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Objective To discuss the clinical features and treatment of isovaleric academia (IVA) patients,and to gain more comprehensive understanding of isovaleryl-CoA dehydrogenase(IVD) mutation in 2 siblings in order to raise awareness to prevent the occurrence of IVA.Methods The clinical history and laboratory test of 2 cases of children with IVA were carried out.The exons and neighboring introns of IVD gene of the whole family were PCR-amplified for DNA sequencing.The literature review of IVA in China was also conducted.Results Organic acid analysis of urine by GC/MS for both siblings showed extremely elevated concentrations of isovaleric glycine.For the older sibling,an acute episode of IVA caused severe metabolic stress and eventually death in the neonatal period.However,the disease was well-controlled for the younger sibling due to timely treatment and follow-up care for 2 years.The DNA sequencing of the IVD gene in the family revealed a novel c.1016G ＞ A(C339Y) heterozygous mutation in mother and both of the siblings.No IVD mutation was detected in father or in any of the 50 cases of healthy controls.According to literature review,15 cases of IVA were reported in recent 15 years in China,including neonatal onset (11 cases),acute episode (12 cases),odor of sweaty feet (12 cases),pancytopenia (9 cases),hyperammonemia (5 cases),hypocalcemia (6 cases),and 6 cases of death were reported.Additionally,5 cases that received treatment of BCAA-free formula milk showed positive outcome.However,only 2 cases were followed up for more than 2 years.Conclusions Two new IVA patients carrying c.1016G ＞ A(C339Y) mutation were reported in China.The mutation may lead to conformational change and functional deficient of the IVD protein.It is also necessary to point out that using direct DNA sequencing can not identify all patients with IVA due to limitations of this technology,and thus clinicians should be aware of the possibility of genetic misdiagnosis.Moreover,there is a trend of increasing IVA in China in recent years.
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Objective To investigate the effect of chronic stress on the expression of uncoupling protein 4 (UCP4) and Bcl-2 protein in hippocampal neurons of rat depression model.Methods Rat depression models were established by chronic unpredicted mild stress.All rats were randomly assigned to 2 groups:control group and model group.Flow cytometry was applied to detect the apoptosis rate and mitochondrial membrane potential.The level of LDH was measured by enzymes labelling instrument.The number of neurons was measured by immunohistochemistry.The expression of UCP4 and Bcl-2 protein was measured by Western blotting.Results After chronic stress,the apoptosis rate((4.35±0.19) ％)and LDH activity ((445.50±91.70) U/mg) in hippocampal tissue in the model group was significantly higher than the control group((0.34±0.06) ％,(167.20±63.40)U/mg).Compared to control group,the number of hippocampal neurons ((72.50±4.25) vs (45.30±2.54)) and the mitochon drial membrane potential decreased in the model group.The expressions of UCP4 and Bcl-2 protein in hippocampal tissue were significantly lower than the control group.Conclusion Chronic unpredicted mild stress can lead to apoptosis in rat hippocampal neurons,which is related with decline of mitochondrial membrane potential and low expression of UCP4 and Bcl-2 protein.
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The effects of microRNA-34a (miR-34a)-regulated Notch1 gene on the proliferation and apoptosis of the human glioma cell line U87 were investigated in this study. The U87 cells were divided into miR-34a mimics, negative control, mock transfection and blank control groups in terms of different treatments. In miR-34a mimics group, human U87 glioma cells were transfected with miR-34a mimics by using lipofectamine 2000. The cells transfected with nonsense microRNA were set up as negative control group. Those treated with lipofectamine 2000 only were designated to the mock tranfection group. In the blank control group, the cells were cultured routinely and no treatment was given. The expression of miR-34a and Notch1 was detected by using real-time RT-PCR. Western blotting was employed to monitor the change in Notch1 protein. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry. The results showed that the proliferative ability of U87 cells was significantly reduced and the apoptotic cells increased in miR-34a mimics group relative to control groups. The expression of miR-34a was significantly up-regulated in mimics group as compared with control groups (P<0.05). Furthermore, Notch1 protein levels were significantly decreased in miR-34a mimics group when compared with control groups (P<0.05), but the mRNA expression of Notch1 showed no significant difference among these groups. It was concluded that miR-34a may suppress the proliferation and induce apoptosis of U87 cells by decreasing the expression of target gene Notch1, suggesting that miR-34a may become a promising gene therapeutic target for brain glioma.
Subject(s)
Humans , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Genetics , Glioma , Pathology , MicroRNAs , Genetics , Receptor, Notch1 , Physiology , TransfectionABSTRACT
The effects of microRNA-34a (miR-34a)-regulated Notch1 gene on the proliferation and apoptosis of the human glioma cell line U87 were investigated in this study. The U87 cells were divided into miR-34a mimics, negative control, mock transfection and blank control groups in terms of different treatments. In miR-34a mimics group, human U87 glioma cells were transfected with miR-34a mimics by using lipofectamine 2000. The cells transfected with nonsense microRNA were set up as negative control group. Those treated with lipofectamine 2000 only were designated to the mock tranfection group. In the blank control group, the cells were cultured routinely and no treatment was given. The expression of miR-34a and Notch1 was detected by using real-time RT-PCR. Western blotting was employed to monitor the change in Notch1 protein. Cell proliferation and apoptosis were measured by CCK-8 and flow cytometry. The results showed that the proliferative ability of U87 cells was significantly reduced and the apoptotic cells increased in miR-34a mimics group relative to control groups. The expression of miR-34a was significantly up-regulated in mimics group as compared with control groups (P<0.05). Furthermore, Notch1 protein levels were significantly decreased in miR-34a mimics group when compared with control groups (P<0.05), but the mRNA expression of Notch1 showed no significant difference among these groups. It was concluded that miR-34a may suppress the proliferation and induce apoptosis of U87 cells by decreasing the expression of target gene Notch1, suggesting that miR-34a may become a promising gene therapeutic target for brain glioma.
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A rapid and sensitive method based on biotin-avidin mediated competitive enzyme-linked immu nosorbent assay(BA-ELISA) was established for the determination of ketamine.The optimal concentration of coated antigen and anti-ketamine monoclonal antibody were found to be 2.0 and 10.2 mg/L.The concentra tions of biotinylated goat anti-mouse IgG(Biotin-IgG) and streptavidin-horseradish peroxidase(SA-HRP) were optimized and the optimum results were found to be 0.29 and 1.0 mg/L, respectively.The linear range of the presented method was from 0.1 to 1000 μg/L, and the limit of detection was found to be 0.03 μg/L.The recoveries of ketamine spiked in human serum and urine were between 94% and 102%.Comparing the result of traditional ELISA, the present BA-ELISA method had a lower detection limit for ketamine.The experimental results indicated that the present BA-ELISA method was specific and sensitive.