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Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 2/sangre , Cetoacidosis Diabética/sangre , Coma Hiperglucémico Hiperosmolar no Cetósico/sangre , Polipéptido alfa Relacionado con Calcitonina/sangre , Adulto , Anciano , Análisis de los Gases de la Sangre , Glucemia/metabolismo , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Cetoacidosis Diabética/diagnóstico , Cetoacidosis Diabética/etiología , Femenino , Humanos , Coma Hiperglucémico Hiperosmolar no Cetósico/diagnóstico , Coma Hiperglucémico Hiperosmolar no Cetósico/etiología , Masculino , Persona de Mediana EdadAsunto(s)
Aldosterona/sangre , Diabetes Mellitus Tipo 2/sangre , Hiperaldosteronismo/diagnóstico , Adulto , Anciano , Artefactos , Análisis Químico de la Sangre/métodos , Análisis Químico de la Sangre/normas , Presión Sanguínea/fisiología , Estudios de Casos y Controles , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/fisiopatología , Técnicas de Diagnóstico Endocrino/normas , Femenino , Humanos , Hiperaldosteronismo/sangre , Hiperaldosteronismo/complicaciones , Masculino , Persona de Mediana Edad , Sistema Renina-Angiotensina/fisiologíaRESUMEN
We report the synthesis of a 5-formyl-2'-deoxyuridine (5fU) phosphoramidite and the preparation of oligonucleotides comprising all known, naturally observed eukaryotic thymidine modifications. Biophysical characterization of the synthetic oligonucleotides indicates that 5fU, but not the other T-derivatives, can alter DNA structures.
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ADN/análisis , ADN/química , Desoxiuridina/análogos & derivados , Oligonucleótidos/análisis , Oligonucleótidos/síntesis química , Compuestos Organofosforados/síntesis química , Timina/química , ADN/síntesis química , Desoxiuridina/síntesis química , Desoxiuridina/química , Oligonucleótidos/química , Compuestos Organofosforados/químicaAsunto(s)
Bilirrubina/sangre , Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/epidemiología , Retinopatía Diabética/epidemiología , Regulación hacia Abajo , Anciano , Antagonistas de Receptores de Angiotensina/efectos adversos , Antagonistas de Receptores de Angiotensina/uso terapéutico , Bilirrubina/antagonistas & inhibidores , Biomarcadores/sangre , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Nefropatías Diabéticas/inducido químicamente , Nefropatías Diabéticas/fisiopatología , Retinopatía Diabética/inducido químicamente , Retinopatía Diabética/fisiopatología , Regulación hacia Abajo/efectos de los fármacos , Femenino , Hospitales de Enseñanza , Humanos , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/uso terapéutico , Japón , Modelos Logísticos , Masculino , Microvasos/efectos de los fármacos , Microvasos/fisiopatología , Persona de Mediana Edad , Factores de Riesgo , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: Cumulative interceptive supportive therapy (CIST) is currently used as a guideline for treating peri-implant diseases. The objectives of this study were to determine the detection rate and measure the number of periodontopathic bacteria in lesions of different CIST levels and thereby characterize peri-implant disease from a bacteriological viewpoint. METHODS: This study included 105 patients who had both residual natural teeth and implants with peri-implant disease. A total of 105 implants were divided into levels A, B, C and D according to the CIST classification. Bacterial samples were collected from peri-implant pockets and four periodontopathic bacteria were measured by PCR and PCR-Invader assay. RESULTS: The number of periodontopathic bacteria increased in line with CIST level, and the detection rate was also associated with CIST level. However, no difference was found in the bacterial detection rate of P. gingivalis and T. denticola between CIST-B and CIST-C. There was a higher detection rate of all periodontopathic bacteria for CIST-D. CONCLUSIONS: The number of periodontopathic bacteria and detection rate increased as peri-implant disease advanced. However, there were no major differences in the detection rate between CIST-B and CIST-C. On the other hand, a higher detection rate of periodontopathic bacteria was seen for CIST-D.
