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Incubation Conditions (incubation + condition)
Selected AbstractsMALDI-TOF mass spectrometric analysis for the characterization of the 5,10,15,20-tetrakis- (m -hydroxyphenyl)bacteriochlorin (m -THPBC) photoproducts in biological environmentJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2005Henri-Pierre Lassalle Abstract Photoproducts formation upon irradiation (739 nm) of 5,10,15,20-tetrakis(m -hydroxyphenyl)bacteriochlorin (m -THPBC) in phosphate buffer saline (PBS) supplemented with human serum albumin (HSA) were studied by means of absorption spectroscopy and MALDI-TOF mass spectrometry. The experiments were performed with a freshly prepared PBS,HSA solution of m -THPBC and with a PBS,HSA m -THPBC solution incubated for 6 h at 37 °C. The incubation of m -THPBC solution leads to the dye monomerisation, whereas in the freshly prepared solution, m -THPBC is under an aggregated form. Regardless of the incubation condition, photobleaching experiments carried out by absorption spectroscopy demonstrate the degradation of the photosensitizer and its phototransformation in m -THPC. Moreover, m -THPC was the sole photoproduct detected using absorption spectroscopy. Together with a degradation of m -THPBC and formation of m -THPC, MALDI-TOF mass spectrometry evidenced several other photoinduced modifications. Photoproducts such as dihydroxy m -THPBC and dihydroxy m -THPC were detected in both conditions; however, the formation of hydroxylated photoproducts was significantly greater in incubated solution. In addition, small molecules arising from the degradation of the photosensitizer and identified as dipyrin derivatives and dipyrrolic synthon were observed. Copyright © 2005 John Wiley & Sons, Ltd. [source] CE-based noncompetitive immunoassay for immunoglobulin G in bovine colostrum productsELECTROPHORESIS, Issue 21 2007Jin Zhao Abstract A CE-based noncompetitive immunoassay for IgG in bovine colostrum products was established. FITC-labeled protein G (FITC-PrG) was tagged through noncovalent bindings to the Fc region of the mouse monoclonal antibovine IgG (Ab). The FITC-PrG, Ab, and IgG formed a sandwiched immunocomplex FITC-PrG-Ab-IgG under optimal incubation conditions. The immunocomplex was separated and analyzed by CZE with LIF detection in less than 2,min in an uncoated fused-silica capillary. Addition of PEG 20,000 (PEG 20M) in the running buffer significantly suppressed analyte adsorption and thus improved the reproducibility and the resolution. The precision of the method was 5.1% (n,=,7). A linear relationship was established for the IgG concentration in the range of 1,5,mg/L with a linear correlation coefficient (r,=,0.9917). The LOD was 0.1,mg/L (S/N,=,3). The method was successfully applied for the determination of IgG in bovine colostrum products and satisfactory results were achieved. [source] Persistence and degradation of maize-expressed vaccine protein, Escherichia coli heat-labile enterotoxin subunit B, in soil and water,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 6 2008Hirofumi Kosaki Abstract Transgenic plants represent an innovative platform for the cost-effective large-scale production of various pharmaceutical proteins. The eventual open-field production of plant-made pharmaceuticals (PMPs) requires risk assessment to determine the potential for harm to the surrounding ecosystem. In the present study, the environmental persistence of a transgenic maize-expressed antigen, Escherichia coli heat-labile enterotoxin subunit B (LTB), was studied under laboratory conditions. To semiquantitatively monitor the persistence of LTB in soil, extraction with a high-salt, high-pH extraction buffer was optimized using the closely homologous Vibrio cholerae enterotoxin subunit B (CTB) as a test substance. The time to dissipation of 50% (DT50) of the extractable fraction of maize-expressed LTB was 4 to 15 d in pond water and 35 to 90 d in soils. Both extraction efficacy and persistence were strongly affected by the matrix type and incubation conditions. In contrast with maize-expressed LTB, the DT50 for bacterially produced LTB and CTB was less than 4 d both in pond water and soil. Although maize-expressed LTB was more stable than bacterially produced analogue, its dissipation was governed by an initial lag, which could be attributed to release from the plant material, followed by rapid decline. [source] Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stressFEBS JOURNAL, Issue 3 2000Barbara Tavazzi To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5,10 mm) of hydrogen peroxide for 1 h at 37 °C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mm H2O2), oxidative stress produced, starting from 1 mm H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mm). