elastic fiber formation that leads to structural weakness and results in various tissue disorders, including cardiovascular and periodontal disease. Notably however, the risk of periodontal disease in MFS patients affected by an aortic aneurysm or dissection has not yet been clarified. METHODS: We investigated the periodontal condition in the following three groups: MFS patients diagnosed with an aortic aneurysm or dissection with a planned aortic surgery (MFS surgery), MFS patients who had already undergone aortic surgery (MFS post-surgery) and healthy control patients (Healthy). The periodontal condition of all of these patients was evaluated at their first visit, reassessed again at two-month after the first visit, and evaluated again at a six-month follow-up after the reassessment. RESULTS: A total of 14 participants, 3 MFS surgery patients, 4 MFS post-surgery patients and 7 healthy control volunteers were examined. Saliva examinations revealed no significant differences between any of the groups at the first visit, reassessment, or follow-up. Interestingly, the MFS surgery cases showed a higher BOP and PISA at the first visit and follow-up compared with the other groups. In contrast, the MFS surgery patients showed an improvement in their LVDd and EF values, both markers of cardiac function, at the reassessment and follow-up compared with the first visit. CONCLUSIONS: MFS associated with an aortic aneurysm or dissection leads to a higher risk of periodontal disease, indicating the need for more frequent oral hygiene maintenance in these patients. In addition, MFS patients who undergo frequent professional cleaning of their teeth show a lower onset of cardiovascular disease, suggesting that professional oral hygiene in these cases contributes to a healthier condition.
Assuntos
Aneurisma Aórtico , Dissecção Aórtica , Síndrome de Marfan , Doenças Periodontais , Dissecção Aórtica/complicações , Dissecção Aórtica/cirurgia , Aneurisma Aórtico/etiologia , Aneurisma Aórtico/cirurgia , Humanos , Síndrome de Marfan/complicações , Doenças Periodontais/complicaçõesRESUMO
Objectives: To assess the physical features and compression characteristics of a newly developed adjustable compression garment, McBoooon (Mc). Methods: Twelve healthy volunteers were recruited to assess the compression characteristics. The interface pressure (IP) was continuously measured to calculate the static (SSI) and dynamic stiffness indices (DSI). Additionally, the peak flow velocity (PV) of the popliteal vein during ankle dorsiflexion was measured using ultrasonography. Each parameter was compared between ASHIKA stockings (AS), Mc applied at the same resting pressure as AS (Mc1), and Mc applied at a resting pressure approximately twice that of Mc1 (Mc2). Results: SSI and DSI were significantly different, increasing in the order AS < Mc1 < Mc2 (p <0.01). Although the PV was significantly higher in the compression group than in the control group (p <0.05), no significant differences were found among the three groups. Conclusion: The physical features and compression characteristics of Mc were clarified. The high stiffness of this garment improves the adherence to compression therapy and contributes to the treatment of chronic venous insufficiency.
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OBJECTIVE AND DESIGN: To clarify the molecular mechanism of polyenylphosphatidylcholine (PPC), we examined the involvement of reactive oxygen species (ROS) and NADPH oxidase 4 (Nox4) in human hepatic stellate cells (HSCs). MATERIAL: Using human LX-2 HSC cells, we examined the effects of PPC on expression of α-smooth muscle actin (α-SMA) and collagen 1, generation of ROS, Nox4 expression, p38 activation and cell proliferation, induced by transforming growth factor ß1 (TGFß1). RESULTS: PPC suppressed ROS which are induced by TGFß1, phosphorylation of p38MAPK, and expression levels of α-SMA and collagen 1 in a dose-dependent manner. Higher concentrations of PPC also suppressed Nox4 levels. CONCLUSION: These results suggest that ROS and Nox4 induced by TGFß1 are the therapeutic targets of PPC in the suppression of human hepatic stellate cell activation.
Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , NADPH Oxidases/metabolismo , Fosfatidilcolinas/farmacologia , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular , Células Estreladas do Fígado/metabolismo , Humanos , NADPH Oxidase 4 , NADPH Oxidases/genética , RNA Mensageiro/metabolismoRESUMO
UNLABELLED: Human bone marrow-derived mesenchymal stem cells (BM-MSCs) are expected to be a potential source of cells for transplantation. Although recent reports have shown that isolated MSCs can differentiate into hepatocytes, the efficiency of differentiation is insufficient for therapeutic application. To circumvent this problem, it is necessary to understand the mechanisms of hepatic differentiation of human BM-MSCs. Hepatocyte nuclear factor 3beta (HNF3beta), a forkhead/winged helix transcription factor, is essential for liver development. In the present study, we established a tetracycline (Tet)-regulated expression system for HNF3beta in UE7T-13 BM-MSCs. HNF3beta expression significantly enhanced expression of albumin, alpha-fetoprotein (AFP), tyrosine amino transferase (TAT) and epithelial cell adhesion molecule (EpCAM) genes. The differentiated cells showed hepatocyte-specific functions including glycogen production and urea secretion. During treatment with the Tet-on system for 8 days, over 80% of UE7T-13 cells turned out to express albumin. Furthermore, the combination of Tet with basic fibroblast growth factor (bFGF) efficiently induced the genes such as albumin and TAT, which are associated with maturity of hepatocytes; however, it suppressed genes such as AFP and EpCAM, which are associated with immaturity of hepatocytes, suggesting that Tet-induced HNF3beta expression sensitizes BM-MSCs to bFGF signals. Finally, the results of the present study suggest that down-regulation of Wnt/beta-catenin signals caused by translocation of beta-catenin to cytoplasmic membrane is associated with hepatic differentiation of human BM-MSCs. CONCLUSION: HNF3beta expression induced efficient differentiation of UE7T-13 human BM-MSCs.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fígado/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Tetraciclina/farmacologia , Albuminas/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Fator 2 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica/efeitos dos fármacos , Glicogênio/biossíntese , Fator 3-beta Nuclear de Hepatócito/genética , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tetraciclina/administração & dosagem , Transfecção , Ureia/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
Retinoids play an important role in the regulation of cell growth and death. Synthetic retinoid CD437 reportedly induces apoptosis in various cancer cell lines. However, the mechanism of inducing apoptosis in hepatocellular carcinoma (HCC) cells by this agent remains to be clarified. In this study, we investigated the signaling pathway by which CD437 induces apoptosis in HCC cell lines. Apoptosis of six human HCC cell lines was induced by treatment with CD437. Caspase-3 and -9 were activated by CD437, suggesting that the apoptosis is mediated by mitochondrial pathways. Consistent with these findings, the treatment with CD437 upregulated Bax protein, downregulated Bcl-2 protein and released cytochrome c into the cytoplasm. Moreover, rhodamine123 staining revealed mitochondrial depolarization in the cells treated with CD437. These data of the present study suggest that CD437 induces apoptosis in HCC cells via mitochondrial pathways.
Assuntos
Apoptose , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Mitocôndrias/efeitos dos fármacos , Retinoides/farmacologia , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Humanos , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
A synthetic retinoid, CD437, has been shown to exert potent anti-tumor activity against various types of cancer cell lines, regardless of their sensitivities to natural retinoids. We herein demonstrate that CD437 induces endoplasmic reticulum (ER) stress, including the up-regulation of CHOP, BIP and GADD34 mRNA through ER stress transducer (PERK and IRE1alpha) activation in an ovarian adenocarcinoma cell line, SKOV3. It was also shown that CD437 induced the CHOP and GADD34 expressions in another four ovarian adenocarcinoma cell lines, indicating that CD437 functions as an ER stress inducer in these cell lines. Moreover, the siRNA-mediated knockdown of inducible CHOP expression prevented the cytotoxic effect of CD437. These results suggest that ER stress plays an important role in the mechanism by which CD437 induces apoptosis in ovarian adenocarcinoma cells.
Assuntos
Adenocarcinoma/metabolismo , Apoptose/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Neoplasias Ovarianas/metabolismo , Retinoides/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Feminino , Humanos , Estresse Oxidativo/efeitos dos fármacosRESUMO
The mechanisms of prevention of the development of liver cancer by NIK-333, an acyclic retinoid (ACR), were investigated. The transgenic mice expressing the dominant negative form of retinoic acid receptor alpha (RARE mice), that produce reactive oxygen species and lead to development of liver tumor were used. The effect of NIK-333 on hepatocarcinogenesis in RARE mice was studied. The RARE mice were examined after feeding 0.03% and 0.06% NIK-333 diets at 12 months of age. In the mice fed 0.06% NIK-333 diet, tumor incidence was greatly suppressed, compared to that of wild type mice (0/9 versus 5/9, P<0.05), but not in the mice fed 0.03% NIK-333 diet. In addition, expression of cytochrome p450 4a14 and acyl-CoA oxidase was normalized, and the percentages of positive cells for 8-hydroxy-2'-deoxyguanosine, 4-hydroxy-2-nonenal and proliferating cell nuclear antigen were decreased. Furthermore, expression of beta-catenin and cyclin D1 was also depressed. These data suggest that NIK-333 suppressed liver tumor in association with repression of oxidative stress.
