9α-hydroxy-4-androstene-3,17-dione (9-OH-AD) is an important intermediate in the steroidal drugs production. 3-ketosteroid-9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the transformation of 4-androstene-3,17-dione (AD) into 9-OH-AD specifically. In the present study, the putative KshA and KshB genes were cloned from Mycobacterium smegmatis mc(2)155 and Gordonia neofelifaecis NRRL B-59395 respectively, and were inserted into the expression vector pNIT, the co-expression plasmids of kshA-kshB were obtained and electroporated into Mycobacterium sp. NRRL B-3805 cells. The recombinants were used to transform steroids, the main product was characterized as 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), showing that kshA and kshB were expressed successfully. Different from the original strain Mycobacterium sp. NRRL B-3805 that accumulates 4-androstene-3,17-dione, the recombinants accumulates 9α-hydroxy-4-androstene-3,17-dione as the main product. This results indicates that the putative genes kshA, kshB encode active KshA and KshB, respectively. The process of biotransformation was investigated and the results show that phytosterol is the most suitable substrate for biotransformation, kshA and kshB from M. smegmatis mc(2)155 seemed to exhibit high activity, because the resultant recombinant of them catalyzed the biotransformation of phytosterol to 9-OH-AD in a percent conversion of 90%, which was much higher than that of G. neofelifaecis NRRL B-59395. This study on the manipulation of the ksh genes in Mycobacterium sp. NRRL B-3805 provides a new pathway for producing steroid medicines.
Androstenedione ; analogs & derivatives ; biosynthesis ; Bacterial Proteins ; genetics ; metabolism ; Biotransformation ; Ketosteroids ; Mixed Function Oxygenases ; genetics ; metabolism ; Mycobacterium ; metabolism ; Mycobacterium smegmatis ; enzymology ; Plasmids

Background Aniridia is a rare congenital hereditary eye disease.Studies determined that PAX6 gene mutation is closely associated with congenital aniridia,but the mutation locus are varied.Objective This study was to identify virulence mutation locus of PAX6 gene of a Chinese family pedigree with autosomal dominant aniridia.Methods A Chinese family affected with autosomal dominant aniridia was collected and examined in Affiliated First Hospital of Zhengzhou University in August 2014.Periphery blood of 10 ml was collected from all the families and 100 unrelated health controls.The genomic DNA was extracted by standardized phenol-chloroform method,and all exons and splicing junctions of PAX6 were amplified by PCR.Real-time fluorescence quantitative PCR was performed to examine the relative expression of PAX6 mRNA in patients and normal phenotype families and heahh controls.This study protocol was approved by Ethic Committee of Affiliated First Hospital of Zhengzhou University and complied with Helsinki Declaration.Written informed consent was obtained from subjects or custodian before any medical examination.Results This Chinese family inclued 3 generations and 9 members,with a classic autosomal dominant inheritance mode.Five patients were found,showing the absence of iris and cataract in 3 adult patients and only absence of the iris in 2 children,and other 4 members showed the normal phenotype.A novel heterozygous PAX6 deletion mutation c.796 del G (p.A266 fs) (GenBank ID:KP255960) in exon 10 was exclusively found in all affected individuals but not in any of the unaffected families or unrelated health controls.PAX6 mRNA level in lymphocytes was about 50％ lower in aniridia patients than in unaffected family members,indicating that this mutation caused nonsense-mediated mRNA decay.Conclusions A novel deletion mutation in PAX6 gene results in an abnormal PAX6 COOH-terminal extension in the Chinese aniridia family.This finding expands the mutation spectrum of PAX6 gene.

Objective To investigate the expression of microRNA-224 in HepG2 cells and analyze its target genes to reveal its role in the carcinogenesis of hepatoma. Methods The genes with differential expression in HepG2 cells and LO2 cells were obtained by gene expression microarray analysis. The up-regulated target genes of microRNA-224 were predicted by bioinformatics method, and their functions were analyzed. Results Compared with LO2 cells, microRNA-224 was highly expressed in HepG2 cells. A total of 264 target genes of microRNA-224 were predicted, including genes involved in cell cycle, signal transduction, cell differentiation, proliferation and apoptosis. Conclusions MicroRNA-224 is highly expressed in HepG2 cells. MicroRNA-224 plays an important role in the carcinogenesis of hepatoma via regulating the expression of its target genes directly or indirectly.

