A Novel Method for the Preparation of Substrate-Free Cytochrome P-45011β

A new method for the removal of the stabilizing substrate, deoxycorticosterone, from adrenal cytochrome P-450_11β, has been developed. Dextran coated charcoal is used for the adsorption of the steroid and the adsorbed steroid is separated from the cytochrome P-450-preparation by low speed centrifugation. The substrate-free enzyme, obtained in this manner, has all the characteristic spectral properties of low-spin cytochrome P-450_11β, and may be converted to the high-spin form by the addition of deoxycorticosterone.

The dextran coated charcoal method has the following advantages over the previously used method of substrate removal. It does not require the addition of the cofactors for cytochrome P-450-dependant hydroxyla-tion of deoxycorticosterone, small amounts of enzyme may be prepared in a short time and the enzyme preparation is not diluted to any great extent during the process.     

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC(50)=1.06μM) and CYP2C9 (IC(50)=3.89μM), minimal inhibition on CYP3A4 (IC(20)=11.94μM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12μM, 2.00μM and 95.03μM, respectively. HM-1 competitively inhibited testosterone 6β-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.     

Radiochemical and radiobiological assessment of a pyridyl-S-cysteine functionalized bombesin derivative labeled with the 99mTc(CO)3+ core

Bombesin is a neuropeptide widely studied due to its ability to target various types of cancers. Technetium-99m on the other hand is ideal for diagnostic tumor targeting. The aim of the present study is the investigation of the coupling of the ligand (S)-(2-(2′-pyridyl)ethyl)-d,l-cysteine with the BN-peptide Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met(CONH₂) through the spacer aminohexanoic acidand the labeling of the resulting derivative MBN with the synthon [M(CO)₃(H₂O)₃]⁺ (M = ^99mTc, Re). The peptide was synthesized according to the SPPS method, purified and characterized by ESI-MS. The new ^99mTc-labeled biomolecule was stable in vitro, showed high affinity for the human GRP receptor expressed in PC3 cells and the rate of internalization was found to be time-dependent tissue distribution of the radiopeptide was evaluated in normal mice and in prostate cancer experimental models and significant radioactivity uptake was observed in the pancreas of normal mice as well as in PC3 tumors. Dynamic studies of the radiopeptide showed satisfactory tumor images.     

A cluster-randomised trial of staff education to improve the quality of life of people with dementia living in residential care: the DIRECT study

Background

The Dementia In Residential care: EduCation intervention Trial (DIRECT) was conducted to determine if delivery of education designed to meet the perceived need of GPs and care staff improves the quality of life of participants with dementia living in residential care.

Methodology/Principal Findings

This cluster-randomised controlled trial was conducted in 39 residential aged care facilities in the metropolitan area of Perth, Western Australia. 351 care facility residents aged 65 years and older with Mini-Mental State Examination ≤24, their GPs and facility staff participated. Flexible education designed to meet the perceived needs of learners was delivered to GPs and care facility staff in intervention groups. The primary outcome of the study was self-rated quality of life of participants with dementia, measured using the QOL-Alzheimer''s Disease Scale (QOL-AD) at 4 weeks and 6 months after the conclusion of the intervention. Analysis accounted for the effect of clustering by using multi-level regression analysis. Education of GPs or care facility staff did not affect the primary outcome at either 4 weeks or 6 months. In a post hoc analysis excluding facilities in which fewer than 50% of staff attended an education session, self-rated QOL-AD scores were 6.14 points (adjusted 95%CI 1.14, 11.15) higher at four-week follow-up among residents in facilities randomly assigned to the education intervention.

Conclusion

The education intervention directed at care facilities or GPs did not improve the quality of life ratings of participants with dementia as a group. This may be explained by the poor adherence to the intervention programme, as participants with dementia living in facilities where staff participated at least minimally seemed to benefit.

Trial Registration

ANZCTR.org.au ACTRN12607000417482     

Esterase catalysis of substrate vapour: enzyme activity occurs at very low hydration

It has been generally accepted that enzyme activity requires a minimal hydration of about 0.2 g H2O g(-1) protein. This fits well with evidence that hydration above this level is associated with the onset of intramolecular motions. The influence of enzyme hydration on the hydrolysis of substrate by Candida rugosa Lipase B and pig liver esterase was investigated. Each enzyme was studied as a powder at various hydration levels, using vapour phase ethyl butyrate as substrate. This procedure allows the separation of those effects that are due to hydration from those arising from diffusional constraints. We found hydrolytic activity in both enzymes at all hydration levels above zero (between 0.054-0.47 and 0.029-0.60 g H2O g(-1) protein, respectively) that were investigated. The lowest hydration level investigated, <0.03 g H2O g(-1) enzyme, corresponded to a water/enzyme mole ratio of 100 and a coverage of about 10% of the enzyme surface by water molecules. The hydrolytic activity of both enzymes was dependent on protein hydration. However, since the hydrolysis of ethyl butyrate requires water as a second substrate, the absence of activity at zero hydration does not rule out the possibility of enzyme activity in the absence of water. These results suggest that the properties conferred on proteins by water, at least above 10% surface coverage (in this case corresponding to a hydration level of 0.03 g H2O g(-1) protein), are not a requirement for enzyme catalysis.