To explore the individual roles of G protein β and γsubunit in the interactions with effectors. Methods We investigated the interactions of Gβ1 and Gβ1γ2 with adenylyl cyclase Ⅱ(ACⅡ) us ing the yeast two-hybrid and three-hybrid systems. Results When assayed for the abilities to activate the reporter gene, the interactions among AD-β1, γ2 and BD-AC ⅡQ in the three-hybrid system were more po tent than the interactions between AD-β1 and BD-AC ⅡQ in the two-hybrid system. The expressions of BD-AC ⅡQ and AD-β1 in transformants coexpressed AD-β1 and BD-AC ⅡQ, and transformants coexpressed AD-β1, γ2 and BD-AC ⅡQ were respectively detected. The comparisons between the reporter activity and the expression levels of BD-AC ⅡQ and AD-β1 in the yeast cells show there was no correlations, i.e.The difference in the reporter activity was not a reflection of differential expression level of the hybrid pro teins. Conclutions All these results suggest that G protein β1 subunit is sufficient to maintain the basic in teraction between Gβ1γ2 and AC ⅡQ, and γ2 subunit plays an important role in the high affinity interaction of Gβ1γ2 with ACⅡQ.

Objective We selected baculovirus expression vector system to express human NT-4 with biological activity. Methods The hNT-4 mature peptide-coding sequence is amplified by polymerase chain reaction (PCR), ligated to baculovirus expression vector PacGP67B, and expressed in the insert sf9 cell line. Results The protein molecular weight of the expressed hNT-4 was about 15000 by SDS-PAGE,and the hNT-4 antibody could react with this protein in the infected supernatant and total cell by western-blot . The activity of hNT-4 determined by PC12 cell line was definite. Conclusions The results may aid for studying the effect of the hNT-4 on basic medical research and clinical application.

Objective To isolate and identify exosomes from serum samples of the patients with polymyositis / dermatomyositis (PM/ DM),and analyze their protein composition preliminarily.Methods Exosomes from serum samples of the patients with PM/DM were isolated and purified by the ExoQuickTM kit.The morphological characteristics and particle size of exosomes were determined by transmission electron microscope (TEM) and NanoSight analyzer,respectively.The surface markers of exosomes such as CD9,CD81 and Flotillin-2 were identified by western blot.The concentration and composition of exosome protein were determined by the BCA method and SDS-PAGE,respectively.Results The exosomes from serum samples of PM/DM patients displayed round or oval vesicles with membrane structure under TEM,and their diameter range was about (92 ± 67) nm.western blot showed that these exosomes expressed CD9,CD81 and Flotillin-2.The total protein concentrations of exosomes in the patients with PM/DM and healthy controls were 14.68 (6.00,32.55) μg/μL and 14.09 (8.00,23.28) μg/μL,respectively.SDS-PAGE showed that high-abundance proteins enriched in 55-70 kD in both PM/DM patients and healthy controls,and that there were different bands in 40-55 kD between them.Conclusion Exosomes are isolated from serum samples of the patients with PM/DM successfully,and their protein concentration and composition are analyzed preliminarily,which provides the experimental evidences for further finding differential proteins.

Objective To analyze effect of the syphilis prevention and control work among female sex workers(FSWs), and provide reference data for target intervention in Guangdong province.Methods Syphilis prevalence among FSWs from 2009 to 2013 were analyzed statistically based on the data from Guangdong Provincial high-risk groups in sexually transmit-ted infection(STI)control program.Results A total of 34 338 FSWs were detected,including 23 816(69.36%)FSWs at the high-tier venues,8 811(25.66%)middle-tier,and 1 711(4.98%)low tier.The syphilis prevalence of FSWs at high-, middle-,and low-tier venues in 2009 was 2.60%,7.36%,and 10.54% respectively,and in 2013 was 1.45%,2.75%,and 21.88% respectively.Chi-square test showed a downward trend in the prevalence of syphilis among the high and middle tier FSWs in 2009-2013(χ2 =12.807,52.333 respectively,both P <0.001),and remained high among FSWs at low-tier venues(χ2 =0.027,P =0.87).The difference in syphilis prevalence in 2013 among FSWs from different types of venues was statistically significant (χ2 =190.64,P <0.001 ).No significant difference in syphilis prevalence was found among FSWs from Pearl River Delta,eastern,western and northern Guangdong(χ2 =5.24,P =0.07).Conclusion Significant difference is found in prevalence of syphilis among FSWs from different types of settings.FSWs from low-tier settings have a relatively lower syphilis testing rate but with higher infection rate.

