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Article in English | WPRIM | ID: wpr-927630


OBJECTIVE@#To determine if ARHGEF10 has a haploinsufficient effect and provide evidence to evaluate the severity, if any, during prenatal consultation.@*METHODS@#Zebrafish was used as a model for generating mutant. The pattern of arhgef10 expression in the early stages of zebrafish development was observed using whole-mount in situ hybridization (WISH). CRISPR/Cas9 was applied to generate a zebrafish model with a single-copy or homozygous arhgef10 deletion. Activity and light/dark tests were performed in arhgef10 -/-, arhgef10 +/-, and wild-type zebrafish larvae. ARHGEF10 was knocked down using small interferon RNA (siRNA) in the SH-SY5Y cell line, and cell proliferation and apoptosis were determined using the CCK-8 assay and Annexin V/PI staining, respectively.@*RESULTS@#WISH showed that during zebrafish embryonic development arhgef10 was expressed in the midbrain and hindbrain at 36-72 h post-fertilization (hpf) and in the hemopoietic system at 36-48 hpf. The zebrafish larvae with single-copy and homozygous arhgef10 deletions had lower exercise capacity and poorer responses to environmental changes compared to wild-type zebrafish larvae. Moreover, arhgef10 -/- zebrafish had more severe symptoms than arhgef10 +/- zebrafish. Knockdown of ARHGEF10 in human neuroblastoma cells led to decreased cell proliferation and increased cell apoptosis.@*CONCLUSION@#Based on our findings, ARHGEF10 appeared to have a haploinsufficiency effect.

Animals , Annexin A5 , Apoptosis , Blotting, Western , CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Cell Line , Cell Proliferation , Cells, Cultured , Flow Cytometry , Genotype , Humans , In Situ Hybridization , Larva/physiology , Phenotype , RNA/isolation & purification , Real-Time Polymerase Chain Reaction/standards , Rho Guanine Nucleotide Exchange Factors/metabolism , Sincalide/analysis , Spectrophotometry/methods , Zebrafish/physiology
Int. arch. otorhinolaryngol. (Impr.) ; 19(2): 116-120, Apr-Jun/2015. tab
Article in English | LILACS | ID: lil-747152


Introduction Thyroid cancer incidence has increased in the previous 2 decades. Preoperative identification of lymph node metastasis is a suggested risk factor associated with recurrence following thyroidectomy. Objectives We aimed to evaluate the accuracy of preoperative radiologic investigations of nodal status in determining the postoperative risk of regional nodal recurrence in cases of well-differentiated thyroid cancer. Methods This is a case series. We retrospectively reviewed data, including preoperative ultrasonography and/or computed tomography results, on patients who underwent total thyroidectomy for thyroid cancer at our hospital between 2006 and 2012. Prognostic factors for predicting recurrence, including age, sex, tumor diameter, and nodal diameter, were evaluated. Results Total thyroidectomy was performed on 24 male and 74 female patients (median age, 43 years). The median follow-up time was 21 months. Sixty-eight patients had papillary thyroid cancer, and 30 had follicular cancer. Nodal recurrence was evident in 30% of patients, and 4% of patients died. Identification of lymph node involvement during preoperative radiologic investigations was strongly prognostic for recurrence: 35.3% of patients with positive preoperative ultrasonography findings and 62.5% of those with positive preoperative computed tomography findings had recurrence (p = 0.01). Conclusions Preoperative identification of lymph node metastasis on radiologic studies was correlated with an increased risk of regional nodal recurrence in well-differentiated thyroid cancer. Computed tomography was superior to ultrasonography in detecting metastatic nodal involvement preoperatively and is therefore recommended for preoperative assessment and postoperative follow-up. .

