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1.
Chinese Medical Journal ; (24): 1835-1844, 2016.
Article in English | WPRIM | ID: wpr-251294

ABSTRACT

<p><b>BACKGROUND</b>Amyloid β (Aβ) has been established as a key factor for the pathological changes in the brains of patients with Alzheimer's disease (AD), and cellular senescence is closely associated with aging and cognitive impairment. However, it remains blurred whether, in the AD brains, Aβ accelerates the neuronal senescence and whether this senescence, in turn, impairs the cognitive function. This study aimed to explore the expression of senescence-associated genes in the hippocampal tissue from young to aged 5XFAD mice and their age-matched wild type (WT) mice to determine whether senescent neurons are present in the transgenic AD mouse model.</p><p><b>METHODS</b>The 5XFAD mice and age-matched wild type mice, both raised from 1 to 18 months, were enrolled in the study. The senescence-associated genes in the hippocampus were analyzed and differentially expressed genes (DEGs) were screened by quantitative real-time polymerase chain reaction. Cognitive performance of the mice was evaluated by Y-maze and Morris water maze tests. Oligomeric Aβ (oAβ) (1-42) was applied to culture primary neurons to simulate the in vivo manifestation. Aging-related proteins were detected by Western blotting analysis and immunofluorescence.</p><p><b>RESULTS</b>In 5XFAD mice, of all the DEGs, the senescence-associated marker p16 was most significantly increased, even at the early age. It was mainly localized in neurons, with a marginal expression in astrocytes (labeled as glutamine synthetase), nil expression in activated microglia (labeled as Iba1), and negatively correlated with the spatial cognitive impairments of 5XFAD mice. oAβ (1-42) induced the production of senescence-related protein p16, but not p53 in vitro, which was in line with the in vivo manifestation.</p><p><b>CONCLUSIONS</b>oAβ-accelerated neuronal senescence may be associated with the cognitive impairment in 5XFAD mice. Senescence-associated marker p16 can serve as an indicator to estimate the cognitive prognosis for AD population.</p>


Subject(s)
Alzheimer Disease , Metabolism , Amyloid Precursor Protein Secretases , Genetics , Metabolism , Amyloid beta-Peptides , Metabolism , Amyloid beta-Protein Precursor , Metabolism , Animals , Aspartic Acid Endopeptidases , Genetics , Metabolism , Brain , Metabolism , Cells, Cultured , Cellular Senescence , Genetics , Physiology , Cognition , Physiology , Cognition Disorders , Metabolism , Disease Models, Animal , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neurons , Metabolism , Pathology , Real-Time Polymerase Chain Reaction
2.
Chinese Medical Journal ; (24): 2845-2852, 2016.
Article in English | WPRIM | ID: wpr-230869

ABSTRACT

<p><b>BACKGROUND</b>Amyloid β (Aβ) deposits and the endoplasmic reticulum stress (ERS) are both well established in the development and progression of Alzheimer's disease (AD). However, the mechanism and role of Aβ-induced ERS in AD-associated pathological progression remain to be elucidated.</p><p><b>METHODS</b>The five familial AD (5×FAD) mice and wild-type (WT) mice aged 2, 7, and 12 months were used in the present study. Morris water maze test was used to evaluate their cognitive performance. Immunofluorescence and Western blot analyses were used to examine the dynamic changes of pro-apoptotic (CCAAT/enhancer-binding protein homologous protein [CHOP] and cleaved caspase-12) and anti-apoptotic factors (chaperone glucose-regulated protein [GRP] 78 and endoplasmic reticulum-associated protein degradation-associated ubiquitin ligase synovial apoptosis inhibitor 1 [SYVN1]) in the ERS-associated unfolded protein response (UPR) pathway.</p><p><b>RESULTS</b>Compared with age-matched WT mice, 5×FAD mice showed higher cleaved caspase-3, lower neuron-positive staining at the age of 12 months, but earlier cognitive deficit at the age of 7 months (all P < 0.05). Interestingly, for 2-month-old 5×FAD mice, the related proteins involved in the ERS-associated UPR pathway, including CHOP, cleaved caspase-12, GRP 78, and SYVN1, were significantly increased when compared with those in age-matched WT mice (all P < 0.05). Moreover, ERS occurred mainly in neurons, not in astrocytes.</p><p><b>CONCLUSIONS</b>These findings suggest that compared with those of age-matched WT mice, ERS-associated pro-apoptotic and anti-apoptotic proteins are upregulated in 2-month-old 5×FAD mice, consistent with intracellular Aβ aggregation in neurons.</p>


Subject(s)
Alzheimer Disease , Metabolism , Amyloid beta-Peptides , Metabolism , Animals , Apoptosis , Physiology , Blotting, Western , Caspase 12 , Metabolism , Endoplasmic Reticulum Stress , Physiology , Frontal Lobe , Metabolism , Heat-Shock Proteins , Metabolism , Immunohistochemistry , Mice , Mice, Transgenic , Neurons , Metabolism , Transcription Factor CHOP , Metabolism , Ubiquitin-Protein Ligases , Metabolism , Unfolded Protein Response , Physiology
3.
National Journal of Andrology ; (12): 53-56, 2015.
Article in Chinese | WPRIM | ID: wpr-319542

