BACKGROUNDCholera toxin B subunit (CTB) was shown to be a potent adjuvant for protein immunogen, especially when inoculated through mucosal route. We aimed to optimize the expression approach for CTB and thereafter to determine the adjuvant effect on DNA vaccine.

METHODSWild type CTB coding gene was amplified and cloned into prokaryotic expression vector pET-30a, and the recombinant CTB was expressed in the presence of different concentration of chloramphenicol and isopropyl β-D-thiogalactoside. Purified recombinant CTB was mixed with HIV-1 AE2f tat-rev-integrase-vif-nef fusion gene DNA vaccine and female BALB/c mice were vaccinated with a DNA priming-recombinant vaccinia vectored vaccine boosting regimen through intramuscular injection. Interferon γ (IFN-γ) enzyme-linked immunospot (Elispot) assay was used to read out the specific T-cell immunity.

RESULTSChloramphenicol was essential for the efficient expression of recombinant CTB (rCTB) in pET-30a/BL21 (DE3) system and could be optimized at the concentration of 0.625 µg/ml in the presence of chloramphenicol. The purified rCTB could bind with GM1 efficiently. INF-γ Elispot data showed the T-cell response induced in CTB adjuvanted group ((734 ± 240) spot forming cells/10(6) splenocytes) was higher than that induced by non-adjuvanted ((520 ± 150) spot forming cells/10(6) splenocytes), all responses against different antigens were enhanced in parallel.

CONCLUSIONCTB could be efficiently expressed in the presence of chloramphenicol and purified CTB is functional and capable of enhancing the specific T cell responses elicited by DNA vaccine, the mechanism needs to be explored in the future.

Adjuvants, Immunologic ; pharmacology ; Animals ; Blotting, Western ; Chloramphenicol ; pharmacology ; Cholera Toxin ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; drug effects ; metabolism ; Female ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Vaccines, DNA ; genetics ; immunology ; metabolism

A series of new coumarin-based benzotriazole derivatives were successfully synthesized via a multi-step sequence of cyclization, etherification and N-alkylation, and were confirmed by 1H NMR, IR, MS spectra as well as elemental analyses. All these synthesized coumarin compounds were evaluated for in vitro antimicrobial activities against four Gram-positive bacteria, four Gram-negative bacteria and three fungi by two fold serial dilution technique. The bioactive assay showed that all these prepared coumarin benzotriazoles could inhibit the growth of the tested bacterial and fungal strains. Title compounds 11a-11e and 13a-13c were more active than chloromycin on Proteus vulgaris ATCC 6896. Coumarin benzotriazoles 11a and 11b displayed comparable antibacterial efficacy against Staphylococcus aureus ATCC 25923 and Micrococcus luteus ATCC 4698 in comparison with reference drug chloromycin. Compared to fluconazole, compounds 11a-11d displayed stronger inhibition on Aspergillus fumigatus ATCC 96918. Moreover, coumarin-based benzotriazoles in combination with antibacterial chloromycin or antifungal fluconazole, showed notable antimicrobial efficacy with less dosage and broader antimicrobial spectrum. More importantly, fluconazole-insensitive A. fumigatus and methicillin-resistant Staphylococcus aureus N 315 (MRSA) were sensitive to these combined drugs.
Anti-Bacterial Agents ; chemical synthesis ; chemistry ; pharmacology ; Antifungal Agents ; chemical synthesis ; chemistry ; pharmacology ; Aspergillus fumigatus ; drug effects ; Chloramphenicol ; pharmacology ; Coumarins ; chemical synthesis ; chemistry ; pharmacology ; Drug Synergism ; Fluconazole ; pharmacology ; Fungi ; drug effects ; Gram-Negative Bacteria ; drug effects ; Gram-Positive Bacteria ; drug effects ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; Staphylococcus aureus ; drug effects ; Triazoles ; chemical synthesis ; chemistry ; pharmacology

