The gene which encodes (R)-specific carbonyl reductase(rCR) from Candida parapsilosis CCTCC M203011 was cloned.The cloned sequence includes an open reading frame (ORF) consisting of 1011bp, encoding a protein of 336 amino acids, with a molecular weight of 35.9 kD.The nucleotide sequence showed 99% similarity to those of the other members of the alcohol dehydrogenase superfamily.A recombinant Escherichia coli JM109 strain harboring the expression plasmid, produced (R)-1-phenyl-1,2-ethanediol(100%e.e,80.4%yield) from ?-hydroxyacetophenone without any additive to regenerate NAD~ + from NADH.

The gene of mutated TNF-?D4 gene was amplified by overlap PCR and cloned into the prokaryotic expressive vector pBV220.TNF-?D4 contains two changes:substitutions of Pro8Arg,Ser9Lys,Asp10Arg,Ile157Phe,Leu29Ser,Arg31Val and a deletion of the N terminal four amino acids.The recombinant vector pBV220-TNF-?D4 was transformated into E.coli strain DH5?,and the high expression strain was obtained by screening monoclones.The level of expression was about 45% of total cell protein.After purification,the purity of fusion protein was above 90% by HPLC and relative ability was 8 ?107.TNF-?D4 was modificated by mPEG-ButyrALD。After purification,the purity of mPEG-TNF-?D4 was above 85% and relative ability was 8.6?107.The in vivo systemic toxicity of mPEG-TNF-?D4,which is indicated by LD50,is lower than that of rhTNF-?.These results strongly supported for the further study and exploitation of TNF-antitumor drug.

Objective To explore the efficacy of Fushe Jiedu Decoction combined with red light on viper bites injury limb swelling and the effects on the inflammatory cytokines. Methods Totally 90 patients were divided into control group and experimental group by using random number table method, with 45 cases in each group. The wounds of the control group were sterilized and given anti-snake venom serum, antibiotics, and tetanus immunoglobulin and supplemented with energy to correct water and electrolyte disturbances. The experimental group was treated with Fushe Jiedu Decoction based on the treatment of control group,150 mL each time,orally,twice a day; Red light was applied at the site of the most obvious swelling of the injured limb, 20 minutes each time, twice a day. The treatment lasted for 6 d. The swelling of injured limbs, serum C-reactive protein (CRP), 5-hydroxytryptamine (5-HT), histamine, tumor necrosis factor-α (TNF-α), interleukin (IL)-6 levels before and after treatment in the two groups were observed. Results Compared with before treatment, the swelling of the limbs disappeared significantly in the experimental group at 3 d and 6 d and in the control group at 6 d, with statistical significance (P<0.01). Compared with the control group at the same time point, the swelling of the limbs in the treatment group was significantly better than that in the control group at 3 d and 6 d, with statistical significance (P<0.01). Compared with before treatment, the levels of CRP, 5-HT, TNF-α, and IL-6 were significantly lower in the two groups, with statistical significance (P<0.05). After treatment, CRP, 5-HT and histamine in the experimental group were significantly better than that in the control group(P<0.05).Conclusion Fushe Jiedu Decoction combined with red light has good efficacy for viper bites injury limb swelling, which can reduce inflammatory cytokines levels of patients.

Objective To study the single nucleotide polymorphisms (SNP) characteristics of Yersinia pestis strains from different natural foci in China.Methods Genome-wide comparison was done to find SNP sites by the Mummer program among 9 Yersinia pestis genome which was downloaded from NCBI.Then 13 genic fragments including 19 SNP sites were amplified by PCR and sequenced in 133 Yersinia pestis strains,and the results were cluster analyzed with the BioNumerics software.Results Three thousand seven hundred and eighty sequence variation sites were found by genome-wide comparison.Using the different combinations of SNP sites,UPGMA cluster analysis revealed obvious geographic regional and eco-aggregation characteristics of Yersinia pestis strains isolated from China.Conclusions As relatively stable genetic markers,SNP can better reflect the genome characteristics of Yersinia pestis in different plague natural foci of China.

OBJECTIVETo investigate the proliferation effects of arsenic trioxide (As2O3) on salivary adenoid cystic carcinoma-2 (ACC-2) cells in vitro and to study the role of Survivin on the apoptosis of ACC-2 induced by As2O3.

METHODSACC-2 cells were treated with different concentration of As2O3 for different time. The inhibitory effects on cell's viability were assayed with methyl thiazolyl tetrazolium (MTT) test. Apoptosis was determined by flow cytometry. The expression of Survivin mRNA and protein were investigated by reverse transcription-polymerase chain raction (RT-PCR) and Western blot analysis respectively.