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Implantes Dentales/microbiología , Bacterias Gramnegativas/clasificación , Periimplantitis/microbiología , Estomatitis/microbiología , Aggregatibacter actinomycetemcomitans/aislamiento & purificación , Carga Bacteriana , Bacteroides/aislamiento & purificación , Estudios Transversales , Placa Dental/microbiología , Femenino , Hemorragia Gingival/microbiología , Humanos , Masculino , Persona de Mediana Edad , Periimplantitis/clasificación , Bolsa Periodontal/clasificación , Bolsa Periodontal/microbiología , Reacción en Cadena de la Polimerasa , Porphyromonas gingivalis/aislamiento & purificación , Estomatitis/clasificación , Diente/microbiología , Treponema denticola/aislamiento & purificaciónRESUMEN
The fractal and the small-world properties of complex networks are systematically studied both in the box-covering (BC) and the cluster-growing (CG) measurements. We elucidate that complex networks possessing the fractal (small-world) nature in the BC measurement are always fractal (small world) even in the CG measurement and vice versa, while the fractal dimensions d{B} by the BC measurement and d{C} by the CG measurement are generally different. This implies that two structural properties of networks, fractality and small worldness, cannot coexist in the same length scale. These properties can, however, crossover from one to the other by varying the length scale. We show that the crossover behavior in a network near the percolation transition appears both in the BC and CG measurements and is scaled by a unique characteristic length ξ.
RESUMEN
Neuropathy is one of the typical features of chronic complications of diabetes mellitus. Recent analyses indicate that subjects with impaired glucose tolerance (IGT) already have disturbance of peripheral nerve function. To test the role of adipocytokines, that tend to be abnormal in IGT subjects, on diabetic neuropathy, we analyzed the relationship between plasma adipocytokine levels (TNFalpha, adiponectin, and leptin) and nerve conduction velocity in 105 type 2 diabetic subjects (M/F = 66/39, age = 60.8 +/- 11.8 years, BMI = 24.7 +/- 5.0kg/m2). Adipocytokines were measured by ELISA, and motor conduction velocity (MCV) and sensory conduction velocity (SCV) in median, ulnar, and tibial nerve were measured by electrical stimulation. Motor conduction velocity and SCV were corrected by age to be 1.0 as the normal value, and the average of three nerves were used to be the representative value. Relationship between corrected MCV or corrected SCV as a dependent variable and the duration of diabetes, HbA1C, BMI, TNFalpha, adiponectin, and leptin concentrations as independent variables were analyzed by multiple regression. Duration of diabetes and HbA1C were highly related with both corrected MCV (P < 0.02 and P < 0.001) and SCV (P < 0.02 and P < 0.05) by this analysis. Only corrected SCV was related significantly with TNFalpha (P < 0.05), and close to significantly with leptin (P = 0.059) concentrations. These results indicate that increased plasma glucose levels and duration of diabetes are the major factors that modulate diabetic neuropathy. However, nerve function may be affected by plasma cytokine levels like TNFalpha, and this effect was more significant on sensory nerves than motor nerves. The present results suggest that adipocytokines may play a role not only on angiopathy but also on neuropathy in diabetics.
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Adipocitos/inmunología , Citocinas/sangre , Diabetes Mellitus Tipo 2/fisiopatología , Neuropatías Diabéticas/fisiopatología , Péptidos y Proteínas de Señalización Intercelular/sangre , Adiponectina , Adulto , Edad de Inicio , Anciano , Anciano de 80 o más Años , Índice de Masa Corporal , Diabetes Mellitus Tipo 2/inmunología , Neuropatías Diabéticas/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Nervios Periféricos/fisiopatologíaRESUMEN
