RESUMEN
In this study, we observed the germination behaviour of airborne conidia from powdery mildews that settle on thalloid surfaces. We inoculated thalli (flat, sheet-like leaf tissues) and gemmae (small, flat, sheet-like leaf tissues that propagate asexually via bud-like structures) of the common liverwort (Marchantia polymorpha) with conidia from tomato powdery mildew (Oidium neolycopersici; KTP-02) and red clover powdery mildew (Erysiphe trifoliorum; KRCP-4N) and examined their germination and subsequent appressorium formation under a high-fidelity digital microscope. Conidial bodies and germ tubes of the inoculated KRCP-4N conidia were destroyed on both the thalli and gemmae. The destruction of these fungal structures was observed only for KRCP-4N conidia inoculated onto M. polymorpha on both leaf surfaces. No differences in destruction of the KRCP-4N fungal structures between thalli and gemmae were observed. At 4 h post-inoculation, destruction of the germ tube tip was observed when it reached the gemmae leaf surface. At 6 h post-inoculation, the conidial bodies and germ tubes were destroyed. In contrast, KTP-02 conidia were not destroyed and formed normal, well-lobed appressoria on the surface of M. polymorpha gemmae.
Asunto(s)
Ascomicetos/patogenicidad , Marchantia/microbiología , Hojas de la Planta/microbiología , GerminaciónRESUMEN
AIMS: Repetitive subcutaneous or intracerebroventricular administration of brain-derived neurotrophic factor (BDNF) ameliorates glucose metabolism and enhances energy expenditure in obese diabetic C57BL/KsJ-db/db mice. To explore the mechanism of action through which BDNF regulates glucose metabolism, we examined the effects of BDNF on glucose utilization and norepinephrine (NE) content in peripheral tissues of diabetic mice. METHODS: [(14)C]2-deoxyglucose ([(14)C]2-DG) uptake into peripheral tissues was analysed after intravenous injection of [(14)C]2-DG in db/db and normal C57BL/6 mice, and [(14)C]2-DG uptake and NE content in peripheral tissues were analysed after subcutaneous administration of BDNF (20 mg/kg) to male db/db and normal mice for 8 days. RESULTS: [(14)C]2-DG uptake in the diaphragm, heart, gastrocnemius, soleus and interscapular brown adipose tissue (BAT) of db/db mice was significantly lower than in normal mice. Repetitive administration of BDNF to db/db mice for 8 days enhanced [(14)C]2-DG uptake in the diaphragm, heart, soleus, BAT and liver. The NE content in heart, skeletal muscle, interscapular BAT and liver of db/db mice given BDNF was high compared with db/db mice given vehicle, whereas no significant change in NE content in peripheral tissues was observed in normal mice given BDNF and those given vehicle. BDNF did not affect [(14)C]2-DG uptake or NE content in the white adipose tissue of db/db mice. CONCLUSIONS: These data indicate that BDNF ameliorates glucose metabolism by enhancement of glucose utilization in muscle and BAT, with this effect caused by modulation of the central and peripheral nervous systems.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Desoxiglucosa/farmacocinética , Diabetes Mellitus Experimental/metabolismo , Tejido Adiposo Pardo/metabolismo , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Radioisótopos de Carbono/farmacocinética , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Norepinefrina/metabolismo , Proteínas Recombinantes/farmacología , Distribución TisularRESUMEN
The formation of conidial pseudochains by the tomato powdery mildew Oidium neolycopersici on tomato leaves was monitored using a high-fidelity digital microscope. Individual living conidiophores that formed mature conidial cells at their apex were selected for observation. The conidial cells were produced during repeated division and elongation by the generative cells of the conidiophores. Under weak wind conditions (0.1 m/s), these conidial cells did not separate from each other to produce a chain of conidial cells (pseudochain). The pseudochains dropped from the conidiophores once four conidial cells were connected. The conidiophores resumed conidium production, followed by another cycle of pseudochain formation. The formation of pseudochains by tomato powdery mildew was not influenced by the ambient relative humidity. On the other hand, the conidial cells produced were easily wind dispersed without forming pseudochains when conidiophores were exposed to stronger winds (1.0 m/s). The present study successfully demonstrated that the pathogen required wind to disperse progeny conidia from the conidiophores and produced conidial pseudochains when the wind was below a critical level, independent of high relative humidity as reported previously.
