ABSTRACT
Objective To investigate the effects ofendothelin-1 (ET-1) and endothelin-3 (ET-3) on the expression of transformation growth factor-beta 1 (TGF-β1) and phosphorylation of Smad 3 in malignant melanoma cell line, A375. Methods Cultured A375 cells were classified into 5 groups, i.e. control group (no stimulation), ET-1 group (stimulated with ET-1), ET-1+BQ123 group (treated with ET-1 and BQ123),ET-1 + BQ788 group (treated with ET-1 and BQ788), ET-3 group (stimulated with ET-3), to receive different stimulation. The working concentrations were 0, 0.1, 1, 10, 100 nmol/L for ET-1 and ET-3, 10 μmol/L for BQ123 and BQ788. After another 12- and 24-hour culture, ELISA, RT-PCR and Western blot were used to detect the expression of TGF-β1 protein and mRNA as well as phosphorylated Smad 3 (P-Smad 3). Results The expression of TGF-β1 in A375 cells was up-regulated by ET-1, but down-regulated by ET-3, and both of the effects were in a concentration-dependent manner. Under the stimulation with ET-1 and ET-3 of 100 nmol/L, the level of TGF-β1 reached 1289.38 ± 89.42 ng/L per 105 cells and 85.09 ± 9.37 ng/L per 105 cells, respectively, significantly different from that in unstimulated cells (both P < 0.05). BQ123 signifi-cantly blocked the up-regnlatory effect of ET-1 on the expression TGF-β1 protein(P < 0.05), but BQ788 had no significant influence on the effect, so was the case with TGF-β1 mRNA. Western blot revealed that ET-1significantly elevated the expression of P-Smad 3 in A375 cells (P <0.05), and the elevation was significantly inhibited by BQ123, but not by BQ788. The expression of P-Smad 3 was statistically decreased by ET-3 in A375 cells (P <0.05). Conclusions The expression of TGF-β1 could be enhanced by ET-1, but suppressed by ET-3. It is likely that endothelin receptor A mediates the phosphorylation of Smad 3 induced by ET-1.
ABSTRACT
In order to analyze the in vivo expression of Candida albicans secreted aspartyl proteinases (SAP) in human vaginal infection, the vaginal secretion from 29 human subjects was collected by vaginal swab, and the expression of SAP1-SAP6 was detected by reverse-transcriptase polymerase chain reaction using specific primer sets. It was found that Sap2 and Sap5 were the most common genes expressed during infection; Sap3 and Sap4 were detected in all subjects and all 6 SAP genes were simultaneously expressed in some patients with vaginal candidiasis. It was suggested that the SAP family is expressed by Candida albicans during infection in human and that Candida albicans infection is associated with the differential expression of individual SAP genes which may be involved in the pathogenesis of vaginal candidiasis.
ABSTRACT
In order to study the expression of intedeukin-22 (IL-22) and S100A7, A8, A9 mRNA in the skin lesions of patients with psoriasis vulgaris and their relationship, the biopsies were taken from skin lesions in 35 patients with psoriasis vulgaris and the skin of 16 normal controls, and the expres- sion levels of IL-22 and S 100A7, A8 and A9 mRNA were detected by semi-quantitative RT-PCR. The results showed that (1) IL-22 and SI00A8, A9 mRNA were positively expressed in the psoriatic skin lesions but negatively expressed in the normal controls; The expression level of S100A7 was (1.133±0.040) in the psoriatic skin lesions, significantly higher than that in the normal controls (0.744±0.037, P<0.01). (2) There were significantly positive correlations between the expression of IL-22/S100A7 mRNA, IL-22/S100A8 mRNA, IL-22/S100A9 mRNA in the psoriasis vulgaris (r1=0.543, r2=0.774, r3=0.621, P<0.01). It was concluded that IL-22 and S100A7, A8, A9 might play important roles in the occurrence and progression of psoriasis.
ABSTRACT
A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria Gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by over-lap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step po-lymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat se-quence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifi-cally designed mutant fragments.