RESULTSCell viability after As2O3 treatment was markedly suppressed and exhibited as a dose- and time-dependent pattern. The apoptotic index showed the similar trend. The results of RT-PCR revealed gene expression of Survivin was suppressed significantly. Through Western blot analysis, a negative correlation between concentration and amount of protein product of Survivin was determined.

CONCLUSIONAs2O3 might markedly suppressed ACC-2 cell's viability in vitro. The inhibition of Survivin gene expression may play a critical role on ACC-2 cell apoptosis induced by As2O3.

Antineoplastic Agents ; Apoptosis ; Arsenicals ; Carcinoma, Adenoid Cystic ; Humans ; Inhibitor of Apoptosis Proteins ; Oxides

ObjectiveTo explore the prevalence and spectrum of β-thalassemia mutations in Fujian province,and to provide a reference for prenatal diagnosis and genetic counseling in this population.Methods Two thousand three hundred and one blood samples were randomly selected from 9 different areas of Fujian province from May 2008 to December 2010.PCR and reverse dot blot hybridization (RDB) were adopted for detection of the 17 common types of mutation,and the frequency of each genotype of β-thalassemia mutations was calculated.The β-globin gene of unknown positive samples were analyzed directly with DNA sequencing.Results Three hundred and fifty-nine cases were detected with β-thalassemia mutations of the 2301 copy blood samples submitted,and the detection rate was 15.60％ (359/2301).Of the mutated genes,12 different mutations were identified,namely IVS-2-654(C→T),CD41-42(-TCTT),CD17(A→T),-28(A→G),CD27-28(+C),CD26(G→A),CD71-72(+A),IVS-1-1(G→T),CD43(G→T),-29(A→G),initiation codon ATG→AGG and CD36(-C).Mutation frequencies were 46.54％ (175/376),33.24％ (125/376),9.31％ (35/376),5.05％ (19/376),2.13％(8/376),1.33％(5/376),0.80％(3/376),0.27％(1/376),0.27％(1/376),0.27％(1/376),0.53％(2/376),and 0.27％(1/376),respectively.The most common mutations were IVS-2-654 (C→T) and CD41-42 (-TCTT),which accounted for 79.78％(300/376) of total genetic mutations.In addition,a novel β-globin gene mutation CD36 (-C) allele was detected for the first time,the deletion of a nucleotide C at code 36 within exon 2 lead to a frameshift mutation that could result in a premature termination at code 60.Conclusions β-thalassemia mutations in Fujian province are complex with significant genetic heterogeneity.We present for the first time the detection of a new β-thalassemia mutation in the population:CD36(-C),which provides valuable information for genetic counseling and prenatal diagnosis in Fujian province.

OBJECTIVETo establish the rabbit VX2 tumor as a model for pyriform sinus carcinoma and to observe its biological features.

METHODSVX2 tumor was implanted into the pyriform sinus of 15 rabbits by direct laryngoscope. Fifteen rabbits were randomized into 3 groups (average of 5 rabbits per group). Observation of the tumor growth and evaluation of the histopathological characterization were taken on one group each at the of time 14, 21 and 28 days after transplantation respectively.

RESULTSTumors were found grown in the pyriform sinus of all 15 rabbits with a success implantation rate of 100%. Dysphagia, body weight loss, rhinorrhea and short of breath appeared in the rabbits 28 days after transplantation. The metastasis rates of deep cervical were 100% in all three groups. The metastasis rates of submandibular lymph nodes were 3/5, 4/5 and 5/5 in 14-day, 21-day and 28-day group respectively. The metastasis rates of paratracheal lymph nodes were 0, 4/5 and 5/5 in 14-day, 21-day and 28-day group respectively. There were opposite side lymph node metastasis of deep cervical, submandibular and paratracheal in 4, 3 and 5 rabbits on 14, 21 and 28 days after transplantation respectively. The median diameter for deep cervical, submandibular and paratracheal neck lymph nodes were 1.50, 0.60 and 0.0 cm in 14 days; 1.60, 0.80 and 0.50 cm in 21 days; 1.80, 0.8 and 0.65 cm in 28 days (P > 0.05).

CONCLUSIONSThe animal model for pyriform sinus carcinoma is established successfully. The metastasis of deep cervical lymph node could be induced from day 14 after VX2 transplantation.

Animals ; Disease Models, Animal ; Hypopharyngeal Neoplasms ; pathology ; Lymphatic Metastasis ; pathology ; Neoplasm Transplantation ; methods ; Pyriform Sinus ; pathology ; Rabbits

OBJECTIVETo establish an effective separation system of 2-DE for the proteome of caudal gland, and provide foundation for revealing the mechanisms of histological development and pharmacological activities.

METHODThe total proteins of caudal gland were extracted by TCA/acetone precipitation, phenol extraction/methanol-ammonium acetate precipitation and trizol-base method respectively and separated by immobilized pH gradient (IPG) strips prior to SDS-PAGE. Loading protein sample size and isoelectric focusing conditions were optimized. The gels were stained with Coomassie brilliant blue, scanned and then analyzed using PDQuest 8.0 analysis software.

