Objective:To establish a method to determine the metabolites in rat kidney tissues by gas chromatography-mass spectrometry (GC-MS) combined with chemometric techniques. Methods:Metabolites were separated and identiifed on HP-5MS column (30 m × 0.25 μm × 0.25 mm). The initial column temperature was 100℃lasting 3 min, and then programmed at 8℃/min to 300℃, maintaining at this temperature for 6 min. hTe internal standard was heptadecanoic acid. hTe grinded kidney tissue was exacted by methanol. hTe supernatant was dried by nitrogen. Atfer the oximation and derivation, the supernatant was analyzed by GC-MS. hTe overlapped peaks were resolved into pure chromatogram and mass spectra with chemometric techniques. Qualitative analysis was performed by comparing the obtained pure mass spectra with those in NIST mass spectra database and certiifcated by the standards and the references. hTe internal method was used for semi-quantitation. Results:A total of 53 compounds were identiifed. hTe main constitutions in the kidney tissue were amino acids, saccharides, fatty acids and urea. Conclusion:hTe combination of methods is rapid and accurate for the analysis of metabolites in the kidney tissue, which provides more information for further study of metabonomics in kidney tissues.

Objective To explore the role of Hippo pathway in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD),and find potential targets for drug therapy.Methods By means of immunofluorescence staining,Western blotting,Real-time PCR,the differences of sublocalization,expression and phosphorylation level about Hippo pathway molecules in Han:SPRD (cy/+) and ADPKD patients compared with the control were observed.Knockdown Yes kinaseassociated protein (YAP),transcriptional coactivator with PDZ binding motif (TAZ) and large tumor suppressor kinase1 (LATS1) in cystic lining epithelium cell line WT9-12 were took by siRNA interference,and then their effects on cell proliferation,apoptosis and cell cycle were assessed.Results In cystic lining epithelium of Han:SPRD(cy/+),decreased expression of LATS1 and increased expression of YAP were found compared with the control,and the immunofluorescence of YAP was distributed both in cytoplasm and nucleus,while distribution and expression level of TAZ were without significant variance.Abnormal mRNA expressions of Hippo pathway components in ADPKD patients were found (P ＜ 0.05).Down-regulation of LATS1 in WT9-12 cells could prohibit phosphorylation of YAP,and prompted proliferation and cell division.Knockdown YAP in WT9-12 cells could inhibited cell proliferation by arresting cell cycle in G0/G1 phase,but down-regulating TAZ showed no significant differences in proliferation and cell cycle.Conclusions Altered Hippo signaling exists in ADPKD,and YAP activation may be one leading cause of autosomal dominant polycystic kidney disease onset.In vitro,knockdown YAP in WT9-12 cells can inhibit cell proliferation by arresting cell cycle and depressing cell division,suggesting the expression level and activity of YAP are potential targets for ADPKD treatment.

Objectives To explore the clinical application of deceleration capacity of rate (DC), acceleration capacity of rate (AC) and heart rate variability (HRV) in children with precardial distress of unknown origin. Methods A total of 56 children with precardial distress of unknown origin and 63 healthy children aged 6 to 17 years were examined by 24 h dynamic elec-trocardiogram, and the indexes of DC and HRV were compared between these two groups. Results DC value of children with precardial distress is less than that of the control group (P<0.05), AC value is greater than that of the control group (P<0.05), and heat rate (HR) is greater than that of the control group (P<0.05). No statistical differences were observed in the indexes of HRV between the two groups. The indexes of DC show a signiifcant positive correlation with HRV in children with precardial distress(r=0.27~0.40, P<0.05), while appear a negative relation with HR (r=-0.46, P=0.000). In contrast, the indexes of AC show a signiifcant negative correlation with HRV (r=-0.57~-0.34, P<0.05), and appears a positive relation with HR(r=0.61, P=0.000). HR value is higher in male children less than 12 years old with precardial distress than that of age-matched males in control group, and RMSSD is lower than the latter. DC value of male children more than 12 years with precardial distress is lower than that of age-matched males in control group, while AC value is higher than that of the latter;DC value is signiifcant lower in fe-male children more than 12 yeares with precardial distress than that of age-matched females in the control group (P<0.05). Con-clusions The activity of vagus nerve in children with precardial distress of unknown origin is decreased. DC value is signiifcantly lower than that of control group, and shows correlation with indexes of HRV. There is no signiifcant difference in DC and HRV value between male and female children with precardial distress. DC value is lower in children aged 12 or older with precardial distress than that of age-matched children in the control group, which indicates adolescents are vulnerable to autonomic nerve functional disorder.