ABSTRACT
In order to investigate the expression of endothelin receptor B (ETR-B) in human ma-lignant melanoma (MM) cells A375 and SK-mel-1 and the proliferative effects of endothelin 3 (ET3) on A375 cells, RT-PCR was applied to detect the expression of ETR-B gene in human MM cells A375 and SK-mel-1. MTT method was used to evaluate the growth enhancing effects of ET3 on A375 cell line in vitro. The results showed that ETR-B gene was expressed in both MM A375 and SK-mel-1 cells. ET3 had stronger ability to enhance the proliferation of A375 cells in vitro in a con- centration-dependent manner. It was suggested that ET3/ETR-B might play an important proliferative role in MM.
ABSTRACT
In order to investigate the expression of endothelin receptor B (ETR-B) in human malignant melanoma (MM) cells A375 and SK-mel-1 and the proliferative effects of endothelin 3 (ET3) on A375 cells, RT-PCR was applied to detect the expression of ETR-B gene in human MM cells A375 and SK-mel-1. MTT method was used to evaluate the growth enhancing effects of ET3 on A375 cell line in vitro. The results showed that ETR-B gene was expressed in both MM A375 and SK-mel-1 cells. ET3 had stronger ability to enhance the proliferation of A375 cells in vitro in a concentration-dependent manner. It was suggested that ET3/ETR-B might play an important proliferative role in MM.
Subject(s)
Cell Line, Tumor , Cell Proliferation/drug effects , Endothelin-3/pharmacology , Melanoma/metabolism , Melanoma/pathology , Receptor, Endothelin B/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by overlap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifically designed mutant fragments.
Subject(s)
Bacterial Proteins/genetics , DNA Fragmentation , DNA, Bacterial/genetics , Mutagenesis, Site-Directed , Neisseria gonorrhoeae/genetics , Neisseria gonorrhoeae/metabolism , Recombination, Genetic , Repressor Proteins/genetics , Sequence Deletion , Transfection , Transformation, BacterialABSTRACT
In order to analyze the in vivo expression of Candida albicans secreted aspartyl proteinases (SAP) in human vaginal infection, the vaginal secretion from 29 human subjects was collected by vaginal swab, and the expression of SAP1-SAP6 was detected by reverse-transcriptase polymerase chain reaction using specific primer sets. It was found that Sap2 and Sap5 were the most common genes expressed during infection; Sap3 and Sap4 were detected in all subjects and all 6 SAP genes were simultaneously expressed in some patients with vaginal candidiasis. It was suggested that the SAP family is expressed by Candida albicans during infection in human and that Candida albicans infection is associated with the differential expression of individual SAP genes which may be involved in the pathogenesis of vaginal candidiasis.
ABSTRACT
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.
ABSTRACT
To study the relationship between mutation of the inverted repeat sequence (IR) in the multiple transferable resistant system (mtr) of Neisseria gonorrhoeae (NG) and itsmultiple 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 susce ptive or five penicillin-resistant strains had no base mutation in IR gene, while all of the 13 strains with multiple-antibiotic-resistance had a singlebase 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.
ABSTRACT
In order to provide a rational research basis for detection of resistance of Neisseria gonorrhoeae to antimicrobial hydrophobic agents and study on the resistant mechanism of multiple transferable resistance (mtr) efflux system, plasmid pET-28a(+) encoding mtrC gene was constructed and the related target protein was expressed in Escherichia coli (E. coli) DE3. The fragments of mtrC gene of Neisseria gonorrhoeae from the standard strains were amplified and cloned into prokaryotic expression plasmid pET-28a(+) with restriction endonuclease to construct recombinant pET-mtrC which was verified by restriction endonuclease and DNA sequencing. The recombinant was transformed into E. coli DE3 to express the protein mtrC induced by IPTG. The results showed mtrC DNA fragment was proved correct through restriction endonuclease and DNA sequencing. Its sequence was 99.5 % homologus to that published on GeneBank (U14993). A 48.5 kD fusion protein which was induced by IPTG was detected by SDS-PAGE. It was concluded that the construction of prokaryotic expression plasmid of mtrC protein of Neisseria gonorrhoeae was correct and the fusion protein was successively expressed in E. coli.