RESULTThe total proteins of caudal gland extracted by trizol-base method were the highest quality and could meet the needs of 2-DE. With 300 microg of proteins loaded on 7 cm pH 3-10 IPG strip followed by isoelectric focusing program II ,a satisfying 2-DE profiles were obtained. The total number of disticted protein spots was 209 with the optimized system.

CONCLUSIONA well-resolved 2-DE patterns of caudal gland were obtained by this optimized system. This method could be applied to prepare other similar tissue sample and 2-DE studies.

Animals ; Deer ; Electrophoresis, Gel, Two-Dimensional ; methods ; Proteins ; chemistry ; Scent Glands ; chemistry

Ligustrazine, one of the major effective components of the Chinese traditional medicinal herb Ligusticum Chuanxiong Hort, has been reported plenty of biological activities, such as protect cardiovascular and cerebrovascular, neuroprotection and anti-tumor, et al. Because of its remarkable effects, studies on structural modification of ligustrazine have attracted much attention. Ligustrazine synthetic derivatives reported in recent decades are mainly derived from four primary intermediates (TMP-COOH, TMP-OH, TMP-NH2, HO-TMP-OH). To explore the neuroprotection activitiy of ligustrazine intermediates, six ligustrazine intermediates (2, 5, 8, 11, 12, 13) were synthesized and their protective effects against CoCl2-induced neurotoxicity in differentiated PC12 cells were studied. The target compounds were prepared via different chemical methods, including oxidation, substitution, esterification and amidation without changing the structure nucleus of ligustrazine. Compared with TMP (EC50 = 56.03 micromol x L(-1)), four compounds (2, 5, 12 and 13) exhibited higher activity (EC50 < 50 micromol x L(-1)) respectively, of which, compound 2 displayed the highest protective effect against the damaged PC12 cells (EC50 = 32.86 micromol x L(-1)), but target compounds 8 and 11 appeared lower activity (EC50 > 70 micromol x L(-1)). By structure-activity relationships analysis, the introduction of carboxyl, amino to the side chain of ligustrazine and appropriately increase the proportion of ligustrazine may contribute to enhance its neuroprotective activity, which provides a reference for the design, synthesis and activity screening of relevant series of ligustrazine derivatives in the future.
Animals ; Cell Differentiation ; drug effects ; Chemistry Techniques, Synthetic ; Cobalt ; toxicity ; Drugs, Chinese Herbal ; chemistry ; Neuroprotective Agents ; chemical synthesis ; chemistry ; pharmacology ; Neurotoxins ; toxicity ; PC12 Cells ; Pyrazines ; chemical synthesis ; chemistry ; pharmacology ; Rats

OBJECTIVETo evaluate the effect of autograft bone, allograft bone, calcium sulfate bone cement, and calcium phosphate bone cement on the repair of tibial plateau defect in rabbits.

METHODSWe used autograft bone, allograft bone, calcium sulfate bone cement, and calcium phosphate bone cement to repair tibial plateau defect in rabbits. Gross and histologic observations, X-ray examination, and biomechanical test were conducted at 1, 2, 4, 8 weeks after operation.

RESULTSX-ray examination found that the bone density was evidently reduced in calcium sulfate group at 8 weeks after operation; there were no marked changes in other groups. The maximal load measurements showed that autograft and allograft groups were greater than calcium sulfate and calcium phosphate groups at 1 and 2 weeks after operation. However at 4 and 8 weeks after operation, no significant difference was found among the four groups. In autograft and allograft groups, there was no significant difference in biomechanical intensity at 2, 4, and 8 weeks, but it was significantly higher than that at 1 week. In calcium sulfate and calcium phosphate groups, the outcome was ranked in descending order as 1 week less than 2 week less than 4 week equal to 8 week. Histologic examination found a great amount of new bones at 8 week in both autograft and allograft groups. In calcium sulfate group, calcium sulfate was almost absorbed and there were numerous bone trabeculations. There was a large amount of unabsorbed calcium phosphate in calcium phosphate group.

CONCLUSIONAt 1-2 weeks postoperatively, the biomechanical intensity is higher in autograft and allograft groups than calcium sulfate and calcium phosphate groups, but after 4-8 weeks, there is no significant difference among groups. At 1-2 weeks, the biomechanical intensity in all groups is increased, but at 4-8 weeks, there is no significant increase. The rates of absorption and bone formation are quicker in calcium sulfate group than calcium phosphate group.

Animals ; Biomechanical Phenomena ; Bone Cements ; Bone Transplantation ; Knee Joint ; Tibia ; Transplantation, Autologous