Assuntos
Neoplasias Hepáticas/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Tretinoína/análogos & derivados , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Estresse Oxidativo/fisiologia , Tretinoína/farmacologia , Tretinoína/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
AIM: Bone marrow-derived mesenchymal stem cells (MSC) are expected to be an excellent source of cells for transplantation. We aimed to study the culture conditions and involved genes to differentiate MSC into hepatocytes. METHODS: The culture conditions to induce the efficient differentiation of human bone marrow-derived UE7T-13 cells were examined using cytokines, hormones, 5-azacytidine and type IV collagen. RESULTS: We found that combination of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) with type IV collagen coating induced hepatic differentiation of UE7T-13 cells at over 30% frequency, where expression of albumin mRNA was increased over 20-fold. The differentiated cells had functions of albumin production, glycogen synthesis and urea secretion as well as expressing hepatocyte-specific genes. In addition, these cellshave binuclear and cuboidal morphology, which is a characteristic feature of hepatocytes. During hepatic differentiation, UE7T-13 cells showed depressed expression of WISP1 and WISP2 genes, members of the CCN family. Conversely, knockdown of WISP1 or WISP2 gene by siRNA stimulated hepatic differentiation. The effect of aFGF/bFGF/HGF/type IV collagen coating and WISP1-siRNA on hepatic differentiation was additive. CONCLUSION: The present study suggests that aFGF/bFGF/HGF/type IV collagen coating is the efficient condition for hepatic differentiation of UE7T-13 cells, and that WISP1 and WISP2 play an important role in hepatic transdifferentiation of these cells.
RESUMO
BACKGROUND/AIMS: Although the importance of reactive oxygen species (ROS) in the pathogenesis of various diseases is stressed, clinical significance of the markers reflecting DNA oxidation such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) remains to be clarified. METHODOLOGY: To examine clinical usefulness of 8-OHdG in healthy individuals in comparison with liver disease patients, urinary excretion of 8-OHdG was measured in 336 healthy individuals and 110 patients with liver disease. RESULTS: In healthy persons, the 8-OHdG excretion was increased in an age-dependent manner. It was positively correlated with cigarettes smoked a day and negatively correlated with body mass index (BMI) (P < 0.05, each). Age, smoking and BMI were independent predictors of urinary 8-OHdG excretion (P < 0.01, P < 0.01 and P < 0.05, respectively). In liver disease, the excretion of 8-OHdG was not changed, as compared with healthy individuals. However, the liver disease patients under the age of 40 had higher values of 8-OHdG than healthy persons. In addition, the urinary excretion of 8-OHdG was higher in patients with hepatitis C virus (HCV) infection than those with hepatitis B virus (HBV) infection. CONCLUSIONS: The results of the present study suggest that measurement of urinary 8-OHdG excretion is useful in assessing DNA oxidation caused by aging, smoking, body composition and liver disease.
Assuntos
Desoxiguanosina/análogos & derivados , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/urina , 8-Hidroxi-2'-Desoxiguanosina , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Índice de Massa Corporal , DNA/metabolismo , Desoxiguanosina/urina , Feminino , Humanos , Neoplasias Hepáticas/diagnóstico , Masculino , Pessoa de Meia-Idade , Oxigênio/metabolismo , Espécies Reativas de Oxigênio , Valores de Referência , Reprodutibilidade dos Testes , FumarRESUMO
BACKGROUND/AIMS: To rescue patients with severe liver injury, it is critical to develop the efficient regulatory system of hepatic stem cell proliferation in vitro. Our aims are to examine whether combination of adenovirus-mediated hepatocyte growth factor (HGF) gene transfer with signal transduction inhibitors can regulate cell proliferation of oval cells. METHODOLOGY: We examined the effects of treatment with adenoviral mediated HGF gene transfer and signal transduction inhibitors including LY294002, rapamycin and U0126 on proliferation OC/CDE22 hepatic oval cells and expression of signal transduction molecules. RESULTS: Infection with pAxCAHGF expanded the cells by 8-fold at 2 days, by 18-fold at 3 days and by 55-fold at 4 days. The addition of inhibitors inhibited pAxCAHGF-induced cell proliferation by LY294002 or rapamycin (P < 0.01, each). U0126 also inhibited growth of hepatic oval cells (P < 0.01). pAxCAHGF treatment induced phosphorylation of AKT. Treatment with rapamycin resulted in enhanced phosphorylation of AKT, and phosphorylation of AKT was induced by pAxCAHGF plus U0126. CONCLUSIONS: Autocrine expression of HGF with signal transduction inhibitors can regulate proliferation of OC/CDE22 hepatic oval cells. In addition, the AKT pathway is important for HGF-stimulated hepatic oval cell proliferation.
