RESUMEN
Animal circadian clocks play a crucial role in regulating behavioral adaptations to daily environmental changes. The fruit fly Drosophila melanogaster exhibits 2 prominent peaks of activity in the morning and evening, known as morning (M) and evening (E) peaks. These peaks are controlled by 2 distinct circadian oscillators located in separate groups of clock neurons in the brain. To investigate the clock neurons responsible for the M and E peaks, a cell-specific gene expression system, the GAL4-UAS system, has been commonly employed. In this study, we re-examined the two-oscillator model for the M and E peaks of Drosophila by utilizing more than 50 Gal4 lines in conjunction with the UAS-period16 line, which enables the restoration of the clock function in specific cells in the period (per) null mutant background. Previous studies have indicated that the group of small ventrolateral neurons (s-LNv) is responsible for controlling the M peak, while the other group, consisting of the 5th ventrolateral neuron (5th LNv) and the three cryptochrome (CRY)-positive dorsolateral neurons (LNd), is responsible for the E peak. Furthermore, the group of posterior dorsal neurons 1 (DN1p) is thought to also contain M and E oscillators. In this study, we found that Gal4 lines directed at the same clock neuron groups can lead to different results, underscoring the fact that activity patterns are influenced by many factors. Nevertheless, we were able to confirm previous findings that the entire network of circadian clock neurons controls M and E peaks, with the lateral neurons playing a dominant role. In addition, we demonstrate that 4 to 6 CRY-positive DN1p cells are sufficient to generate M and E peaks in light-dark cycles and complex free-running rhythms in constant darkness. Ultimately, our detailed screening could serve as a catalog to choose the best Gal4 lines that can be used to rescue per in specific clock neurons.
Asunto(s)
Ritmo Circadiano , Criptocromos , Proteínas de Drosophila , Drosophila melanogaster , Neuronas , Proteínas Circadianas Period , Animales , Drosophila melanogaster/fisiología , Drosophila melanogaster/genética , Criptocromos/genética , Criptocromos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Neuronas/fisiología , Neuronas/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Relojes Circadianos/genética , Relojes Circadianos/fisiología , Actividad Motora , Fotoperiodo , Proteínas del OjoRESUMEN
The circadian clock in Drosophila is governed by a neural network comprising approximately 150 neurons, known as clock neurons, which are intricately interconnected by various neurotransmitters. The neuropeptides that play functional roles in these clock neurons have been identified; however, the roles of some neuropeptides, such as Trissin, remain unclear. Trissin is expressed in lateral dorsal clock neurons (LNds), while its receptor, TrissinR, is expressed in dorsal neuron 1 (DN1) and LNds. In this study, we investigated the role of the Trissin/TrissinR signaling pathway within the circadian network in Drosophila melanogaster. Analysis involving our newly generated antibody against the Trissin precursor revealed that Trissin expression in the LNds cycles in a circadian manner. Behavioral analysis further demonstrated that flies with Trissin or TrissinR knockout or knockdown showed delayed evening activity offset under constant darkness conditions. Notably, this observed delay in evening activity offset in TrissinRNAi flies was restored via the additional knockdown of Ion transport peptide (ITP), indicating that the Trissin/TrissinR signaling pathway transmits information via ITP. Therefore, this pathway may be a key regulator of the timing of evening activity offset termination, orchestrating its effects in collaboration with the neuropeptide, ITP.
Asunto(s)
Relojes Circadianos , Proteínas de Drosophila , Neuropéptidos , Animales , Drosophila melanogaster/metabolismo , Ritmo Circadiano/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Transducción de Señal , Relojes Circadianos/fisiología , Neuropéptidos/metabolismoRESUMEN
High-throughput sequencing has identified vast numbers of variants in genetic disorders. However, the significance of variants at the exon-intron junction remains controversial. Even though most cases of Mowat-Wilson syndrome (MOWS) are caused by heterozygous loss-of-function variants in ZEB2, the pathogenicity of variants at exon-intron junction is often indeterminable. We identified four intronic variants in 5/173 patients with clinical suspicion for MOWS, and evaluated their pathogenicity by in vitro analyses. The minigene analysis showed that c.73+2T>G caused most of the transcripts skipping exon 2, while c.916+6T>G led to partial skipping of exon 7. No splicing abnormalities were detected in both c.917-21T>C and c.3067+6A>T. The minigene analysis reproduced the splicing observed in the blood cells of the patient with c.73+2T>G. The degree of the exon skipping was concordant with the severity of MOWS; while the patient with c.73+2T>G was typical MOWS, the patient with c.916+6T>G showed milder phenotype which has been seldom reported. Our results demonstrate that mRNA splicing assays using the minigenes are valuable for determining the clinical significance of intronic variants in patients with not only MOWS but also other genetic diseases with splicing aberrations and may explain atypical or milder cases, such as the current patient.