ABSTRACT

<p><b>OBJECTIVE</b>To identify the predictors of the positive results of transrectal ultrasound (TRUS)-guided biopsy for prostate cancer.</p><p><b>METHODS</b>We performed univariate and multivariate logistic regression analyses on the relevant data on 385 male patients that underwent TRUS-guided biopsy for prostate cancer, including such potential predictors as age, body mass index (BMI), symptoms, results of digital rectal examination (DRE), tPSA, fPSA, free/total PSA ratio (f/tPSA), prostate volume (PV), and PSA density (PSAD) for identification of the risk factors related to the positive rate of biopsy. Then we constructed a scoring system as a tool for predicting prostate cancer in repeat biopsies and determined the sensitivity of the system by calculating the false positive rate using the receiver operating characteristic curve.</p><p><b>RESULTS</b>Among the 385 patients, 139 (36.1%) were diagnosed with prostate cancer. On multivariate analysis, age (P < 0.01), DRE (P < 0.01), tPSA (P < 0.01), fPSA (P < 0.01), f/tPSA (P < 0.01), PV (P < 0.01), and PSAD (P < 0.01) were all significant predictors of prostate cancer. Multivariate logistic regression analysis showed age, tPSA, f/tPSA, PV, and PSAD to be independent predictors, with ORs and 95% CIs of 1.07 (1.05-1.16), 1.05 (1.02-1.15), 0.97 (0.86-0.99), 0.98 (0.87-0.96), and 1.79 (1.48-2.06), respectively. Moreover, patients with the risk score of 3-5 had a significantly higher rate of prostate cancer than those with 0-2 (64% vs 11%, P < 0.001).</p><p><b>CONCLUSION</b>The scoring system on the key predictors of prostate cancer can help urologists to identify the men in need of prostatic biopsy.</p>


Subject(s)
Aged , Digital Rectal Examination , Humans , Image-Guided Biopsy , Methods , Logistic Models , Male , Middle Aged , Prostate , Pathology , Prostate-Specific Antigen , Prostatic Neoplasms , Chemistry , Pathology , ROC Curve , Risk Factors , Ultrasonography, Interventional , Methods
4.
Article in Chinese | WPRIM | ID: wpr-236337

ABSTRACT

<p><b>OBJECTIVE</b>Eplets mismatch based on HLAMatchmaker software evaluates the clinical application of kidney transplantation.</p><p><b>METHODS</b>In 239 cases of renal transplant,merits of methods of the traditional HLA six antigen matcheing criteria, cross reaction groups standard and Eplets mismatch based on HLAMatchmaker standard were compared respectively.</p><p><b>RESULTS</b>The number of mismatchs with three methods in 239 cases, were grouped according to low-high mismatchs. The results revealed that HLAMatchmaker algorithm could significantly increase the number of low mismatchs group 54 (22.6%), compared with the HIA group 19(7.9%) and CREGs group 32 (13.4%). The comparison was discovered statistical significance among the three groups (P<0.001), so the comparison between each group was.</p><p><b>CONCLUSION</b>HLAMachmaker of donor-recipients matching, is a more efficient, time-saving and high sensitivity matching solution to allograft renal transplantation.</p>


Subject(s)
Adolescent , Adult , Aged , Female , Histocompatibility Testing , Methods , Humans , Kidney Transplantation , Male , Middle Aged , Software , Transplantation, Homologous , Young Adult
5.
Chinese Journal of Virology ; (6): 410-414, 2013.
Article in Chinese | WPRIM | ID: wpr-339936

ABSTRACT

To establish a fluorescent quantitative PCR method (FQ-PCR) with TaqMan probe for simultaneous detection of polyomavirus (BKV) and cytomegalovirus (CMV) and to evaluate its clinical application in the renal transplantation recipients. The conservative sequences of BKV and CMV were targeted and amplified by nested PCR technique. The PCR products were cloned into the plasmids pcDNA3. 1(+). The recombinant plasmid containing target sequences of BKV and CMV were constructed as external standards. The TaqMan-based assay was optimized. For evaluating the assay, the sensitivity was determinated by diluted standard (5 X 103-10icopies/mL), and the specificity was verified by negative control and positive control, and the precision was assessed by intra-assay coefficient of variation (ICV) through detecting standard repeatedly (20 times). A total of 480 blood samples of renal transplantation recipients were used to detect BKV and CMV DNA simultaneously with FQ-PCR, and the concentrations of FK506 were measured by ELISA. The association of DNA copy and concentrations of FK506 was analyzed. The cloned target BKV and CMV DNA was confirmed by sequencing and analysis. The sensitivity of the FQ-PCR assay reached 5 X 103 copies/ml in detecting BKV or CMV DNA. Control DNA verified the assay specifically detecting target DNA. The precision of the assay to quantif target DNA copies was acceptable (Intra-assay CV was 3.44% for BKV and 2.23% for CMV; Inter-assay CV was 4. 98% for BKV and 3.76% for CMV;). Of 480 samples, 130 samples (27. 08%) were CMV DNA positive, significantly higher than the BKV DNA positive (13.33%, 64/480, P<0.05). The positive BKV or CMV DNA was found to be associated with high concentrations of FK506 (P<0. 05). In conclusion, the developed real-time PCR assay for detecting both CMV and BKV DNA simultaneously was s high sensitive, precise and time-effectiveand could be applied in the monitoring of the CMV and BKV infection in the renal transplantation recipients.