RESUMEN
Two-step PCR (RT-PCR and nested PCR) was used to detect gene expression in powdery mildew pathogen-infected cells of detached inner epidermis of barley coleoptiles. Cellular contents of infected cells were microscopically suctioned with a micropipette and subjected to PCR. Triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase genes involved in the glycolytic pathway and a stimulus-induced endochitinase gene were targeted, and their expression was determined by detecting cDNAs derived from spliced transcripts. The two gycolysis-related genes were constantly expressed in the tissue irrespective of pathogen inoculation. In contrast, chitinase gene expression was induced in non-infected inner epidermis after detachment. After inoculation, this expression was selectively suppressed in pathogen-invaded cells, in spite of continuous expression in non-invaded cells of the same epidermis. Thus, the present method enabled us to directly analyze transcripts in individual cells at the infection site and assess the capability of the pathogen to regulate host gene expression.
Asunto(s)
Quitinasas/genética , Cotiledón/genética , Cotiledón/microbiología , Regulación Enzimológica de la Expresión Génica/genética , Hordeum/genética , Hordeum/microbiología , Bioensayo/métodos , Cotiledón/enzimología , ADN Complementario/análisis , ADN Complementario/genética , ADN de Plantas/análisis , ADN de Plantas/genética , Regulación de la Expresión Génica de las Plantas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Hordeum/enzimología , Intrones/genética , Enfermedades de las Plantas/genética , Epidermis de la Planta/genética , Epidermis de la Planta/metabolismo , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , ARN Mensajero/genética , ARN de Planta/análisis , ARN de Planta/genética , Triosa-Fosfato Isomerasa/genéticaRESUMEN
AIMS: To establish a rapid and efficient method for detecting Enterobacter cloacae based on chitinase gene transformation and lytic infection by virulent bacteriophages. METHODS AND RESULTS: A phylloplane strain of E. cloacae was isolated from tomato leaves and transformed with a chitinase gene. Transformed bacteria were collected from single colonies and infected with newly isolated, virulent bacteriophages in the presence of the chitinase substrate 4-methylumbelliferon (4MU)-(GlcNac)3. To assay chitinase activity in the lysates, the product 4MU was measured spectrofluorophotometrically or visibly detected under u.v. irradiation. Chitinase gene-transformed bacteria obtained from single colonies could be specifically identified in 30 min by the emission of 4MU fluorescence following lysis caused by phage infection. CONCLUSIONS: The chitinase gene was used as a reporter gene to construct a new system for easy and rapid monitoring of transgenic strains of E. cloacae released in the environment, in combination with specific recognition by virulent bacteriophages. SIGNIFICANCE AND IMPACT OF THE STUDY: The assay is simple, rapid, inexpensive, easy to perform and applicable to other strains. The system can be used for the routine monitoring of bacteria, which is important because of the increased use of transgenic strains of E. cloacae as an antagonistic biological control agent for plant diseases.