Animals , Humans , Hematopoiesis/genetics , Leukemia, Myeloid, Acute/genetics , Zebrafish Proteins/physiology , Zebrafish/physiology , /physiology , Amino Acid Sequence , Animals, Genetically Modified , Conserved Sequence , Embryo, Nonmammalian , Molecular Sequence Data , Protein Structure, Tertiary/genetics , Sequence Homology, Amino Acid , Tandem Repeat Sequences , Transcriptome , Zebrafish Proteins/chemistry , Zebrafish/embryology , /chemistry
Braz. j. biol ; 70(2): 425-434, May 2010. graf, tab
Article in English | LILACS | ID: lil-548247


In order to verify the influence of chronic and acute ambient oxygen levels from egg to adult stage of the zebrafish, in vivo oxygen consumption (MO2), critical tensions of oxygen (Pcrit), heart rate (fH) and total body lactate concentration (Lc) were determined for Danio rerio (Hamilton, 1822) raised at 28 °C under normoxic (7.5 mgO2.L-1 or 80 mm.Hg-1) and hypoxic conditions (4.3 mgO2.L-1) and exposed to acute hypoxia during different developmental stages. Our findings confirmed that very early stages do not respond effectively to ambient acute hypoxia. However, after the stage corresponding to the age of 30 days, D. rerio was able to respond to acute hypoxia through effective physiological mechanisms involving aerobic and anaerobic metabolism. Such responses were more efficient for the fishes reared under hypoxia which showed that D. rerio survival capability increased during acclimation to mild hypoxia. Measurements of body mass and length showed that moderate hypoxia did not affect growth significantly until the fish reached the stage of 60 days. Moreover, a growth delay was verified for the hypoxic-reared animals. Also, the D. rerio eggs-to-larvae survival varied from 87.7 to 62.4 percent in animals reared under normoxia and mild hypoxia, respectively. However, the surviving animals raised under moderated hypoxia showed a better aptitude to regulate aerobic and anaerobic capacities when exposed to acute hypoxia.

A influência de diferentes níveis de oxigênio no desenvolvimento (ovos a adulto) do peixe paulistinha Danio rerio (Hamilton, 1822) foi verificada por meio de medidas experimentais de consumo de oxigênio (MO2), tensões críticas de oxigênio (Pcrit), taxa de batimentos cardíacos (fH) e concentração total de lactato nos tecidos (Lc), para os animais mantidos a 28 ºC sob níveis normóxicos de oxigênio (7.5 mgO2.L-1 ou 80 mmHg) e hipóxicos (4.3 mgO2.L-1) e submetidos a hipóxia ambiental aguda, em diferentes estágios de desenvolvimento. Os resultados obtidos indicam que os peixes em estágios iniciais do desenvolvimento não variam suas respostas fisiológicas em função das oscilações ambientais nos níveis de oxigênio, visto que tais respostas iniciaram-se somente no estágio de 30 dias de vida. A partir deste estágio D. rerio apresentou capacidade em responder à hipóxia aguda por meio de mecanismos fisiológicos efetivos envolvendo metabolismo aeróbico e anaeróbico. Tais respostas foram mais efetivas para os peixes mantidos sob hipóxia, o que mostrou que a capacidade de sobrevivência de D. rerio aumentou durante o período de aclimatação à hipóxia moderada. As medidas de massa e comprimento corpóreos mostraram que a permanência dos peixes em hipóxia durante o desenvolvimento não afetou esses parâmetro até os peixes atingirem o estágio de 60 dias. A partir deste estágio foi observado ligeiro atraso no crescimento dos espécimes mantidos sob hipóxia. A taxa de sobrevivência de D.rerio variou de 87,7 a 62,4 por cento para os animais mantidos respectivamente sob níveis normóxicos e hipóxicos. No entanto, os animais mantidos sob hipóxia moderada, que sobreviveram, mostraram maior capacidade em regular seu metabolismo aeróbico e anaeróbico quando expostos à hipóxia ambiental aguda.