Subject(s)
Adolescent , Adult , Aged , Conserved Sequence , Cytomegalovirus , Genetics , Cytomegalovirus Infections , Diagnosis , Virology , DNA, Viral , Blood , Female , Humans , Immunosuppressive Agents , Blood , Kidney Transplantation , Male , Middle Aged , Polyomavirus , Genetics , Polyomavirus Infections , Diagnosis , Virology , Real-Time Polymerase Chain Reaction , Methods , Reproducibility of Results , Sensitivity and Specificity , Species Specificity , Tacrolimus , Blood , Time Factors , Tumor Virus Infections , Diagnosis , Virology , Viral Load , Young Adult
6.
Chinese Journal of Cardiology ; (12): 870-875, 2013.
Article in Chinese | WPRIM | ID: wpr-356477

ABSTRACT

<p><b>OBJECTIVE</b>To explore the impact and related mechanisms of stromal cell-derived factor-1α (SDF-1α) on serum deprivation-induced apoptosis of cardiac stem cells (CSCs).</p><p><b>METHODS</b>CSCs were isolated from adult mouse heart tissue and cultured in vitro. Obtained cells were purified using magnetic-activated cell sorting (MACS) with c-kit magnetic beads. C-kit(+)CSCs were divided into five groups: normal control group, serum deprivation group, serum deprivation+SDF-1α group, serum deprivation+SDF-1α+AMD3100 group, serum deprivation+SDF-1α+LY294002 group. Cell apoptosis was assessed using the DeadEnd Colorimetric TUNEL System and flow cytometry analyses with an Annexin V-FITC Apoptosis Detection Kit. The viability of CSCs was assessed by CCK-8. The protein expression of Bcl-2 and phosphorylated Akt were detected by Western blot. The caspase-3 activity was determined using caspase-3 Colorimetric Assay Kit.</p><p><b>RESULTS</b>After magnetic separation, more than 85% of cardiosphere derived cells were positive for c-kit expression. Compared with the normal control group, the apoptosis rate of serum deprivation group was significantly increased[(27.03 ± 0.80)% vs. (1.51 ± 0.54)%, P < 0.01], which could be significantly reduced by SDF-1α in a concentration dependent manner and peak effect was seen with 100 ng/ml SDF-1α[(10.67 ± 1.06)% vs. (27.03 ± 0.80)%, P < 0.01]. The expressions of p-Akt and Bcl-2 were significantly increased and the activity of caspase-3 was significantly decreased in serum deprivation+SDF-1α group compared to serum deprivation group (P < 0.01). Further more, the expression of p-Akt and Bcl-2 were significantly decreased and the activity of caspase-3 was increased in both serum deprivation+SDF-1α+AMD3100 group and serum deprivation+SDF-1α+LY294002 group compared to serum deprivation+SDF-1α group (P < 0.01).</p><p><b>CONCLUSIONS</b>SDF-1α reduces serum deprivation induced CSCs apoptosis via modulating PI3K/Akt signaling pathway.</p>


Subject(s)
Animals , Apoptosis , Caspase 3 , Metabolism , Cells, Cultured , Chemokine CXCL12 , Pharmacology , Culture Media , Chemistry , Mice , Myocardium , Cell Biology , Proto-Oncogene Proteins c-akt , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Signal Transduction , Stem Cells
7.
Article in Chinese | WPRIM | ID: wpr-326266

ABSTRACT

<p><b>OBJECTIVE</b>We aimed to establish a quantified method for the 17 phthalate acid esters (PAE) in edible vegetable oil by gas chromatography-mass spectrometry (GC-MS) with the pretreatment of acetonitrile extraction and silica/N-(n-propyl)ethylenediamine (silica/PSA) mixed solid phase extraction column and evaluated the PAE of 25 edible oil samples from supermarkets in Hangzhou city.</p><p><b>METHODS</b>The internal standard solution (D4-DEHP) was added in edible vegetable oil sample. The analytes were extracted by acetonitrile with 1 min vortex, and centrifuged at 3050×g for 5 min. The supernatant was then cleaned with silica/PSA column, and eluted with acetonitrile. The elution was dried with N2 flow at 50°C and diluted to 1.0 ml with hexane. Then, 17 PAE were tested by GC-MS and quantified with internal standards. The repeatability and sensitivity of the assay were evaluated. PAE were then determined in 25 plastic buckets of edible vegetable oil from supermarkets in Hangzhou city.</p><p><b>RESULTS</b>By the quantification of internal standard of D4-DEHP, a good linearity range of related 17 PAE was observed. The correlation coefficient was 0.994-1.000 and the standard lowest quantified level was 0.05-0.15 µg/ml. The spiking recoveries of 17 PAE were 78.3%-108.9% with the RSD of 4.3%-12.1% (n=6). The method detection limits were 0.1-0.2 mg/kg. In 25 plastic buckets of edible vegetable oil from Hangzhou, DMP, DEP, DIBP, DBP and DEHP were detected at the range of <0.1-1.8 mg/kg and the detection rates were 12% (3/25), 24% (6/25), 100% (25/25), 96% (24/25) and 100% (25/25), respectively. Other 12 PAE was not detected. For DBP with the level of <0.1 to 1.3 mg/kg, the results of 16% (4/25) samples exceeded the regular migrating limit of 0.3 mg/kg. For DEHP of <0.2-1.8 mg/kg, the data of 12% (3/25) samples were beyond the regular migrating limit of 1.5 mg/kg.</p><p><b>CONCLUSION</b>The pretreatment by silica/PSA mixed solid phase extraction column can satisfy the PAE determination requirements in edible vegetable oils. The DMP, DIBP, DEP, DBP and DEHP were detected from the survey of 25 edible oil samples in Hangzhou city.</p>