Asunto(s)
Quitinasas/genética , Enterobacter cloacae/aislamiento & purificación , Microbiología Ambiental , Transformación Bacteriana , Tipificación de Bacteriófagos , Genes Reporteros , Solanum lycopersicum , Hojas de la PlantaRESUMEN
AIM: It has been reported previously that brain-derived neurotrophic factor (BDNF) regulates blood glucose metabolism in rodent obese diabetic models such as C57BL/KsJ-leprdb/leprdb (db/db) mice. BDNF further regulates energy expenditure, possibly through the central and autonomous nervous systems. In this study, we evaluated the effect of BDNF on both lipid and glucose metabolisms to clarify its action mechanism. METHODS: To control the energy intake, we used a pellet pair-feeding apparatus to synchronize food intake precisely between BDNF-treated and vehicle-treated db/db mice. BDNF (50 mg/kg/week) was subcutaneously injected to male db/db mice twice weekly for 3 weeks, and blood glucose, serum biochemical lipid parameters and tissue weights were measured. Liver triglyceride contents were measured and liver sections were histologically analysed. RESULTS: Twice weekly BDNF treatment for 3 weeks significantly lowered blood glucose compared with pellet pair-fed, vehicle-treated db/db mice (294 +/- 109 vs. 529 +/- 91 mg/dL). Serum non-esterified free fatty acid (726 +/- 72 vs. 999 +/- 220 microEq/l), total cholesterol (125 +/- 8 vs. 151 +/- 23 mg/dL) and phospholipid levels (215 +/- 13 vs. 257 +/- 36 mg/dL) of the BDNF-treated db/db mice decreased significantly. Liver weights (1.51 +/- 0.11 vs. 2.05 +/- 0.11 g), liver triglyceride contents (17.5 +/- 1.4 vs. 26.1 +/- 2.1 mg/g) and fatty liver in histological appearance were reduced with BDNF treatment. There were no significant differences in body weights and white adipose tissue weights between the two groups. CONCLUSIONS: Taken together with the accelerating effect of BDNF on energy metabolism, these findings indicate that BDNF improves glucose and lipid metabolism in obese diabetic animals without enlarging liver or adipose tissues.
Asunto(s)
Glucemia/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diabetes Mellitus/sangre , Metabolismo de los Lípidos , Obesidad , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Animales , Glucemia/efectos de los fármacos , Colesterol/sangre , Cruzamientos Genéticos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Factores de TiempoRESUMEN
We experienced with a case of mycotic descending thoracic aortic aneurysm (MTAA) complicated by pyothorax postoperatively. The graft replacement accompanied with wrapping by omental pedicle flap (OPF) was performed in this case. We considered that causative organism for MTAA was Salmonella species, and discussed effectiveness of the application of OPF to MTAA.
Asunto(s)
Aneurisma Infectado/cirugía , Aneurisma de la Aorta Torácica/cirugía , Implantación de Prótesis Vascular , Empiema Pleural/etiología , Complicaciones Posoperatorias , Infecciones por Salmonella/etiología , Empiema Pleural/microbiología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: We recently demonstrated that chronic treatment with brain-derived neurotrophic factor (BDNF) regulates energy expenditure in obese diabetic C57BL/KsJ-db/db mice. In this study, we investigated the acute effects of BDNF on energy expenditure. DESIGN: After BDNF was singly administered to male db/db mice (aged 10-12 weeks), their body temperature and whole body glucose oxidation were measured. Their norepinephrine (NE) turnover and uncoupling protein (UCP) 1 expression in interscapular brown adipose tissue (BAT) were also analyzed. RESULTS: Even though the body temperatures of hyperphagic db/db mice dropped remarkably in a 24 h period after food deprivation, only a single subcutaneous administration of BDNF significantly prevented the reduction of body temperature. BDNF was also observed to have similar efficacy in cold exposure experiments at 15 degrees C. Respiratory excretion of (14)CO(2) after intravenous injection of D-[(14)C(U)]-glucose was significantly increased by BDNF administration, indicating that BDNF increases whole-body glucose oxidation. BDNF administered intracerebroventricularly was also able to prevent the reduction of body temperature of db/db mice. To clarify the BDNF action mechanism we examined NE turnover in BAT. Four hours after a single administration, BDNF reduced NE content in the presence of the tyrosine hydroxylase inhibitor, alpha-methyl-P-tyrosine methyl ester, indicating enhanced NE turnover in BAT. BDNF also increased the expression of the UCP1 mRNA and protein in BAT. CONCLUSION: These data indicate that BDNF rapidly regulates energy metabolism in obese diabetic animals, partly through activating the sympathetic nervous system and inducing UCP1 gene expression in BAT.