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mm H2O2 -treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mm H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress. [source] Analysis of microbial community functional diversity using sole-carbon-source utilisation profiles , a critiqueFEMS MICROBIOLOGY ECOLOGY, Issue 1 2002Juliet Preston-Mafham Abstract Information on functional diversity (metabolic potential) is essential for understanding the role of microbial communities in different environments. Variations of the commercially available BIOLOG bacterial identification system plates are now widely used to assess functional diversity of microorganisms from environmental samples, based on utilisation patterns of a wide range (up to 95) of single carbon sources. There are many problems as well as benefits of using the approach, but the former are often disregarded. Here the basis of the approach is summarised, including type of plate to use, treatment of samples, replication, incubation conditions, monitoring of plates, and statistical analysis. The pros and cons of its use are critically assessed, inherent biases and limitations are pointed out and methodological difficulties are considered. Possible ways of overcoming some of the difficulties are suggested. [source] Female zebra finches compromise clutch temperature in energetically demanding incubation conditionsFUNCTIONAL ECOLOGY, Issue 5 2010Andreas Nord Summary 1.,Avian embryos depend on the incubating parent to provide a thermal environment suitable for embryogenesis, but as the maintenance of optimal incubation temperatures is energetically costly, an incubating bird often must trade off embryonic investment against self-maintenance. 2.,We manipulated the energetic cost of incubation in female zebra finches (Taeniopygia guttata Vieillot) by varying ambient temperature and clutch size during nocturnal incubation and recorded the corresponding effects on incubation metabolic rate and clutch temperature. 3.,Females increased their night-time incubation metabolic rate more than twofold when incubating at 10 °C compared to when incubating close to thermoneutrality (28 °C). Furthermore, clutch enlargement caused females to elevate their metabolic rate with 2·8% per additional egg added to the clutch. 4.,However, despite spending more energy, females did not fully cover the increased costs of incubation, because clutch temperature decreased with decreasing ambient temperature and increasing clutch size. 5.,These findings suggest that parental investment in incubation can be energetically constrained and sometimes result in clutch temperatures below the optimal level for embryonic development, at least during nocturnal incubation. [source] Characterization of an enrichment culture debrominating tetrabromobisphenol A and optimization of its activity under anaerobic conditionsJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2010L. Iasur-Kruh Abstract Aim:, To study the effects of incubation conditions on the microbial community structure and activity of a TBBPA-debrominating enrichment culture composed of bacterial and archaeal species. Methods and Results:, The effects of the methanogen inhibitor 2-bromoethanesulfonate (BES), of the antibiotic ampicillin, of substrate (tetrabromobisphenol A, TBBPA) omission and availability of different electron donors on microbial community structure and activity were examined under anaerobic conditions. Debromination of TBBPA was blocked in the presence of ampicillin, while long-term incubation with BES resulted in delayed debromination activity. The results suggest that the bacterial species responsible for the debromination of TBBPA, while archaeal species involved in electron donor metabolism. The enrichment culture lost its debromination activity after cultivation for 9 months without TBBPA, concomitantly with the disappearance of two DNA bands in a denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments corresponding to Pelobacter carbinolicus and Sphaerochaeta sp. TQ1 that were present in the original culture. When butyrate was used as an electron donor, TBBPA debromination activity was attenuated. When acetate was used as the electron donor, no debromination was observed and in addition, there was a decrease in the abundance of the mcrA gene. Conclusions:, The results indicate that to maintain a high rate of TBBPA debromination activity, it is essential to preserve the microbial community structure (bacterial and archaeal members) of this culture and supply an electron donor that produces high amounts of hydrogen when fermented. Significance and Impact of the Study:, The study provides important information for the management of cultures to be used in bioremediation of TBBPA contaminated sites. [source] Simultaneous purification and immobilization of bitter gourd (Momordica charantia) peroxidases on