Asunto(s)
Empalme del ARN , Humanos , Intrones , Virulencia , ExonesRESUMEN
Intracellular Ca2+-mediated mechanisms for pacemaker depolarization were studied in sinus node tissue preparations from mice and guinea pigs. Microelectrode recordings revealed that the sinus node of the mouse, which had a higher beating rate, had a steeper slope of the pacemaker depolarization than that of the guinea pig. BAPTA and ryanodine, agents that interfere with intracellular Ca2+, significantly decreased the slope of the pacemaker depolarization in both species. In contrast, SEA0400, a specific inhibitor of the Na+-Ca2+ exchanger (NCX), as well as change to low Na+ extracellular solution, significantly decreased the slope in the mouse, but not in the guinea pig. Niflumic acid, a blocker of the Ca2+ activated Cl- channel, decreased the slope in both species. Confocal microscopy revealed the presence of spontaneous Ca2+ oscillations during the interval between Ca2+ transients; such phenomenon was more pronounced in the mouse than in the guinea pig. Thus, although intracellular Ca2+-mediated mechanisms were involved in the pacemaker depolarization of the sinus node in both species, the NCX current was involved in the mouse but not in the guinea pig.
Asunto(s)
Marcapaso Artificial , Nodo Sinoatrial , Potenciales de Acción , Animales , Calcio/metabolismo , Cobayas , Ratones , Nodo Sinoatrial/metabolismo , Intercambiador de Sodio-CalcioRESUMEN
Instantaneous orthostatic hypotension (INOH) is one of the main types of orthostatic dysregulation in children and adolescents. In patients with INOH arterial pressure drops considerably after active standing and is slow to recover. We investigated changes in cerebral oxygenation in the bilateral prefrontal cortex during an active standing test in juvenile INOH patients to evaluate changes in cerebral oxygen metabolism. We enrolled 82 INOH patients (mean age 13.8 ± 2.2 years, 52 mild and 30 severe patients) at Nihon University Itabashi Hospital from October 2013 to April 2018. We measured cerebral oxygenated hemoglobin, deoxygenated hemoglobin, and total hemoglobin levels in the bilateral prefrontal cortex using near-infrared spectroscopy during an active standing test. In severe INOH patients, cerebral oxygenation of the right prefrontal cortex remained constant when blood pressure dropped; however, de-oxy-Hb significantly increased. These findings confirm that there is asymmetrical autoregulation between the right and left prefrontal cortex.
Asunto(s)
Circulación Cerebrovascular , Hipotensión Ortostática , Adolescente , Circulación Cerebrovascular/fisiología , Niño , Homeostasis , Humanos , Hipotensión Ortostática/fisiopatología , Oxihemoglobinas , Espectroscopía Infrarroja CortaRESUMEN
Understanding of the pathogenesis of nonalcoholic steatohepatitis (NASH)-associated fibrosis has been hampered by the lack of a comprehensive and physiological small animal model of NASH with fibrosis. Feeding a high-fat and high-cholesterol (HFC) diet supplemented with cholic acid to rats is known to replicate human NASH pathology, and it induces fibrosis earlier than with an HFC diet alone. In the present study, physiological and histopathological observations from 65 Sprague-Dawley (SD) rats fed an HFC diet with or without cholic acid for 9 or 18 weeks in our laboratory between January 2013 and February 2018 were retrospectively reviewed. The liver weight/body weight ratio at the end of the rearing period was higher in rats fed an HFC diet than in rats fed a normal diet in a cholesterol dose-, cholic acid dose-, or rearing period dependent manner. Dietary fat, cholesterol and/or cholic acid and rearing period affected the histopathologic severity of NASH. Overall, 56 (86.2%) of 65 SD rats fed an HFC diet for 9 or 18 weeks developed histopathologically proven NASH. It is noted that the SD rats fed an HFC diet supplemented with 2% (w/w) cholic acid for 18 weeks frequently developed advanced fibrosis, including cirrhosis. Thus, this diet-induced NASH rat model is likely to be a highly reproducible.
