(C) 2009 Elsevier Inc. All rights reserved.”
“Cytochrome b(5) (b(5)) has been shown to modulate many cytochrome P450 (CYP)-dependent reactions. In order to elucidate the mechanism of such modulations, it is necessary to evaluate not only the effect of native b(5) on CYP-catalyzed reactions, but also that of the apo-cytochrome b(5) (apo-b(5)). Therefore, the apo-b(5) protein was prepared using a heterologous expression in Escherichia coli. The gene for rabbit b(5) was constructed from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and

amplified in DH5 alpha cells. The gene sequence was verified by DNA sequencing. The sequence coding b(5) was cleaved from pUC19 by Ndel and Xhol restriction endonucleases and subcloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) GSK621 price Cold cells by heat shock. Expression of b(5) was induced with isopropyl beta-D-1-thiogalactopyranoside (IPTG). The b(5) protein, produced

predominantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE-Sepharose. Using such procedures, the homogenous preparation of apo-b(5) protein was obtained. BAY 80-6946 cell line Oxidized and reduced forms of the apo-b(5) reconstituted with heme exhibit the same absorbance spectra as native b(5). The prepared recombinant apo-b(5) reconstituted with heme can be reduced by NADPH:CYP reductase. The

reconstituted apo-b5 is also fully biologically active, exhibiting the comparable stimulation effect on the CYP3A4 enzymatic activity towards oxidation of 1-phenylazo-2-hydroxynaphthalene (Sudan I) as native rabbit and human b(5). (C) 2009 Published by Elsevier Inc.”
“This study developed an approach to quantify frailty with a frailty index (FI) and investigated whether age-related changes in contractions, calcium transients, PAK5 and ventricular myocyte length were more prominent in mice with a high FI. The FI combined 31 variables that reflect different aspects of health in middle-aged (similar to 12 months) and aged (similar to 30 months) mice of both sexes. Aged animals had a higher FI than younger animals (FI = 0.43 +/- 0.03 vs 0.08 +/- 0.02, p < .001, n = 12). Myocyte hypertrophy increased by 30%-50% as the FI increased in aged animals. Peak contractions decreased more than threefold from lowest to highest FI values in aged mice (p < .037), but calcium transients were unaffected. Similar results were seen with an FI based on eight noninvasive variables identified as underlying factors. These results show that an FI can be developed for murine models and suggest that age-associated changes in myocytes are more prominent in animals with a high FI.