bioaffinity supportJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 2 2005Suhail Akhtar Abstract This paper demonstrates the construction of an inexpensive bioaffinity adsorbent by simply incubating Sephadex G 50 matrix with jack bean meal extract at room temperature. Sephadex G 50 adsorbed 17 mg Con A (concanavalin A) per g of the matrix. Con A-adsorbed Sephadex was employed for the immobilization of glycoenzymes directly from ammonium sulfate-fractionated proteins of bitter gourd. The obtained bioaffinity support was very efficient for high yield immobilization of peroxidases from bitter gourd and it bound nearly 425 enzyme units per g of the matrix. Bitter gourd peroxidase immobilized on lectin,Sephadex support showed a very high effectiveness factor, ,,,' of 1.25. Immobilized BGP preparation was quite stable against the denaturation induced by pH, heat, urea, Triton X 100, Tween 20, SDS, Surf Excel and water-miscible organic solvents: dimethyl sulfoxide and dimethyl formamide. Low concentration of detergents like SDS, Tween 20, and Triton X 100 enhanced the activity of soluble and immobilized bitter gourd peroxidase. Peroxidase bound to the bioaffinity support exhibited very high resistance to proteolysis caused by the trypsin treatment. Con A,Sephadex-bound bitter gourd peroxidase retained 85% of its initial activity after treatment with 2.5 mg trypsin per cm3 of incubation mixture for 1 h at 37 °C while the soluble enzyme lost nearly 40% of the initial activity under similar incubation conditions. Immobilized bitter gourd peroxidase preparation appeared to be more rigid to proteolysis mediated by trypsin compared with soluble bitter gourd peroxidase. Copyright © 2004 Society of Chemical Industry [source] Novel method for clearing red blood cell debris from BacT/ALERT® blood culture medium for improved microscopic and antimycobacterial drug susceptibility test resultsJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 4 2007Krishnamoorthy Gopinath Abstract Even though automation in mycobacterial culture has immensely improved the detection of organisms, identification of species and antimycobacterial susceptibility testing from blood culture bottles remain cumbersome and error-prone due to the presence of intact red blood cells (RBCs). The removal or lysis of these RBCs and excessive protein from the blood components could theoretically help improve this process. The present study reports an effective method that uses ammonium chloride (NH4Cl) and Triton X-100 to lyse the RBCs in blood culture medium. The method was optimized by preparing various concentrations of NH4Cl and Triton X-100, and incubation conditions, leading to eight protocols. The lysis protocol with a concentration of 150,mM of NH4Cl, 0.5% Triton X-100, and 1% potassium bicarbonate, pH 7.0, and incubation at 37°C for 15,min was found to be optimal. This method not only made the culture medium clear, the protein concentration decreased from 753.5±39.4 to 53.2±4.2,mg/mL in the M. tuberculosis -spiked culture medium and in the blood culture medium inoculated with the blood from tuberculosis patients. The method had no adverse effect on mycobacteria, and no depletion of M. tuberculosis colony-forming units was found. The lysate could be used for antimycobacterial susceptibility testing with no difficulty in setting the mycobacterial concentration of inoculum to 0.5 McFarland standards. Furthermore, this method had the added advantage in the microscopy and molecular methods for the speciation of Mycobacterium sp. J. Clin. Lab. Anal. 21:220,226, 2007. © 2007 Wiley-Liss, Inc. [source] Stability of intravesical epirubicin infusion: a sequential temperature studyJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 5 2003G. J. Sewell PhD Summary Objective:, To investigate the stability of epirubicin bladder instillation, prepared from two different epirubicin formulations, under refrigerated storage, transportation and clinical use conditions. Method:, A sequential study design was used. Epirubicin instillation (1 mg/mL) in polypropylene syringes was sequential incubated for periods of 84 days at 8°C followed by 2 h at 25°C and 1 h at 37°C, the latter two temperatures replicating transport and intravesical conditions, respectively. Results:, The instillation was both chemically and physically stable under those incubation conditions. The formulation of epirubicin used to prepare the instillation infusions did not affect stability. [source] Phytate content and phytate degradation by endogenous phytase in pea (Pisum sativum)JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 12 2001Mattias Fredrikson Abstract In order to rapidly reduce the content of inositol tri,hexaphosphates in pea flour by action of the endogenous phytase, raw materials as well as incubation conditions have been evaluated. The