Salmonella enterica serovar Typhimurium is one of the most important bacterial pathogens causing diarrhea. The resistance of S. typhimurium to antimicrobial agents, which has recently been isolated from patients, is causing serious problems. We investigated the effects of salicylic acid (Sal) and acetyl salicylate (AcSal) on the susceptibility of S. typhimurium to cephalosporin antibiotics, which are known to increase resistance to cephalosporin and quinolone antibiotics. The MIC of cephalosporin antibiotics was higher than that of the media without Sal. The rate of accumulation of ethidium bromide (EtBr) in the bacteria by the outer membrane protein (Omp) was not different from that of the bacteria cultured in the medium containing Sal. However, Carbonyl cyanide-m-chlorophenylhydrazone (CCCP), an inhibitor of bacterial efflux pumps, significantly reduced the rate of accumulation of EtBr in bacteria cultured on Sal containing medium. In the medium containing CCCP, the MIC of the antimicrobial agent tended to decrease as compared with the control. In addition, the MIC of the bacteria treated with CCCP and Sal was higher than that of the antimicrobial agent against the CCCP treated experimental bacteria. These results suggest that Sal decreases the expression of OmpF in the Omp of S. typhimurium and reduces the permeability of cephalosporin antibiotics to bacteria, which may induce tolerance to cephalosporin antibiotics.
Anti-Bacterial Agents ; Anti-Infective Agents ; Bacteria ; Carbonyl Cyanide m-Chlorophenyl Hydrazone ; Cephalosporin Resistance ; Cephalosporins ; Diarrhea ; Ethidium ; Humans ; Membrane Proteins ; Permeability ; Salicylic Acid ; Salmonella enterica ; Salmonella typhimurium ; Salmonella ; Serogroup

OBJECTIVETo study the radiobiological characteristics of a HepG2 cell line with mitochondrial DNA (mtDNA) deletion.

METHODSHepG2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The HepG2 cell line with mtDNA deletion (ρ(0)HepG2 cells) were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mtDNA deletion in the cells was confirmed by PCR. The radiosensitivity of HepG2 and ρ(0)HepG2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X-ray exposure with Hochest33342 staining, and the invasiveness of ρ(0)HepG2 cells was measured by Transwell assay.

RESULTSHepG2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium. PCR confirmed total mtDNA deletion from ρ(0)HepG2 cells, whose α/β value was significantly lower than that of HepG2 cells. ρ(0)Hep-G2 cells showed an obviously lowered cell apoptosis rate following X-ray exposure with enhanced cell invasiveness.

CONCLUSIONHepG2 cells can be induced by ethidium bromide into ρ(0)HepG2 cells with an increased radiation resistance, anti-apoptosis ability and cell invasiveness.

Apoptosis ; Culture Media ; chemistry ; DNA, Mitochondrial ; genetics ; Ethidium ; chemistry ; Hep G2 Cells ; radiation effects ; Humans ; Radiation Tolerance ; genetics ; Sequence Deletion ; X-Rays

OBJECTIVES: Group B Streptococcus (GBS), a common bowel commensal, is a major cause of neonatal sepsis and an emerging cause of infection in immune-compromised adult populations. Fluoroquinolones are used to treat GBS infections in those allergic to beta-lactams, but GBS are increasingly resistant to fluoroquinolones. Fluoroquinolone resistance has been previously attributed to quinolone resistance determining regions (QRDRs) mutations. We demonstrate that some of fluoroquinolone resistance is due to efflux-mediated resistance. METHODS: We tested 20 GBS strains resistant only to norfloxacin with no mutations in the QRDRs, for the efflux phenotype using norfloxacin and ethidium bromide as substrates in the presence of the efflux inhibitor reserpine. Also tested were 68 GBS strains resistant only to norfloxacin not screened for QRDRs, and 58 GBS strains resistant to ciprofloxacin, levofloxacin or moxifloxacin. Isolates were randomly selected from 221 pregnant women (35-37 weeks of gestation) asymptomatically carrying GBS, and 838 patients with GBS infection identified in South Korea between 2006 and 2008. The VITEK II automatic system (Biomerieux, Durham, NC, USA) was used to determine fluoroquinolone resistance. RESULTS: The reserpine associated efflux phenotype was found in more than half of GBS strains resistant only to norfloxacin with no QRDR mutations, and half where QRDR mutations were unknown. No evidence of the efflux phenotype was detected in GBS strains that were resistant to moxifloxacin or levofloxacin or both. The reserpine sensitive efflux phenotype resulted in moderate increases in norfloxacin minimum inhibitory concentration (average=3.6 fold, range=>1-16 fold). CONCLUSIONS: A substantial portion of GBS strains resistant to norfloxacin have an efflux phenotype.
Adult ; beta-Lactams ; Ciprofloxacin ; Ethidium ; Female ; Fluoroquinolones ; Humans ; Korea ; Levofloxacin ; Microbial Sensitivity Tests ; Norfloxacin* ; Phenotype ; Pregnant Women ; Reserpine ; Sepsis ; Streptococcus*