Asunto(s)
Glucemia/metabolismo , Temperatura Corporal/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/administración & dosificación , Diabetes Mellitus/metabolismo , Metabolismo Energético/efectos de los fármacos , Norepinefrina/metabolismo , Obesidad , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Animales , Glucemia/efectos de los fármacos , Northern Blotting , Western Blotting , Factor Neurotrófico Derivado del Encéfalo/farmacología , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Regulación de la Expresión Génica/efectos de los fármacos , Inyecciones Intraventriculares , Inyecciones Subcutáneas , Canales Iónicos , Cinética , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Proteínas Mitocondriales , ARN Mensajero/análisis , Termogénesis/efectos de los fármacos , Proteína Desacopladora 1RESUMEN
It has been previously demonstrated that brain-derived neurotrophic factor (BDNF) regulates glucose metabolism and energy expenditure in rodent diabetic models such as C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice. Central administration of BDNF has been found to reduce blood glucose in db/db mice, suggesting that BDNF acts through the central nervous system. In the present study we have expanded these investigations to explore the effect of central administration of BDNF on energy metabolism. Intracerebroventricular administration of BDNF lowered blood glucose and increased pancreatic insulin content of db/db mice compared with vehicle-treated pellet pair-fed db/db mice. While body temperatures of the pellet pair-fed db/db mice given vehicle were reduced because of restricted food supply in this pair-feeding condition, BDNF treatment remarkably alleviated the reduction of body temperature suggesting the enhancement of thermogenesis. BDNF enhanced norepinephrine turnover and increased uncoupling protein-1 mRNA expression in the interscapular brown adipose tissue. Our evidence indicates that BDNF activates the sympathetic nervous system via the central nervous system and regulates energy expenditure in obese diabetic animals.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Diabetes Mellitus Tipo 2/fisiopatología , Diabetes Mellitus/fisiopatología , Metabolismo Energético/efectos de los fármacos , Obesidad , Animales , Regulación de la Temperatura Corporal , Factor Neurotrófico Derivado del Encéfalo/administración & dosificación , Ventrículos Cerebrales/efectos de los fármacos , Ventrículos Cerebrales/fisiología , Ventrículos Cerebrales/fisiopatología , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Inyecciones Intraventriculares , Insulina/sangre , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Valores de Referencia , Temperatura Cutánea/fisiología , Factores de TiempoRESUMEN
We previously reported that brain-derived neurotrophic factor (BDNF) regulates both food intake and blood glucose metabolism in rodent obese diabetic models such as C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice. To elucidate the effect of BDNF on glucose metabolism, we designed a novel pellet pair-feeding apparatus to eliminate the effect of appetite alteration on glucose metabolism. The apparatus was used to synchronize food intake precisely between BDNF-treated and vehicle-treated db/db mice. It was shown using this pellet pair-feeding apparatus that BDNF administered daily (20 mg x kg(-1) x day(-1)) to db/db mice significantly lowered blood glucose compared with pellet pair-fed db/db mice. To evaluate the effect of BDNF on insulin action, we used streptozotocin-induced type 1 diabetic mice. In this case, BDNF did not lower blood glucose concentration but rather enhanced the hypoglycemic action of insulin. In hyperglycemic db/db mice, pancreatic insulin content was reduced and glucagon content was increased compared with normoglycemic db/m mice. BDNF administered to db/db mice significantly restored both pancreatic insulin and glucagon content. Histological observations of aldehyde-fuchsin staining and immunostaining