phytate (inositol hexaphosphate) content was analysed in 27 pea varieties; the influence of storage time and the difference in phytate content between the germ and the cotyledon were determined. Furthermore, degradation of inositol phosphates by the endogenous phytase enzyme was studied in pea flour, germ and cotyledon. To find the maximum phytate degradation, the effects of temperature and pH during pea flour incubation were investigated. The most efficient phytate degradation in pea flour incubation was achieved at pH 7.5 and 45,°C. At this condition an almost complete degradation of phytate and a 66% reduction in the sum of inositol hexa-, penta-, tetra- and triphosphates were reached in 10,h. The storage time of pea seeds or removal of the germ did not have a major effect on the phytate content. Since several inositol pentaphosphate isomers were produced during phytate degradation, it can be concluded that peas contain several phytate-degrading enzymes, or one phytate-degrading enzyme with unspecific initial hydrolysation pattern. © 2001 Society of Chemical Industry. [source] Food uptake in the mixotrophic Dinophysis acuminataTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2005LUCIE MARANDA Evidence of food uptake in the photosynthetic genus Dinophysis comes solely from the presence of food vacuoles, as no photosynthetic cells have ever been observed in the act of feeding. We examined the feeding ecology of D. acuminata in natural populations and under laboratory conditions. Using depth-integrated sampling of the water column, we determined the frequency of food vacuolated cells at 2-h intervals over a 24-h period in a shallow marine embayment. Food vacuoles in preserved cells were enumerated using Nomarski differential interference contrast microscopy; ultrastructural characters were recorded by transmission electron microscopy. A peak in the feeding activity was observed toward dusk for an abundant June population, with 26% of cells with at least one food vacuole. Mechanisms of concurrent carbon acquisition were evident from the presence of chloroplasts with starch grains and food vacuoles within the same cell. Vacuole content could not be identified. In a preliminary 2-wk long simulated grazing experiment, a mixture of two hypothesized preys, Rhodomonas salina and Dunaliella tertiolecta, was offered to D. acuminata; the Dinophysis populations decreased steadily and at the same rate, whether food was present or not. The evaluation of the food vacuole frequency will be repeated in the coming season to verify the observed pattern, while grazing experiments will include a variety of food items and incubation conditions. Our current inability to successfully culture any photosynthetic Dinophysis limits ecophysiological approaches, either at the population or cellular level, to manipulation of field samples. Supported by National Institutes of Health Grant GM62126-01A1. [source] Bacteriocin production by Shigella sonnei isolated from faeces of children with acute diarrhoeaAPMIS, Issue 2 2010MIREILLE ÂNGELA BERNARDES SOUSA Sousa MÂB, Mendes EN, Apolônio ACM, Farias LM, Magalhães PP. Bacteriocin production by Shigella sonnei isolated from faeces of children with acute diarrhoea. APMIS 2010; 118: 125,35. Shigella is a common agent of diarrhoea, a worldwide major health problem. The bacterium produces bacteriocins; however, the role of these substances as a virulence factor is completely unknown. With the aim to search for colicin production by Shigella sonnei, to evaluate the influence of culture conditions on bacteriocin expression, and to characterize the substance partially, 16 S. sonnei strains isolated from children with diarrhoea were tested for antagonism against members of the intestinal microbiota or agents of diarrhoea. Nine strains exhibited isoantagonism and heteroantagonism against S. flexneri and diarrhoeagenic Escherichia coli. Autoantagonism and antagonism against the intestinal microbiota were not detected. Culture medium and incubation conditions influenced antagonism expression. Antagonism resulting from bacteriophages, low pH, fatty acids, hydrogen peroxide, and chloroform was excluded. The activity of the intracellular fraction obtained with 75% ammonium sulphate was preserved at pH 1.0,11.0, and was found to be reduced by organic solvents and affected by high temperatures and proteases. The antagonistic spectrum and the in vitro conditions for better antagonism expression suggest that the role of colicin in S. sonnei virulence, if any, would be expressed prior to infection, and may regulate population density of enteropathogens by helping in organism transmission. [source] Candida glabrata, an emerging fungal pathogen, exhibits superior relative cell surface hydrophobicity and adhesion to denture acrylic surfaces compared with Candida albicansAPMIS, Issue 