Glycyrrhiza uralensis (or licorice) is a widely used Oriental herbal medicine from which the phenylflavonoids dehydroglyasperin C (DGC), dehydroglyasperin D (DGD), and isoangustone A (IsoA) are derived. The purpose of the present study was to evaluate the antioxidant properties of DGC, DGD, and IsoA. The three compounds showed strong ferric reducing activities and effectively scavenged DPPH, ABTS+, and singlet oxygen radicals. Among the three compounds tested, DGC showed the highest free radical scavenging capacity in human hepatoma HepG2 cells as assessed by oxidant-sensitive fluorescent dyes dichlorofluorescein diacetate and dihydroethidium bromide. In addition, all three compounds effectively suppressed lipid peroxidation in rat tissues as well as H2O2-induced ROS production in hepatoma cells. This study demonstrates that among the three phenylflavonoids isolated from licorice, DGC possesses the most potent antioxidant activity, suggesting it has protective effects against chronic diseases caused by reactive oxygen species as well as potential as an antioxidant food additive.
Animals ; Benzopyrans ; Carcinoma, Hepatocellular ; Chronic Disease ; Ethidium ; Flavonoids ; Fluorescent Dyes ; Food Additives ; Glycyrrhiza ; Glycyrrhiza uralensis ; Hep G2 Cells ; Herbal Medicine ; Humans ; Isoflavones ; Lipid Peroxidation ; Rats ; Reactive Oxygen Species ; Singlet Oxygen

PURPOSE: Troglitazone (TRO), a PPAR-gamma agonist, can reduce heat shock protein (HSP) 70 and increase the antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, which might affect thermal sensitivity. Here, we investigated whether TRO modifies thermal sensitivity in uterine cervical cancer cells, which is most commonly treated by hyperthermia (HT). MATERIALS AND METHODS: HeLa cells were treated with 5microM TRO for 24 hours before HT at 42degrees C for 1 hour. Cell survival was analyzed by clonogenic assay. The expression of HSPs was analyzed by Western blot. SOD and catalase activity was measured and reactive oxygen species (ROS) was measured using 2',7'-dichlorofluorescin diacetate and dihydroethidium. RESULTS: The decreased cell survival by HT was increased by preincubation with TRO before HT. Expression of HSP 70 was increased by HT however, it was not decreased by preincubation with TRO before HT. The decreased Bcl-2 expression by HT was increased by preincubation with TRO. SOD and catalase activity was increased by 1.2 and 1.3 times,respectively with TRO. Increased ROS by HT was decreased by preincubation with TRO. CONCLUSION: TRO decreases thermal sensitivity through increased SOD and catalase activity, as well as scavenging ROS in HeLa cells.
Blotting, Western ; Catalase ; Cell Survival ; Cervix Uteri ; Chromans ; Ethidium ; Female ; Fever ; Fluoresceins ; Heat-Shock Proteins ; HeLa Cells ; Humans ; Reactive Oxygen Species ; Superoxide Dismutase ; Thiazolidinediones ; Uterine Cervical Neoplasms

OBJECTIVETo observe the inhibition of flavonoids extract (FE) from Inula britannica on oxidative stress in rat aorta after balloon injury.