AIMS: Adipocytokines, products from adipose tissue, have biological activities on the vascular system, and may affect diabetic angiopathy. In this study, we assessed the relationship between adiposity and plasma adipocytokine levels, and investigated the clinical significance of adiposity and plasma adipocytokine levels on diabetic micro- and macroangiopathy in Type 2 diabetic subjects. METHODS: We studied 231 Japanese Type 2 diabetic subjects (135 men and 96 women, aged 60.4 +/- 12.3 years, body mass index 24.8 +/- 5.2 kg/m2). We measured adipocytokine [adiponectin, leptin, resistin, and tumour necrosis factor (TNF)-alpha] levels, lipid profiles and urine albumin excretion. We also performed optic fundus examination and measured carotid intramedia thickness (IMT) using B-mode ultrasonography, and the localization of fat with abdominal computed tomography. A group of 93 subjects with microalbuminuria or overt proteinuria was compared with the other 148 to assess the effect on nephropathy. A group of 191 eyes with simple retinopathy or more advanced changes was compared with 263 eyes without retinal changes to assess the effect on retinopathy. RESULTS: Plasma adiponectin level was negatively correlated with both visceral (r = -0.37, P < 0.01) and subcutaneous (r = -0.25, P < 0.01) fat areas. Resistin concentration was positively related with visceral fat area (r = 0.15, P < 0.05). Adiponectin concentration was positively correlated with age (r = 0.26, P < 0.01). TNF-alpha was correlated with IMT (r = 0.16, P < 0.05) after correction for age. Logistic regression analysis indicates a 4085 times greater chance of having retinopathy with a one unit increase in TNF-alpha (pmol/l) and a 30.64 times greater chance of having nephropathy with one unit increase in leptin (nmol/l). CONCLUSIONS: The present observations suggest that visceral and subcutaneous fat has an impact on microangiopathy as well as macroangiopathy, possibly through different adipocytokines.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/patología , Angiopatías Diabéticas/patología , Adiponectina , Índice de Masa Corporal , Diabetes Mellitus Tipo 2/metabolismo , Angiopatías Diabéticas/metabolismo , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lípidos/sangre , Masculino , Persona de Mediana EdadRESUMEN
In order to investigate the role of interleukin-6 (IL-6) and interleukin-6 soluble receptor (sR) in human ovulation, we evaluated the concentrations in human follicular fluid and analyzed the correlation of IL-6 and IL-6 sR with oocyte maturation. The oocytes were obtained from the follicular fluid of 45 women undergoing in vitro fertilization and embryo transfer. The concentrations of IL-6 and IL-6 sR in follicular fluid were measured by ELISA. In addition, granulosa cells obtained from the follicular fluid were cultured and treated with forskolin and 12- o-tetradecanoylphorbol 13-acetate for 24-48 h. The concentration of IL-6 was significantly higher in the follicular fluid than in the serum (P<0.01). In contrast, the concentration of IL-6 sR was significantly lower in the follicular fluid than in the serum (P<0.001). The concentrations of IL-6 and IL-6 sR were significantly higher in the follicular fluid containing mature oocytes than in fluid containing immature oocytes (P<0.05). The production of IL-6 was markedly increased over the basal level after 24 h of treatment with forskolin (P<0.001) and 48 h of treatment (P<0.01) with cultured granulosa cells. Our data suggest that IL-6 and IL-6 sR may play an important role in follicular growth and development in human preovulatory processes. It is possible that IL-6 in particular may be regulated by cAMP. IL-6 and IL-6 sR might also be valuable biochemical markers in the evaluation of oocyte maturation.
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Líquido Folicular/metabolismo , Interleucina-6/fisiología , Receptores de Interleucina-6/fisiología , Células Cultivadas , Colforsina/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Fertilización In Vitro , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Humanos , Interleucina-6/sangre , Interleucina-6/metabolismo , Receptores de Interleucina-6/sangre , Receptores de Interleucina-6/metabolismoRESUMEN
Our previous genetic analysis of synaptic mechanisms in Drosophila identified a temperature-sensitive paralytic mutant of the voltage-gated calcium channel alpha1 subunit gene, cacophony (cac). Electrophysiological studies in this mutant, designated cac(TS2), indicated cac encodes a primary calcium channel alpha1 subunit functioning in neurotransmitter release. To further examine the functions and interactions of cac-encoded calcium channels, a genetic screen was performed to isolate new mutations that modify the cac(TS2) paralytic phenotype. The screen recovered 10 mutations that enhance or suppress cac(TS2), including second-site mutations in cac (intragenic modifiers) as well as mutations mapping to other genes (extragenic modifiers). Here we report molecular characterization of three intragenic modifiers and examine the consequences of these mutations for temperature-sensitive behavior, synaptic function, and processing of cac pre-mRNAs. These mutations may further define the structural basis of calcium channel alpha1 subunit function in neurotransmitter release.