Objective:To explore the relationship between pulmonary function and coronary artery disease (CAD) with the severity of coronary artery lesions in relevant patients.
Methods:A total of 200 patients received coronary angiography (CAG) in our hospital were studied. The patients were divided into 2 groups: Non-CAD group, n=88 and CAD group, n=112. The degree of coronary stenosis was assessed by GENSINI score;the pulmonary function, echocardiography and fasting blood level of brain natriuretic peptide(BNP) were examined in all patients.
Results:Forced expiratory volume in 1 second (FEV1) in CAD group (2.33±0.54) L/1s was lower than Non-CAD group (2.63±0.39) L/1s, P=0.04. Multivariate logistic regression analysis indicated that decreased FEV1 was the independent risk factor for CAD (OR=2.9, 95%CI 1.89-4.23, P<0.01). Spearman correlation analysis showed that FEV1 was negatively related to blood level of BNP (r=-0.54, P<0.01), positively related to the ratio of E/A (r=0.27, P=0.03). GENSINI score was positively related to smoking (r=0.31, P=0.01), diabetes (r=0.19, P=0.03) and negatively related to FEV1 (r=-0.40, P<0.01). With adjusted variables, partial correlation analysis presented that FEV1 was negatively related to GENSINI score (r=-0.21, P=0.01).
Conclusion:Decreased FEV1 is not only related to CAD occurrence, but also related to the degree of coronary stenosis in relevant patients.

[ ABSTRACT] AIM: To explore the relationship between FRAS 1 protein and brain metastases of non-small cell lung cancer (NSCLC).METHODS:The mRNA expression of FRAS1 in the brain metastatic tumor tissues and primary tumor tissues of NSCLC was detected by qPCR .The protein expression of FRAS 1 in the tumor tissues and normal tissues adjacent to tumor tissues of NSCLC was measured by SP method of immunohistochemistry .The protein expression of FRAS 1 in NSCLC primary tumor tissues with or without brain metastases was also determined .RESULTS:The mRNA expression of FRAS1 in the brain metastatic zone was nearly 10 times higher than that in the primary tumor tissues , and there was sig-nificant difference between the 2 groups (P<0.05).FRAS1 protein was expressed in the NSCLC primary tumor tissues , but was not found in the normal tissues adjacent to primary tumor tissues .The protein expression of FRAS 1 in the NSCLC with brain metastases was significantly higher than that without brain metastases ( P<0.01 ) .CONCLUSION: FRAS1 protein may be associated with the occurrence of NSCLC .The over-expression of FRAS1 protein may be related to brain metastases with NSCLC .

Based on IHE technical framework, this paper proposes the HIS and RIS/PACS integration strategy in combination with our hospital's practice. And it has been proved to be efficient in our practice.
Hospital Information Systems ; Integrated Advanced Information Management Systems ; Systems Integration

Objective　To investigate the effect of cardiopulmonary bypass (CPB) on vascular endothelial cell injury and plasma endothelin-1 and nitric oxide equilibrium in patients undergoing cardiovascular operation with CPB. Methods　A total of 20 patients with congenital heart disease (Group Ⅰ) and 20 with valvular problem (group Ⅱ) were operated on under CPB respectively. Blood samples were collected from central vein before skin incision, before CPB, 30 min after CPB, at the end of CPB, and end of operation, the first morning and third morning after operation. The levels of plasma thrombomodulin(TM), endothelin-1(ET-1) and nitric oxide(NO) were measured. Results　The plasma TM level was significantly elevated during CPB (P<0.01, P<0.05) and 1 d after operation, reached its peak as (4.88±1.12) ng/ml in Group Ⅰand (8.34±1.84) ng/ml in group Ⅱ at the end of surgery and came back to the level as before operation. The plasma level of ET-1 was also increased significantly after CPB and reached peak as (129.04±22.29) in Group Ⅰ and (156.62±29.66) in Group Ⅱ at the end of operation. And the level was still higher than before operation in 2 groups 3 d after operation. No change was found on the level of NO in 2 groups. Conclusion　CPB may cause extensive acute endothelial cells damage for about 24-48 h and recovered about 72 h and it may also cause an imbalance of ET-1 and NO.