METHODThe model of vascular intimal hyperplasia was established by balloon injury. The rats were randomly divided into four groups: control, model, FE and captopril (CAP, positive control). The FE group was treated by intragastric administration with FE in dose of 12.5, 25, 50 mg x kg(-1) x d(-1). The level of malondialdehyde (MDA) and superoxide dismutase (SOD) in serum were detected by thiobarbituric acid (TAB) method and xanthine oxidase method respectively, and superoxide anion (O2-) in vessel was detected by dihydroethidium (DHE) staining. The histochemistry, immunohistochemistry and Western blot were used to observe the changes of appearance and SOD expression in vascular tissues after balloon injury.

RESULTThe best concentration of FE to rats was 50 mg x kg(-1) x d(-1). The neointima thickness in the model group was significantly higher than that in the FE group (50 mg x kg(-1) x d(-1)) and control group at 14 days after balloon injury (P < 0.01). The lever of MDA in serum of FE group was decreased (P < 0.01) and SOD was increased (P < 0.05) in both serum and vascular tissues. The level of O2*- in the drug group was lower than that in the model group.

CONCLUSIONFE can enhance the antioxidation capacities of vessel tissues by suppressing the formation of O2- induced by injury, by which FE inhibites neointima formation after balloon injury in rat.

Animals ; Antioxidants ; administration & dosage ; chemistry ; Aorta ; drug effects ; metabolism ; Blood Vessels ; drug effects ; metabolism ; Catheterization ; adverse effects ; Down-Regulation ; Ethidium ; analogs & derivatives ; blood ; metabolism ; Flavonoids ; administration & dosage ; chemistry ; Inula ; chemistry ; Male ; Oxidative Stress ; drug effects ; Random Allocation ; Rats ; Superoxide Dismutase ; blood ; metabolism

PURPOSE: The aim of this study was to determine the prevalence of asymptomatic nasopharyngeal carriages in children using a multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) assay kit. METHODS: We obtained nasopharyngeal swabs from 33 children without any underlying disease from July 25 to July 28, 2008. The children were free from the signs of respiratory tract infections at the time of sampling. DNA was extracted from the swabs and subjected to multiplex RT-PCR using a primer set for the detection of pneumococci (Seeplex(R) PneumoBacter ACE Detection Seegene, Seoul, Korea). The amplified PCR products were separated on 2% agarose gels and stained with either ethidium bromide or screen tape system (Lab901 Scotland, UK). RESULTS: A total of 33 children (male, 15 female, 18) aged between 3.2 and 16.3 (median, 8.2) years were included in this study. The mRT-PCR detected colonized bacteria (Streptococcus pneumoniae, Hemophilus influenzae, Chlamydia pneumoniae, and Bordetella pertussis) in 30 children (90.9%). Of these, 13 children (39.4%) showed more than 2 bacteria: 12 children were positive for 2 bacteria (S. pneumoniae and H. influenzae) and 1 child was positive for 3 bacteria (S. pneumoniae, H. influenzae, and C. pneumoniae). CONCLUSION: mRT-PCR was found to be a sensitive tool for the detection of asymptomatic nasopharyngeal carriages. Clinical significances of the bacteria detected by mRT-PCR will have to be evaluated in the future.
Aged ; Bacteria ; Bordetella ; Child ; Chlamydophila pneumoniae ; Colon ; DNA ; Ethidium ; Female ; Gels ; Haemophilus influenzae ; Humans ; Influenza, Human ; Nasopharynx ; Pneumonia ; Polymerase Chain Reaction ; Prevalence ; Respiratory Tract Infections ; Scotland ; Sepharose ; Streptococcus pneumoniae