Asunto(s)
Canales de Calcio/genética , Proteínas de Drosophila/genética , Drosophila/genética , Mutación , Temperatura , Animales , Secuencia de Bases , Canales de Calcio/metabolismo , Cruzamientos Genéticos , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Parálisis/genética , Parálisis/metabolismo , Edición de ARN/genética , Edición de ARN/fisiología , Precursores del ARN/metabolismoRESUMEN
PROBLEM: In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and macrophage chemoattractant protein-1 (MCP-1) in human ovulation, we measured the concentrations of M-CSF and MCP-1 in human follicular fluids (FFs) and correlated them with oocyte maturation. METHOD OF STUDY: The oocytes were obtained from the FFs of 46 women undergoing in vitro fertilization and embryo transfer (IVF ET). The concentrations of M-CSF and MCP-1 in the FFs were measured by enzyme-linked immunosorbent assay. In addition, granulosa cells obtained from the FFs of IVF patients were cultured and treated with forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 48 hr. RESULTS: Concentrations of M-CSF and MCP-1 were significantly higher in the FFs than in the serum (P < 0.01). M-CSF concentrations tended to be higher, while MCP-1 concentrations were significantly higher in the FFs containing mature oocytes than in FFs containing immature oocytes (P < 0.05). The production of M-CSF was markedly increased over the basal level after treatment with forskolin (10 microM) for 24 (P < 0.02) and 48 hr (P < 0.01); however, the production of MCP-1 was unchanged. CONCLUSIONS: Our data suggest that M-CSF and MCP-1 may play an important role in human preovulatory processes and that M-CSF, in particular, may be regulated by cyclic adenosine monophosphate. M-CSF and MCP-1 may also be valuable biochemical markers in the evaluation of oocyte maturation.
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Quimiocina CCL2/fisiología , Líquido Folicular/metabolismo , Factor Estimulante de Colonias de Macrófagos/fisiología , Adulto , Quimiocina CCL2/sangre , Femenino , Células de la Granulosa/metabolismo , Humanos , Factor Estimulante de Colonias de Macrófagos/sangre , Oocitos/fisiologíaRESUMEN
The GTPase dynamin is involved in endocytosis in many cell types, as first revealed by temperature-sensitive paralytic mutations in the Drosophila dynamin gene, shibire (shi), which disrupt synaptic vesicle endocytosis and deplete synaptic terminals of vesicles. Here we report that shi synapses exhibit a fast synaptic fatigue phenotype within 20 ms of repetitive stimulation, which cannot be explained by vesicle depletion, as we confirmed by electron microscopy. These results suggest that, in addition to its well-characterized role in synaptic vesicle recycling, dynamin may be required for short-term maintenance of the readily releasable pool of synaptic vesicles.
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Membrana Celular/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/metabolismo , GTP Fosfohidrolasas/metabolismo , Fatiga Muscular/fisiología , Vesículas Sinápticas/metabolismo , Animales , Axones/metabolismo , Axones/ultraestructura , Dinaminas , GTP Fosfohidrolasas/genética , Microscopía Electrónica , Neuronas Motoras/metabolismo , Neuronas Motoras/ultraestructura , Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Vesículas Sinápticas/ultraestructura , Factores de TiempoRESUMEN
Differentiated thyroid carcinoma (DTC) is one of the least aggressive cancers of the human malignancies, however, recurrent disease is occasionally found and is difficult to determine the risk for recurrence only by clinicopathological features. In the present study, we investigated argyrophilic nucleolar organizer regions (AgNORs) score, a well-known indicator for proliferative potential of the cancer cells, in 89 cases of DTC. In tumors with capsular invasion, or with extended contra-lateral lymph node metastasis, AgNORs score was significantly higher than in tumors without them, while was not correlated with age or gender of the patients, nor the histological type of the tumor (papillary or follicular). Disease recurrence was observed in one-third of the patients in high AgNOR score group (scored more than mean value +/- standard deviation) when the patients were divided into four groups according to the AgNOR score, and a significantly higher risk for disease recurrence was demonstrated in those cases with high AgNOR score than cases with lower score. These results clearly indicated the usefulness of AgNOR score in selecting the patients at high risk for disease recurrence. All 5 recurrent cases displayed local recurrence in high AgNOR score group, while 5 of 7 cases with low AgNOR score did in the manner of hematological metastasis. In conclusion, this method was technically simple and accurate giving an exact value in individual cases. Thus, we believe AgNOR score might be clinically applicable as a useful indicator for disease recurrence in DTC.