with anti-insulin indicated that insulin-positive pancreatic beta-cells were extensively regranulated by BDNF administration. We also studied the effect of BDNF on KK mice, normoglycemic animals with impaired glucose tolerance. In these mice, BDNF administration improved insulin resistance in the oral glucose tolerance test. To elucidate how blood glucose was metabolized in BDNF-treated animals, we investigated the effect of BDNF on the energy metabolism of db/db mice. Body temperature and oxygen consumption of the pellet pair-fed vehicle-treated mice were remarkably lower than the ad libitum-fed vehicle-treated mice. Daily BDNF administration for 3 weeks completely ameliorated both of the reductions. Finally, to clarify its action mechanism, the effect of intracerebroventricular administration of BDNF on db/db mice was examined. Here, a small dose of BDNF was found to be effective in lowering blood glucose concentration. This indicates that BDNF regulates glucose metabolism by acting directly on the brain.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Diabetes Mellitus/fisiopatología , Metabolismo Energético/efectos de los fármacos , Glucosa/metabolismo , Animales , Glucemia/análisis , Temperatura Corporal/efectos de los fármacos , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patología , Diabetes Mellitus Experimental/sangre , Ingestión de Alimentos/efectos de los fármacos , Privación de Alimentos/fisiología , Inyecciones Intraventriculares , Islotes Pancreáticos/patología , Ratones , Ratones Endogámicos C57BL/genética , Ratones Mutantes , Páncreas/efectos de los fármacos , Páncreas/patologíaRESUMEN
Musk melon is the favorite fruit with a high market value in Japan, and the fragrance is one of the major factors determining the fruit quality of melon. In this study, mutant melon hairy roots which had been induced by means of the T-DNA insertion mutagenesis were found to produce volatile compounds with the fruity fragrance of mature melon. The volatile compounds were extracted and identified by GLC-mass spectrometry. Some essential oils such as (Z)-3-hexenol, (E)-2-hexenal, 1-nonanol, and (Z)-6-nonenol were stably synthesized by these hairy roots despite the increased number of subcultures. The productivity of these compounds by the best hairy root line was shown to be considerably higher than naturally ripened melon fruits.
Asunto(s)
Cucurbitaceae/fisiología , Odorantes/análisis , Aceites Volátiles/análisis , Raíces de Plantas/fisiología , Cucurbitaceae/genética , ADN Bacteriano , ADN de Cadena Simple , Cromatografía de Gases y Espectrometría de Masas , Mutagénesis Insercional , Aceites Volátiles/metabolismoRESUMEN
We have previously shown that brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, interacts with the endocrine system in obese diabetic mice, and systemic peripheral administration of BDNF regulates glucose metabolism in this model. Results from the present study show that the hypoglycemic effect induced by 2 weeks' daily administration of BDNF (20 mg/kg/d) to db/db mice lasts for several weeks after treatment cessation, irrespective of food reduction. On the other hand, the antidiabetic agent, metformin had no lasting effect. This duration of the BDNF hypoglycemic action prompted us to examine the efficacy of BDNF intermittent administration on glucose metabolism. BDNF administered once or twice per week (70 mg/kg/wk) to db/db mice for 3 weeks significantly reduced blood glucose concentrations and hemoglobin A(1c), (HbA(1c)) as compared with ad libitum-fed phosphate-buffered saline (PBS)-treated and pair-fed PBS-treated groups. This suggests that BDNF not only temporarily reduced blood glucose concentrations but also ameliorated systemic glucose balance in this obese diabetic mouse model during the experimental period. Our results indicate that BDNF could be a novel hypoglycemic agent with an exceptional ability to normalize glucose metabolism even with treatment as infrequently as once per week.
