Sequence analysis of small cryptic plasmids isolated from Selenomonas ruminantium S20

The degradation kinetics of five glutamine dipeptides in aqueous solution, i.e. glycyl-L-glutamine (Gly-Gln), L-alanyl-L-glutamine (Ala-Gln), L-valyl-L-glutamine (Val-Gln), L-leucyl-L-glutamine (Leu-Gln) and L-isoleucyl-L-glutamine (Ile-Gln), were studied. Stability tests were performed using a stability-indicating high-performance liquid chromatographic assay. Two different Ala-Gln degradation routes, i.e. the cleavage of a peptide bond and the deamination of an amide group, were observed. The degradation was adequately described by pseudo-first-order kinetics. The maximum stability of Ala-Gln was obtained at an approximate pH of 6.0. The pH-rate profile described by specific acid-base catalysis and hydrolysis by water molecules agreed with the experimental results. The activation energy of Ala-Gln at pH 6.0 was determined to be 27. 1kcal mol-1, and the shelf-life (90% remaining) at 25 and 40 degrees C was predicted to be 5.3 years and 7.1 months, respectively. The rate constants of the glutamine dipeptides were influenced by the N-terminal amino acid residue and decreased in the order: Gly-Gln, Ala-Gln, Leu-Gln, Val-Gln and Ile-Gln.     

Comparison of methods to enumerate bacteria in dental unit water lines

Millipore HPC samplers are simple, self-contained test devices that can be used by personnel in dental offices who do not have microbiologic training to easily and economically monitor dental unit water quality without laboratory support. This study evaluated the correlation of HPC samplers to R2A agar for enumerating planktonic bacteria in dental unit treatment water. Eight different dental units were sampled. Five replicates were performed for each media at each dilution. The Pearson correlation coefficient between the R2A agar and HPC sampler is 0.89. These data suggest HPC samplers correlate with conventional laboratory-based R2A culture techniques for determining dental unit water line contamination.     

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation

1. To determine kinetic parameters (Vmax, K(m)) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike's Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model (F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2. For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (Vmax = 1.2 nmol min-1 mg-1, K(m) = 24 microM) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; Vmax = 2.1 nmol min-1 mg-1, K' = 70 microM; Hill exponent n = 2.34), was favoured according to AIC and the F-test. 3. Data generated by incubating AMI under the same conditions but in the presence of 10 microM ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (Vmax = 0.74 nmol min-1 mg-1, K(m) = 186 microM, K1 = 0.0028 microM-1). 4. Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4'-hydroxylation in humans, had no important effect on this reaction. 5. Incubation of AMI with 50 microM SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (Vmax = 0.72 nmol min-1 mg-1, K(m) = 54 microM) the other with 'Hill-kinetics' (Vmax = 2.1 nmol min-1 mg-1, K' = 195 microM; n = 2.38). 6. A fourth data-set was generated by incubating AMI with 10 microM KET and 50 microM SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (Vmax = 0.048 nmol min-1 mg-1, K(m) = 7 microM) and the other obeying MM kinetics but with substrate inhibition (Vmax = 0.8 nmol min-1 mg-1, K(m) = 443 microM, K1 = 0.0041 microM-1). 7. The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.     

多关节测量臂的小生境混沌优化校准

多关节测量臂是一种便携式的坐标测量设备,它由一系列的旋转关节组成。为了提高多关节测量臂的测量精度和可重复性水平,必须对其运动学参数进行校准。首先,利用小生境的混沌优化算法提出了一种新的运动学校准方法以及一种混合目标的运动学校准函数,它考虑了诸如单点测量实验、容积性测量实验等多种性能测量实验的实验结果,然后,采用Levenberg Marquardt(L-M)算法和小生境混沌优化算法应用于运动学参数校准。小生境混沌优化算法显示出了优于L-M算法的性能。实验结果表明:使用NCOA算法校准后,测量误差的标准差始终优于LMA算法,并且校准前后多关节测量臂的测量精度提高了40倍。采用L-M算法和小生境混沌优化算法应用于运动学参数校准。小生境混沌优化算法显示出了优于L-M算法的性能。     

Dysprosium doped Ga:La:S glass for an efficient optical fibreamplifier operating at 1.3 μm

The authors present experimental results which indicate that a Dy ³⁺-doped Ga:La:S glass fibre amplifier may be operated efficiently at 1.3 μm with an optimum device length significantly shorter than a comparable Pr³⁺-doped glass fibre amplifier. Moreover, the efficiency exceeds that obtained from the Pr³⁺-doped ZBLAN devices currently available     

