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Increased levels of the protein kinase C (PKC) isoenzymes alpha and theta occur in conjunction with MDR1 gene expression in cells and tissues that have acquired a multidrug resistance (MDR) phenotype. Studies using PKC activators or antisense strategies against PKC suggest that activation of PKC engenders MDR1 gene transcription. In this study the potential roles of PKC-alpha and PKC-theta in MDR1 gene transcriptional regulation were explored. Human-derived MCF-7 breast cancer cells that lack constitutive expression of PKC-alpha or PKC-theta at detectable levels were transfected with full-length PKC-alpha or PKC-theta genes driven by the ecdysone promoter. Stable transfectants were selected by use of the appropriate antibiotics. Treatment of these cells with ponasterone A induced expression of PKC that was catalytically active and underwent translocation and down-regulation on exposure to 12-O-tetradecanoyl-13-phorbol acetate (TPA). These cells were used to analyse PKC-mediated regulation of the MDR1 promoter by further transient transfection with either 1073 bp of the MDR1 gene promoter or deletion fragments thereof to -8 bp, each linked to a chloramphenicol acetyl transferase (CAT) reporter gene. In PKC-alpha expressing cells TPA caused activation of all promoter fragments to -29 bp. This finding suggests that TPA-inducible MDR1 transcription mediated through the TPA responsive factor early growth response 1 (EGR-1) in this region of the promoter may be due to activation of PKC-alpha. In contrast, PKC-theta activated only two MDR1 fragments, -982 and -612 bp. The effect of TPA on reporter gene expression was attenuated by the PKC inhibitor GF 109203X. These data suggest that MDR1 promoter transcription can be regulated by PKC-alpha and PKC-theta. The results support the search for therapeutic strategies directed specifically against PKC-alpha to ameliorate resistance of tumours against cytotoxic agents.
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A total of 44 morphologically different colonies were obtained from water samples and 20 types of colonies (45.5%) could be identified using API test strips. The mean TVC values were 4.36 log CFU ml(-1) for source waters, 4.95 log CFU ml(-1) for 3-in-1 syringe samples, 4.91 log CFU ml(-1) for air rotor samples and 3.66 log CFU cm(-2) for biofilm samples. Susceptibilities of the isolates were tested against piperacillin, ampicillin, ceftazidime, meropenem, gentamicin, tetracycline, ofloxacin and chloramphenicol by using microdilution method according to NCCLS. The meropenem and ofloxacin have shown the broadest spectrum against to the tested isolates.
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A small resistance plasmid of Mannheimia varigena was analysed with regard to its gene organization and the development of a multiresistance gene cluster.
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Ninety-five percent or more of the isolates were susceptible to amikacin, nitrofurantoin, colistin, tigecycline, and fosfomycin. CTX-M group 9 was more susceptible than CTX-M group 1 to ciprofloxacin, gentamicin, and tobramycin. Sixty-eight percent of the isolates were multi-resistant, and the most common multi-resistance pattern was ESBL phenotype with decreased susceptibility to trimethoprim, trimethoprim-sulfamethoxazole, ciprofloxacin, gentamicin, and tobramycin. Only 1 isolate carried a qnrS1 gene, but 37% carried aac(6')-Ib-cr.
The potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) induces activator protein-1 (AP-1) transcription factors, early response genes involved in a diverse set of transcriptional regulatory processes, and protein kinase C (PKC) activity. This work was designed to explore the signal transduction pathways involved in TPA regulation of 5-aminolevulinate synthase (ALAS) gene expression, the mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. We have previously reported that TPA causes repression of ALAS gene, but the signaling pathways mediating this effect remain elusive. The present study investigates the role of different cascades often implicated in the propagation of phorbol ester signaling. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in human hepatoma HepG2 cells. In these experimental conditions, we analyzed TPA action upon endogenous ALAS mRNA levels, as well as the promoter activity of a fusion reporter construct, harboring the TPA-responsive region of ALAS gene driving chloramphenicol acetyl transferase gene expression. We demonstrated that the participation of alpha isoform of PKC, phosphatidylinositol 3-kinase (PI3K), extracellular-signal regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) is crucial for the end point response. Remarkably, in this case, ERK activation is achieved in a Ras/Raf/MEK-independent manner. We also propose that p90RSK would be a convergent point between PI3K and ERK pathways. Furthermore, we elucidated the crosstalk among the components of the cascades taking part in TPA-mediated ALAS repression. Finally, by overexpression of a constitutively active p90RSK and the coactivator, cAMP-response element protein (CREB)-binding protein (CBP), we reinforced our previous model, that implies competition between AP-1 and CREB for CBP.
Cross sectional laboratory based study.