Assuntos
Técnicas de Cultura de Células , Proliferação de Células , Fator de Crescimento de Hepatócito/genética , Hepatócitos/fisiologia , Adenoviridae/genética , Butadienos/farmacologia , Cromonas/farmacologia , Inibidores Enzimáticos/farmacologia , Técnicas de Transferência de Genes , Hepatócitos/efeitos dos fármacos , Humanos , Morfolinas/farmacologia , Nitrilas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , beta-Galactosidase/análise , beta-Galactosidase/metabolismoRESUMO
We investigated age-related changes in intestinal intraepithelial lymphocyte (IEL) subsets in mice by flow cytometric analysis and their functional preservation as affected by feeding Spirulina, a cyanobacterium that is known to possess various therapeutic effects, including immune modulation activity. The number of cells possessing the leukocyte-common antigen CD45(+) cells in mice (43 weeks old) of the aged group, used as a representative marker for IELs, was significantly lower than that of adult mice (5 weeks old). Either the proportion or the number of CD45(+)CD8(+) cells of the aged mice was significantly lower than that of adult mice, corresponding to previous reports. Proportions and numbers of CD4(+)CD8(+) cells in aged mice, on the other hand, were higher than those in adult mice. Increased or decreased levels of the cell surface antigens observed in the aged mice tended to be restored in aged mice fed Spirulina (aged-SP group), which ingested a hot water extract of Spirulina (SpHW). In fact, the proportions of CD45(+)CD8(+) cells and CD45(+)TCRgammadelta(+) cells in the aged-SP group significantly increased in comparison to the control aged group, which ingested ordinary chow and water ad libitum. These results suggest that ingestion of SpHW in the aged-SP group may contribute to the functional preservation of the intestinal epithelium as a first line of mucosal barrier against infectious agents through retaining the number of certain IELs. Changes in the number of other IEL subsets and blood cells are also discussed.
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Envelhecimento/imunologia , Produtos Biológicos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Spirulina , Subpopulações de Linfócitos T/efeitos dos fármacos , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Feminino , Citometria de Fluxo , Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Antígenos Comuns de Leucócito/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Receptores de Antígenos de Linfócitos T gama-delta/efeitos dos fármacos , Subpopulações de Linfócitos T/citologiaRESUMO
A novel biomass-energy process for the production of methane from sewage sludge using a subcritical water (sub-CW) hydrolysis reaction as pretreatment is proposed. The main substances of sewage sludge hydrolyzed by sub-CW at 513 K for 10 min were acetic acid, formic acid, pyroglutamic acid, alanine, and glycine. Fermentation experiments were conducted in an anaerobic-sludge reactor for two different samples: real sewage sludge and a model solution containing components typically produced by the sub-CW pretreatment of sewage sludge. In the experiment for the sub-CW pretreatment of sewage sludge, methane generation was twice that for non-pretreatment after 3 days of incubation. In the model experiment, the methane conversion was about 40% with the application of mixture of organic acids and amino acids after 5 days of incubation. Furthermore, the methane conversion was about 60% for 2 days when only organic acids, such as acetic acid and formic acid, were applied. Because acetic acid is the key intermediate and main precursor of the methanogenesis step, fermentation experiments were conducted in an anaerobic-sludge reactor with high concentrations of acetic acid (0.01-0.1M). Nearly 100% of acetic acid was converted to methane and carbon dioxide in 1-3 days.