BACKGROUND: The aim of this study was to determine the prevalence of asymptomatic nasopharyngeal carriages in immunocompromized children by using a multiplex reverse transcriptase-polymerase chain reaction (mRT-PCR) assay kit. METHODS: We obtained clinical samples by nasopharyngeal swabs from 42 patients with underlying immune deficiency from May 20, 2008 to May 22, 2008. The children were free from signs of respiratory tract infections at the time of sampling. Isolated cDNA was extracted and after this the DNA was examined using a multiplex primer set for pneumonial bacteria detection (Seeplex(R) PneumoBacter ACE Detection, Seegene, Seoul, Korea). The amplified PCR products were separated on 2% agarose gels and stained with ethidium bromide and a screentape system (Lab901, Scottland, UK) and then they were compared. The nasopharyngeal swab culture was done simultaneously and this was compared with the results of mRT-PCR. RESULTS: A total of 42 patients (males: 24, females: 18) aged between 1.2 and 16.3 years (median: 9.2 years) were included in this study. The mRT-PCR detected bacteria (Streptococcus pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae, and Bordetella pertussis) in 28 patients (66.6%). Of these 28 patients, 4 patients (14.3%) showed more than 2 bacteria: 2 patients were positive for two bacteria (S. pneumoniae and H. influenzae, H.influenzae and B. pertussis) and 2 patients were positive for three bacteria (S. pneumoniae, B. pertussis and C. pneumoniae, C. Pneumoniae, H. influenzae and B. pertussis). S. pneumoniae was cultured in one patient (2.4%). Conclusions: The mRT-PCR is a sensitive tool for the detection of the asymptomatic nasopharyngeal carriages. The clinical significance of the bacteria detected in immunocompromized patients by mRT-PCR will need further evaluation.
Aged ; Bacteria ; Bordetella ; Child ; Chlamydophila pneumoniae ; DNA ; DNA, Complementary ; Ethidium ; Gels ; Haemophilus influenzae ; Humans ; Influenza, Human ; Nasopharynx ; Pneumonia ; Polymerase Chain Reaction ; Prevalence ; Respiratory Tract Infections ; Reverse Transcriptase Polymerase Chain Reaction ; Sepharose ; Whooping Cough

AIMTo determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes.

METHODSAcrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used.

RESULTSAlthough sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality.

CONCLUSIONThe ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality.

Acrosome ; drug effects ; physiology ; Acrosome Reaction ; drug effects ; Annexin A5 ; metabolism ; Anti-Bacterial Agents ; pharmacology ; Calcimycin ; pharmacology ; Ethidium ; Flow Cytometry ; Fluorescent Dyes ; Humans ; In Vitro Techniques ; Male ; Microscopy, Confocal ; Pancreatic Elastase ; metabolism ; Phosphatidylserines ; metabolism ; Phospholipases A2, Secretory ; metabolism ; Semen ; cytology ; drug effects ; Spermatozoa ; drug effects ; enzymology

OBJECTIVE: Hypertensive disorder in pregnancy is one of the most serious obstetrical complications. Apolipoprotein E (Apo E) genotype is polymorphic and recently it was reported to be associated with preeclampsia. We undertook this study to clarify whether polymorphic marker of apolipoprotein E is associated with the development of hypertensive disorder in pregnancy. METHODS: Retrospective chart review was done. DNA was extracted from maternal blood of 124 healthy pregnant women and 253 women with hypertensive diseases in pregnancy. Hypertensive disorders complicating pregnancy include gestational hypertension (n=44), preeclampsia (n=158; mild=56, severe=102), preeclampsia superimposed preeclampsia (n=30), and chronic hypertension (n=21). Apo E allele was amplified by polymerase chain reaction, size fractionated on 4% agarose gels, and stained with ethidium bromide. RESULTS: We found all six possible genotypes of Apo E. However, three genotypes, E2/2, E2/4 and E4/4, were uncommon in Korean. We analysed the data according to each allele. Allele frequencies of the Apo epsilon2, epsilon3 and epsilon4 were as follows: 6.8%, 83.0%, 10.2% in women with gestational hypertension, 4.5%, 84.8%, 10.7% in women with mild preeclampsia, 5.9%, 85.3%, 8.8% in women with severe preeclampsia, 1.7%, 91.7%, 6.7% in women with superimposed preeclampsia, 0.0%, 90.5%, 9.5% in women with chronic hypertension, and 5.2%, 86.5%, 8.5% in normal controls. No significant difference in Apo E alllele frequency was found among these groups (P>0.05). CONCLUSIONS: The study suggests that specific allele of apolipoprotein E does not appear to be a risk factor for hypertensive disorders complicating pregnancy in Korean population.
Alleles ; Apolipoproteins ; Apolipoproteins E ; DNA ; Ethidium ; Female ; Gels ; Gene Frequency ; Genotype ; Humans ; Hypertension ; Hypertension, Pregnancy-Induced ; Polymerase Chain Reaction ; Pre-Eclampsia ; Pregnancy ; Pregnant Women ; Retrospective Studies ; Risk Factors ; Sepharose