9 2002G. Luo Oral candidosis is a common opportunistic infection in debilitated individuals and Candida glabrata is the second most frequently isolated species from this condition, after Candida albicans. Candidal adherence to various biological or non-biological surfaces is considered a prerequisite for colonization, and pathogenesis of candidal infections, and their relative cell surface hydrophobicity (CSH) is likely to be a possible contributory force involved in this process. Whereas a large body of data on the latter features of C. albicans is available, there is surprisingly little information on C. glabrata. As a comprehensive database on the relative adhesion and CSH of Candida spp. is instructive and useful, we investigated in vitro the latter attributes of 34 oral isolates of C. glabrata and 15 isolates of C. albicans. There were remarkable intraspecies differences in both the CSH and the adhesive ability of C. glabrata strains (p<0.001). Compared with C. albicans, C. glabrata demonstrated a four-fold greater CSH value (30.63±11.20% vs 7.23±3.56%, p<0.0001) and a two-fold greater tendency to adhere to denture acrylic surfaces (75.18±39.96 vs 30.36±9.21, p<0.0001). A significant positive correlation between CSH and adhesion was also noted for both C. glabrata (r=0.674, p<0.0001) and C. albicans (r=0.636, p<0.05). When the effect of different incubation conditions on the relative CSH and adherence of C. glabrata was examined, CSH and the adherence to acrylic surfaces of four of six C. glabrata isolates were significantly affected by a reduction of the culture temperature (from 37 °C to 25 °C). A positive relationship also emerged when the temperature-induced variations in the adherence values were correlated with their relative CSH. These data provide hitherto unavailable archival information on important pathogenic attributes of the two most common oral Candida species that may help explain their predominance in this milieu. [source] Effects of nest temperature and moisture on phenotypic traits of hatchling snakes (Tropidonophis mairii, Colubridae) from tropical AustraliaBIOLOGICAL JOURNAL OF THE LINNEAN SOCIETY, Issue 1 2006GREGORY P. BROWN Previous research on developmentally plastic responses by reptile embryos has paid relatively little attention to tropical species, or to possible interactions between the effects of thermal and hydric regimes. In the present study, eggs of keelback snakes (Tropidonophis mairii), from a tropical area with strong temporal and spatial variation in soil temperatures and moisture levels, were incubated. The phenotypic traits of hatchling snakes (body size, shape, muscular strength) were affected by moisture content of the incubation medium (vermiculite plus 100% vs. 50% water by mass), by mean incubation temperatures (25.7 vs. 27.9 °C) and by diel thermal variation (diel range 6.0 vs. 8.4 °C). Interactions between these factors were negligible. Cooler, more thermostable, moister conditions resulted in larger offspring, a trait under strong selection in this population. Thermal and hydric conditions covary in potential nest-sites (e.g. deeper nests are more thermostable as well as moister). This covariation may influence the evolution of reaction norms for embryogenesis. For example, if moister nests enhance offspring fitness and are cooler, then selection will favour the ability to develop in cool as well as moist conditions. Thus, the evolution of optimal incubation conditions with respect to one variable (e.g. temperature) may be driven by patterns of association with another variable (e.g. soil moisture) among natural nest-sites. Perhaps for this reason, the thermal optimum for incubation is surprisingly low in this tropical species. © 2006 The Linnean Society of London, Biological Journal of the Linnean Society, 2006, 89, 159,168. [source] New method for the simultaneous estimation of intrinsic hepatic clearance and protein binding by matrix inhibitionBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 1 2008Takahide Uchimura Abstract The purpose of this study was to develop a method for estimating the hepatic clearance (CLh) without using a protein binding test. This method allows the simultaneous evaluation of the intrinsic hepatic clearance (CLint) with a correction for microsomal binding, and the free fraction in the serum (fu). It uses the decrease in metabolic velocity achieved by decreasing the free fraction of a compound in the incubation mixture (fuinc) by the addition of serum, and by changing the microsomal protein concentration. This method is denoted as the ,matrix inhibition method', because it uses the inhibition of the metabolic velocity by the incubation matrix. The metabolic rates of eight compounds (diazepam, imipramine, warfarin, and compounds A,E) were evaluated under several incubation conditions