ABSTRACT
In order to investigate the correlation between the expression of the apoptotic regulatory proteins (Fas, Bcl-2) in peripheral blood lymphocytes (PBLC) and the level of IFN-gamma and IL-4 in serum of the patients with condyloma acuminata (CA) in the immune pathogenesis of CA, indirect immunofluorescence labeling method of flow cytometer and solid sandwich ELISA method were performed for detecting the expression of Fas, Bcl-2 in PBLC and the level of IFN-gamma and IL-4 in serum of 60 cases of CA. The results showed the expression level of Fas in PBLC of CA was significantly higher than in the normal control group, but the expression level of Bcl-2 was significantly lower (both P<0.01). The level of IFN-gamma in serum of CA was significantly lower than in the normal control group (P<0.01), but IL-4 was significantly lower (both P<0.01). The expression of Fas in PBLC had a negative correlation with the level of IFN-gamma in serum of patients with CA, but had a positive correlation with the level of IL-4; The expression of Bcl-2 had a positive correlation with the level of IFN-gamma, but had a negative correlation with the level of IL-4. All the correlation coefficients had significant difference by t test (P<0.01). It was suggested abnormal apoptosis in PBLC, the suppressed secretion of the TH1-associated cytokines (eg: IFN-gamma) and the increased secretion of the TH2-associated cytokines (eg: IL-4) existed in the patients with CA and might play an important role in the immune pathogenesis of CA.
Subject(s)
Adolescent , Adult , Female , Humans , Male , Middle Aged , Apoptosis , Condylomata Acuminata , Blood , Flow Cytometry , Interferon-gamma , Blood , Interleukin-4 , Blood , Lymphocytes , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Blood , fas Receptor , BloodABSTRACT
In order to investigate the susceptibility of mixed infection of Ureaplasma Urealyticum (UU) and Mycoplasma Hominis (MH) to 7 kinds of antimicrobial agents and comparison with that of UU infection in NGU patients, the in vitro susceptibility was determined by using microdilution method. The positive results were analyzed. The results showed that the sequence of susceptibility to 7 kinds of antimicrobial agents for both UU infection group and UU-MH mixed infection group was almost the same from the highest susceptibility to the lowest accordingly: Josamycin, Doxycycline, Minocycline, Sparfloxacin, Roxithromycin, Ofloxacin and Azithromycin. The total drug resistance rate for UU-MH mixed infection group (97.67%) was significantly higher than that for UU infection group (44.67%, P < 0.01). The highest drug resistance rate in UU group and UU-MH mixed infection group was 31.33% (Ofloxacin) and 90.48% (Azithromycin) respectively. UU-MH mixed infection showed an increased drug resistance and changes of drug resistance spectrum.
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Anti-Bacterial Agents , Pharmacology , Azithromycin , Pharmacology , Doxycycline , Pharmacology , Drug Resistance, Bacterial , Josamycin , Pharmacology , Minocycline , Pharmacology , Mycoplasma Infections , Microbiology , Mycoplasma hominis , Ofloxacin , Pharmacology , Superinfection , Ureaplasma Infections , Microbiology , Ureaplasma urealyticumABSTRACT
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.
ABSTRACT
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.
ABSTRACT
The telomerase activity in condyloma acuminatum (CA) tissue with human papillomavirus (HPV) types of 6/11 and 16/18 was detected to investigate the function of telomerase in the occurrence, development and carcinogenesis of genital CA. Forty-two biopsies from patients with genital CA and 30 control tissue samples were tested for telomerase activity, HPV presence and types. The telomerase activity was determined by modified telomerase repeat amplification protocol (TRAP) assay and HPV typing by polymerase chain reaction (PCR) with typing-specific primers. Results showed that HPV-DNA was negative and the expression rate of telomerase was 16.7% in all normal skin samples. All CA samples were positive for HPV (6/11 type was found in 32 cases, 16/18 in 3 and mixed type in 7). Telomerase activity was detectable in all CA patients. The telomerase activity in CA of 16/18 type was apparently higher than in CA of 6/11 type. It was concluded that the hyperplasia in CA might be increased as a result of HPV infection, suggesting that the activation of telomerase by HPV, especially by 16/18 type may play a role in the etiology and carcinogenesis of genital CA.