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Adenocarcinoma Folicular/patología , Carcinoma Papilar/patología , Región Organizadora del Nucléolo/patología , Neoplasias de la Tiroides/patología , Adenocarcinoma Folicular/mortalidad , Adenocarcinoma Folicular/cirugía , Adulto , Carcinoma Papilar/mortalidad , Carcinoma Papilar/cirugía , Supervivencia sin Enfermedad , Femenino , Humanos , Escisión del Ganglio Linfático , Metástasis Linfática , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Pronóstico , Recurrencia , Factores de Riesgo , Neoplasias de la Tiroides/mortalidad , Neoplasias de la Tiroides/cirugía , Factores de TiempoRESUMEN
Neurotransmission at chemical synapses involves regulated exocytosis of neurotransmitter from the presynaptic terminal. Neurotransmitter release is thought to be triggered by calcium influx through specific classes of voltage-gated calcium channels. Here we report genetic and functional analysis implicating a specific calcium channel gene product in neurotransmitter release. We have isolated a temperature-sensitive paralytic allele of the Drosophila calcium channel alpha1 subunit gene, cacophony (cac). This mutant, referred to as cac(TS2), allows functional analysis of synaptic transmission after acute perturbation of a specific alpha1 subunit. Electrophysiological analysis at neuromuscular synapses revealed that neurotransmitter release in cac(TS2) is markedly reduced at elevated temperatures, indicating that cac encodes a primary alpha1 subunit functioning in synaptic transmission. These observations further define the molecular basis of voltage-gated calcium entry at synapses and provide a new starting point for further genetic analysis of synaptic mechanisms.
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Canales de Calcio/genética , Drosophila melanogaster/fisiología , Mutación , Sinapsis/fisiología , Animales , Canales de Calcio/fisiología , Drosophila melanogaster/genética , Unión Neuromuscular/fisiología , Parálisis , Fenotipo , TemperaturaRESUMEN
Bacillus thuringiensis serovar israelensis, a bacterium which possesses plasmid transfer ability after mating, has been lysogenized by plasmid integrative phage J7W-1. The induction of phage in this J7W-1 lysogen was observed after mating with phage-insensitive strains, such as B. thuringiensis serovar thuringiensis, B. cereus and B. subtilis, as well as the phage-sensitive strain serovar israelensis. The phage induction was not observed after mating with B. thuringiensis strains AF101, serovar dendrolimus and serovar indiana. Because these strains are naturally associated with J7W-1 or its related phage, the data strongly suggest a constitutive expression of the repressor encoded by the prophage in these strains. However, the phage induction was observed in B. thuringiensis serovar aizawai, although it contained the J7W-1 DNA homologous region(s).
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Fagos de Bacillus/crecimiento & desarrollo , Bacillus thuringiensis/fisiología , Bacillus thuringiensis/virología , Factores de Tiempo , Ensayo de Placa Viral , Activación ViralRESUMEN
We report a case of torsion of an ovarian follicular cyst that developed during treatment with tamoxifen for breast cancer. A 40-year-old Japanese woman was admitted complaining of acute lower abdominal pain. Eight months earlier, she had undergone a partial mastectomy and local irradiation for ductal carcinoma of her left breast, estrogen receptor-positive stage I (T(1a) N(1b) M(0)). The administration of tamoxifen, 20 mg/day, and doxifluridine, 600 mg/day, were started immediately postoperatively. Pelvic examination after admission revealed the left ovarian cyst and enlarged uterus. Transvaginal ultrasonography and computed tomography revealed a multilocular cystic mass in the pelvic cavity. The pathological diagnosis of the tumor after total hysterectomy and bilateral salpingo-oophorectomy was a typical follicular cyst with torsion and uterine leiomyoma. This ovarian cyst was believed to have developed during tamoxifen administration.