Objective To investigate the clinical significance of measuring resting energy expenditure (REE) for guiding an accurate nutritional support in elderly bedridden patients with nasal feeding.Methods The REE of 32 elderly bedridden patients with nasal feeding was assessed by using the Cosmed K4b2 portable telemetric gas analysis system.The waist-hip ratio, serum levels of albumin, transferrin, prealbumin and retinol-binding protein were determined to assess comprehensive nutrition status.The energy intakes were calculated, and the correlation of REE and the difference between the energy intakes and consumption with nutritional index were analyzed.Results The resting energy expendture was lower in the patients with waist-hip ratio≥0.95 than in patients with waist-hip ratio ＜0.95 (t=3.622, P＜0.01).The waist-hip ratio was reduced and serum albumin and transferrin levels were decreased along with the increase of REE in elderly patients (r=-0.55,-0.36 and-0.593, respectively, P=0.001, 0.043, ＜0.001).The difference between the energy intake and expenditure was higher in patients with waist-hip ratio≥0.95 than those with waist-hip ratio＜0.95 (t =5.643, P＜ 0.001).Serum albumin, prealbumin, transferrin and retinol-binding protein levels were increased along with the increase of the difference between the energy intake and expenditure, which showed the positive correlations (r=0.525, 0.409, 0.624, 0.414, respectively,P=0.002, 0.02, ＜0.001, 0.019).Conclusions Precise determination of REE and energy intake guided by REE are the important guarantees for the reasonable nutrition support in the elderly.

Objective To observe the clinical effect of combination of Tongxiening granule and Mesalazine on treating mild and moderate ulcerative colitis(UC) .Methods Totally 380 patients with mild‐to‐moderate UC diagnosed through endoscopy were allocated to the control group (n=190) and observation group(n=190) .For the observation group ,patients were remedied with the combination of Tongxiening Granules and the Mesalazine by oral administration for eight weeks ,meanwhile the control group only received the Mesalazine for eight weeks .The total effective rate of the two groups were statistically analyzed ,and the levels of ser‐um MMP‐2 and MMP‐9 before and after treatment in the two groups were measured .The expression of S100A12 and RAGE were detected by immunohistochemistry SP method .Results The total effective rate of the observation group and the control group was 94 .74% and 89 .47% respectively ,and the difference was statistically significant(P<0 .01) .After treatment ,the expression levels of MMP‐2 and MMP‐9 in the two groups were decreased ,additionally the expression levels in the observation group was lower than those in the control group ,and the difference was statistically significant (P< 0 .01) .After treatment ,the expression levels of RAGE and S100A12 in the observation group were decreased ,and there was a significant difference when compared with the control group(P<0 .01) .Conclusion Combined application of Tongxiening Granules and Mesalazine in treating patients with mild‐to‐mod‐erate UC could better improve clinical symptoms and bring better therapeutic effect than single use of Mesalazine .

Objective To analyze the distribution and subcellular localization of NOR1 alternative splice isoforms in human tissues and cell lines. Methods NOR1 open reading frame(ORF) was amplified from human fetal brain cDNA library and subcloned into pCMV/myc vector. The level of NOR1 mRNA in human tissues was determined by real-time RT-PCR. Region spanning exon 2 was amplified from cDNA or genomic DNA by specific primers and sequenced. The expression plasmids of NOR1 were transfected into cells and immunofluoresence assay was performed to determine the subcellular localization of NOR1 protein isoforms in human cells. Results The expression of NOR1 mRNA was high in human adult testis, moderate in human fetus nasopharynx, trachea, brain, and kidney tissues, and weak or undetectable in other tissues. Two splice variants of human NOR1 gene resulted from alternative splicing at exon 2 were identified. Both of isoform 1 and isoform 2 mRNA were detected in human fetus brain. Isoform 2 was the sole isoform in other tissues but brain. Only isoform 2 mRNA was detected in cell lines used in this study, though no genomic deletion of exon 2 could be found in all these cell lines. Immunofluoresence assay showed both isoform 1 and isoform 2 proteins were distributed in cytoplasm. Conclusion Alternative splice isoforms of tumor suppressor gene NOR1 are identified. NOR1 isoform 1 and isoform 2 are both detected in fetus brain. NOR1 isoform 2 lacking of exon 2 is the sole isoform in multiple tissues except for brain. The exon 2 encoded peptide does not affect the subcellular location of NOR1 protein.

Objective To explore the relationship between heart rate variability (HRV) and deceleration capacity (DC) in children with idiopathic ventricular premature contraction of different origins. Methods The clinical data from 155 children with idiopathic ventricular premature contraction were retrospectively analyzed. According to the age, the children were divided into young children group (

OBJECTIVETo determine the roles of mitochondrial apoptosis and energy metabolism in hepatocytes during the pathogenic process of acute renal failure (ALF) by assessing disease-related differential activities of several key mitochondrial enzymes, including citrate synthase (CS), carnitine palmitoyltransferase-1 (CPT-1) and cytochrome c oxidase (COX).

METHODSThirty-two male Sprague Dawley rats were given D-galactosamine followed by and lipopolysaccharide (LPS) to induce acute liver failure and sacrificed after 4 (4 h group), 8 (8 h group) 12 (12 h group) and 24 hours (24 h group) of treatment. Eight unmodeled rats served as controls. Effects related to apoptosis were evaluated by pathological analysis of hepatic tissues and TUNEL staining. Ultrastructural changes in mitochondria were assessed by electron microscopy. The activity and expression of CS, CPT-1 and COX were measured.

