Objective:To obtain a large amount of erythromycin B and to investigate the activity site in eryK. Methods:The key sequence of the BC loop region in eryK gene was knocked out and the eryK gene with 101 bp deleted was amplified by overlapping PCR,and cloned into vector pWHM3 to construct recombinant plasmid. The Saccharopolyspora erythraea mutant AK17 was constructed through chromosomal homologous recombination technique.Results and Conclusions:The S.erythraea mutant AK17 was constructed. The results of TCL and MS analysis showed that the major fermentation product of AK17 is erythromycin B.

Objective: To investigate the effects of plateau environment exposure on the reproductive system of male rats, so as to provide the reference for mechanisms of reproductive damage in plateau environment. Methods: Sixty SPF-grade 12-week-old male Wistar rats were randomly divided into the plain-exposed group, the 1 day-, 3 day-, 7 day-, 14 day- and 28 day- plateau-exposed groups. The rats in the plain-exposed group were raised under normal conditions for 28 days, while the rats in the plateau-exposed groups were raised in a simulated high-altitude plateau chamber. After the completion of the designated feeding periods, the rats were sacrificed under anesthesia, and testicular tissue and abdominal aortic blood were collected to detect the testicular index and evaluate sperm quality. Histological and cellular morphologies of the testicular tissue were analyzed. Additionally, the levels of malondialdehyde (MDA), superoxide dismutase (SOD) and reactive oxygen (ROS) in the testicular tissue were determined, along with serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T). Conclusions Plateau environment may cause a decrease in testicular index and sperm quality, impair mitochondrial function, induce oxidative stress, and thus affect reproductive system of male rats. However, there are signs of self-repair in the reproductive system with the increase of exposure duration.

Objective:To express rationally engineered antibodies against EGFR and assess their affinity to EGFR and anti-tumor cell migration effect. Methods:L and V_H genes of humanized antibodies against EGFR were designed and synthesized. Genes encoding V_H and C_H were connected and then cloned into a pIRES based bicistronic expression vector. Gene encoding the corresponding L gene was also cloned into the same vector. 293T cells were transfected with the recombinant plasmid and the antibody expression was confirmed by Western blotting. The antibodies were purified by protein A based affinity chromatography. Binding of the humanized antibody to the EGFR was assessed by Surface Plasmon Renainance with Biacore3000, and the biological activity of the humanized antibody was determined by tumor cell invasion test.Results:Three expression vectors were constructed and the humanized anti-EGFR antibodies were expressed and purified successfully. In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25×10~3and 50×10~3, respectively. Western blot assay showed that the humanized antibodies had recognition specificity to goat-human IgG antiserum. Biacore assay revealed that the humanized antibody C3 binds to EGFR with high affinity(6.13×10~(-10)M). Cell migration test showed that C2,C3 and C5 could suppress growth and migration of tumor cells.Conclusion:Three anti-EGFR humanized antibodies (C2,C3 and C5) have been constructed and expressed successfully, and the C3 antibody retained high affinity for EGFR and showed improved inhibitory effect on tumor cell growth and migration.

Genetic engineering on macrolide antibiotics was a new field in recent years and more than 100 novel polyketides had been produced until then. Using genomic DNA of S. erythraea A226 as a template, about 3.2 kb DNA fragment without KR6 domain was amplified by overlapping PCR technique and cloned into pWHM3 carrier, which resulted in the construction of homologous recombinant plasmid pWHM2201. Plasmid pWHM2201 was introduced into protoplasts of S. erythraea A226 by PEG-mediated transformation and integrated into the gene locus for erythromycin biosynthesis. After integrants grew for two generations on R3M media without Tsr, they were protoplasted and grown on R3M plates. By PCR identification, 8 mutants without KR6 domain were selected out and named S. erythraea M(1-8). With the identification of mass spectrometry, it was proved that S. erythraea M1 synthesized a novel ketolide compound 3-deoxy-3-oxo-erythronolide B.
Anti-Bacterial Agents ; biosynthesis ; chemistry ; Chromosomes, Bacterial ; Erythromycin ; analogs & derivatives ; biosynthesis ; chemistry ; Genetic Engineering ; Ketolides ; Molecular Structure ; Multienzyme Complexes ; genetics ; Saccharopolyspora ; enzymology ; genetics ; metabolism

Adult ; Antigens, CD20 ; metabolism ; Cervical Vertebrae ; pathology ; Eosinophilic Granuloma ; complications ; metabolism ; pathology ; surgery ; Follow-Up Studies ; Hodgkin Disease ; complications ; metabolism ; pathology ; surgery ; Humans ; Ki-1 Antigen ; metabolism ; Male ; Middle Aged ; Spinal Neoplasms ; complications ; metabolism ; pathology ; surgery ; Thoracic Vertebrae ; pathology

OBJECTIVETo investigate the role of integrin α4β7 in the development of ulcerative colitis (UC) in rats.

METHODSSixty Sprague-Dawley rats were randomly divided into the control group (acetone enema), the model group (2,4-dinitrochlorobenzene, DNCB enema), and the α4 intervention group. Colonic mucosa of different groups was observed and compared in terms of pathology and cytokine changes(IL-2 and IL-6) using ELISA. Semi-quantitative RT-PCR was used to detect the colon α4β7 expression. Integrin α4β7(+) lymphocytes in the portal vein of rats were determined by flow cytometry.

RESULTSThe expression of α4 mRNA was 0.68±0.24 in the model group and 0.58±0.37 in the intervention group, and the expression of β7 mRNA was 0.84±0.37 in the model group and 0.65±0.30 in the intervention group, which were all significantly higher as compared to those in the control group(0.15±0.13 for α4 and 0.24±0.62 for β7, P<0.01). The proportions of integrin α4β7 positive lymphocytes in the portal vein in the model group and intervention group were significantly higher than that in the control group [(76.7±8.2)% and (68.2±7.6)% vs. (14.7±6.7)%, P<0.01]. The expression of IL-2 and IL-6 and the result of macroscopic and microscopic scores in the intervention group were lower than those in the model group(P<0.05).