Objective To explore the most effective route of mesenchymal stem cell transplantation (MSCs) in D-galactosamine (D-gal) induced porcine model with acute liver failure (ALF) and the potential mechanism.Methods BA-MA mini-pigs with D-gal-induced ALF were transplanted with porcine mesenchymal stem cells (MSCs) through four routes:intraportal injection (InP),peripheral intravenous injection (PV),hepatic intra-arterial injection (AH) and intrahepatic injection (IH).The survival time was recorded.The blood samples before and after MSC transplantation were collected for detecting liver function.Liver histology was interpreted and scored.Hepatic apoptosis and regeneration were detected by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay and Ki-67 assay.The protein expression of cleaved caspase-3,survivin,AKT and ERK were analyzed by Western blot.Results The average survival time in each group was (10.7 ±1.6) days (InP),(6.0 ±0.9) days (AH),(4.7 ±1.4) days (PV),(4.3 ± 0.8) days (IH) when compared with D-gal group [(3.8 ± 0.8) days].The histopathological scores revealed a significantly decrease in InP group (3.17 ± 1.04,P ＜0.05) and AH group (8.17 ± 0.76,P ＜ 0.05) when compared with that in D-gal group (11.50 ± 1.32).The apoptosis rate in InP group (25.0 ± 3.4％,P ＜ 0.05) and AH group (40.5 ± 1.0％,P ＜ 0.05) was lower than that in D-gal group (70.6 ± 8.5％).The expression of active caspase-3 was inhibited,while the expression of survivin,AKT and ERK was elevated in InP group.Conclusions The intraportal injection was superior to other pathways for MSCs transplantation.Intraportal MSC transplantation could improve liver function,inhibit cell apoptosis,promote cell proliferation and prolong the survival in porcine ALF model.

Objective To explore the expressions and significance of miRNA - 101,enhancer of ZESTE homo-log 2(EZH2)and transforming growth factor(TGF)- β1 in the kidneys with complete uniateral ureteral obstruction (CUUO). Methods Thirty male SD rats,(6 ± 1)weeks old,weighted(150 ± 10)g,were divided into sham group, 7 - day group with CUUO and 14 - day group with CUUO by using random number table method,10 rats in each group. The obstructed kidney samples were collected in 7 and 14 days,respectively,for detecting the expression of miRNA -101 by real time - polymerase chain reaction(RT - PCR)and TGF - β1 and EZH2 protein by Western blot,immuno-histochemistry and hematoxylin - eosin staining. Their correlated expressions were analyzed. Results RT - PCR results showed that the expressions of miRNA - 101 in sham group and 7 - day group with CUUO were(12. 69 ± 1. 60)times and(3. 74 ± 1. 24)times which were higher than those of 14 - day group with CUUO,respectively,there was a signifi-cant difference among these 3 groups(P ﹤ 0. 05). The expressions of TGF - β1 and EZH2 proteins were 1. 14 ± 0. 12, 1. 04 ± 0. 14,0. 76 ± 0. 18 and 1. 04 ± 0. 04,0. 89 ± 0. 03,0. 73 ± 0. 02 in 14 - day group with CUUO,7 - day group with CUUO and sham group,respectively. There was a negative correlation between miRNA - 101 with EZH2( r =- 0. 92,P ﹤ 0. 05),and negative correlation with TGF - β1(r = - 0. 63,P ﹤ 0. 05),and positive correlation between EZH2 and TGF - β1(r = 0. 67,P ﹤ 0. 05);the expressions of miRNA - 101,EZH2 and TGF - β1 were associated with each other in obstruction renal in different time periods. Conclusions With the extension of obstruction time, miRNA - 101 expression decreased,EZH2 and TGF - β1 expression increased,evidently,which indicates that the de-velopment of renal interstitial fibrosis may be affected through regulating EZH2 expression of renal obstruction through miRNA - 101 in the young rats.

Objective: To review the systematic reviews of acupuncture for diabetic peripheral neuropathy (DPN) and to provide evidence for clinical decisions. Methods: Published systematic reviews targeting acupuncture treatment of DPN were searched using computer through both Chinese and English databases till July 1, 2019. Two researchers screened the papers based on inclusion and exclusion criteria and conducted report quality evaluation, methodological quality assessment and evidence quality grading using the preferred reporting items for systematic reviews and meta-analyses (PRISMA), assessment of multiple systematic review 2 (AMSTAR 2) and grading of recommendations assessment, development and evaluation (GRADE). Results: Ten systematic reviews were included, involving 11 outcome measures. According to PRISMA, 6 items were sufficiently reported while 1 item was not; AMSTAR 2 appraised that all the included systematic reviews were of low quality in the methodological evaluation; according to GRADE, of the 30 clinical evidences, only 5 were graded moderate while the remained were graded low or extremely low. Descriptive analysis showed that acupuncture can significantly improve DPN symptoms, accelerate the conduction velocities of sensory and motor nerves, and up-regulate the content of plasma nitric oxide (NO), while the adverse reaction rate was low. Conclusion: Acupuncture can produce satisfactory clinical efficacy in treating DPN, but the existing problems, such as low-quality evidence, unitary outcome measures, poor methodological quality of systematic reviews and nonstandard reporting, need to be treated cautiously; meanwhile, more high-quality clinical trials are required to elevate the level of evidence.