Subject(s)
Esters , Gas Chromatography-Mass Spectrometry , Methods , Phthalic Acids , Plant Oils
8.
Article in Chinese | WPRIM | ID: wpr-266120

ABSTRACT

<p><b>OBJECTIVE</b>To observe the ethyl carbamate concentrations in different commercial fermented foods in Hangzhou in 2010.</p><p><b>METHODS</b>In 2010, 237 commercial fermented food samples of eight categories, including yellow wine, white spirit, wine, beer, cooking wine, sauce, vinegar and fermented bean curd, were purchased from 3 different size markets respectively in Hangzhou. The ethyl carbamate was measured by gas chromatography-mass spectrometry in selection ion mode, after the samples were coupled with D5-ethyl carbamate, and purified by diatomite solid phase extraction column.</p><p><b>RESULTS</b>The results showed that ethyl carbamate was detected in all samples analyzed (100%) with the range from 2.0 µg/kg to 515.0 µg/kg. The ethyl carbamate average (median) levels in 8 food categories were descending with fermented red bean curd (182.2 µg/kg (161.2 µg/kg)), yellow wine (159.6 µg/kg (121.0 µg/kg)), cooking wine (86.8 µg/kg (95.6 µg/kg)), white spirit (72.0 µg/kg (60.5 µg/kg)), soy sauce (47.2 µg/kg (40.7µg/kg)), vinegar (26.7 µg/kg (31.8 µg/kg)), wine (15.7 µg/kg (16.8 µg/kg)) and beer (2.2 µg/kg (2.3 µg/kg)).</p><p><b>CONCLUSION</b>The ethyl carbamate was detected in all fermented foods in Hangzhou in 2010, and the levels of ethyl carbamate in red bean curd and yellow wine were higher than others.</p>


Subject(s)
Acetic Acid , Alcoholic Beverages , Beer , China , Fermentation , Food Analysis , Food Contamination , Food Inspection , Gas Chromatography-Mass Spectrometry , Methods , Urethane , Wine
9.
Acta Pharmaceutica Sinica ; (12): 853-859, 2010.
Article in Chinese | WPRIM | ID: wpr-354564

ABSTRACT

This study is to explore whether the Wnt/beta-catenin signaling pathway is involved in the process of tripchlorolide (T4) protecting against oligomeric Abeta(1-42)-induced neuronal apoptosis. Primary cultured cortical neurons were used for the experiments on day 6 or 7. The oligomeric Abeta(1-42) (5 micromol x L(-1) for 24 h) was applied to induce neuronal apoptosis. Prior to treatment with Abeta(1-42) for 24 h, the cultured neurons were pre-incubated with T4 (2.5, 10, and 40 nmol x L(-1)), Wnt3a (Wnt signaling agonists) and Dkk1 (inhibitors) for indicated time. Then the cell viability, neuronal apoptosis, and protein levels of Wnt, glycogen synthase kinase 3beta (GSK3beta), beta-catenin and phospho-beta-catenin were measured by MTT assay, TUNEL staining and Western blotting, respectively. The result demonstrated that oligomeric Abeta(1-42) induced apoptotic neuronal cell death in a time- and dose-dependent manner. Pretreatment with T4 significantly increased the neuronal cell survival and attenuated neuronal apoptosis. Moreover, oligomeric Abeta(1-42)-induced phosphorylation of beta-catenin and GSK3beta was markedly inhibited by T4. Additionally, T4 stabilized cytoplasmic beta-catenin. These results indicate that tripchlorolide protects against the neurotoxicity of Abeta by regulating Wnt/beta-catenin signaling pathway. This may provide insight into the clinical application of tripchlorolide to Alzheimer's disease.