BACKGROUND: Bacteroides fragilis group organisms are the most frequently isolated anaerobes in human infections. Increasing resistance to various antimicrobial agents is a significant problem in choosing appropriate antimicrobial agents to treat anaerobic infections. Periodic monitoring of the regional resistance trends of B. fragilis group isolates is needed. METHODS: A total of 466 nonduplicate clinical isolates of B. fragilis group organisms (276 B. fragilis, 106 Bacteroides thetaiotaomicron, and 84 other B. fragilis group organisms) were collected during the 8-yr period from 1997 to 2004 in a Korean university hospital. Minimum inhibitory concentrations to various antimicrobial agents were determined by the CLSI agar dilution method. RESULTS: Eight isolates were resistant to imipenem. Additionally, the resistance rates to cefotetan were decreased in B. thetaiotaomicron, while those for clindamycin were significantly increased compared to the rates found in previous studies. Depending on species, resistance rates were 1-4% for imipenem, 1-6% for piperacillin-tazobactam, 4-11% for cefoxitin, 33-49% for piperacillin, 14-60% for cefotetan, and 51-76% for clindamycin. No isolates were resistant to chloramphenicol or metronidazole. CONCLUSIONS: Piperacillin-tazobactam, cefoxitin, imipenem, chloramphenicol, and metronidazole are still active against B. fragilis group isolates, while clindamycin no longer has a value as an empirical therapeutic agent in Korea. Furthermore, this study identified the first imipenem-resistant B. fragilis group isolates in Korea.
Anti-Bacterial Agents/pharmacology ; Bacteroides/classification/*drug effects/isolation & purification ; Bacteroides fragilis/drug effects/isolation & purification ; Cefoxitin/pharmacology ; Chloramphenicol/pharmacology ; *Drug Resistance, Multiple, Bacterial ; Humans ; Imipenem/pharmacology ; Metronidazole/pharmacology ; Microbial Sensitivity Tests ; Penicillanic Acid/analogs & derivatives/pharmacology ; Piperacillin/pharmacology ; Republic of Korea

Osteoblasts can synthesize the insulin-like growth factors (IGFs) and the IGF-binding proteins (IGFBPs), which may either enhance or attenuate IGF-stimulated bone cell proliferation. Since estrogen induced osteoblastic differentiation and proliferation through an estrogen-responsive gene in target cells, we investigated the effects of estrogen on IGFBP-6 expression in the human osteoblastic-like cell line SaOS-2. Expressions of IGFBP-6 protein and mRNA increased 2.8 and 2-fold, respectively, in the presence of 17-beta-estradiol (E2) (0.01 to 1 micrometer) and estrogen receptor (ER) in SaOS-2 cells. On the other hand, E2 induced a 2-fold increase in SaOS-2 cell proliferation. To identify genomic sequences associated with estrogen responsiveness, the 5'-promoter region (-44 to +118) of the IGFBP-6 gene was cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. E2 induced a 3-fold increase in CAT activity in SaOS-2 cells transiently transfected with this construct. Identification of the estrogen-responsive element (ERE) [5'-CCTTCA CCTG-3'] (-9 to +1) in this IGFBP-6 gene promoter region was confirmed using electromobility shift assays and deletion analysis. This functional ERE was important for E2-induced trans-activation of the IGFBP-6 gene. These results demonstrate that E2 exhibits a positive effect on IGFBP-6 gene transcription through estrogen-liganded ER binding to the functional ERE in the IGFBP-6 gene promoter in SaOS-2 cells.
Blotting, Western ; Cell Proliferation ; Chloramphenicol O-Acetyltransferase/metabolism ; Electrophoretic Mobility Shift Assay ; Estradiol/*pharmacology ; Estrogen Receptor alpha/genetics/metabolism ; Estrogens/pharmacology ; Humans ; Insulin-Like Growth Factor Binding Protein 6/*genetics/metabolism ; Osteoblasts/*drug effects/metabolism ; Promoter Regions, Genetic/*genetics ; RNA, Messenger/genetics/metabolism ; Response Elements ; Reverse Transcriptase Polymerase Chain Reaction ; *Transcriptional Activation ; Tumor Cells, Cultured

We made fusion protein of fastatin and FIII 9-10, termed tetra-cell adhesion molecule (T-CAM) that can interact simultaneously with alphavbeta3 and alpha5beta1 integrins, both playing important roles in tumor angiogenesis. T-CAM can serve as a cell adhesion substrate mediating adhesion and migration of endothelial cells in alphavbeta3 and alpha5beta1 integrin-dependent manner. T-CAM showed pronounced anti-angiogenic activities such as inhibition of endothelial cell tube formation, endothelial cell proliferation, and induction of endothelial cell apoptosis. T-CAM also inhibited angiogenesis and tumor growth in mouse xenograft model. The anti-angiogenic and anti-tumoral activity of molecule like fastatin could be improved by fusing it with integrin-recognizing cell adhesion domain from other distinct proteins. The strategy of combining two distinct anti-angiogenic molecules or cell adhesion domains could facilitate designing improved anticancer agent of therapeutic value.
Angiogenesis Inhibitors/chemistry/*pharmacology ; Animals ; Antineoplastic Agents/chemistry/*pharmacology ; Base Sequence ; Benzocaine/chemistry/*pharmacology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Chloramphenicol/chemistry/*pharmacology ; DNA Primers ; Drug Combinations ; Factor VIII/chemistry/*pharmacology ; Humans ; Integrin alpha5beta1/*physiology ; Integrin alphaVbeta3/*physiology ; Male ; Mice ; Mice, Inbred BALB C ; Nitrofurazone/chemistry/*pharmacology ; Recombinant Fusion Proteins/chemistry/*pharmacology