Asunto(s)
Glucemia/metabolismo , Factor Neurotrófico Derivado del Encéfalo/farmacología , Hipoglucemiantes/farmacología , Animales , Peso Corporal/efectos de los fármacos , Diabetes Mellitus/sangre , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Ingestión de Alimentos/efectos de los fármacos , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Ratones , Ratones Obesos , Obesidad , Factores de TiempoRESUMEN
Bone, one of the favored sites for tumor metastasis, is a dynamic organ undergoing formation and resorption. We found bone metastasis with osteolytic lesion in the bone marrow of the femur by injecting BW5147 T-lymphoma cells into the tail vein of AKR mice. To understand this bone destruction, we constructed a cDNA library from BW5147 with a cloning vector that allowed in vitro synthesis of mRNAs, and then identified a particular cDNA clone by adding the conditioned medium from Xenopus oocytes following injection of the mRNA synthesized in vitro to primary bone marrow heterogeneous cell populations on hydroxyapatite thin films. By means of this method, we isolated a factor with 16% leucine residues, termed neurochondrin, that induces hydroxyapatite resorptive activity in bone marrow cells resistant to bafilomycin A1, an inhibitor of macrophage- and osteoclast-mediated resorption. Expression of the gene was localized to chondrocyte, osteoblast, and osteocyte in the bone and to the hippocampus and Purkinje cell layer of cerebellum in the brain. This may provide insights into the molecular mechanisms underlying bone resorption with potential implications for the activation of cells other than macrophages and osteoclasts in bone marrow cells.
Asunto(s)
Antibacterianos/farmacología , Células de la Médula Ósea/metabolismo , Durapatita , Macrólidos , Proteínas del Tejido Nervioso/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células de la Médula Ósea/química , Células de la Médula Ósea/efectos de los fármacos , Resorción Ósea/fisiopatología , Huesos/metabolismo , Encéfalo/metabolismo , ADN Complementario/química , Resistencia a Medicamentos , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Oocitos , Transfección , Xenopus laevisRESUMEN
45Ca2+ influx in rat glioma C6 cells induced by 0.3 nM maitotoxin (MTX) was markedly inhibited by brevetoxin A (PbTx1) and brevetoxin B (PbTx2), with EC50 values of 16 and 13 microM, respectively. This inhibition was observed immediately after addition of MTX when monitored with fura-2, which suggests that PbTx2 directly blocks the action of MTX. This blockade by PbTx2 was not affected by tetrodotoxin, which excludes the involvement of voltage-sensitive sodium channels. The depolarizing effects of these brevetoxins were also not a likely cause of this inhibition, because melittin, a channel-forming peptide, did not significantly block MTX-induced 45Ca2+ influx. Instead, this inhibition was thought to be mediated by blockade of an MTX-binding site by the brevetoxins, based on the fact that these toxins, particularly PbTx2, closely mimic the partial structure of MTX. Synthetic fragments of MTX corresponding to the hydrophilic EF-GH rings (200 microM) and LM-NO rings (500 microM) of MTX significantly reduced MTX-elicited Ca2+ influx. The observation that the effects of MTX were inhibited by structural mimics of both its hydrophobic and hydrophilic portions implies that both portions of the MTX molecule recognize its target.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Glioma/metabolismo , Toxinas Marinas/farmacología , Oxocinas , Fragmentos de Péptidos/farmacología , Animales , Electrofisiología , Colorantes Fluorescentes , Fura-2 , Glioma/patología , Glioma/fisiopatología , Toxinas Marinas/química , Conformación Molecular , Fragmentos de Péptidos/síntesis química , Ratas , Células Tumorales Cultivadas/metabolismoRESUMEN
Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family. However, it is not yet known if BDNF works on the endocrine system itself. Here we report that BDNF improves hyperglycemia in obese diabetic animals. BDNF reduced the blood glucose level in obese db/db diabetic mice in which the effect of BDNF was age-dependent and high under the condition of hyperinsulinemia, while BDNF showed no effect on non-diabetic db/m mice. These results suggest that BDNF ameliorates insulin resistance by enhancing insulin action in peripheral tissues. Furthermore, BDNF was found to reduce the plasma insulin level in db/db mice. Among the neurotrophin family, NT-3 also reduced the blood glucose level in db/db mice. These results provide a novel insight that neurotrophin functions on the endocrine system as well as the nervous system.
