Reaction of cytochrome P450 with cumene hydroperoxide: ESR spin-trapping evidence for the homolytic scission of the peroxide O-O bond by ferric cytochrome P450 1A2

ESR spin trapping was used to investigate the reaction of rabbit cytochrome P450 (P450) 1A2 with cumene hydroperoxide. Cumene hydroperoxide-derived peroxyl, alkoxyl, and carbon-centered radicals were formed and trapped during the reaction. The relative contributions of each radical adduct to the composite ESR spectrum were influenced by the concentration of the spin trap. Computer simulation of the experimental data obtained at various 5,5-dimethyl-1-pyrroline N-oxide (DMPO) concentrations was used to quantitate the contributions of each radical adduct to the composite ESR spectrum. The alkoxyl radical was the initial radical produced during the reaction. Experiments with 2-methyl-2-nitrosopropane identified the carbon-centered adducts as those of the methyl radical, hydroxymethyl radical, and a secondary carbon-centered radical. The reaction did not require NADPH-cytochrome P450 reductase or NADPH. It is concluded that the reaction involves the initial homolytic scission of the peroxide O-O bond to produce the cumoxyl radical. Methyl radicals were produced from the beta-scission of the cumoxyl radical. The peroxyl adduct was not observed in the absence of molecular oxygen. We conclude that the DMPO peroxyl radical adduct detected in the presence of oxygen was due to the methylperoxyl radical formed by the reaction of the methyl radical with oxygen. At a higher P450 concentration, a protein-derived radical adduct was also detected.     

EEG spectral abnormalities and psychosis as predictors of cognitive and functional decline in probable Alzheimer's disease

We examined whether either psychotic features (e.g., delusions and hallucinations) or EEG abnormalities are associated with more rapid progression of Alzheimer's disease (AD). AD patients with psychosis have exhibited more EEG abnormalities than those without psychosis, and both abnormal EEG and psychosis have been noted to be predictors of functional and cognitive decline in AD. Ninety-five probable AD patients participating in a longitudinal study of dementia had an EEG and a semistructured psychiatric interview at baseline. Using EEG spectral analysis, we classified records as normal/abnormal based on the parasagittal mean frequency. Patients with abnormal EEGs were more functionally (e.g., Blessed Rating Scale for activities of daily living) and cognitively (e.g., Mini-Mental State) impaired than patients with normal EEG. AD patients with psychosis were only more functionally impaired than patients without psychosis. A two-factor analysis showed no interaction between abnormal EEG and psychosis. In addition, using a Cox proportional hazard model adjusted for age and education, the presence of an abnormal EEG or psychotic symptom at study entry was associated with higher risk of reaching severe cognitive and functional impairment during follow-up. Neither abnormal EEG nor the presence of psychosis predicted death. These results indicate that both abnormal EEG and psychosis are independent predictors of disease progression but not of physical survival.     

Effects of citrate on the different phases of calcium oxalate crystallization

Urinary citrate appears to be an important factor in the crystallization process of calcium oxalate and calcium phosphate. The urinary excretion of citrate was found to be significantly lower in patients with calcium oxalate stone disease as compared with normal subjects, and about 30 per cent of the calcium stone formers can be considered as hypocitraturic. The lowest excretion of citrate was recorded in urine collected during the night. Citrate has significant effects on supersaturation with respect to both calcium oxalate and calcium phosphate, it also inhibits the growth of these crystals. In addition, citrate appears to be capable of inhibiting the aggregation of crystals composed of calcium oxalate, brushite, and hydroxyapatite. The heterogenous growth of calcium oxalate on calcium phosphate is also counteracted by citrate. As a consequence of the crucial role of citrate in these processes, stone prevention with alkaline citrate has become an attractive form of treatment in patients with recurrent stone formation. Single evening dose administration of sodium potassium citrate resulted in an of sodium potassium citrate resulted in an increased excretion of citrate, reduced levels of the calcium/citrate ratio as well as supersaturation with respect to calcium oxalate and a decreased rate of stone formation. However, conflicting results of stone preventive treatment with alkaline citrate have been reported by different groups, and long-term follow-up of patients treated in a randomized way is necessary to definitely assess the efficacy of alkaline citrate.     

Investigations of delamination criticality and the transferability of growth criteria

This article describes a study of delamination growth along 0 °/0 °, 0 °/ 5 °, ± 5 °, and 0 °/90 ° interfaces sandwiched between unidirectional carbon fibre/epoxy composite material. Relationships between damage criticality, growth rate and acoustic emission activity for delamination growth have been studied and the transferability of results from laboratory coupons to composite structural elements has been examined. Two types of coupon tests, conventional delamination beams and rigidly loaded single edge notched strips, have been compared for different mode ratios. Comparative tests have been made on buckling-induced delamination in plates. A graphite crack gauge has been used to measure delamination length and growth rate, ranging from 0.05–2000 m/s. Damage growth was also followed using visual, ultrasonic C-scan, X-ray radiography, macro-video and acoustic emission measurements. Empirical evaluations of interlaminar toughness for delamination beams are made using the Irwin-Kies relation. Unstable growth is analysed using elasto-dynamic moving finite elements. Bucklinginduced delamination is analysed using plate/shell FE methods with growth/remeshing algorithms.     

