Objective To study the relationship between the mutation of the inverted repeat (IR) gene in the multiple transferable resistant (mtr) system and multiple antibiotic resistance of Neisseria gonorrhoeae. Methods The antimicrobial susceptibilities of isolated strains were tested. An agar plate dilution method was used to determine the minimum inhibitory concentrations. The target genes were amplified by PCR and subjected to sequencing. Results No mutation was found in the IR gene of either of 2 sensitive or 5 penicillin-resistant Neisseria gonorrhoeas strains. Among the 17 multiple-antibiotic-resistant strains, a strain with both azithromycin- and penicillin-resistance had T/A and T/A insertions, and another had A/T deletion. Conclusion Mutations in the IR gene of the mtr system of Neisseria gonorrhoeae might result in multiple antibiotic resistance.

Objective To investigate the effect of cold atmospheric plasma (CAP) on the healing of skin ulcers using Balb/c mice. Methods Wounds with a diameter 6 mm were created on each side of the backs of BalB/c mice ( n = 150) using a punch bioptome. The mice were assigned randomly into a control group ( wounds healed naturally), a laser group (wounds treated with a He-Ne laser for 10 min daily) and a CAP group (wounds treated with CAP for 10 min daily). Wound healing was evaluated on postoperative days (PODs) 4, 7, 10 and 14 in terms of percent wound closure. Ten mice per group were sacrificed on each of the evaluation days. Both wounds were removed and a histological examination was conducted. A scoring system was used to evaluate the wounds. The expression of vascular endothelial growth factors (VEGFs) in the wounded tissue was detected by using immunohistochemical methods on POD 7. The results were quantified using an HPIAS-1000 system. Results Compared with the control group, the average percentage of wound healing was significantly greater in the CAP group on PODs 7 and 10. The average scores on the histological examination were significantly higher in the CAP group on PODs 7, 10 and 14. Compared with the other two groups, the expression of VEGF was up-regulated significantly in the CAP group.Conclusions CAP can positively affect the wound healing process. This might be related to the up-regulation of VEGF in the wounded tissues.

Objective To construct a prokaryotic expression plasmid pET-28a(+) encoding the multiple transferable resistance C (mtrC) gene of N. gonorrhoeae and express it in E.coli DE3, in order to provide a model to study the pathogen's resistance mechanisms to antimicrobial hydrophobic agents. Methods The mtrC gene of N. gonorrhoeae was amplified by polymerase chain reaction from reference strains,cleaved with restriction endonuclease, and then cloned into the prokaryotic expression plasmid pET-28a (+) to construct the recombinant pET-mtrC. This was confirmed by cleavage of restriction endonuclease and DNA sequencing. The recombinant pET-mtrC was transformed into E.coli DE3 to express the protein MtrC with induction by IPTG. Results The mtrC gene in the recombinant pET-mtrC showed 99.5% homology with the reference sequence in GeneBank (U14993). A 48.5 kD fusion protein was identified by SDS-PAGE. Conclusions The successful construction of a prokaryotic plasmid encoding the mtrC gene of N. gonorrhoeae and its expression in E.coli may facilitate the development of a monoclonal antibody to the MtrC protein and help to investigate the mechanism of the mtr efflux system of N. gonorrhoeae.

Objective To research the distribution and the characteristics of the plasmid mediated quinolone resistant genes in Shewanella algae. Methods The qnr, qepA, aac(6')-Ib-cr genes were amplified by PCR, then the positive PCR products were sequenced to determine the gene type. The transferability of plasmid mediated quinolone resistance was ensured by conjugation experiment. MICs were measured by E-test. qnrA gene was mapped to plasmids to locate it. Results The qnrA gene were detected in the Shewanella algae, this is a newfound subgroup qnrA7, the GenBank accession no. was GQ463707, qnrB, qnrS,qnrC, qnrD, qepA and aac(6')-Ib-cr genes were not detected. qnrA7 reside in a plasmid about 33 kb, conjugation experiment was unsuccessful. The strain was susceptible to quinolones. Conclusion It deserves paying close attention to the report of an original qnrA subgroup in an isolate of water-borne species of Shewanella algae.

Objective: To construct the insulin-like growth factor binding protein 7 (IGFBP7) expression plasmid (pEGFC1-IGFBP7) and to investigate the effect of IGFBP7 on the apoptosis of SK-MEL-28 (human malignant melanoma cell line) cells. Methods: The pEGFC1-IGFBP7 plasmid was constructed; pEGFC1-IGFBP7 and empty plasmids were transfected into SK-MEL-28 cells separately. The transfection efficiency was observed under fluorescence microscope. Apoptosis of SK-MEL-28 cells after transfection was detected by Annexin-FITC/PI staining. Results: The pEGFC1-IGFBP7 plasmid was successfully constructed and was effectively transfected into SK-MEL-28 cells by Effectene reagent, with the transfection rate being 61%. The results of flow cytometry showed that pEGFC1-IGFBP7 significantly induced apoptosis of SK-MEL-28 cells, with the apoptotic rates of pEGFC1-IGFBP7, empty vector, and non-transfected plasmid groups being (28.4±2.57)%, (5.8±0.44)%, and (6.4±0.71)% 24 h after transfection, respectively (F=406.138, P<0.05). Conclusion: pEGFC1-IGFBP7 can effectively induce apoptosis of malignant melanoma SK-MEL-28 cells, which provides an experimental basis for IGFBP7 gene-based therapy of malignant melanoma.

