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The angiotensinogen(AGT) expression and angiotensin Ⅱ (AngⅡ ) secretion levels in cultured SD rat mesangial cells were determined. High glucose up-regulated AGT mRNA(0. 29±0.07 vs 0. 20±0. 05,P< 0.05)and protein(0.66±0.23 vs 0.37±0. 15,P<0.05) expression and Ang Ⅱ secretion [(9.85±2.08 vs 7.50± 1. 51) pg/ml,P<0. 05]levels, which were down-regulated by pyrrolidine dithiocarbamate( PDTC) treatment via inhibiting NF-κB activity.
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BACKGROUND: The experimental results showed that insulin sensitivity and glucose uptake in skeletal muscle could be improved by activating adenosine monophosphate-activated protein kinase a2 (AMPKα2). AMPKa2 is expected to become a new physiological and pharmacological target for the prevention and treatment of type 2 diabetes mellitus. OBJECTIVE: To clone human AMPKa2 subunit gene and to construct its wild-type and mutant eukaryotic expression vectors. DESIGN: A single sample observation.TIME AND SETTING: The experiment was performed in the Clinical Molecular Biology Laboratory, the Second Affiliated Hospital of Sun Yet-sen University from April 2007 to January 2008.MATERIALS: QuikChange II Site-Directed Mutagenesis Kit was produced by Stratagene. Eukaryotic expression vector pcDNA3.1(+) and E. coll DH5a were provided by the laboratory. Human skeletal muscle tissue was from patients who received amputation surgery in the Second Affiliated Hospital of Sun Yat-sen University. Informed consent was obtained from the patients, and fresh samples were collected and frozen in liquid nitrogen.METHODS: The human AMPKo2 subunit gone was amplified from human skeletal muscle by RT-PCR, cloned into T vector, and the recombinant plasmid was confirmed by sequencing. In vitro site-directed mutagenesis was carded out with Quickchange site-directed mutagenesis kit. The wild-type and mutant coding genes were subcloned into eukaryotic expression vector pcDNA3.1, and the recombinant plasmids were validated by enzyme digestion and sequencing.MAIN OUTCOME MEASURES: ①The cloning of aim gone; ②site-directed mutagenesis; ③ eukaryotic expression plasmid. RESULTS: The human AMPKα2 subunit gene (about 1700 bp) was successfully cloned, with 99％ homology to the reported AMPK α2 gene. A GenBank accession number was EF056019, The achieved mutation of the 45<'th> Lysine (AAA) was found to Arginine(AGA). The wild-type and mutant pcDNA-AMPKα2 recombinant plasmids were constructed successfully. CONCLUSIONS: The human AMPKα2 subunit gene was cloned successfully from human skeletal muscle and its wild-type and mutant eukeryotic expression vectors were constructed successfully in the experiment.
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The relationship between C936T polymorphism at 3'-untranslated region of vascular endothelial growth factor (VEGF) gene and diabetic nephropathy (DN) was analysed in 194 type 2 diabetic patients. The frequencies of genotype CC and allele C were significantly higher in DN group than those in non-DN group and control group. Allele C and genotype CC of VEGF may be a genetic marker susceptible to DN.
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The study of glomerular mesangial cells of normal rats showed that angiotensin Ⅱ (ATⅡ) down-regulated the expression of MMP-2 mRNA, did not have significant effect on TIMP-2 mRNA. And in consequence, ATⅡ down-regulated the ratio of MMP-2 mRNA to TIMP-2 mRNA.
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OBJECTIVE To establish SYBR Green Ⅰ fluorescent quantitative PCR assay for the detection of West Nile virus(WNV),which could be used for early laboratory diagnosis.METHODS A fragment of WNV gene was amplified by PCR,then cloned into pMD-18 T vector.The combinant plasmid was sequenced and analyzed by means of BLAST program,and used as the positive DNA in place of WNV.The SYBR GreenⅠfluorescent quantitative PCR assay was established based on positive plasmid.The sensitivity and specificity of the assay were performed.RESULTS The combinant plasmid was confirmed by sequencing and the fragment belonged to WNV.Ten copies of WNV RNA were detected by SYBR GreenⅠfluorescent quantitative RT-PCR assay.Results of the other members of Flaviviridae were negative,which indicated this assay was specific for WNV.CONCLUSIONS SYBR GreenⅠfluorescent quantitative PCR assay established in this study is highly sensitive and specific,and so it can be used for early diagnosis of WNV infection.