Subject(s)
Amyloid beta-Peptides , Toxicity , Animals , Apoptosis , Cell Survival , Cells, Cultured , Cerebral Cortex , Cell Biology , Diterpenes , Pharmacology , Female , Fetus , Glycogen Synthase Kinase 3 , Metabolism , Glycogen Synthase Kinase 3 beta , Neurons , Cell Biology , Neuroprotective Agents , Pharmacology , Peptide Fragments , Toxicity , Phenanthrenes , Pharmacology , Phosphorylation , Plants, Medicinal , Chemistry , Pregnancy , Rats , Rats, Sprague-Dawley , Signal Transduction , Tripterygium , Chemistry , Wnt Proteins , Metabolism , beta Catenin , Metabolism
10.
Chinese Medical Journal ; (24): 1133-1138, 2010.
Article in English | WPRIM | ID: wpr-352604

ABSTRACT

<p><b>BACKGROUND</b>Familial hypercholesterolemia (FH) is an autosomal disorder associated with elevated plasma low density lipoprotein (LDL) levels leading to premature coronary heart disease (CHD). As a result of long-term hyperlipemia, FH patients will present endarterium thickening and artherosclerosis. In the present study we scanned the related gene of a clinically diagnosed autosomal genetic hypercholesterolemia family for the possible mutations and established eukaryotic expression vector of mutation of proprotein convertase subtilisin/kexin type 9 (PCSK9) gene with gene recombination technique to investigate the contributions of the variation on low density lipoprotein receptor (LDL-R) metabolism and function alternation.</p><p><b>METHODS</b>Mutation detection was conducted for LDL-R, apolipoprotein B(100) (apoB(100)) and PCSK9 gene with nucleotide sequencing in a Chinese FH family. The full-length cDNA of wild type PCSK9 gene (WT-PCSK9) was obtained from Bel-7402. Site mutagenesis was used to establish the recombinant eukaryotic expression vector carrying pathogenic type of PCSK9 gene and the inserted fragment was sequenced. With the blank vector as control, liposome transfection method was used to transfect the Bel-7402 cells with recombinant plasmid. The expression of LDL-R mRNA was examined by RT-PCR. PCSK9 and the expression of LDL-R protein were determined by Western blotting.</p><p><b>RESULTS</b>The G-->T mutation at the 918 nucleotide of PCSK9 gene resulted in the substitution of the arginine by a serine at the codon 306 of exon 6. After sequencing, it was confirmed that the inserted fragment of established expression vector had correct size and sequence and the mutant was highly expressed in Bel-7402 cells. There was no significant variation in the levels of LDL-R mRNA. LDL-R mature protein was decreased by 57% after the cells were transfected by WT-PCSK9 plasmid. Mature LDL-R was significantly decreased by 12% after the cells were transfected by R306S mutant as evidenced by gray scale scanning, suggesting that the new mutant R306S can significantly decrease the expression of mature LDL-R protein.</p><p><b>CONCLUSIONS</b>A novel missense mutation of PCSK9 gene, R306S, was found and the eukaryotic expression vectors of mutant and wild-type of PCSK9 gene were established. There was no significant variation in the levels of LDL-R mRNA. The R306S mutation could significantly lead to the decrease of LDL-R mature protein expression, which might be the pathogenic gene of the FH family.</p>


Subject(s)
Adolescent , Adult , Female , Humans , Hyperlipoproteinemia Type II , Genetics , Lipids , Blood , Male , Mutation , Pedigree , Proprotein Convertase 9 , Proprotein Convertases , Serine Endopeptidases , Genetics
11.
Article in Chinese | WPRIM | ID: wpr-329534

ABSTRACT

Objective To develop a Simple,accurate,rapid,economic,large-scale detection method for the detection of singe nucleotide polymorphisms (SNPs) metabolic enzymes,using polymerase chain reaction with confronting two-pair primers (PCR-CTPP).Methods The primers of CYP1A1 (A4889G),EPHX1 (A416G) and NQO1 (C609T) were designed for PCR-CTPP,and the PCR conditions were optimized.The results of genotyping were verified by DNA sequencing.The above SNPs were detected by the PCR-CTPP detection method in a randomly selected 183 healthy individuals of Han ethnicity.The genotype frequencies were analyzed and compared with people from other ethnicities.Results The allele-specific bands of CYP1A1 (A4889G),EPHX1 (A416G) and NQO1 (C609T) were successfully amplified by PCR-CTPP under the optimal conditions and the results of genotyping were consistent with DNA sequencing.Among 183 healthy Han individuals,the genotypic distributions of CYP1A1 (A4889G),EPHX1 (A416G) and NQO1 (C609T) showed that the wild-type,homozygous variants,and heterozygotes were 103 (56.3%),8 (4.4%),72 (39.3%) and 142 (77.6%),4 (2.2%),37(20.2% ),60(32.8% ),32 (17.5%),91 (49.7%) respectively.The distributions of genotypes were all in accordance with the Hardy-Weinberg equilibrium (P>0.05),with statistical differences and with other ethnic populations(P<0.05).Conclusion The SNPs of metabolic enzymes can be detected by PCR-CTPP method which is simple,accurate,rapid,economic and with large scale.PCR-CTPP can be used for large scale clinical and epidemiological screening.