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Quistes Ováricos/diagnóstico , Enfermedades del Ovario/diagnóstico , Tamoxifeno/efectos adversos , Adulto , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma Ductal de Mama/tratamiento farmacológico , Trompas Uterinas/cirugía , Femenino , Humanos , Histerectomía , Leiomioma/diagnóstico , Quistes Ováricos/inducido químicamente , Quistes Ováricos/cirugía , Enfermedades del Ovario/diagnóstico por imagen , Enfermedades del Ovario/cirugía , Ovariectomía , Tamoxifeno/uso terapéutico , Tomografía Computarizada por Rayos X , Anomalía Torsional/diagnóstico , Anomalía Torsional/diagnóstico por imagen , Anomalía Torsional/cirugía , Ultrasonografía , Neoplasias Uterinas/diagnósticoRESUMEN
The N-ethylmaleimide sensitive fusion protein (NSF) was originally identified as a cytosolic factor required for constitutive vesicular transport and later implicated in synaptic vesicle trafficking as well. Our previous work at neuromuscular synapses in the temperature-sensitive NSF mutant, comatose (comt), has shown that the comt gene product, dNSF1, functions after synaptic vesicle docking in the priming of vesicles for fast calcium-triggered fusion. Here we investigate whether dNSF1 performs a similar function at central synapses associated with the well-characterized giant fiber neural pathway. These include a synapse within the giant fiber pathway, made by the peripherally synapsing interneuron (PSI), as well as synapses providing input to the giant fiber pathway. The latency (delay) between stimulation and a resulting muscle action potential was used to assess the function of each class of synapses. Repetitive stimulation of the giant fiber pathway in comt produced wild-type responses at both 20 and 36 degrees C, exhibiting a characteristic and constant latency between stimulation and the muscle response. In contrast, stimulation of presynaptic inputs to the giant fiber (referred to as the "long latency pathway") revealed a striking difference between wild type and comt at 36 degrees C. Repetitive stimulation of the long latency pathway led to a progressive, activity-dependent increase in the response latency in comt, but not in wild type. Thus the giant fiber pathway, including the PSI synapse, appears to function normally in comt, whereas the presynaptic inputs to the giant fiber pathway are disrupted. Several aspects of the progressive latency increase observed in the long latency pathway can be understood in the context of the activity-dependent reduction in neurotransmitter release we observed previously at neuromuscular synapses. These results suggest that repetitive stimulation causes a progressive reduction in neurotransmitter release by presynaptic inputs to the giant fiber neuron, resulting in an increased latency preceding a giant fiber action potential. Thus synapses presynaptic to the giant fiber appear to utilize dNSF1 in a manner similar to the neuromuscular synapse, whereas the PSI chemical synapse may differ with respect to the expression or activity of dNSF1.
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Proteínas Portadoras/fisiología , Ganglios de Invertebrados/fisiología , Unión Neuromuscular/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Proteínas de Transporte Vesicular , Animales , Axones/fisiología , Encéfalo/fisiología , Proteínas Portadoras/genética , Drosophila , Estimulación Eléctrica , Femenino , Interneuronas/fisiología , Masculino , Fusión de Membrana , Potenciales de la Membrana , Modelos Neurológicos , Neuronas Motoras/fisiología , Proteínas Sensibles a N-Etilmaleimida , Fibras Nerviosas/fisiología , Vías Nerviosas/fisiología , Tiempo de ReacciónRESUMEN
N-Ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic protein thought to play a key role in vesicular transport in all eukaryotic cells. Although NSF was proposed to function in the trafficking of synaptic vesicles responsible for neurotransmitter release, only recently have in vivo experiments begun to reveal a specific function for NSF in this process. Our previous work showed that mutations in a Drosophila NSF gene, dNSF1, are responsible for the temperature-sensitive paralytic phenotype in comatose (comt) mutants. In this study, we perform electrophysiological and ultrastructural analyses in three different comt alleles to investigate the function of dNSF1 at native synapses in vivo. Electrophysiological analysis of postsynaptic potentials and currents at adult neuromuscular synapses revealed that in the absence of repetitive stimulation, comt synapses exhibit wild-type neurotransmitter release at restrictive (paralytic) temperatures. In contrast, repetitive stimulation at restrictive temperatures revealed a progressive, activity-dependent reduction in neurotransmitter release in comt but not in wild type. These results indicate that dNSF1 does not participate directly in the fusion of vesicles with the target membrane but rather functions in maintaining the pool of readily releasable vesicles competent for fast calcium-triggered fusion. To define dNSF1 function further, we used transmission electron microscopy to examine the distribution of vesicles within synaptic terminals, and observed a marked accumulation of docked vesicles at restrictive temperatures in comt. Together, the results reported here define a role for dNSF1 in the priming of docked synaptic vesicles for calcium-triggered fusion.