Objective To evaluate the targeted killing of malignant melanoma cells by aclarubicin liposomes conjugated with vascular endothelial growth factor(ADM-VEGF-SSL)in vitro.Metheds To detect the binding abilitv of liposomes to malignant melanoma(MM)cells,the human malignant melanoma cell line A375 was cultured in the presence of ADM-VEGF-3H-SSL or ADM-3H-SSL for 2 days followed by the detection of radioactivity of these cells.Then.A375 cells were cultured with various concentrations(0.01,0.1,1,10,100 mol/L)of ADM-VEGF-SSL,ADM-SSL or free ADM for 48 hours in the 48-hour cytotoxity test,or for 0.5 hour followed by another 48-hour culture in drug-free medium in the 0.5-hour cytotoxity test.After that,MTT assay was used to detect the survival rate of these cells.Results ADM-VEGF-SSL could specifically bind to and kill A375 cells.The binding rate of ADM-VEGF-SSL was 2.15 folds as high as that of ADM-SSL.The survival rate of A375 cells after being treated with ADM-VEGF-SSL for 48 hour was similar to that with flee ADM(P＞0.05).but lower than that with ADM-SSL(P＜0.05),while the survival rate of melanocytes treated with ADM-VEGF-SSL was higher than that with free ADM or ADM-SSL(both P＜0.05).As shown by the 0.5-hour cytotoxity test.shortening the treatment course did not attenuate the effect of ADM-VEGF-SSL on A375 cells.Conclusions ADM-VEGF-SSL can specifically recognize A375 cells.efficiently deliver adriamycin into tumor cells,markedly inhibit the proliferation of A375 cells,and eventually,a targeted kill of these cells is realized.

Objective To study the relationship of symptoms of female gonococcal infections to Chlamydia trachomatis infection, serum sex hormone levels, etc. Methods A total of 136 gonorrhea female patients without obvious symptoms were recruited in this study together with 45 gonorrhea patients with obvious symptoms as the controls. Serum progesterone (P) and estradiol (E2) levels were measured by radio immunoassay (RIA). Cervical swabs were obtained from the subjects and eluted into isotonic saline solution, the elution was divided into 2 portions and tested for the levels of TNF-α and IL-1β by ELISA and for the DNA of C. Trachomatis and N. Gonorrhea with PCR. Statistical analysis was carried out by SPSS for Windows (version 12.0). Results There was no statistical correlation between C. Trachomatis infection and asymptomatic status of female gonococcal infection (χ2 = 0.016, P > 0.05). However, the decrease in the level of TNF-α and IL-1β significantly correlated with the increase in serum progestogen (r = -0.8798, -0.8935, respectively, both P < 0.01). Conclusion The high serum level of progesterone may be associated with the asymptomatic status of gonococcal infection.

To study the relationship between mutation of the inverted repeat sequence (IR) in the multiple transferable resistant system (mtr) of Neisseria gonorrhoeae (NG) and its multiple antibiotic resistance, minimal inhibitory concentrations (MICs) for the clinically isolated strains were tested by agar-dilution-method. The mtr system's IR gene of NG was sequenced after amplification by polymerase chain reaction (PCR). Either two susceptive or five penicillin-resistant strains had no base mutation in IR gene, while all of the 13 strains with multiple-antibiotic-resistance had a single-base deletion (A/T). The result suggests that a single-base deletion of the thirteen-base IR sequence in mtr system of NG might result in multiple antibiotic resistance but is not associated with single antibiotic resistance.

Progesterone has nongenomic effects on inducible nitric oxide synthase (iNOS), which is mediated by mitogen activated protein kinase (MAPK) pathways. This effect is supposed to have some potential association with asymptomatic gonococcal infections in women by immunological depression. In this study, polymorphonuclear leukocytes (PMNs) challenged by gonococci were used to study the nongenomic effects of progesterone. The activation of iNOS was assessed by measuring [(3)H] L-arginine converses to [(3)H] L-citrulline, and the activity of MAPK was detected by Western blot. It was found that the activity of iNOS and the yields of NO were enhanced significantly in gonococci-challenged PMNs compared with the controls (P<0.01). Progesterone could repress the activation of iNOS through P38MAPK pathway within PMNs (P<0.05), which could be blocked by SB203580 (P<0.01), but not by actinomycin D (P>0.05). It was also found subsequently that in the serum specimens collected from gonococci-infected but asymptomatic women, the progesterone level was higher than that in women with severe symptoms (P<0.01). Moreover, the yield of NO had an inverse correlation with progesterone. With these results it suggested that the rapid nongenomic effects of progesterone may inhibit iNOS activation and NO yields mediated by P38MAPK pathways, which were supposed to be concerned with asymptomatic women infected with gonococci.

In this study, the sterilizing effect of atmospheric pressure nonequilibrium plasmas (APNPs) on Neisseria gonorrhoeae (N. gonorrhoeae) was preliminarily examined and the possible mechanisms were explored. N. gonorrhoeae FA1090, FA19 and MS11 were treated by APNPs and their survival rate was analyzed by using CFUs counting and structurally studied by laser scanning confocal microscopy. The morphological changes of bacterial cell membrane and wall were studied under TEM. Our results showed that APNPs had strong sterilizing effect on N. gonorrhoeae. The survival rate of MS11 in N. gonorrhoeae liquid medium was 60.65% after disinfection with the APNPs for 5 min, whereas, the survival rate of FA19 was 92.60% and the rate of FA1090 was 96.40%. The survival rate of MS11 was 21.13% after exposure to APNPs for 6 min, whereas the survival rate of FA19 was 31.60% and the rate of FA1090 was 91.00%. N. gonorrhoeae was structurally damaged after treatment with APNPs. It is concluded that APNPs is able to effectively and quickly kill the N. gonorrhoeae, and the killing effect is related to the architectural damage of cell membrane.