12.
Article in Chinese | WPRIM | ID: wpr-356282

ABSTRACT

<p><b>AIM</b>To explore the possible mechanism of tert-butyl hydroperoxide (t-BHP)-induced apoptosis in murine MIN6 pancreatic beta-cells.</p><p><b>METHODS</b>MIN6 cells were cultured in vitro. Cell damage was evaluated by epifluorescence microscopy after staining with AO-EB. The percentage of cell apoptosis was determined by flow cytometric assay after Annexin- V-PI staining. Nitric oxide levels were measured by Griess assay. Inducible nitric oxide synthase(iNOS) protein and NF-kappaBp65 fragment were detected by Western blot.</p><p><b>RESULTS</b>Exposure of 25 micromol/L t-BHP to MIN6 cells for 60 min, cell viability was reduced and the percentage of apoptosis was increased significantly. The levels of cytoplasmic iNOS protein and nitrite were elevated. Meanwhile, treatment with t-BHP resulted in nucleus NF-kappaBp65 fragment peaking at 20 min. Both L-NAME and N-Acetyl-l-cysteine (NAC) attenuated the elevated levels of nitrite and percentage of apoptosis due to t-BHP alone.</p><p><b>CONCLUSION</b>NF-kappa-iNOS-nitric oxide signalling pathway can mediated t-BHP induced apoptosis in MIN6 cells .</p>


Subject(s)
Animals , Apoptosis , Cell Line , Insulin-Secreting Cells , Cell Biology , Mice , NF-kappa B , Metabolism , Nitric Oxide , Metabolism , Nitric Oxide Synthase Type II , Metabolism , Oxidative Stress , Physiology , Signal Transduction , tert-Butylhydroperoxide , Pharmacology
13.
Acta Pharmaceutica Sinica ; (12): 690-694, 2008.
Article in Chinese | WPRIM | ID: wpr-277811

ABSTRACT

To explore the effect of glucagon-like peptide-1 receptor agonist--Exendin-4 (Ex-4) on murine MIN6 pancreatic beta-cells apoptosis induced by oxidative stress, the morphological changes of cell damage were evaluated by epifluorescence microscopy after staining with AO-EB. The percentage of cell apoptosis was determined by flow cytometric assay after Annexin-V-FITC-PI staining. Nitric oxide level was measured by Griess reagent assay. Inducible nitric oxide synthase (iNOS) protein and NF-kappaBp65 fragment were detected by Western blotting. Ex-4 inhibited the increase of nitrite level and percentage of apoptosis induced by t-BHP in MIN6 cells. Furthermore, Ex-4 partly reduced the expression of iNOS protein and the ratio of NF-kappaBp65 protein in nucleus:cytosol induced by t-BHP. These results suggest that Ex4 protects MIN6 pancreatic kappa-cells from oxidative stress-induced apoptosis via down-regulation of NF-kappaB-iNOS-nitric oxide pathway.


Subject(s)
Animals , Apoptosis , Down-Regulation , Glucagon-Like Peptide-1 Receptor , Hypoglycemic Agents , Pharmacology , Incretins , Insulin-Secreting Cells , Cell Biology , Metabolism , Lizards , Mice , Nitric Oxide , Metabolism , Nitric Oxide Synthase Type II , Metabolism , Oxidative Stress , Peptides , Pharmacology , Receptors, Glucagon , Signal Transduction , Transcription Factor RelA , Metabolism , Venoms , Pharmacology , tert-Butylhydroperoxide , Pharmacology
14.
Chinese Medical Journal ; (24): 776-781, 2008.
Article in English | WPRIM | ID: wpr-258593

ABSTRACT

<p><b>BACKGROUND</b>Familial hypercholesterolemia (FH), caused by low density lipoprotein (LDL) receptor (LDL-R) gene mutations, is associated with increased risk of premature coronary heart disease. Until now, limited molecular data concerning FH are available in China. The present study described the clinical profiles and cell biological defects of a Chinese FH kindred with novel LDL-R gene mutation.</p><p><b>METHODS</b>The patient's LDL-R gene coding region was sequenced. The patient's lymphocytes were isolated and the LDL-R expression, binding and up-take functions were observed by immunohistochemistry staining and flow cytometry detection. The patient's heart and the major large vessels were detected by vessel ultrasound examination and myocardial perfusion imaging (MPI).</p><p><b>RESULTS</b>The patient's LDL-R expression, LDL binding and up-take functions were significantly lower than normal control (39%, 63% and 76% respectively). A novel homozygous 1439 C-->T mutation of the LDL-R gene was detected in the patient and his family. ECG showed atypical angina pectoris. Echocardiogram showed stenosis of the coronary artery and calcification of the aortic valve and its root. Blood vessel ultrasound examination showed the thickness of large vessel intima, and the vessel lumen was narrowed by 71%. MPI showed ischemic changes.</p><p><b>CONCLUSIONS</b>The LDL-R synthesis dysfunction of FH patients leads to arterial stenosis and calcification, which are the major phenotype of the clinical disorder. The mutation of the LDL-R gene is determined. These data increase the mutational spectrum of FH in China.</p>


Subject(s)
Adult , Child, Preschool , Homozygote , Humans , Hyperlipoproteinemia Type II , Genetics , Middle Aged , Mutation , Receptors, LDL , Genetics , Physiology
15.
Acta Pharmaceutica Sinica ; (12): 1179-1185, 2008.
Article in Chinese | WPRIM | ID: wpr-232621