Animals , Hypoxia/physiopathology , Oxygen Consumption/physiology , Zebrafish/physiology , Aerobiosis/physiology , Anaerobiosis/physiology , Hypoxia/metabolism , Body Mass Index , Heart Rate/physiology , Lactic Acid/analysis , Zebrafish/growth & development , Zebrafish/metabolism
Braz. j. med. biol. res ; 42(1): 128-134, Jan. 2009. ilus, tab
Article in English | LILACS | ID: lil-505415


The aim of the present study was to determine the effect of the histaminergic precursor L-histidine and the H3 receptor antagonist thioperamide on the learning process of zebrafish submitted or not to confinement stress. On each of the 5 consecutive days of experiment (D1, D2, D3, D4, D5), animals had to associate an interruption of the aquarium air supply with food offering. Non-stressed zebrafish received an intraperitoneal injection of 100 mg/kg L-histidine, 10 mg/kg thioperamide or saline after training. Stressed animals received drug treatment and then were submitted to confinement stress for 1 h before the learning procedure. Time to approach the feeder was measured (in seconds) and was considered to be indicative of learning. A decrease in time to approach the feeder was observed in the saline-treated group (D1 = 141.92 ± 13.57; D3 = 55 ± 13.54), indicating learning. A delay in learning of stressed animals treated with saline was observed (D1 = 217.5 ± 25.66). L-histidine facilitated learning in stressed (D1 = 118.68 ± 13.9; D2 = 45.88 ± 8.2) and non-stressed (D1 = 151.11 ± 19.20; D5 = 62 ± 14.68) animals. Thioperamide inhibited learning in non-stressed (D1 = 110.38 ± 9.49; D4 = 58.79 ± 16.83) and stressed animals (D1 = 167.3 ± 26.39; D5 = 172.15 ± 27.35). L-histidine prevented the increase in blood glucose after one session of confinement (L-histidine = 65.88 ± 4.50; control = 53 ± 3.50 mg/dL). These results suggest that the histaminergic system enhances learning and modulates stress responses in zebrafish.

Animals , Avoidance Learning/drug effects , /pharmacology , Histidine/pharmacology , Piperidines/pharmacology , Zebrafish/physiology , Avoidance Learning/physiology , Blood Glucose/analysis , Blood Glucose/drug effects , Dose-Response Relationship, Drug , Injections, Intraperitoneal , Stress, Physiological , Zebrafish/blood
Article in English | WPRIM | ID: wpr-65395


Although rodents have previously been used in ecotoxicological studies, they are expensive, time-consuming, and are limited by strict legal restrictions. The present study used a zebrafish (Danio rerio) model and generated data that was useful for extrapolating toxicant effects in this system to that of humans. Here we treated embryos of the naive-type as well as a transiently transfected zebrafish liver cell line carrying a plasmid (phAhREEGFP), for comparing toxicity levels with the well-known aryl hydrocarbon receptor (AhR)-binding toxicants: 3,3',4,4',5-pentachlorobiphenyl (PCB126), 2,3,7,8-tetrachlorodibenzo-p-dioxin, and 3-methylcholanthrene. These toxicants induced a concentration-dependent increase in morphological disruption, indicating toxicity at early life-stages. The transient transgenic zebrafish liver cell line was sensitive enough to these toxicants to express the CYP1A1 regulated enhanced green fluorescent protein. The findings of this study demonstrated that the zebrafish in vivo model might allow for extremely rapid and reproducible toxicological profiling of early life-stage embryo development. We have also shown that the transient transgenic zebrafish liver cell line can be used for research on AhR mechanism studies.

Animals , Benz(a)Anthracenes/toxicity , Cell Line , Green Fluorescent Proteins , Hepatocytes/cytology , Larva/drug effects , Lethal Dose 50 , Polychlorinated Biphenyls/toxicity , Polychlorinated Dibenzodioxins/toxicity , Water Pollutants, Chemical/adverse effects , Zebrafish/physiology