Objective To investigate the effects of leptin on the oxidative damage in human retinal pigment epithelial (RPE) cells.Methods Human RPE cells (ARPE-19) were cultured in vitro,and randomly divided into control group and insulin resistance group.RPE cells were treated with 0,10,100 ng/mL leptin for 24,48,72 hours respectively.Then the levels of reactive oxygen species (ROS) expression in RPE cells were detected by 2',7'-dichlorofluorescin-diacetate (DCFH-DA),and the levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG) expression in RPE cells were observed by immunocytochemistry (ICC),and the levels of human 8-oxoguanine DNA glycosylase 1 (hOGG1) expression in lysate were measured by Western blot.Results After 24,48,72 hours,the level of ROS (Control group:F=37.136,37.178,49.634; P＜0.05.Insulin resistance group:F=9.822,28.881,71.150;P＜0.05),8-OHdG (Control group:F =88.643,390.920,1039.276; P ＜ 0.05.Insulin resistance group:F =273.311,299.155,82.237;P＜0.05) and hOGGl (Control group:F=470.062,1073.113,295.456;P＜0.05.Insulin resistance group:F =240.032,592.389,527.760 ; P＜0.05) expression increased significantly with the increase of leptin concentration in control group and insulin resistance group.Under the same leptin concentration,the level of 8-OHdG has a trend that it was higher in the insulin resistance group than the control group.After 24 hours,the difference of hOGGl expression between control group and insulin resistance group was not significant (F=23.392,P＞0.05).After 72 hours,the level of hOGGl expression was significantly higher in the insulin resistance group than the control group (F=129.394,P＜0.05).The level of hOGGl expression was significantly higher at 48 hours than that at 24 hours and 72 hours (P＜ 0.05).Conclusion Leptin could induce the oxidative damage of RPE cells in normal and insulin resistance status.With the increase of leptin concentration and time extended,the degree of oxidative damage and its repair were both increased.The degree of oxidative repair increased with the increase of leptin concentration,but decreased with time extended.

Porphyromonas gingivalis is a specific causative agent of human chronic periodontitis. This anaerobe produce collagenase PrtC encoded by prtC. To constructed the prokaryotic expression system for the prtC gene from P.gingivalis so as to be used to explore antigenicity and immunoreactivity of prtC gene product as well as the relationship between the local level of PrtC and the periodontitis damage, the entire length of prtC genes fragment from the ATCC-33277 and 47A-1 strains of P.gingivalis was amplified by PCR. After T-A cloning and sequencing, the prokaryotic expression system for prtC was constructed by using pET32a plasmid and E.coli BL21DE3 strain. The expression of the target recombinant PrtC protein was induce with different concentrations of IPTG. Western blot assay was used to detect the antigenicity and immunoreactivity of PrtC protein, and ELISA assay was used to detect the PrtC level in the subgingival plaque samples of patients with chronic periodontitis. The experimental results showed that the nucleotide sequences of the prtC genes from ATCC-33277 and 47A-1 strains of P.gingivalis were entirely identical, and the sequence similarities of nucleotides and amino acids were 98.46% and 99.07% respectively. Under the induction of different concentration of IPTG, the output of recombinant expressed product PrtC may reach up to 50% of the total bacterial proteins. It was also proved that the recombinant PrtC could bind with antibody against the whole cell of P.gingivalis, and could induce the production of specific antibodies in rabbit. In 91.39% of the subgingival plaque samples, the PrtC could be detectable, in which the level of positive rates of detection was higher in severe cases of chronic periodontitis than that in the mild cases. So far, a prokaryotic expression system for the prtC gene from P.gingivalis with high expression efficiency was successfully constructed in the present study, and the expressed product PrtC possesses well antigenicity and immunoreactivity, suggesting the possibility to be used as the candidate antigen for developing the serological kit and P.gingivalis vaccine.

In the present study, the fusion gene lipL32/1-lipL41/2 from Leptospira interrogans was constructed by using PCR with linking primers, expressed in prokaryotic expression system. And the immuno-reactivity of its expressed product was determined. Meanwhile, SDS-PAGE and BioRad agarose image analyzer system was used to examine the expression output of the target recombinant protein rLipL32/1-LipL41/2, and the immune-reactivity of this recombinant protein was identified by Western blot assay. ELISA was used to detect the level of antibodies against the recombinant proteins in sera of patients with leptospirosis. It was demonstrated that the percentages of similarity in nucleotide and the putative amino acid sequences of the fusion gene lipL32/1-lipL41/2 with the corresponding sequences previously reported were 99. 9% and 98. 9%respectively. The expression output of the target recombinant protein was approximately 10% of the total bacterial proteins.Both the rabbit antisera against rLipL32/1 and rLip41/2 could recognize and combine to the recombinant fusion protein rLip32/1-Lip41/2 as well. The positive rates of antibodies in 228 leptospirosis patients with the recombinant proteins rLip32/1,rLip41/2 and rLip32/1-Lip41.2 were 97.4%, 78. 5% and 99. 1% respectively. The results of the present study leads to the conclusion that the fusion gene lipL32/1-lipL41/3 with its prokaryotic expression system is successively constructed and the expressed recombinant protein shows excellent immuno-reactivity, thus suggesting that it may be used as the antigenic candidate for the development of the leptospiral genus-specific engineering vaccine and for the preparation of diagnostic kits.