ABSTRACT

Extracts of Chinese herb Tripterygium wilfordii Hook. f. (TWHF) have been found to have potent anti-inflammatory and immunosuppressive functions and widely used in China for treatment of rheumatoid arthritis. Also they have been considered to be the potential drugs in the treatment of tumor and acute graft rejections. With the progress of neuroimmunological research on neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson disease (PD) and multiple sclerosis (MS), the neuroprotective strategies to rescue neurons from immunological injury are currently being explored. Recently, studies have indicated that extracts of TWHF such as triptolide, tripchlorolide and (5R)-5-hydroxytriptolide are able to attenuate progression of these neuroimmunologic disorders in vitro and in vivo. Accumulating evidence has shown that they can promote neuronal survival and neurite growth and facilitate functional recovery of brain injury by invoking distinct mechanisms that are related to their neuroprotective roles as inhibitor of neuroinflammatory toxicity of activated-microglia, antioxidants, calcium channel blockers, neurotrophic actions, modulating T cell functions, inhibitor of transcriptional activation of NF-kappaB on genes and signaling. Significant pharmaceutical strategies against neuroimmunologic disorders will hopefully be discovered by understanding the valuable components of TWHF.


Subject(s)
Animals , Anti-Inflammatory Agents , Pharmacology , Antioxidants , Pharmacology , Diterpenes , Pharmacology , Therapeutic Uses , Drugs, Chinese Herbal , Pharmacology , Therapeutic Uses , Epoxy Compounds , Pharmacology , Therapeutic Uses , Humans , NF-kappa B , Metabolism , Neurodegenerative Diseases , Drug Therapy , Phenanthrenes , Pharmacology , Therapeutic Uses , Structure-Activity Relationship , Tripterygium , Chemistry
16.
Article in English | WPRIM | ID: wpr-359384

ABSTRACT

We investigated the effects of fish protein hydrolysate (FPH) on growth performance and humoral immune response of the large yellow croaker (Pseudosciaena crocea R.). One thousand and two hundred large yellow croakers [initial average weight: (162.75+/-23.85) g] were divided into four groups and reared in floating sea cages (3 m x 3 m x 3 m). The animals were fed with 4 diets: basal diet only (control) or diets supplemented with 5%, 10% and 15% (w/w) FPH. The results show that dietary FPH levels significantly influenced the growth and immunity of the large yellow croaker. Compared with the control group, total weight gain (TWG) in all treatment groups, relative weight gain (RWG) and specific growth rate (SGR) in fish fed with diets supplemented with 10% and 15% FPH were significantly increased (P<0.05). Similar results were observed in immune parameters [lysozyme activity, serum complements, immunoglobulin M (IgM)]. Lysozyme activity, complement C4 and IgM were also significantly increased (P<0.05) in fish fed with diets supplemented with 10% and 15% FPH, while complement C3 level was significantly increased (P<0.05) in all treatment groups. In general, with the supplementation of FPH, particularly at dose of 10%, the growth performance and immunity of the large yellow croaker can be improved effectively.


Subject(s)
Administration, Oral , Animals , Antibody Formation , Allergy and Immunology , Dietary Supplements , Fish Products , Gadiformes , Metabolism , Perciformes , Allergy and Immunology , Protein Hydrolysates
17.
Acta Pharmaceutica Sinica ; (12): 828-832, 2007.
Article in Chinese | WPRIM | ID: wpr-268571

ABSTRACT

This study is to explore the effect of ginsenoside Rb1 on the process of beta-amyloid peptide(25-35) (Abeta(25-35)) -induced hyperphosphorylation of tau protein, and on the level of cyclin-dependent kinase 5 activator, p25/p35. Western blotting and/or immunocytochemical staining were used to detect the levels of phosphorylation of tau protein at the sites of Thr205, Ser396, Ser404 in hippocampal neurons, cdk5 and p25/p35. After exposure to Abeta(25-35) (20 micromol x L(-1)) for 12 h, the levels of tau protein phosphorylation at the sites of Thr205, Ser396, Ser404 were enhanced, the level of p25 was increased, but the level of protein cdk5 was not changed markedly. Pretreatment with ginsenoside Rb1 reduced Abeta(25-35) -induced hyperphosphorylation of tau protein and decreased the lever of p25, but had no effect on cdk5. Ginsenoside Rb1 can attenuate Abeta(25-35) -induced hyperphosphorylation of tau protein through CDK5 signal pathway.