using rat serum and microsomes. The correlation of CLint evaluated using the method and using equilibrium dialysis after the CLint was corrected for microsomal binding was r,=,0.968. The correlation of fu,·,CLint was r,=,0.996. Although the method required a high enough fu and fumicrosomes difference among the reaction conditions for each compound, it could evaluate CLint and fu simultaneously and easily by adding additional reaction conditions to the metabolic stability tests performed in ADME screening. Copyright © 2007 John Wiley & Sons, Ltd. [source] In vitro,in vivo correlations for drugs eliminated by glucuronidation: Investigations with the model substrate zidovudineBRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Issue 5 2002Sam Boase Aims, To investigate the effects of incubation conditions on the kinetic constants for zidovudine (AZT) glucuronidation by human liver microsomes, and whether microsomal intrinsic clearance (CLint) derived for the various conditions predicted hepatic AZT clearance by glucuronidation (CLH) in vivo. Methods, The effects of incubation constituents, particularly buffer type (phosphate, Tris) and activators (Brij58, alamethacin, UDP-N-acetylglucosamine (UDP-NAcG)), on the kinetics of AZT glucuronidation by human liver microsomes was investigated. AZT glucuronide (AZTG) formation by microsomal incubations was quantified by h.p.l.c. Microsomal CLint values determined for the various experimental conditions were extrapolated to a whole organ CLint and these data were used to calculate in vivo CLH using the well-stirred, parallel tube and dispersion models. Results, Mean CLint values for Brij58 activated microsomes in both phosphate (3.66 ± 1.40 µl min,1 mg,1, 95% CI 1.92, 5.39) and Tris (3.79 ± 0.74 µl min,1 mg,1, 95% CI 2.87, 4.71) buffers were higher (P < 0.05) than the respective values for native microsomes (1.04 ± 0.42, 95% CI 0.53, 1.56 and 1.37 ± 0.30 µl min,1 mg,1, 95% CI 1.00, 1.73). Extrapolation of the microsomal data to a whole organ CLint and substitution of these values in the expressions for the well-stirred, parallel tube and dispersion models underestimated the known in vivo blood AZT clearance by glucuronidation by 6.5- to 23-fold (3.61,12.71 l h,1vs 82 l h,1). There was no significant difference in the CLH predicted by each of the models for each set of conditions. A wide range of incubation constituents and conditions were subsequently investigated to assess their effects on GAZT formation, including alamethacin, UDP-NAcG, MgCl2, d -saccharic acid 1,4-lactone, ATP, GTP, and buffer pH and ionic strength. Of these, only decreasing the phosphate buffer concentration from 0.1 m to 0.02 m for Brij58 activated microsomes substantially increased the rate of GAZT formation, but the extrapolated CLH determined for this condition still underestimated known AZT glucuronidation clearance by more than 4-fold. AZT was shown not to bind nonspecifically to microsomes. Analysis of published data for other glucuronidated drugs confirmed a trend for microsomal CLint to underestimate in vivo CLH. Conclusions, AZT glucuronidation kinetics by human liver microsomes are markedly dependent on incubation conditions, and there is a need for interlaboratory standardization. Extrapolation of in vitro CLint underestimates in vivo hepatic clearance of drugs eliminated by glucuronidation. [source] Mueller,Hinton agar is superior to PDM blood agar for detection of methicillin-resistant Staphylococcus aureusCLINICAL MICROBIOLOGY AND INFECTION, Issue 1 2003T. Monsen The aim of this study was to compare the expression of oxacillin resistance in methicillin-resistant Staphylococcus aureus (MRSA) on Paper Disc Method agar supplemented with 5% defibrinated blood (PDM blood agar) and Mueller,Hinton agar supplemented with 2% NaCl (MH NaCl agar) using different susceptibility tests. Fifty mecA- containing isolates of S. aureus, exhibiting 46 different pulsed-field gel electrophoresis patterns, were comparatively tested using the E test, the single disk diffusion test, and the multipoint inoculation technique, under various culture conditions. The E test incubated at 35 °C for 24 h (breakpoint of resistance ,2.0 mg/L) detected 94% of the isolates on MH NaCl agar, compared with 28% for PDM blood agar (P < 0.05). The disk diffusion test (breakpoint ,,10 mm in diameter) under these incubation conditions detected resistance in 100% of the isolates on MH NaCl agar and in 80% of the isolates on PDM blood agar (P < 0.05). The multipoint technique (breakpoint ,1 mg/L), applied at 35 °C for 24 h, detected 100% on MH NaCl agar and 46% on PDM blood agar (P < 0.05). Irrespective of the method of susceptibility testing evaluated, MH NaCl agar was superior to PDM blood agar for the detection of oxacillin resistance in mecA -containing S. aureus. [source] | ||||||||||||||||