Asunto(s)
Glucemia/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diabetes Mellitus Experimental/sangre , Animales , Glucemia/análisis , Femenino , Ratones , Ratones ObesosRESUMEN
We investigated the signaling pathways exerted by brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in relation to their survival-promoting effects on dissociated cultures of cerebellar granule cells prepared from postnatal 9-day-old rats. Granule neuron survival in culture was supported by BDNF, but not significantly by either nerve growth factor (NGF) or NT-3. BDNF and NT-3 resulted in not only the respective autophosphorylation of the Trk receptors, TrkB or TrkC, but also tyrosine phosphorylation of SHC, a protein involved in controlling p21ras activity, and phosphatidylinositol-3' (PI-3') kinase. NGF does not result in TrkA phosphorylation. In parallel, c-fos was induced within 30 min, in response to BDNF and NT-3. NT-3 induced the phosphorylation of these proteins to a lesser extent than BDNF. BDNF also induced the tyrosine phosphorylation of phospholipase C gamma (PLC gamma), but the NT-3-induced one was not detected. We postulate that no survival promotion by NT-3 is due to lesser level of trkC expression and of the NT-3-induced signaling in the cultured cerebellar granule neurons. Wortmannin, a specific inhibitor of PI-3' inhibited the BDNF effect on neuronal survival. PI-3' kinase-dependent pathways might be involved in the promotion of cerebellar granule cell survival by BDNF.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Factor Neurotrófico Derivado del Encéfalo/farmacología , Cerebelo/citología , Factores de Crecimiento Nervioso/farmacología , Transducción de Señal/fisiología , Androstadienos/farmacología , Animales , Northern Blotting , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/enzimología , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/fisiología , Isoenzimas/metabolismo , Neurotrofina 3 , Fosfatidilinositol 3-Quinasas , Fosfolipasa C gamma , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , ARN Mensajero/análisis , Ratas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismo , WortmaninaRESUMEN
Basal forebrain cholinergic neurons respond in vitro and in vivo to nerve growth factor (NGF) and to brain-derived neurotrophic factor (BDNF). It is not clear to what extent the neurons that respond to these two factors, or to neurotrophin-3 or -4/5 (NT-3; NT-4/5) are identical or only partially overlapping populations. We have addressed this issue in cultures of basal forebrain neurons derived from 2-week-old postnatal rats, using choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) as cholinergic markers. Cholinergic neuron survival was enhanced in the presence of NGF, BDNF and NT-4/5. NT-4/5 was as effective as BDNF. NT-3 was without effect at this age, although in cultures derived from embryonic forebrain, cholinergic differentiation was induced by NT-3. Cotreatment with NGF and BDNF resulted in small, but consistent increases in the number of ChAT-positive neurons, compared with either factor alone. NT-4/5 was also found to be additive with NGF, whereas cotreatment with BDNF and NT-4/5 showed no additivity. NT-3 had no additive effects with any other neurotrophin on any cholinergic parameters in postnatal cultures. Taken together, the results indicate the existence in postnatal rat brain of a large overlapping population of cholinergic neurons that are responsive to ligands for the neurotrophin receptors TrkA (NGF) and TrkB (BDNF and NT-4/5), but not TrkC (NT-3), and small distinct populations that show specificity for NGF or BDNF but not both. We hypothesize that cholinergic neurons projecting into different regions of the hippocampus may derive trophic support from distinct neurotrophins.