Objective To explore the protein factors that could interact with Gβ subunit within the G protein sig nal transducing pathway. Methods The highly sensitive protein-protein interaction system——Yeast Two-hybrid System was applied to screen the human cDNA library with constructed “Bite plasmid” contain ing Gβ subunit gene fragment. And then the faulse positive test was adapted. Results Three positive gene fragments were obtained. One codes for “Actin bundling protein”. The other two are new ones and their GeneBank accession numbers are AF288405 and AF288406 respectively. Conclusions It is the first time to find that among human brain tissue, Gβ subunit becomes a structural or functional unit interacts with actin bundling protein and the other two unknown protein factors to activate the following pathway. This re sult may be important to understand the relationship between the actin cytoskeleton and G proteins.

This article reviews the resultsof the basic research about epidermal growth factor and its receptor,and the development of the novel drug, EGF eyedrop, that containing chemically synthesised EGF gene,the construction of EGF expression vector, the transformation of the host cells , the purification of the recombinant protein EGF, the preparation of three batches of the EGF product and identification, the preclinical and clinical trials. Relevant studies show that recombinant EGF consisting of 51 amino acids can be secreted into the medium under the control of the α factor leading sequence in the yeast cells.The EGF can accelerate the growth of corneal-limbal epithelial cells and the healing of an alkali burned corneal. The EGF can be used in curing oral cavity ulcer and skin burned wound. And it has the preventive effects on experimental duodenal ulcer of rat. The antiserum was made for test of the con- centration of blood EGF and urine EGF by RIA. Data from studies demonstrate the inhibition effect of EGF on the growth of tumor cells, such as A431 and BT325 cells in the presence of high EGF concentration (＞10 ng/ml). The expression of EGFR and DNA ploidy in renal carcinoma has clinical significance. Crystallization and preliminary x-ray diffraction studies of the EGF has been made. The MW of the EGF product is 6000, and the pI is about 4. 6 and it has correct N-terminal amino acids sequences , immunogenicity and biological activity. There is no vestige of the DNA of the yeast cells. Animal experiments reveal that there is no cumulation of the EGF in the body, and EGF can promote corneal epithelial healing. There is no toxicological effect during cornea wound healing of rabbit. A randomized, double-blind, placebo-controlled, multi-center clinical trial was conducted in four hospitals to assess safety,ocular tolerance and efficacy of an ophthalmic solution of EGF for 200 cases of cornea transplantation and 247 cases of nebulae. Unequivocal results were obtained as the eyedrop really accelerate the wounded cornea healing. So, the EGF eyedrop as a novel drug of class I is approved by the National Drug Administration, and this is the first gene engineering drug that come from yeast expression system in China.

To analyze the reasons of impaired vision after LASlK and explore its preventive treatment measures preliminarily.METHODS: ln this retrospective study, 175 eyes of 134 patients whose vision was decreased after LASlK were included. The constituent ratio of every reason was counted and uncorrected visual acuity ( UCVA ) between pre-treatment and post-treatment were compared by paired t-test respectively.RESULTS:The overall incidence of impaired vision after LASlK was 1. 86%. The constituent ratio of regression was 51. 43% and UCVA increased from 0. 61±0. 22 to 0. 90±0. 38 (t=8. 00, P<0. 001) after treatment. The constituent ratio of punctate corneal epithelial defect was 32. 57% and UCVA increased from 0. 60±0. 19 to 1. 20±0. 24 (t=20. 00, P<0. 001 ) after treatment. The constituent ratio of accommodative spasm was 5. 14% and UCVA increased from 0.76±0. 21 to 1. 32±0. 22 (t=8. 14, P<0. 001) after treatment. The constituent ratio of corneal flap shift and gauffer was 4% and UCVA increased from 0. 29 ± 0. 26 to 1. 24 ± 0. 28 ( t = 6. 33, P<0. 001 ) after treatment. The constituent ratio of corticosteroid - induced ocular hypertension was 4% and UCVA increased from 0. 57±0. 05 to 1. 0 ± 0. 16 ( t= 2. 53, P<0. 05 ) after treatment. The constituent ratio of fundus lesions and diffuse lamellar keratitis ( DLK) was 2. 86% and UCVA all increased by different degrees after treatment.CONCLUSlON: The reasons of impaired vision after LASlK are many and varied. These cases could recover their vision by discovery and treatment in time, and the appropriate preventive measures were essential.