In this study, a comparison of different methods to predict drug-polymer solubility was carried out on binary systems consisting of five model drugs (paracetamol, chloramphenicol, celecoxib, indomethacin, and felodipine) and polyvinylpyrrolidone/vinyl acetate copolymers (PVP/VA) of different monomer weight ratios. The drug-polymer solubility at 25 °C was predicted using the Flory-Huggins model, from data obtained at elevated temperature using thermal analysis methods based on the recrystallization of a supersaturated amorphous solid dispersion and two variations of the melting point depression method. These predictions were compared with the solubility in the low molecular weight liquid analogues of the PVP/VA copolymer (N-vinylpyrrolidone and vinyl acetate). The predicted solubilities at 25 °C varied considerably depending on the method used. However, the three thermal analysis methods ranked the predicted solubilities in the same order, except for the felodipine-PVP system. Furthermore, the magnitude of the predicted solubilities from the recrystallization method and melting point depression method correlated well with the estimates based on the solubility in the liquid analogues, which suggests that this method can be used as an initial screening tool if a liquid analogue is available. The learnings of this important comparative study provided general guidance for the selection of the most suitable method(s) for the screening of drug-polymer solubility.
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High levels of resistance found to penicillin, co-trimoxasole and macrolides in isolated pneumococcus strains of healthy carriers in all studied regions, and their association to a previous use of antibiotics, represent a significant public health problem in our country. This emphasizes the need to implement nationwide strategies to reduce the irrational use of antibiotics, especially among children. It is necessary to complement data of resistance to penicillin with the determination of minimal inhibitory concentration to make proper therapeutic recommendations.
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We evaluated the interaction between Punica granatum (pomegranate) methanolic extract (PGME) and antibiotics against 30 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). Susceptibility testing of the isolates to PGME and antibiotics was performed by the broth dilution method. Synergic activity was detected between PGME and the 5 antibiotics tested, chloramphenicol, gentamicin, ampicillin, tetracycline, and oxacillin, ranging from 38% to 73%. For some isolates, PGME did not interfere with the action of any of the antibiotics tested. The bactericidal activity of PGME (0.1 x MIC) in combination with ampicillin (0.5 x MIC) was assessed using chosen isolates by time-kill assays, and they confirmed the synergic activity. Using this combination, cell viability was reduced by 99.9% and 72.5% in MSSA and MRSA populations, respectively. PGME increased the post-antibiotic effect (PAE) of ampicillin from 3 to 7 h. In addition, PGME demonstrated the potential to either inhibit the efflux pump NorA or to enhance the influx of the drug. The detection of in vitro variant colonies of S. aureus resistant to PGME was low and they did not survive. In conclusion, PGME dramatically enhanced the activity of all antibiotics tested, and thus, offers an alternative for the extension of the useful lifetime of these antibiotics.
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Francisella tularensis cerebrospinal fluid culture, antibiotic sensitivity, transmission source, and outcome.
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Plasma disposition of florfenicol in channel catfish was investigated after an oral multidose (10 mg/kg for 10 days) administration in freshwater at water temperatures ranging from 24.7 to 25.9 °C. Florfenicol concentrations in plasma were analyzed by means of liquid chromatography with MS/MS detection. After the administration of florfenicol, the mean terminal half-life (t(1/2)), maximum concentration at steady-state (Css (max)), time of Css (max) (T(max)), minimal concentration at steady-state (Css (min)), and Vc /F were 9.0 h, 9.72 μg/mL, 8 h, 2.53 μg/mL, and 0.653 L/kg, respectively. These results suggest that florfenicol administered orally at 10 mg/kg body weight for 10 days could be expected to control catfish bacterial pathogens inhibited in vitro by a minimal inhibitory concentration value of <2.5 μg/mL.
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The prevalence and degree of antibiotic resistance of faecal indicator bacteria, Escherichia coli and enteroccoci, were determined in 1321 faecal samples collected from pigs at five abattoirs in The Netherlands and in 100 samples from Swedish pigs. In the Dutch samples a high prevalence of resistance was observed in E. coli for three commonly used antibiotics in pig medicine, amoxycillin (70-94%), oxytetracycline (78-98%) and trimethoprim (62-96%). Also, the prevalence for chloramphenicol (55-67%) and neomycin (38-67%) was relatively high. For the other compounds tested the prevalence was less than 10%. The percentage of samples with a high degree of resistant E. coli showed the same tendency in all Dutch abattoirs although significant differences between the abattoirs were observed. The percentage of Swedish samples with a high degree of resistant E. coli was significantly lower for all antibiotics except nitrofurantoin, gentamicin, flumequin and ciprofloxacin. All enterococci were susceptible to amoxycillin and high-level resistance to gentamicin was observed in 4-6% of the Dutch samples. A high prevalence of resistance and a high degree of resistance was found for erythromycin and oxytetracycline. The prevalence of resistance to dalfopristin-quinupristin ranged from 6 to 8% and for vancomycin from 24 to 46%. Significant differences between the abattoirs were found for all compounds tested except amoxycillin. In the Swedish population both the prevalence and degree of resistance in enterococci were significantly lower except for amoxycillin and gentamicin. This point prevalence study showed that the prevalence and degree of antibiotic resistance in indicator bacteria, E. coli and enterococci, in faecal samples from pigs differed between two countries and reflected differences in antibiotic usage in pigs. To analyse the differences observed between the slaughterhouses, additional information about the farms of origin and antibiotic consumption is necessary.