Subject(s)
Amyloid beta-Peptides , Animals , Cyclin-Dependent Kinase 5 , Metabolism , Fetus , Ginsenosides , Pharmacology , Hippocampus , Cell Biology , Nerve Tissue Proteins , Metabolism , Neurons , Metabolism , Panax , Chemistry , Phosphorylation , Plants, Medicinal , Chemistry , Rats , Rats, Sprague-Dawley , Signal Transduction , tau Proteins , Metabolism
18.
Article in Chinese | WPRIM | ID: wpr-640320

ABSTRACT

Objective To screen the mutation of certain gene of a 10-years-old boy with multiple xanthomas and very high level of cholesterol who could be diagnosed as homozygous familial hypercholesterolemia (FH),to explore the relationship between the genotype and phenotype,and to discuss the molecular pathologic mechanism.Methods The basic information of life styles were asked from the boy and his familial members.The blood was drown to examine the lipid and genes.The boy was examined with electrocardiogram examination,ultrasonography and coronary CT angiography (CTA) to evaluate the degree of atherosclerosis.Peripheral blood DNA of the boy and his parents were extracted by phenol-chloroform method and investigated for mutations of promoter and all 18 exons of low density lipoprotein receptor(LDLR) gene.Screening was carried out by using Touch-down polymerase chain reaction (PCR) and single strand conformation polymorphism(PCR-SSCP),combined with DNA sequence analysis.In addition,the apolipoprotein B100 gene(apoB100) for known mutations (R3500Q) which caused familial defective apoB100 was screened by PCR-DNA sequence analysis.Results 1.The level of cholesterol of his parents were higher than the normal.2.Several clinical manifestations of atherosclerosis were detected from that boy.Increased intima-media thickness and plaques were detected in the common carotid artery.Mitral valve regurgitation was found by echocardiography.Coronary stenosis was confirmed by CTA.3.No mutations R3500Q of apoB100 was observed.4.A homozygous mutation in exon13 of the LDLR gene (D601Y) were identified in the boy and his parents harbour D601Y heterozygous mutation due to a single base pair substitution of G for T in the codon for residue 1864.Conclusions The final diagnosis of the boy with multiple xanthomas was homozygous FH.His disease was caused by D601Y homozygous mutation in exon13 of the LDLR gene inherited from his heterozygous parents.

19.
Article in Chinese | WPRIM | ID: wpr-640015

ABSTRACT

Objective To identify mutations site and clinical characteristics of a familial hypercholesterolemia(FH) proband diagnosed clinically through DNA sequencing and family analysis in the proband and his family members of 3 generations.Methods Blood samples and clinical data of the kindred of total 29 from 3 generations members were collected.Proband had a physical examination electrocar-diogrom and vascular ultrasound.The proband and his family members took routine clinical exams,and genomic DNA was isolated.The promoter region and the 18 exons of low density liporotein receptor(LDLR) gene were screened by Touch down polymerase chain reaction -single strand conformation polymorphism(PCR-SSCP) and DNA sequencing.The result of sequencing were matched gene sequence published in the BLAST database.Results 1.Increased intima-media thickness and plaque were detected in the common carotid artery,right subclavian artery of the proband.Aortic valve regurgitation was found by echocardiography.2.No mutation R3500Q of ApoB100 was observed.3.Two heterozygous mutations in exon 10 and 13 of LDLR gene (W462X and A606T) were identified.The proband and 5 members of paternal relatives showed W462X heterozygosis mutation in exon 10 of LDLR gene which introduced the change from tryptophone to a new stop codon.The proband's mother and grandmother harboured A606T heterozygous mutation in exon 13 of LDLR gene due to a single base pair substitution of G for A in the codon for residue 1 879.Conclusions Disease causing mutations of proband are W462X and A606T compound heterozygosis mutation in exon 10 and 13 of LDLR gene inherited from mother and father.Proband shows homozyous phenotype though the genotype analysis indicates heterozygous mutations.

20.
Article in Chinese | WPRIM | ID: wpr-329409

ABSTRACT

<p><b>OBJECTIVE</b>To identify the mutation of low density lipoprotein receptor(LDLR) gene in a large Chinese family with familial hypercholesterolemia(F H) and make a discussion on the pathogenesis of FH at the molecular level.</p><p><b>METHODS</b>Investigations were made on a patient with the clinical phenotype of homozygous FH and his parents for mutations of promoter and all 18 exons of LDLR gene. Screening was carried out using Touch down PCR and a g arose gel electrophoresis, combined with DNA sequence analysis. The results were compared with the normal sequences in GenBank and FH database (www.ucl.uk/fh) t o find the mutation. Then the mutation was identified in other members of the family. In addition, the authors screened the apolipoprotein B(100) (apoB(100)) gene f or known mutations (R3500Q) that cause familial defective apoB(100) (FDB) by PCR-RFLP.</p><p><b>RESULTS</b>A novel homozygous IN III 5' GT --> AT mutation in the splice donor of LDLR intron 3 was detected in the homozygote propositus with FH. The mutation was also identified in four heterozygous carriers in his family. No mutations R3500Q of apoB(100)were observed.</p><p><b>CONCLUSION</b>A homozygous G --> A splice mutation in LDLR gene was first reported. The change of the splice donor in LDLR intron 3 may cause skipping of exon 3, which is responsible for FH. Perhaps it is a particular pathogenesis for Chinese people.</p>


Subject(s)
Adolescent , Adult , Alternative Splicing , Genetics , Base Sequence , Child , China , DNA , Chemistry , Genetics , DNA Mutational Analysis , Female , Homozygote , Humans , Hyperlipoproteinemia Type II , Blood , Genetics , Pathology , Lipids , Blood , Male , Middle Aged , Mutation , Pedigree , Point Mutation , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Receptors, LDL , Genetics
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