Medical-resource use for suspected tuberculosis in a New York City hospital

OBJECTIVE: To compare resource use by diagnostic outcome among hospital admissions during which tuberculosis (TB) was suspected. DESIGN: Retrospective study based on chart review and microbiology laboratory data. SETTING: The department of medicine in a municipal hospital serving central Brooklyn, New York. PARTICIPANTS: We identified all adult admissions in 1993 during which TB was suspected. We assigned each admission to one of four mutually exclusive groups defined by the results of microbiological tests (acid-fast bacilli [AFB] smear and culture): culture-positive and smear-positive (C+S+); culture-positive and smear-negative (C+S-); culture-negative and smear-positive (C-S+); or culture-negative and smear-negative (C-S-). Each admission was divided into two separate periods to which the utilization of medical resources was assigned: the diagnostic and the postdiagnostic periods, which were separated by the date of receipt of the first definitive culture report. RESULTS: Data on 519 admissions (93 C+S+; 57 C+S-; 30 C-S+; and 339 C-S-) were analyzed. Although C+S+ were more likely than other groups to have an admitting diagnosis of TB, approximately one quarter of the admissions without TB (C-S+, C-S-) were admitted with the principal diagnosis of TB. For the four groups, C+S+, C+S-, C-S+, and C-S-, the respective rates of TB isolation and anti-TB treatment, and median lengths of isolation were 98%, 87%, and 34 days; 74%, 74%, and 7 days; 83%, 83%, and 15 days; and 44%, 29%, and 0 days. During the diagnostic period, the rate and length of isolation were similar in the AFB-smear-positive groups (C+S+ and C-S+). We estimated that admissions without culture-proven TB (C-S+ and C-S-) accounted for 3,174 (36%) of the 8,712 days of TB isolation expended and for 65% of the 16,671 days of anti-TB treatment. The vast majority of this resource consumption (2,737 [86%] of 3,174 days of isolation) occurred during the diagnostic period before a definitive culture result was known. CONCLUSIONS: Our results suggest that prolonged diagnostic uncertainty and misclassification of cases due to false-positive and false-negative smears are associated with substantial medical-resource consumption. New diagnostic modalities that reduce the period of diagnostic uncertainty could reduce the utilization of resources later found to be unnecessary.     

Multiple human cytochromes contribute to biotransformation of dextromethorphan in-vitro: role of CYP2C9, CYP2C19, CYP2D6, and CYP3A

Cytochromes mediating the biotransformation of dextromethorphan to dextrorphan and 3-methoxymorphinan, its principal metabolites in man, have been studied by use of liver microsomes and microsomes containing individual cytochromes expressed by cDNA-transfected human lymphoblastoid cells. In-vitro formation of dextrorphan from dextromethorphan by liver microsomes was mediated principally by a high-affinity enzyme (Km (substrate concentration producing maximum reaction velocity) 3-13 microM). Formation of dextrorphan from 25 microM dextromethorphan was strongly inhibited by quinidine (IC50 (concentration resulting in 50% inhibition) = 0.37 microM); inhibition by sulphaphenazole was approximately 18% and omeprazole and ketoconazole had minimal effect. Dextrorphan was formed from dextromethorphan by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, -2C19, and -2D6 but not by those expressing CYP1A2, -2E1 or -3A4. Despite the low in-vivo abundance of CYP2D6, this cytochrome was identified as the dominant enzyme mediating dextrorphan formation at substrate concentrations below 10 microM. Formation of 3-methoxy-morphinan from dextromethorphan in liver microsomes proceeded with a mean Km of 259 microM. For formation of 3-methoxymorphinan from 25 microM dextromethorphan the IC50 for ketoconazole was 1.15 microM; sulphaphenazole, omeprazole and quinidine had little effect. 3-Methoxymorphinan was formed by microsomes from cDNA-transfected lymphoblastoid cells expressing CYP2C9, -2C19, -2D6, and -3A4, but not by those expressing CYP1A2 or -2E1. CYP2C19 had the highest affinity (Km = 49 microM) whereas CYP3A4 had the lowest (Km = 1155 microM). Relative abundances of the four cytochromes were determined in liver microsomes by use of the relative activity factor approach. After adjustment for relative abundance, CYP3A4 was identified as the dominant enzyme mediating 3-methoxymorphinan formation from dextromethorphan, although CYP2C9 and -2C19 were estimated to contribute to 3-methoxymorphinan formation, particularly at low substrate concentrations. Although formation of dextrorphan from dextromethorphan appears to be sufficiently specific to be used as an in-vitro or in-vivo index reaction for profiling of CYP2D6 activity, the findings raise questions about the specificity of 3-methoxymorphinan formation as an index of CYP3A activity.     

RP-HPLC binding domains of proteins

Procedures have been developed to identify the chromatographic binding domains of horse heart cytochrome c (Cyt c) and bovine growth hormone (bGH) during their interaction with reversed-phase sorbent materials. The procedure involves adsorption of the protein solute to the chromatographic sorbent, followed by proteolytic cleavage. Comparison of the proteolytic map obtained for Cyt c and bGH in free solution with the corresponding map obtained when these proteins are adsorbed to the chromatographic sorbent revealed significant differences in the digestion pattern. Following characterization of the peptides generated in both maps, the results indicated that specific regions on the surface of both Cyt c and bGH are inaccessible to tryptic cleavage when adsorbed to the hydrophobic surface of both a C-4 and a C-18 sorbent. Based on the assumption that the region of the protein surface that is in contact with the sorbent remains intact and bound to the sorbent during the digestion step, while the protein surface that is exposed to the solvent is accessible to proteolysis, the regions that were inaccessible to tryptic digestion were found to correspond to hydrophobic domains on the protein surface. These results also suggest that the three-dimensional structures of these proteins remain largely intact upon adsorption to the hydrophobic surface.