Asunto(s)
Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Sistema Nervioso Parasimpático/citología , Prosencéfalo/citología , Acetilcolinesterasa/metabolismo , Animales , Animales Recién Nacidos , División Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colina O-Acetiltransferasa/metabolismo , Inmunohistoquímica , Neuronas/enzimología , Sistema Nervioso Parasimpático/efectos de los fármacos , Sistema Nervioso Parasimpático/enzimología , Fenotipo , Prosencéfalo/efectos de los fármacos , Prosencéfalo/enzimología , RatasRESUMEN
Cerebellar granule neurons obtained from 9-day-old rats were grown in vitro for 4 days in high K+ (26 mM) medium. The culture medium was then replaced with that containing low K+ (5 mM) which caused a large number of granule neurons to die. The death of granule neurons has been characterized as apoptosis. In this study, we investigated the effects of various neurotrophins on neuronal survival using the above system. We found that brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) but not nerve growth factor (NGF) can protect these neurons from apoptosis in low K+. Neurotrophin-3 (NT-3) had a small effect on neuronal survival as reported. To determine whether the granule neurons respond directly to BDNF, we analyzed the induction of the Fos protein in these neurons. Individual cells that synthesize Fos protein after exposure to neurotrophin can be recognized using antibodies to Fos. Immunocytochemical staining of the cultures demonstrated that a relatively large number of cerebellar granule neurons showed immunoreactivity in response to BDNF, but few of them were immunoreactive in the absence of BDNF or in the presence of NGF. Our results suggested that BDNF has a direct effect on mature cerebellar granule neurons and can protect these neurons from apoptosis in low K+.
Asunto(s)
Apoptosis/efectos de los fármacos , Cerebelo/efectos de los fármacos , Proteínas del Tejido Nervioso/farmacología , Animales , Anticuerpos , Factor Neurotrófico Derivado del Encéfalo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/metabolismo , Cicloheximida/farmacología , Dactinomicina/farmacología , Inmunohistoquímica , Neuronas/efectos de los fármacos , Potasio/farmacología , Proteínas Proto-Oncogénicas c-fos , RatasRESUMEN
We found depolarization-dependent promotion of survival of cultured basal forebrain cholinergic neurons from postnatal 2-week-old rats. Over 30 mM KCl (high K+) as well as nerve growth factor (NGF) induced considerably high choline acetyltransferase (ChAT) activity and the increase was potentiated by the addition of BAY K8644, a Ca2+ channel agonist. The increase in ChAT activity by high K+ was due to the increased number of viable acetylcholinesterase-positive and ChAT-positive neurons. Also, a cyclic AMP analog gave the same effect as high K+, but its ability to induce the ChAT activity was higher than that of high K+. On the other hand, both high K+ and NGF had very little effects on the survival of the cultured cholinergic neurons from 10-week-old rats. Cyclic AMP analog induced considerable increase in ChAT activity and promotion of survival of cholinergic neurons in the 10-week-old culture. These findings showed that the neuronal death occurring just of the end of synapse formation in rat basal forebrain cholinergic neurons could be prevented by NGF and high K+, but the death of older cholinergic neurons could not. We propose the possibility that the death of postnatal 2-week-old basal forebrain cholinergic neurons in culture might be programmed cell death.
Asunto(s)
Acetilcolina/fisiología , AMP Cíclico/análogos & derivados , Neuronas/efectos de los fármacos , Potasio/farmacología , Prosencéfalo/efectos de los fármacos , Tionucleótidos/farmacología , Envejecimiento/patología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colina O-Acetiltransferasa/efectos de los fármacos , Medios de Cultivo , AMP Cíclico/farmacología , Femenino , Masculino , Factores de Crecimiento Nervioso/farmacología , Prosencéfalo/citología , Ratas , Ratas WistarRESUMEN
We have established a primary culture technique for neuronal cells from rat basal forebrain from postnatal day 58 (P58) to study the effects of neurotrophic factors on the neurons. The survival of acetylcholinesterase (AChE)-positive neurons of 2-week-old rat septum has already been reported to be strongly supported by nerve growth factor (NGF) in culture. In this culture study of neurons from adult rat brains, the survival of AChE-positive neurons from P58 rat septum was slightly improved by NGF, although low affinity NGF receptor expression was also observed on cultured P58 rat septum neurons as well as on those from 2-week-old rats. The addition of basic fibroblast growth factor (bFGF) improved the survival of AChE-positive neurons cultured from P58 rat septum, but did not promote the survival of neurons from P12 rat septum. These results suggest that NGF changes to a maintenance factor in adult rat brain from a survival factor in postnatal 2-week-old rats. The survival of cholinergic neurons in culture of adult rat septum might be supported by factor(s) other than NGF, such as bFGF.