Asunto(s)
Colesterol en la Dieta/toxicidad , Ácido Cólico/toxicidad , Grasas de la Dieta/toxicidad , Modelos Animales de Enfermedad , Enfermedad del Hígado Graso no Alcohólico/patología , Animales , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Cirrosis Hepática Experimental/etiología , Cirrosis Hepática Experimental/patología , Ratas , Ratas Sprague-DawleyRESUMEN
Nonalcoholic steatohepatitis (NASH), a subtype of nonalcoholic fatty liver disease (NAFLD), has a potentially progressive course that can lead to liver cirrhosis. Age is strongly associated with the development and progression of NAFLD/NASH, but the natural history of pediatric NAFLD is still not fully understood. Here, we evaluated the age-related alterations of NASH in 5-, 9- and 13-wk-old male Sprague-Dawley rats that were fed a high-fat and high-cholesterol diet (30% fat, 1.25% cholesterol and 0.5% sodium cholate, w/w) for 9 wk (6 rats/group). Our results showed that the cumulative energy intake, body weight gain and food efficacy during the 9-wk rearing period were highest in the youngest group and lowest in the oldest group. Serologically, almost all parameters including the serum triglyceride and total cholesterol were similar regardless of age. Histopathological findings, such as hepatic steatosis, lobular inflammation and hepatocyte ballooning, were also similar regardless of age, but hepatic fibrosis was more evident in the oldest group. Also, the mRNA expression levels of some fibrogenic, inflammatory, oxidative stress and cholesterol or lipid metabolism-related genes in the liver were highest in the oldest group and lowest in the youngest group, although the difference was not statistically significant. These results indicated that aging is likely associated with the development of NASH. Because the cumulative energy intake and daily food intake/body weight were not similar among groups in the present study, further studies designed with an equivalent daily food intake/body weight among groups are needed in order to interpret the exact nutritional effect.
Asunto(s)
Envejecimiento/fisiología , Colesterol en la Dieta/efectos adversos , Dieta Alta en Grasa/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Animales , Colesterol en la Dieta/administración & dosificación , Ingestión de Alimentos , Ingestión de Energía , Expresión Génica , Inflamación/genética , Metabolismo de los Lípidos/genética , Lípidos/sangre , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/patología , Masculino , Enfermedad del Hígado Graso no Alcohólico/etiología , Estrés Oxidativo/genética , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Confusional migraine is a rare type of migraine presenting as an acute confusional state. However, the mechanism of this confusional state remains unclear. SUBJECT AND METHODS: We examined an 11-year-old girl with confusional migraine, using electroencephalography, brain magnetic resonance imaging, cerebrovascular magnetic resonance angiography, and single-photon emission computed tomography to investigate cerebral blood flow changes. RESULTS: Our findings revealed vessel narrowing in the left middle and posterior cerebral artery territory, indicating vasospasm and suggesting that the confusion was caused by hypoperfusion. However, abnormal increased cerebral blood flow in the left middle and posterior cerebral artery territory was observed during the non-confusional state. CONCLUSION: The recorded cerebral blood flow changes are similar to those associated with migraine attacks, gradually changing from abnormally low to abnormally high during the confusional and post-confusional state.
Asunto(s)
Circulación Cerebrovascular/fisiología , Trastornos Migrañosos/fisiopatología , Niño , Confusión/fisiopatología , Electroencefalografía/métodos , Femenino , Humanos , Angiografía por Resonancia Magnética/métodos , Imagen por Resonancia Magnética/métodos , Trastornos Migrañosos/sangre , Trastornos Migrañosos/complicaciones , Tomografía Computarizada de Emisión de Fotón Único/métodosRESUMEN
Acute focal bacterial nephritis (AFBN) is a localized bacterial infection of the kidney presenting as an inflammatory mass without frank abscess formation. In children, most patients with AFBN present with nonspecific conditions, such as fever, vomiting, and abdominal pain. A small number of reported cases are accompanied by neurological symptoms, including meningeal irritation, unconsciousness, and seizures. We experienced 2 rare cases of AFBN associated with central nervous system lesions. The first case was a 3-year-old girl who had neurological symptoms, including unconsciousness and seizures, with AFBN associated with acute reversible encephalopathy. The second case was a 5-year-old girl who had neurological symptoms, including unconsciousness, with AFBN accompanied by clinically mild encephalitis/encephalopathy with a reversible splenial lesion.