Maple syrup urine disease (MSUD) is an autosomally recessively inherited disorder of branched-chain amino acid (BCAA) metabolism caused by the defective activity of branched-chainα-ketoacid dehydrogenase complex (BCKD). The disease is characterized by severe ketoacidosis, mental retardation, and neurological impairments. So far, application of tandem mass spectrom-etry and HPLC has allowed newborn screening and early detection, but some patients with variant forms of the disorder will escape detection. Liver transplantation is an effective treatment. However, the rareness of liver donation limits the liver transplantation. In this review, the screening, diagnosis and therapy of MSUD will be discussed.

objective To determine the existence of virulence-associated invA gene in different genospeeies of Leptospira interrogans reference strains in China.and to understand the alterations of invA gene transcription and expression of L.interrogans strain Lai before or after infecting cells.Methods PCR was applied to detect the invA gene of four L.interrogans strains belonging to four different genospecies and L.biflexa strain Patoc Ⅰ.The entire invA genes from the L.interrogans strains were cloned and then sequenced.The prokaryotic expression system of invA gene of L.interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai was constructed.Using Ni-NTA affinity chromatography,the target recombinant protein rInvA was extracted and purified.Rabbits were immunized with rInvA to obtain antiserum and the titer of antiserum was determined by immunodiffusion test.A model of L interrogans strain Lai infecting human embryo kidney cell line HEK293 was established to detect the alterations of invA gene transcription and expression of the leptospiral strain before or after infecting the host cells by real-time fluorescent quantitative RTPCR and western blot assay.Results All the four tested L.interrogans strains had invA gene whereas L.biflexa strain Patoc Ⅰ not.The similarity of nucleotide and putative amino acid sequences of invA genes from the four L.interrogans strains belonging four different genospecies were 99.33%-100%and 98.66%-100%,respectively.The constructed prokaryotic expression system could efficiently express rInvA and the immunodiffusion titer of rabbit anti-rInvA serum was 1:16.After L.interrogans strain Lai infecting HEK293 cells for 30 min or above,the microbe could adhere the surface of the cells.On the 30 min after the infection,the mRNA level of invA gene of L.interrogans strain Lai was remarkably upregulated,and on the 45 min after infection the mRNA level presented a peak value and then graduated decreased.On the 45 min or 60 min after L.interrogans strain Lai infecting HEK293 cells,InvA protein could be detectable but before infection or after infection for over 90 min the InvA protein expression was negative.Conclusion The invA gene is a unique gene for pathogenic L.interrogans.The invA gene of L interrogans has characteristics of cell-touched expression and transient expression,which may have a close correlation with adhering and invading host cells.

Animals ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Male ; Myocytes, Cardiac ; metabolism ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley ; Swimming

Objective: To compare the effects of ultra-fast track anesthesia and traditional anesthesia on blood levels of high sensitivity C-reactive protein (Hs-CRP), C-reactive protein (CRP) and procalcitonin (PCT) in patients after pediatric cardiac surgery.
Methods: A total of 101 patients received pediatric cardiovascular surgery by a same anesthesiologist in our hospital from 2013-09 to 2014-05 were retrospectively reviewed. The patients were studied in 2 anesthesia groups:Ultra-fast track group, in which the extubation was conducted in operating room, n=40 and Traditional group, n=44. Blood levels of Hs-CRP, CRP and PCT at pre-operation (T0), 1st day post-operation (T1) and 2nd day post-operation (T2) were compared.
Results: ①Hs-CRP levels were higher at T1 and T2 than T0 in both groups, all P<0.05;Hs-CRP level in Ultra-fast track group was lower than Traditional group at T1 time point, P<0.05. ②CRP levels were similar among 3 time points in Ultra-fast track group;while in Traditional group, CRP level at T2 was higher than T1, P<0.05; CRP level was higher in Traditional group than Ultra-fast track group at T2, P<0.05.③PCT levels at 3 time points were similar in the same group;while PCT level in Ultra-fast track group was lower than Traditional group at T1 time point, P<0.05.
Conclusion: Compared With traditional anesthesia, ultra-fast track anesthesia could decrease the post-operative elevations of Hs-CRP, CRP and PCT in patients after pediatric cardiac surgery.