A marine unicellular green alga, Platymonas subcordiformis, was demonstrated to photobiologically produce hydrogen gas from seawater. The objective of this study was to localize and identify the hydrogenase isolated from P. subcordiformis. Adaptation in the presence of inhibitors of protein biosynthesis indicated that the hydrogenase was much more inhibited by cycloheximide than that by chloramphenicol. The result suggested that the hydrogenase isolated from P. subcordiformis is probably synthesized in cytoplasmic ribosomes. Both Western blot analysis and immunogold electron microscopy demonstrate that the P. subcordiformis hydrogenase is mainly located in the chloroplast stroma. The proteins that reacted specifically with the antibodies against the iron hydrogenase isolated from Chlamydomonas reinhardtii were concentrated by immunoprecipitation. The separated protein bands were cut out of the SDS-PAGE gel, in-gel digested by trypsin, and analyzed by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Mascot was employed for analysis of the MALDI data using the public databases NCBInr. The hydrogenase isolated from P. subcordiformis was identified to be the Fe-hydrogenase.
Algal Proteins ; isolation & purification ; metabolism ; Biocatalysis ; drug effects ; Blotting, Western ; Chloramphenicol ; pharmacology ; Chlorophyta ; enzymology ; Cycloheximide ; pharmacology ; Cytoplasm ; enzymology ; ultrastructure ; Electrophoresis, Polyacrylamide Gel ; Hydrogenase ; antagonists & inhibitors ; isolation & purification ; metabolism ; Immunoprecipitation ; methods ; Iron-Sulfur Proteins ; antagonists & inhibitors ; isolation & purification ; metabolism ; Kinetics ; Microscopy, Immunoelectron ; Protein Synthesis Inhibitors ; pharmacology ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods

Seventy Escherichia coli isolates recovered from diseasedchickens diagnosed with colibacillosis in Henan Province,China, between 2004 and 2005 were characterized forantimicrobial susceptibility profiles via a broth doublingdilution method. Overall, the isolates displayed resistanceto trimethoprim-sulfamethoxazole (100%), oxytetracycline(100%), ampicillin (83%), enrofloxacin (83%), and ciprofloxacin(81%), respectively. Among the phenicols, resistance wasapproximately 79% and 29% for chloramphenicol andflorfenicol, respectively. Molecular detection revealed thatthe incidence rates of the floR, cmlA, cat1, cat2 and cat3were 29, 31, 16, 13, and 0%, respectively. Additionally,10% of the isolates were positive for both floR and cmlA.As these antimicrobial agents may potentially inducecross-resistance between animal and human bacterialpathogens, their prudent use in veterinary medicine ishighly recommended.
Animals ; Anti-Bacterial Agents/*pharmacology ; *Chickens ; China/epidemiology ; Chloramphenicol/pharmacology ; DNA, Bacterial/chemistry/genetics ; Drug Resistance, Multiple, Bacterial ; Escherichia coli/*drug effects/growth & development ; Escherichia coli Infections/epidemiology/microbiology/*veterinary ; Microbial Sensitivity Tests/veterinary ; Polymerase Chain Reaction/veterinary ; Poultry Diseases/epidemiology/*microbiology ; Thiamphenicol/analogs & derivatives/pharmacology

The aim is to evaluate the effect of ciprofloxacin and chloramphenicol on anti-BSA antibody production triggered by bovine albumin encapsulated in non-ionic surfactant vesicle, niosomes. Reverse phase evaporation method was adopted to entrap the antigen in colloidal carrier composed of Span 80 and Span 85 followed by simultaneous characterization for particle size, entrapment efficiency and in vitro release. The protein content was determined by Bradford method using UV Visible Spectrophotometer at 595 nm. Humoral immune response was measured in terms of systemic IgG antibody titre by ELISA method. Experimental data indicated that 7 : 3 molar ratio of Span 80 and cholesterol based niosomal formulation possessed maximum (39.8 +/- 2.9)% of soluble protein. Ciprofloxacin markedly (P < 0.05) decreased the antibody titre. In contrast, chloramphenicol did not reduce the antibody titre significantly in comparison to control group (P > 0.05). It is necessary to explore the effect of a vaccine antigen when a candidate is medicated with a therapeutic agent, which might help in programming a new drug management and vaccination programme.
Animals ; Antibody Formation ; drug effects ; Chloramphenicol ; administration & dosage ; pharmacology ; Ciprofloxacin ; administration & dosage ; pharmacology ; Drug Carriers ; Hexoses ; Immunoglobulin G ; blood ; Liposomes ; Male ; Particle Size ; Rats ; Rats, Wistar ; Serum Albumin, Bovine