·AIM: To analyze the clinical manifestation of Alport syndrome, especially the ocular features.·METHODS: The physical, ophthalmologic and audiologic examination results of thirty two patients with Alport syndrome were analyzed retrospectively.·RESULTS: Thirty (93.7%) patients had some family history. All patients had renal disease: eighteen(56.3%) patients with chronic renal failure, four(12.5%) patients with renal insufficiency, and the other ten(31.3%) patients with hematuria. Twenty (62.5%) patients had sensorineural deafness. Thirteen (40.6%) patients had ocular deformity, five(15.6%) patients had typical ocular changes: three patients with anterior lenticonus, and two patients with macular flecks.·CONCLUSION: Ocular anomalies are not requisite for the diagnosis of Alport syndrome. But its typical ocular features should be recognized by the ophthalmologists which supports the diagnosis.

AIM To observe the effects of Zuogui Pills and Yougui Pills on the proliferation of BMSCs and the expression of apoptosis after osteogenic and adipogenic differentiation in ovariectomized rats.METHODS Of sixty female SD rats,forty rats followed by bilateral ovariectomy were randomly divided into ovariectomy group (1.0 g/kg distilled water),Zuogui Pills group (9.45 g/kg Zuogui Pills) and Yougui Pills group (10.26 g/kg Yougui Pills) and Bujiale group (0.09 mg/kg estradiol valerate),another ten rats as control group (1.0 g/kg distilled water),ten rats bilateral excision of a small amount of fat around the ovary was treated as sham operation group (1.0 g/kg distilled water).After 12 weeks of administration,the rats were killed,BMSCs were cultured in vitro.Cell proliferation was detected by MTT assay.Western blot was used to detect the protein expressions of Caspase-3 and Bcl-2.RE-SULTS MTT assay showed that the proliferation of BMSCs were promoted in Zuogui and Yougui Pills groups,and the proliferation effect in Zuogui Pills group was better than that in Yougui Pills group.After osteogenic differentiation,as compared with the control group,the Caspase-3 expression of ovariectomy group was up-regulated (P ＜0.05) and the expression of Bcl-2 was down-regulated (P ＜0.01).As compared with ovariectomy group,the expression of Caspase-3 was decreased and the expression of Bcl-2 was up-regulated in Zuogui Pills group and Yougui Pills group.After adipogenic differentiation,as compared with the control group,the expressions of Caspase-3 were significantly up-regulated and Bcl-2 were down-regulated in the ovariectomy group,Zuogui Pills group and Yougui Pills group (P ＜ 0.05).After osteogenic and adipogenic differentiation Zuogui Pills group and Yougui Pills group were significant different (P ＜ 0.05).CONCLUSION Zuogui Pills and Yougui Pills both can inhibit the apoptosis of BMSCs after osteogenic and adipogenic differentiation in ovariectomized rats.Zuogui Pills can promote BMSCs osteogenesis differentiation while Yougui Pills can promote adipogenic differentiation.

Objective To explore the efficacy of excision of pterygium combined with fresh amniotic membrane transplantation for treatment of recurrent pterygium.Methods Pteryga of 27 patients(32 eyes) were excised and transplanted with fresh amniotic membrane.Patients were followed up for 6～36 months.Results Pterygium recurred in only 2 eyes during the period of follow-up.The curative rate of the operation for recurrent pterygium was 93.75%,and the recurrence rate was 6.25%.Conclusion Excision of pterygium combined with fresh amniotic membrane transplantation is an effective therapeutic method for recurrent pterygium.