RESULTSHepatocyte apoptosis was present in the 4 h treatment group and was increased obviously in the 8 h treatment group. Hepatocyte necrosis was first observed in the 12 h treatment group and was significantly higher in the 24 h treatment group, with inflammatory cell invasion. Ultrastructural changes in mitochondria were present in the 4 h treatment group, and the 24 h treatment group showed mitochondria with completely destroyed outer membranes, which resulted in mitochondrial collapse. Activity and protein expression of CS, CPT-1 and COX were increased in the 4 h group (vs. controls), were at their peak in the 8 h group (CS:t =1.481, P less than 0.01; CPT-1:t =2.619, P less than 0.05; COX:t =1.014, P less than 0.01) and showed a decreasing trend in the 12 h group. In addition, the activities of CS, CPT-1 and COX were enhanced at the stage of hepatocyte apoptosis, suggesting that these enzymes were involved in the initiation and development of ALF.

CONCLUSIONEnergy metabolism plays an important role in hepatocyte apoptosis during ALF.

Animals ; Apoptosis ; Carnitine O-Palmitoyltransferase ; metabolism ; Citrate (si)-Synthase ; metabolism ; Disease Models, Animal ; Electron Transport Complex IV ; metabolism ; Hepatocytes ; cytology ; enzymology ; Liver Failure, Acute ; metabolism ; pathology ; Male ; Mitochondria ; ultrastructure ; Rats ; Rats, Sprague-Dawley

OBJECTIVE: To explore the effect of the oxidored nitro domain containing protein 1 (NOR1) gene knockdown on the biological behavior of HeLa cells in cervical carcinoma. METHODS: The recombinant plasmids pSUPER-shNOR1-1, pSUPER-shNOR1-2 and pSUPERscramble, which targeted to NOR1 gene, were constructed by pSUPER.neo+GFP vector, transfected into HeLa cells respectively using Lipofectamine 2000 reagent, and followed by G418 selection. The expression level of NOR1 mRNA and protein were determined by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium (MTT) assay was performed to determine the growth curve of cell viability. The stable transfectants were treated with H₂O₂ and cell apoptosis was determined by Hoechst 33258 staining and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) assay. The expression levels of Bcl-2, cleaved caspase 9 and poly ADP-ribose polymerase (PARP) were measured by Western blot. RESULTS: NOR1- knockdown HeLa cells were successfully constructed by transfection of pSUPER-shNOR1-1 or pSUPER-shNOR1-2 plasmids into HeLa cells. MTT assay showed that the silence of endogenous NOR1 in HeLa cells could lead to the increase in cell viability and proliferation, and the inhibition of H₂O₂-induced apoptosis compared with the negative control. Western blot showed that the expression level of active caspase 9 and cleaved PARP was inhibited in NOR1-knockdown cells when they were treated with H₂O₂ while the expression level of Bcl-2 protein increased. CONCLUSION Silence of endogenous NOR1 facilitates the cell viability and growth of HeLa cells, and attenuates HeLa cells apoptosis induced by H₂O₂, which might be mediated by up-regulation of Bcl-2 level and down-regulation of the cleaved caspase 9 cascade.
Apoptosis ; Caspase 9 ; metabolism ; Cell Survival ; Down-Regulation ; Gene Knockdown Techniques ; Genetic Vectors ; HeLa Cells ; Humans ; Hydrogen Peroxide ; Membrane Transport Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; Transfection ; Up-Regulation

OBJECTIVE: To optimize the induction condition of human NOR1 gene expression in E.coli. and purify NOR1 recombinant proteins. METHODS: A full-length cDNA of human NOR1 was inserted into the corresponding region of pET28b expression vector to yield recombinant prokaryotic expression vector pET28b-NOR1. The prokaryotic expression vector pET28b-NOR1 was introduced into the bacterial host E.coli Rosettablue(DE3). Recombinant NOR1 protein was induced at different conditions. Induction condition was optimized to obtain high yield of recombinant protein. At last, the recombinant NOR1 protein was purified by Ni-IDE chromatography resin. RESULTS: Recombinant NOR1 protein was induced by IPTG in a dose-dependent manner. Increase of kanamycin concentration and induction temperature resulted in high yield of recombinant protein. The most recombinant protein was found in inclusion bodies. The recombinant His-NOR1 protein was purified with Ni-IDE chromatography resin under denature condition. CONCLUSION IPTG, kanamycin concentration and temperature can affect the expression of recombinant NOR1 protein in pET28b system. High yield of recombinant NOR1 protein is achieved by inducing 1 mmol/L IPTG and 200 μg/mL kanamycin at 37 degree. Recombinant His-NOR1 protein with high purity is purified.
Base Sequence ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Isopropyl Thiogalactoside ; pharmacology ; Membrane Transport Proteins ; biosynthesis ; genetics ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Tumor Suppressor Proteins ; biosynthesis ; genetics