The aim of this study was to investigate antimicrobial susceptibilities and macrolide resistance mechanisms of beta-hemolytic viridans group streptococci (VGS) in a tertiary Korean hospital. Minimum inhibitory concentrations (MICs) of seven antimicrobials were determined for 103 beta-hemolytic VGS isolated from various specimens. The macrolide resistance mechanisms of erythromycin-resistant isolates were studied by the double disk test and polymerase chain reaction (PCR). The overall resistance rates of beta-hemolytic VGS were found to be 47.5% to tetracycline, 3.9% to chloramphenicol, 9.7% to erythromycin, and 6.8% to clindamycin, whereas all isolates were susceptible to penicillin G, ceftriaxone, and vancomycin. Among ten erythromycin-resistant isolates, six isolates expressed a constitutive MLSB (cMLSB) phenotype, and each of the two isolates expressed the M phenotype, and the inducible MLSB (iMLSB) phenotype. The resistance rates to erythromycin and clindamycin of beta-hemolytic VGS seemed to be lower than those of non-beta-hemolytic VGS in our hospital, although cMLSB phenotype carrying erm(B) was dominant in beta-hemolytic VGS.
Ceftriaxone/pharmacology ; Chloramphenicol/pharmacology ; Clindamycin/pharmacology ; Cross Infection/*genetics ; *Drug Resistance, Bacterial ; Erythromycin/pharmacology ; Humans ; Immunoenzyme Techniques ; Korea ; Macrolides/*pharmacology ; Penicillin G/pharmacology ; Phenotype ; Polymerase Chain Reaction ; Tetracycline/pharmacology ; Vancomycin/pharmacology ; Viridans Streptococci/*genetics/*metabolism

BACKGROUNDIt is still unclear whether viral genetic variability influences response to interferon (IFN)-alpha treatment. Recent reports suggest that IFN-alpha effects may be associated with hepatitis B virus (HBV) post-transcriptional regulation. This study was designed to explore the heterogeneity of HBV post-transcriptional regulatory elements (HPRE) and the relationship between the diversity of HPRE and the response to IFN-alpha treatment.

METHODSThe HPRE sequences from 31 Chinese patients infected with HBV were determined by directly sequencing of polymerase chain reaction (PCR) product, and comparing them to those from Caucasian patients. Subsequently, eukaryotic expression vectors containing HPRE at various points were constructed and transfected into HepG2 cells, which were then exposed to recombinant human cytokines.

RESULTSThe T to C point mutation at nt 1504 and the C to T (G) at nt 1508 in HPRE were found in 21 and 19 patients with chronic hepatitis B, respectively; the C to T point mutation at nt 1509 was found in 17 patients. These point mutations did not exist in the HPRE of the Caucasian patients. The activity of the CAT gene obviously increased in the case of T to C point mutation at nt 1504, but did not change in the case of the C to T (G) mutations at nt 1508 and 1509. The activity of the CAT gene at these point mutations of HPRE could be inhibited by IFN-alpha/gamma and tumor necrosis factor (TNF)-alpha except for the point mutations at nt 1508 of HPRE which may escape the suppression role of IFN-alpha on HPRE.

CONCLUSIONSThere are point mutations between the HPRE of Chinese and Caucasian HBV patients, which might be correlated with response to IFN-alpha. The variation of HPRE might affect the function of HPRE and influence the regulative function of IFN-alpha other than that of IFN-gamma or TNF-alpha on HPRE.

Chloramphenicol O-Acetyltransferase ; metabolism ; Genes, Regulator ; Hepatitis B virus ; drug effects ; genetics ; Hepatitis B, Chronic ; drug therapy ; virology ; Humans ; Interferon-alpha ; pharmacology ; Interferon-gamma ; pharmacology ; Plasmids ; Point Mutation ; Tumor Necrosis Factor-alpha ; pharmacology