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Condylomata Acuminata , Virology , Genital Diseases, Female , Virology , Genital Diseases, Male , Virology , Papillomaviridae , Classification , Papillomavirus Infections , Virology , Telomerase , Metabolism , Tumor Virus Infections , VirologyABSTRACT
In order to investigate the role of the expression of the Fas/FasL in peripheral blood lymphocytes (PBLC) and the level of IL-2 in serum of patients with condyloma acuminata (CA) in the immune pathogenesis of CA, flow cytometry, indirect immunofluorescence labeling and ELISA were performed to detect the expression of the apoptotic regulatory proteins Fas/FasL in PBLC and the level of IL-2 in serum of 60 patients with different course of CA. The results showed that the expression of Fas/FasL in PBLC of the group of short course in CA was significantly higher than that of the normal controls (P<0.05); the expression of Fas, FasL in PBLC of the group of long course CA was significantly higher than that of the group of short course and the normal controls (P<0.05 and P<0.01, respectively); the level of IL-2 in serum of the group of short and long course CA was significantly lower than that of the group of normal controls (P<0.01); the negative relation was revealed between the expression of Fas/FasL in PBLC and the level of IL-2 in serum of patients with CA (r=－0.76, P<0.01). It was suggested that the abnormal apoptosis in PBLC and decreased level of IL-2 in serum of CA might play an important role in the course of CA.
ABSTRACT
In order to investigate the role of the expression of the Fas/FasL in peripheral blood lymphocytes (PBLC) and the level of IL-2 in serum of patients with condyloma acuminata (CA) in the immune pathogenesis of CA, flow cytometry, indirect immunofluorescence labeling and ELISA were performed to detect the expression of the apoptotic regulatory proteins Fas/FasL in PBLC and the level of IL-2 in serum of 60 patients with different course of CA. The results showed that the expression of Fas/FasL in PBLC of the group of short course in CA was significantly higher than that of the normal controls (P<0.05); the expression of Fas, FasL in PBLC of the group of long course CA was significantly higher than that of the group of short course and the normal controls (P<0.05 and P<0.01, respectively); the level of IL-2 in serum of the group of short and long course CA was significantly lower than that of the group of normal controls (P<0.01); the negative relation was revealed between the expression of Fas/FasL in PBLC and the level of IL-2 in serum of patients with CA (r=－0.76, P<0.01). It was suggested that the abnormal apoptosis in PBLC and decreased level of IL-2 in serum of CA might play an important role in the course of CA.
ABSTRACT
OBJECTIVE:To approach the therapeutic effect of SNMC in treating dermatosis of enzema and dermati-tis.METHODS:190patients with dermatosis of enzema and dermatitis were randomly allocated to therapy group and control group in terms of specific diseases.The control group were only treated with drug for external use,while the therapy group were treated with SNMC as well as drug for external use.RESULTS:The recovery rate and effective rate in the therapy group significantly(P
ABSTRACT
Objective To investigate the relationship between procoagulation of HFGL 2 and abnormality of coagulation in patients with systemic lupus erythematosus (SLE) by detecting the expression of HFGL2/fibroleukin in peripheral blood mononuclear cell(PBMC) of SLE patients. Methods A polyclonal antibody against HFGL2 was applied to detecting the expression of HFGL2 protein in 32 SLE patients and 15 healthy volunteers by immunohistochemistry. Semi-quantitative measurement of HFGL2 expression in the blood specimen was done with high multiple image analytical system(HMIAS). Results The expression of HFGL2 in PBMC from 23 active SLE patients was significantly higher than that in the controls, which showed a positive correlation with the disease activity. Conclusion The expression of HFGL2 in PBMC is probably correlated with the pathogenesis and disease activity of SLE.