Structural analysis of three His32 mutants of DsbA: Support for an electrostatic role of His32 in DsbA stability

DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys3O-Pro31-His32-Cys33). Unlike most protein disulfides, the active-site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH. His32, one of the two residues between the active-site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of similar to 5-7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild-type protein. Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 Variants are very similar to the wild-type oxidized structure, including conservation of solvent atoms near the active-site residue, Cys3O. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature.

New nickel catalysts supported on highly porous alumina intercalated laponite for methane reforming with CO2

Alumina intercalated laponite (Al-laponite) was prepared with a polyethylene oxide (PEO) surfactant and used as supports of nickel catalysts for the carbon dioxide reforming reaction with methane to synthesis gas. The effects of the supports of intercalated laponite and catalyst preparation on catalytic activity, stability and carbon deposition were investigated for the above reforming reaction. We found that the pore structure of the Al-laponite supports can be tailored with the surfactant and the catalyst with well-developed porosity exhibited higher catalytic activity and a longer time of catalyst stability. (C) 2001 Elsevier Science B.V. All rights reserved.

Acoustic signature of the normal swallow: Characterization by age, gender, and bolus volume

Despite growing clinical use, cervical auscultation suffers from a lack of research-based data. One of the strongest criticisms of cervical auscultation is that there has been little research to demonstrate how dysphagic swallowing sounds are different from normal swallowing sounds, In order to answer this question, however, one first needs to document the acoustic characteristics of normal, nondysphagic swallowing sounds, This article provides the first normative database of normal swallowing sounds for the adult population. The current investigation documents the acoustic characteristics of normal swallowing sounds for individuals from 18 to more than 60 years of age over a range of thin liquid volumes. Previous research has shown the normal swallow to be a dynamic event. The normal swallow is sensitive to aging of the oropharyngeal system, and also to the volume of bolus swallowed. The current investigation found that the acoustic signals generated during swallowing were sensitive to an individual's age and to the volume of the bolus swallowed. There were also some gender-specific differences in the acoustic profile of the swallowing sound, It is anticipated that the results will provide a catalyst for further research into cervical auscultation.

Metabolism of tamoxifen by recombinant human cytochrome P450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization oftrans-4-hydroxytamoxifen

The cytochrome P450 (P450)-mediated biotransformation of tamoxifen is important in determining both the clearance of the drug and its conversion to the active metabolite, trans-4-hydroxytamoxifen. Biotransformation by P450 forms expressed extrahepatically, such as in the breast and endometrium, may be particularly important in determining tissue-specific effects of tamoxifen. Moreover, tamoxifen may serve as a useful probe drug to examine the regioselectivity of different forms. Tamoxifen metabolism was investigated in vitro using recombinant human P450s. Forms CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7 were coexpressed in Escherichia coli with recombinant human NADPH-cytochrome P450 reductase. Bacterial membranes were harvested and incubated with tamoxifen or trans-4-hydroxytamoxifen under conditions supporting P450-mediated catalysis. CYP2D6 was the major catalyst of 4-hydroxylation at low tamoxifen concentrations (170 +/- 20 pmol/40 min/0.2 nmol P450 using 18 muM tamoxifen), but CYP2B6 showed significant activity at high substrate concentrations (28.1 +/- 0.8 and 3.1 +/- 0.5 nmol/120 min/0.2 nmol P450 for CYP2D6 and CYP2B6, respectively, using 250 muM tamoxifen). These two forms also catalyzed 4'-hydroxylation (13.0 +/- 1.9 and 1.4 +/- 0.1 nmol/120 min/0.2 nmol P450, respectively, for CYP2B6 and CYP2D6 at 250 muM tamoxifen; 0.51 +/- 0.08 pmol/40 min/0.2 nmol P450 for CYP2B6 at 18 muM tamoxifen). Tamoxifen N-demethylation was mediated by CYP2D6, 1A1, 1A2, and 3A4, at low substrate concentrations, with contributions by CYP1B1, 2C9, 2C19 and 3A5 at high concentrations. CYP1B1 was the principal catalyst of 4-hydroxytamoxifen trans-cis isomerization but CYP2B6 and CYP2C19 also contributed.

Dryout phenomena in a three-phase fixed-bed reactor

Understanding the mechanism of liquid-phase evaporation in a three-phase fixed-bed reactor is of practical importance, because the reaction heat is usually 7-10 times the vaporization heat of the liquid components. Evaporation, especially the liquid dryout, can largely influence the reactor performance and even safety. To predict the vanishing condition of the liquid phase, Raoult's law was applied as a preliminary approach, with the liquid vanishing temperature defined based on a liquid flow rate of zero. While providing correct trends, Raoult's law exhibits some limitation in explaining the temperature profile in the reactor. To comprehensively understand the whole process of liquid evaporation, a set of experiments on inlet temperature, catalyst activity, liquid flow rate, gas flow rate, and operation pressure were carried out. A liquid-region length-predicting equation is suggested based on these experiments and the principle of heat balance.