Asunto(s)
Infecciones Bacterianas/complicaciones , Encefalopatías/complicaciones , Sistema Nervioso Central/patología , Encefalitis/complicaciones , Nefritis/microbiología , Antibacterianos/uso terapéutico , Infecciones Bacterianas/tratamiento farmacológico , Encefalopatías/tratamiento farmacológico , Sistema Nervioso Central/diagnóstico por imagen , Líquido Cefalorraquídeo/química , Líquido Cefalorraquídeo/citología , Preescolar , Imagen de Difusión por Resonancia Magnética/métodos , Femenino , Humanos , Nefritis/diagnóstico , Tomografía Computarizada por Rayos X/métodos , Resultado del TratamientoRESUMEN
Highly metastatic cells, especially in the lungs, are known to be resistant to nitric oxide (NO)-mediated cytotoxicity, compared with poorly or non-metastatic cells. However, the precise mechanisms connecting NO and metastasis remain to be determined. To clarify the role of NO in the characteristic changes in NO-resistant cells in response to inflammatory cytokines, we used Lewis lung tumor (LLT) cells, which are known to be highly metastatic NO-resistant cells, and determined the changes in cell deformability and the gene expression profile after the cells were stimulated using cytokine mixture or an NO donor. Both exogenous NO and endogenous NO via inducible NO synthase produced by cytokines decreased cell deformability by enhancing actin polymerization. The expression of several genes associated with actin polymerization was changed so as to increase actin filaments in the cells by enhancing actin polymerization and by suppressing actin depolymerization, actin filament severing, and barbed-end actin filament capping. In conclusion, inflammatory cytokine stimulation reduces deformability of LLT cells and enhances actin polymerization which is mainly controlled by the same genes induced by NO.
Asunto(s)
Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/ultraestructura , Citocinas/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico/metabolismo , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animales , Citocinas/farmacología , Ratones , Nitratos/metabolismo , Óxido Nítrico/farmacología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Nitritos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Estadísticas no ParamétricasRESUMEN
Growth of FRM cells was inhibited by the addition of pyridoxine in a dose-dependent manner. Use of 5 mM pyridoxine caused an almost complete arrest of cell growth. Pyridoxal was as effective as pyridoxine, but pyridoxamine showed weak inhibitory action. Electron-microscopic examination of control cells revealed large nuclei and cellular membranes with villi, but, in pyridoxine-treated cells, condensed or degraded nuclei were observed. Many vacuoles and cholesterol crystals were widely distributed inside the cellular membrane of pyridoxine-treated cells. One of the vacuoles was identified as a lipid droplet. The DNA ladder was observed in the pyridoxine-treated cells. It is suggested that pyridoxine treatment of FRM cells causes cytolysis of cells by apoptosis.
Asunto(s)
Neoplasias de la Mama/patología , Vitamina B 6/farmacología , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/ultraestructura , Gatos , Línea Celular Tumoral , Microscopía ElectrónicaRESUMEN
BACKGROUND: Autonomic neuropathy is a common complication of diabetes mellitus (DM). METHOD: To clarify the relationship between cardiovascular autonomic function and gastric emptying rate, we investigated the gastric emptying and coefficient of R-R interval variation (CV(RR)) of 84 type 2 diabetic patients: 28 cases without peripheral neuropathy and 56 cases with peripheral neuropathy. All patients were subjected to a gastric emptying test according to the marker method (administration of a capsule containing 20 pieces of radiopaque marker during breakfast, followed by abdominal X-ray imaging 3 and 5 h later). Patients had their CV(RR) assessed at rest and during deep breathing. RESULTS: Gastric emptying scores were significantly correlated with CV(RR) during deep breathing and with the duration of DM, but neither age nor CV(RR) at rest in all patients. Gastric emptying scores and CV(RR) at rest and during deep breathing in patients with peripheral neuropathy were significantly deteriorated than those in patients without peripheral neuropathy. A significant correlation between gastric emptying and CV(RR) during deep breathing could be observed in the patients with peripheral neuropathy, but not in those without it. CONCLUSIONS: These findings showed that CV(RR) during deep breathing might be a good indicator of diabetic gastropathy and that peripheral neuropathy was closely related with cardiac and gastric autonomic neuropathy in the type 2 diabetic patients.
Asunto(s)
Enfermedades Cardiovasculares/diagnóstico , Diabetes Mellitus Tipo 2/complicaciones , Electrocardiografía/métodos , Vaciamiento Gástrico , Gastroparesia/diagnóstico , Anciano , Enfermedades del Sistema Nervioso Autónomo/diagnóstico , Enfermedades del Sistema Nervioso Autónomo/etiología , Enfermedades Cardiovasculares/etiología , Neuropatías Diabéticas/etiología , Técnicas de Diagnóstico del Sistema Digestivo , Femenino , Gastroparesia/etiología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The effect of high-dose pyridoxine (PN) on mammary tumorigenesis was examined in female Sprague-Dawley rats. The first mammary tumors appeared between 84 and 90 days after 7,12-dimethylbenzanthracene treatment. There was no effect of PN level on tumor incidence at 90 days but at 98, 104, and 111 days. Tumor incidence was lower in the high-dose group (35 mg PN/kg diet) compared with the controls (7 mg PN/kg diet). All tumors were identified as adenocarcinoma and most as papillary type. The number of microcarcinomas in mammary glands of the 35-mg PN group tended to be reduce than that of the 7-mg group. The number of proliferating Ki67-positive cells was significantly reduced by supplementation with PN.