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Ácido Acético/metabolismo , Bactérias Anaeróbias/metabolismo , Metano/metabolismo , Modelos Biológicos , Esgotos/microbiologia , Gerenciamento de Resíduos/métodos , Água/química , Simulação por Computador , HidróliseRESUMO
Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to be an excellent source of cells for transplantation. In addition, the stem cell plasticity of human UCBMSCs, which can transdifferentiate into hepatocytes, has been reported. However, the mechanisms involved remain to be clarified. To identify the genes and/or signals that are important in specifying the hepatic fate of human UCBMSCs, we analyzed gene expression profiles during the hepatic differentiation of UCBMSCs with human telomerase reverse transcriptase, UCBMSCs immortalized by infection with a retrovirus carrying telomerase reverse transcriptase, but whose differentiation potential remains unchanged. Efficient differentiation was induced by 5-azacytidine (5-aza)/hepatocyte growth factor (HGF)/oncostatin M (OSM)/fibroblast growth factor 2 (FGF2) treatment in terms of function as well as protein expression: 2.5-fold increase in albumin, 4-fold increase in CCAAT enhancer-binding protein alpha, 1.5-fold increase in cytochrome p450 1A1/2, and 8-fold increase in periodic acid-Schiff staining. Consequently, we found that the expression of Wnt/beta-catenin-related genes downregulated, and the translocation of beta-catenin was observed along the cell membrane and in the cytoplasm, although some beta-catenin was still in the nucleus. Downregulation of Wnt/beta-catenin signals in the cells by Fz8-small interference RNA treatment, which was analyzed with a Tcf4 promoter-luciferase assay, resulted in similar hepatic differentiation to that observed with 5-azacytidine/HGF/OSM/FGF2. In addition, the subcellular distribution of beta-catenin was similar to that of cells treated with 5-azacytidine/HGF/OSM/FGF2. In conclusion, the suppression of Wnt/beta-catenin signaling induced the hepatic differentiation of UCBMSCs, suggesting that Wnt/beta-catenin signals play an important role in the hepatic fate specification of human UCBMSCs.
Assuntos
Diferenciação Celular/fisiologia , Sangue Fetal/fisiologia , Fígado/citologia , Fígado/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Proteínas Wnt/fisiologia , beta Catenina/fisiologia , Primers do DNA , Sangue Fetal/citologia , Humanos , Recém-Nascido , Reação em Cadeia da Polimerase , Transdução de Sinais , Ureia/análiseRESUMO
Both nerve growth factor (NGF) and pituitary adenylate cyclase activating polypeptide (PACAP) have neurotrophic effects on basal forebrain cholinergic neurons. They promote differentiation, maturation, and survival of these cholinergic neurons in vivo and in vitro. Here we report on the cooperative effects of NGF and PACAP on postnatal, but not embryonic, cholinergic neurons cultured from rat basal forebrain. Combined treatment with NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4), and PACAP induced an additive increase in choline acetyltransferase (ChAT) activity. There were no cooperative effects on the number of cholinergic neurons, suggesting that ChAT mRNA expression had been induced in each cholinergic neuron. Further analysis revealed that NGF and PACAP led to complementary induction of different ChAT mRNA species, thus enhancing total ChAT mRNA expression. These results explain the cooperative neurotrophic action of NGF and PACAP on postnatal cholinergic neurons.
Assuntos
Colina O-Acetiltransferase/metabolismo , Regulação Enzimológica da Expressão Gênica , Fator de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Neuropeptídeos/farmacologia , Prosencéfalo/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Células Cultivadas , Colina O-Acetiltransferase/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Camundongos , Fatores de Crescimento Neural/farmacologia , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Neurotrofina 3/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Prosencéfalo/citologia , RNA Mensageiro/metabolismo , RatosRESUMO
Ubiquitin (Ub) ligation is implicated in active protein metabolism and subcellular trafficking and its impairment is involved in various neurologic diseases. In rat brain, we identified two novel Ub ligases, Momo and Sakura, carrying double zinc finger motif and RING finger domain. Momo expression is enriched in the brain gray matter and testis, and Sakura expression is more widely detected in the brain white matter as well as in many peripheral organs. Both proteins associate with the cell membranes of neuronal and/or glial cells. We examined their Ub ligase activity in vivo and in vitro using viral expression vectors carrying myc-tagged Momo and Sakura. Overexpression of either Momo or Sakura in mixed cortical cultures increased total polyubiquitination levels. In vitro ubiquitination assay revealed that the combination of Momo and UbcH4 and H5c, or of Sakura and UbcH4, H5c and H6 is required for the reaction. Deletion mutagenesis suggested that the E3 Ub ligase activity of Momo and Sakura depended on their C-terminal domains containing RING finger structure, while their N-terminal domains influenced their membrane association. In agreement, Sakura associating with the membrane was specifically palmitoylated. Although the molecular targets of their Ub ligation remain to be identified, these findings imply a novel function of the palmitoylated E3 Ub ligase(s).