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BACKGROUND: Nowadays, angiotensin Ⅱ plays an important role in onset of diabetic nephropathy. Therefore, the nuclear factor-κB may have adjustive effects on angiotonin system of kidney tissue of diabetic rats. OBJECTIVE: To observe the relationship of activity of inhibitive nuclear factor-κB with angiotensin Ⅱ and its type 1 receptor mRNA expression of renal tissue of diabetic rats. DESIGN: Completely randomized group design, control experiment. MATERIALS: The experiment was conducted at the Experimental Animal Center, Sun Yat-sen University of Medical Sciences between March and April 2000. Fifty-one pure breed clean grade male Wistar rats were select ed. METHODS: ①Models were established in 39 rats. Streptozotocin dissolv ing in citric acid buffer (0.1 mmol/L,pH=4.5) were given to establish dia betic models with 60 mg/kg intraperitoneal injection. If the fasting blood glucose maintained above 13.9 mmol/L, the establishment of models was successful. The thirty-nine rats were randomly assigned into 3 groups: model group (n=17, without other interventional measure, feeding normally) and pyrrolidine dithiocar2. Bamate (PDTC) (active inhibitor of nuclear fac tor-κB) interventional group [n=22, PDTC at the dose of 20 mg/kg were given with intraperitoneal injection, twice a day]. Other 12 rats were as normal control group, did not make into diabetic models with normal breeding. ②After feeding for 18 weeks kidneys were got in every group. The activity of nuclear factor-κB was detected with electrophoretic mobility shift assay. The expression of type 1 receptor mRNA of angiotensin Ⅱ was measured with reverse transcription polymerase chain reaction (RT-PCR). Contents of angiotonin Ⅰ and angiotensin Ⅱ were tested with Radio Im munoassay (RIA). Activity of rennin was referred to that the result of the level of angiotonin Ⅰ at 37 ℃ water bath subduced to that at 4 ℃. ③Dif ference of measurement data was compared with single factor analysis of variance. After normal transformation, the non-normal distribution data were conducted with statistical disposal. MAIN OUTCOME MEASURES: Comparison of contents of angiotensin Ⅰ and Ⅱ, activities of rennin and nuclear factor-κB and expression of type 1 receptor mRNA of angiotensin Ⅱ in renal tissues of rats of each group. RESULTS: In the normal control group, model group and PDTC interven tional group 1, 6 and 13 rats were dropped out, respectively, so 11, 11 and 9 rats in each group were involved in the result analysis. ①Activity of nu clear factor-κB: It was higher significantly in the model group than that in the normal control group and PDTC interventional group (P ＜ 0.01 ). It was similar between the normal control group and the PDTC interventional group. ②Activity of rennin of renal tissue: It was similar among the 3 groups. ③Content of angiotonin Ⅰ of renal tissue: It was higher obviously in the model group that that in the normal control group and the PDTC interventional group (P ＜ 0.01 ). ④Content of angiotensin Ⅱ in renal tissue: It was similar between the model group and the normal control group. It was lower markedly in the PDTC interventional group than that in the model group and the normal control group (P ＜ 0.01 ). Expression of type 1 receptor mRNA of angiotensin Ⅱ: It was lower remarkably in the model group than that in the normal control group (P ＜ 0.01 ). It was lower dis tinctly in the PDTC interventional group than that in the model group and the normal control group (P ＜ 0.01 ). CONCLUSION: The increase of activity of nuclear factor-κB in renal tissue of diabetic rats can inhibit the activity of nuclear factor-κB, which will induce the reduction of the level of angiotensin Ⅱ and expression of type 1 receptor mRNA of angiotensin Ⅱ in renal tissue of diabetic rats.
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AIM: To evaluate the pulmonary function in patients with type 2 diabetes mellitus in order to identify whether the lung is a target organ of chronic pathologic changes in diabetes mellitus. METHODS: Pulmonary ventilation function and diffusion capacity were studied in 107 patients with type 2 diabetes mellitus and 61 healthy subjects matched for age and sex. Glycosylated hemoglobin (HbA1c), urine albumin excretion rate (AER), fundus examination and nerve conduction velocity were included as parameters of glycemic control and diabetic microangiopathies. RESULTS: Pulmonary ventilation function was similar in type 2 diabetic group and the control. Compared with the control, carbon monoxide diffusion capacity (DLCO) and DLCO corrected by alveolar volume (DLCO/VA) were significantly lower in type 2 diabetic group (P<0.05). DLCO and DLCO/VA were inversely correlated with microangiopathy score (r: -0.291, -0.324, respectively, P<0.01). Furthermore, DLCO/VA was negatively correlated with age and duration of diabetes mellitus (r: -0.269, -0.236, respectively, P<0.05). CONCLUSIONS: Pulmonary ventilation function is normal in patients with type 2 diabetes mellitus, but their diffusion capacity is impaired. It suggests that the lung may also be the target organ of the chronic pathologic changes of diabetes mellitus.
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Objective To study the relationship between large multifunctional proteasome (LMP) 7 gene polymorphism and susceptibility of type 1 diabetes mellitus (DM). Methods The genotyping of LMP7 gene was determined by polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) in 71 type 1 DM patients and 86 healthy persons (as controls). Furthermore, the type 1 DM patients were divided into 3 groups according to the age of diabetic onset. Group A was ≤14 years, group B 15～30 years, group C≥31 years.Results The frequency of LMP7 B/B was decreased significantly (39% vs 58%, P