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Proteínas Portadoras/fisiología , Unión Neuromuscular/fisiología , Unión Neuromuscular/ultraestructura , Neurotransmisores/metabolismo , Proteínas de Transporte Vesicular , Animales , Proteínas Portadoras/genética , Drosophila , Estimulación Eléctrica , Femenino , Técnicas In Vitro , Masculino , Microscopía Electrónica , Músculos/fisiología , Mutación , Proteínas Sensibles a N-Etilmaleimida , Técnicas de Placa-Clamp , Terminales Presinápticos/ultraestructura , Transmisión Sináptica/genética , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestructura , TemperaturaRESUMEN
In abdominal muscles 202 and 203 of the cricket, Gryllus bimaculatus, large and small excitatory junctional potentials (l- and s-EJPs) with similar durations can be recorded from the same muscle fibers. At the normal extracellular calcium ion concentration ([Ca(2+)](o)) of 5mM, the amplitudes of l-EJPs in both muscles were larger than the threshold membrane potential for muscle action potentials, which is about -40mV. Below 0.75mM [Ca(2+)](o), the amplitudes became much smaller and were below the firing level for the action potentials. At 0.5mM, they fluctuated and decreased to 10.3 and 1.9mV in muscles 202 and 203, respectively, and at 0.25mM frequent failures occurred. The amplitudes of s-EJPs at 5mM [Ca(2+)](o) were 13.3 and 5.1mV in muscles 202 and 203, respectively, and the fluctuating amplitudes were far below the threshold for muscle action potentials. Below 0.75mM, s-EJPs were rarely observed. The relation between log(EJP amplitude) and log([Ca(2+)](o)) was linear within a certain range of [Ca(2+)](o) and the slopes of the lines for l-EJPs were about twice as steep as those for s-EJPs in both muscles. In muscle 202, the amplitude distribution of l-EJPs obtained at 0.25mM and that of s-EJPs at 0.75mM both showed peaks at once and twice the voltage at the first peak, which were coincident with the voltages at the peaks of amplitude distributions of miniature EJPs recorded simultaneously. The reversal potentials for l- and s-EJPs in muscle 202 were +1.02 and +0.22mV, respectively. In muscle 202, the decreases in amplitude of both EJPs by L-glutamate were similar and concentration-dependent. The results suggest that the difference in amplitude between l- and s-EJPs is attributable mainly to the difference in quantal contents.
RESUMEN
Detailed morphological and physiological studies on the insect abdominal muscles, including their innervation and neuromuscular transmission, are essential for understanding their important role in respiratory movements. There are both longitudinal and transverse muscles in the ventral abdominal segments of the cricket, Gryllus bimaculatus. Muscle 202 was selected as an example of a longitudinal muscle. This muscle is, on average, 1.4 mm long, paired on both sides of the abdomen, and consists of 127 fibers whose mean maximum diameter is 32 microns; the average sarcomere length is 8.1 microns. It is innervated by two ipsilateral motoneurons in the second abdominal ganglion, the axons of which run in the ipsilateral first nerve root of the third abdominal ganglion. Two motor axons run in parallel from the two cell bodies and innervate in close proximity. Accordingly, large and small excitatory junctional potentials (EJPs) are recorded from the same fiber with slightly different thresholds when the first nerve root of the third abdominal ganglion is stimulated. Muscle 203, which is a transverse muscle that extends across the fifth abdominal sternum and is located over the fourth abdominal ganglion and muscle 202 on both sides, is, on average, 2.9 mm long and consists of 86 fibers with a maximum diameter of 33 microns. The average sarcomere length is 7.9 microns. The right or left half of the muscle is innervated mainly by a contralateral motoneuron in the third abdominal ganglion through the ipsilateral first nerve root of the third abdominal ganglion. Nerve branches of the first nerve root also reach muscles 188 and 218. Muscle 203 is additionally innervated by the first nerve roots of abdominal ganglia 1, 2, and 4. These innervations were ascertained both electrophysiologically and histologically. Individual muscle fibers of muscle 203 produced small EJPs in response to stimulation of the first nerve roots of abdominal ganglia 2, 3, and 4 and large EJPs in response to stimulation of the root from the first abdominal ganglion. The large and small EJPs in muscle 203 have properties similar to those in muscle 202.