CONCLUSIONSHigh expression of α4β7 may play an important role in experimental colon mucosa inflammation in rats with ulcerative colitis. The blockade of integrin α4β7 may be a potential target to reduce colonic mucosa inflammation.

Animals ; Colitis, Ulcerative ; metabolism ; pathology ; Colon ; metabolism ; pathology ; Disease Models, Animal ; Female ; Integrins ; metabolism ; physiology ; Interleukin-2 ; metabolism ; Interleukin-6 ; metabolism ; Intestinal Mucosa ; pathology ; Rats

In order to study the relationship between lengths of homologous fragments and chromsomic recombination rate in Saccharopolyspora erythraea, three homologous sequences, with mutant loci and different flanking sequences, (26bp + 27bp), (500bp + 576bp) and (1908bp + 1749bp), were synthesized by chemical reaction or PCR amplification, and cloned into pWHM3 to construct homologous recombination plasmids, pWHM1113, pWHM1116 and pWHM1119. When the plasmids were transformed into protoplast of Saccharopolyspora erythraea A226 under PEG mediated, on an average 30, 69 and 170 transformants grew on each plate for the three plasmids respectively, but chromosomic integration frequency were 0, 2% and 19% among corresponding transformants. Both pWHM1116 and pWHM1119 could take double crossover recombination, and exchange the mutant loci in the chromosome. It was concluded that when the flanking sequences were equal or more than (500bp + 576bp), they could take effective single and double recombination with Saccharopolyspora erythraea chromosome.
Chromosomes, Bacterial ; genetics ; Polymerase Chain Reaction ; Recombination, Genetic ; genetics ; Saccharopolyspora ; genetics

BACKGROUNDPremature ventricular contractions (PVCs) are common in the general population, and frequent PVCs may result in the poor quality of life or even the damage of cardiac function. We examined the efficacy and safety of a traditional Chinese medicine Wenxin Keli for the treatment of frequent PVCs among a relatively large Chinese cohort.

METHODSWe performed a randomized, double-blind, placebo-controlled, parallel-group, multicenter trial. A total of 1200 eligible participants were randomly assigned in a ratio of 1:1 to receive Wenxin Keli or the placebo for 4 weeks. The primary and secondary endpoint was the change of PVC numbers and PVC-related symptoms after a 4-week treatment compared with baseline, respectively. In addition, vital signs, laboratory values, and electrocardiographic parameters were assessed in a safety analysis.

RESULTSAt the initial evaluation, no significant differences in the baseline characteristics were observed between the Wenxin Keli group and the placebo group. A smaller number of PVCs was observed after the 4-week treatment than at baseline, in both the Wenxin Keli group (5686 ± 5940 vs. 15,138 ± 7597 beats/d, P < 0.001) and the placebo group (10,592 ± 8009 vs. 14,529 ± 5929 beats/d, P < 0.001); moreover, the Wenxin Keli group demonstrated a significantli greater reduction in the frequency of PVCs than the placebo group (P < 0.001). In a full analysis set, patients in the Wenxin Keli group exhibited significantly higher total effective responses in the reduction of PVCs compared to those in the placebo group (83.8% vs. 43.5%,P < 0.001). The per-protocol analysis yielded similar results (83.0% vs. 39.3%,P < 0.001). Treatment with Wenxin Keli also demonstrated superior performance compared to the placebo with respect to PVC-related symptoms. No severe adverse effects attributable to Wenxin Keli were reported.

CONCLUSIONSWenxin Keli treatment effectively reduced the overall number of PVCs and alleviated PVC-related symptoms in patients without structural heart diseases and had no severe side effects.

Adult ; Aged ; Double-Blind Method ; Drugs, Chinese Herbal ; adverse effects ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Quality of Life ; Treatment Outcome ; Ventricular Premature Complexes ; drug therapy

OBJECTIVETo investigate the effect of different treatment options for the flat type of sudden hearing loss.

METHODSProspective, multi-center clinical study was carried out using internationally used standardized clinical research method. Patients with the flat type of sudden hearing loss between 18 and 65 years old, within two weeks duration, and without any medical treatment were recruited. Treatment options were randomly selected according to the designed random table.

RESULTSFrom August 2007 to October 2011, 402 patients with the flat type of sudden hearing loss who met the criteria (account for 39.26% of the total number of patients) from the 33 hospitals were collected; the total effective rate was 82.59%, and no significant difference was detected between different treatments, (χ(2) = 10.95, P = 0.28). In the 402 cases, 139 were cured (34.58%); 118 were markedly improved (29.35%); 75 were effective (18.66%); 70 were invalid (17.41%).

CONCLUSIONSThe therapeutic efficacy of flat type of sudden hearing loss overall is good; the treatment of improving the inner ear blood rheology and/or reducing blood fibrinogen has clinical significance; the therapeutic efficacy of using glucocorticoid systemically is good as well; there is no obvious difference between combination and single medication.

Adult ; Aged ; Ear, Inner ; Glucocorticoids ; therapeutic use ; Hearing Loss, Sudden ; therapy ; Humans ; Middle Aged ; Prospective Studies ; Treatment Outcome ; Young Adult

Animals ; Bacterial Vaccines ; Brucella suis ; genetics ; Brucellosis ; epidemiology ; microbiology ; veterinary ; China ; epidemiology ; DNA Fingerprinting ; DNA, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Nucleic Acid Amplification Techniques ; methods ; Time Factors