Objective To construct a ciaR gene-knockout (ΔciaR) mutant of Streptococcus pneu-moniae ( S. pneumoniae) and to investigate the effects of CiaR in CiaH/CiaR, a streptococcal two-component signal-transducing system, on the expression of genes encoding penicillin-binding proteins ( pbps genes) and cia-dependent small RNAs (csRNAs). Methods Electrophoretic mobility shift assay (ESMA) was per-formed to detect the recombinant CiaR (rCiaR)-binding pbps genes. A suicide plasmid pEVP3ciaR for ciaR gene knockout was constructed and then aΔciaR mutant was obtained through homologous recombination and insertion inactivation of the suicide plasmid, and screening with chloromycin. The mutant was identified using PCR and sequencing analysis. E-test was used to detect the minimal inhibitory concentrations ( MIC) of penicillin ( PCN) and cefotaxime ( CTX) against S. pneumoniae strains. Changes and differences in the expression of pbps genes and csRNAs in theΔciaR mutant and its wild-type strain before and after treatment with 1/4 MIC of PCN or CTX were detected using real-time quantitative RT-PCR. Results The rCiaR could bind to the promoter regions in pbp1a, pbp1b and pbp2b genes of S. pneumoniae. The ciaR gene in ΔciaR mutant was inactivated by insertion according to the results of PCR and sequencing analysis. After treatment with 1/4 MIC of PCN or CTX, the expression of pbps genes at mRNA level ( pbps-mRNAs) in theΔciaR mu-tant was significantly increased (P<0. 05), but the levels of csRNAs were significantly decreased (P<0. 05);whereas a significantly decreased pbps-mRNAs (P<0. 05) and increased csRNAs (P<0. 05) were observed in its wild-type strain. The result of E-test showed that the MICs of PCN and CTX against ΔciaR mutant were increased by 250-fold as compared with those against its wild-type strain. Conclusion The CiaR can enhance the drug resistance of S. pneumoniae to PCN and CTX through down-regulating the expres-sion of PBP1a, PBP1b and PBP2b and up-regulating the expression of csRNAs to inhibit the expression of PBPs.

Objective To develop a recognition method of liver steatosis degree on type-B ultrasonic images based on multi-fractal spectrum texture analysis method and pattern recognition. Methods Features of singularity strength width and multi-spectrum area were extracted from the curve of multi-fractal spectrum of each liver ultrasonic images. These two features and the feature of mean intensity ratio comprised a three-dimensional feature vector, which would be classified by BP neural network. Results The classification accuracy was 96.00% for normal liver, 80.00% for mild fatty liver, 88.00% for moderate fatty liver and 92.00% for severe fatty liver. Conclusion Feature vector combined with BP neural network can identify the steatosis degree of liver on the ultrasonic images and can be used as an assistant diagnostic method.

In order to increase antigenicity of H. pylori-specific antigens and decrease the cost of further industrial production, we used a special PCR with linking primers to construct a fusion gene containing H.pylori ureB and hpaA genes and E.coli ltB gene, and to costract its prokaryotic expression system pET42a-ltB-ureB-hpaA-E.coliBL21DE3. The sequencing result indicated the 100% nucleotide sequence homology of the constructed ltB-ureB-hpaA fusion gene compared to those of the original separated genes. Output of the target recombinant protein rLTB-UreB-HpaA was approximate 15% of the total bacterial proteins measured by SDS-PAGE. The rLTB-UreB-HpaA could induce the immunized rabbits to produce specific antibodies with immunodiffusion titer of 1∶8, and could combine to the commercial rabbit antibody against the whole cell of H.pylori as well as rabbit anti-UreB and anti-HpaA sera by using Western bolt assays. Using GM1-ELISA, the ability of rLTB-UreB-HpaA binding to bovine GM1 was confirmed.And rLTB-UreB-HpaA (200 μg per mouse) could prevent 100% of the immunized BaLb/C mice from H.pylori strain SS1 infection. The co-administration with 10 μg rLTB, the rUreB or rHpa could increase its protective rates in the immunized mice from 66.7% to 81.8% and 83.3%, respectively. All these data leads a conclusion that rLTB-UreB-HpaA is a great potential as a practical genetic engineering vaccine to prevent H.pylori infection.