Comparison of copper emission rates from wood treated with different preservatives to the environment

This study describes a laboratory method for the estimation of emission from preservative-treated wood in the different situations where emissions could enter the environment for use classes 3 (not in contact with ground) and 4 and 5 (in contact with the ground, fresh water or sea water) according to OECD Guidelines. Samples of scotch pine sapwood (Pinus sylvestris L.) were treated with CCA (1% and 2%), ACQ-1900 (3% and 7%), ACQ-2200 (2%), Tanalith E 3491 (2% and 2.8%), and Wolmanit CX-8 (2%).     

Cost-effectiveness of preoperative localization studies in primary hyperparathyroid disease

OBJECTIVE: To evaluate the effect of preoperative localization studies on the surgical management of patients with primary hyperparathyroid disease (PHPT). SUMMARY BACKGROUND DATA: Reported cure rates of initial surgical exploration for PHPT are close to 95%. Preoperative localization studies are frequently obtained to improve surgical success and decrease operative time. METHODS: Initial cervical exploration was performed in 113 patients with PHPT from 1981 to 1993. Twenty-four patients (21%) had surgery without preoperative localization studies. The remaining 89 patients (79%) had 132 noninvasive preoperative localization studies. Success of the localization studies in tumor localization, pathologic findings, postoperative serum calcium levels, and operative times were compared. Patient costs of the studies were calculated. RESULTS: Disease was identified during operation in 23 of 24 patients (96%) having cervical exploration without preoperative localization studies, and they had normal calcium levels after surgery. Eighty-seven of 89 patients (98%) having preoperative localization studies were surgically cured. The highest sensitivity rate (60%) and highest positive predictive value (79%) of the localization studies were found with thallium-technetium scintiscanning. Average cost of the localization studies was $901 per patient. Combination studies were obtained in 32 patients at an average cost of $1,314 per patient without improving sensitivity. Mean operating time did not differ for localized and nonlocalized patients. CONCLUSIONS: Preoperative localization studies did not improve parathyroid localization or cure rate and did not substantially shorten operating time in initial cervical exploration for PHPT. The economic burden of routine preoperative localization studies in these patients is not justified.     

Inhibition of Listeria monocytogenes on cold-smoked salmon by nisin and carbon dioxide atmosphere

The heat-stable enterotoxin of Escherichia coli binds to an intestinal receptor, guanylyl cyclase-C, and produces cGMP to induce diarrhea. Guanylin is an endogenous ligand of this receptor. In the present in vivo study, the intestinal water and ion secretion induced by mucosal application of 2 nmol/ml guanylin or 5 or 10 units/ml heat-stable enterotoxin into closed loops was compared in the rat. The characteristics of secretion induced by cAMP following intravenous perfusion of 1.2 nmol/100 g per h vasoactive intestinal peptide were compared to those induced by cGMP. Unidirectional Na+ and Cl- fluxes were estimated by addition of 22Na into the loop and i.v. injection of 36Cl. Guanylin induced less water and ion secretion than that produced by heat-stable enterotoxin in the colon, confirming the results of in vitro studies, and also in duodenum and ileum. The cAMP- or cGMP-mediated response had a similar pattern, i.e., an inhibition of Na+ absorption and an increase in anion secretion.     

Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement

We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon protease digestion, with fluorescence increases proportional to enzyme activity. These quenched substrates are suitable for the continuous assay of enzymatic activity using standard fluorometers, filter fluorometers, or fluorescence microplate readers using either fluorescein excitation and emission wavelengths to measure BODIPY FL casein hydrolysis or Texas Red wavelengths to detect proteolysis of BODIPY TR-X casein. Most current techniques for detecting protease activity, such as the fluorescein thiocarbamoyl casein (FTC-casein) protease assay, require extensive manipulation, including separation steps, and are therefore labor intensive and error-prone. In comparison, we found the BODIPY dye-labeled casein protease assays to be simple and precise and to have greater sensitivity and a broader dynamic range of detection than the FTC-casein assay. We were able to sensitively detect the activities of a wide variety of enzymes with these new substrates, including serine, acid, sulfhydryl, and metalloproteases. We also found the assay suitable for quantitating protease inhibitor concentrations and for real-time analysis of proteolysis.