Search

This manuscript describes the development and validation of measures assessing patient knowledge about the risks and benefits of prostate cancer (CaP) screening. The measures described include a 10-item knowledge index and four single-item measures, used in previous studies, that assess knowledge of: CaP natural history and treatment efficacy, expert disagreement over the value of CaP screening, and the accuracy of the prostate specific antigen (PSA) test for CaP. We assessed the validity and reliability of these measures on a sample of 1152 male veteran patients age 50 and older. All knowledge index items had acceptable levels of discrimination, difficulty, and reliability. The index demonstrated strong evidence for construct and criterion validity. Much weaker validity evidence was found for the four single-item knowledge questions. The 10-item index developed in this study provides a valid and reliable tool for assessing patient knowledge of the risks and benefits of CaP screening.

The plasma reduced glutathione (GSH) selenoperoxidase is a highly conserved enzyme. Furthermore, a small clinical study reported that patients with severe atherosclerosis had low peroxidase activities. Together these observations suggest that the peroxidase is important in preventing atherosclerosis. Yet others have reported that when the assay was run in Tris buffer, it was inactive with the concentrations of GSH found in the plasma. Second, it is known that hyperhomocysteinemia increases the rate of atherogenesis. Because there is some homology between homocysteine and the cysteine in GSH, the question is whether the hyperhomocysteinemia effect may be due to inhibition of the peroxidase. We purified the peroxidase from human plasma and determined its activity by a coupled spectrophotometric assay and a substrate disappearance chemiluminescence assay. When the peroxidase activity was determined in phosphate-buffered saline solution (PBS), there was significant activity with the reported plasma GSH concentrations (5 to 20 micromol/L). The peroxidase was exclusively in the HDL fraction. There was no correlation between the peroxidase activity and the HDL or LDL cholesterol concentrations. Finally, at physiologic concentrations of GSH (9 micromol/L), the peroxidase was inhibited by physiologic, free homocysteine concentrations (1 to 5 micromol/L). These data suggest that the peroxidase is active in vivo and may be important in protecting the endothelium from atherosclerosis by preventing oxidant injury. The homocysteine inhibition of the peroxidase suggests a possible biochemical basis for the observed association between hyperhomocysteinemia and cardiovascular disease. Our studies imply that low concentrations of this peroxidase may be an independent risk factor for atherosclerosis.

The ratio of urinary 2-hydroxyestrone (2-OHE1) to 16alpha-hydroxyestrone (16alpha-OHE1) has been suggested as a potential biomarker for breast cancer risk. We evaluated within-person variability of this biomarker in ten healthy Caucasian women aged 23-58 years. Each study participant was asked to provide an overnight fasting morning urine sample once a week for an average of 8 weeks. These urine samples were assayed for 2-OHE1 and 16alpha-OHE1 by using competitive enzyme immunoassay kits purchased from the ImmunaCare Corporation. The coefficients of variation for urinary 2-OHE1/16alpha-OHE1 over the study period ranged from 13.7 to 59.6% (mean, 33.3%) in our study participants. There was a good correlation between the level of the urinary 2-OHE1/16alpha-OHE1 ratio in any single urine sample and the average ratio over the 8-week study period from the same woman, with the mean correlation coefficient of 0.85. These results indicated that the within-person variation of the 2-OHE1 to 16alpha-OHE1 ratio for most women was moderate and the level of this ratio in a single urine sample, in general, reflects reasonably well the level of this biomarker over a 2-month period.

Objective: The objective of this study was to assess the longer term (up to 7 years) functional status and quality of life outcomes from lower extremity revascularization., Methods: This study was designed as a cross-sectional telephone survey and chart review at the University of Minnesota Hospital. The subjects were patients who underwent their first lower extremity revascularization procedure or a primary amputation for vascular disease between January 1, 1989, and January 31, 1995, who had granted consent or had died. The main outcome measures were ability to walk, SF-36 physical function, SF-12, subsequent amputation, and death., Results: The medical records for all 329 subjects were reviewed after the qualifying procedures for details of the primary procedure (62.6% arterial bypass graft, 36.8% angioplasty, 0.6% atherectomy), comorbidities (64% diabetics), severity of disease, and other vascular risk factors. All 166 patients who were living were surveyed by telephone between June and August 1996. At 7 years after the qualifying procedure, 73% of the patients who were alive still had the qualifying limb, although 63% of the patients had died. Overall, at the time of the follow-up examination (1 to 7.5 years after the qualifying procedure), 65% of the patients who were living were able to walk independently and 43% had little or no limitation in walking several blocks. In a multiple regression model, patients with diabetes and patients who were older were less likely to be able to walk at follow-up examination and had a worse functional status on the SF-36 and a lower physical health on the SF-12. Number of years since the procedure was not a predictor in any of the analyses., Conclusion: Although the long-term mortality rate is high in the population that undergoes lower limb revascularization, the survivors are likely to retain their limb over time and have good functional status.

The insecticide methoxychlor is estrogenic in birds and mammals and interferes with sexual development and reproduction, but it is not known whether this toxicity is due solely to its estrogenicity. We now have found that during hepatic, microsomal metabolism of [ring-14C]- or [3H-OCH3]methoxychlor, their metabolite primarily binds to iodothyronine 5'-monodeiodinase, type I (5'-ID1). The purified, radiolabeled protein reacted with antibodies against protein disulfide isomerase, isoform Q5, which is highly homologous to 5'-ID1. Sequencing of the radiolabeled tryptic peptide indicated that methoxychlor bound to cysteine 372 or 375 or to lysine 376 of 5'-ID1. Treatment of rats with methoxychlor for 4 days decreased hepatic, microsomal 5'-ID1 activity from 2.94 to 2.20 nmol/min-mg prot (P < 0.02). Since 5'-ID1 catalyzes thyroxine conversion to the biologically active triiodothyronine, these data suggest that methoxychlor may interfere with thyroid hormone metabolism. This may be an additional factor in its environmental toxicity.

We have previously shown that the cDNA sequence published for the rat gene encoding phosphatidylinositol specific phospholipase C alpha (1) may be identical to the sequence of a new isoform of protein disulfide isomerase, Isozyme Q2 (2). To determine whether the presumed phospholipase C alpha gene encodes phospholipase C or protein disulfide isomerase, we have now expressed the cDNA in Escherichia coli and find that the 55.5 kDa protein that it synthesized contains no phospholipase C activity. Moreover, an extract of Escherichia coli containing the expressed protein shows a concentration dependent increase in protein disulfide isomerase activity. The expressed protein is identical to protein disulfide isomerase, Isozyme Q2 on SDS-PAGE and by Western blot analysis. These current studies confirm that the published sequence of phospholipase C alpha is, in fact, the sequence of protein disulfide isomerase, Isozyme Q2.

The effects of selenium, vitamin E, and DL-methionine deficiency on the acute lethality and hepatotoxicity of acetaminophen in male CD-1 mice were studied. Vitamin E and selenium deficiencies led to an increase in the acute lethality of acetaminophen, with a decrease in the LD50 from 376 to 84 mg/kg. These dietary deficiencies impaired the inducibility of the hepatic microsomal mixed function oxidase system by phenobarbital, but on the basis of the covalent binding of acetaminophen to microsomes, these treatments did not alter the activation of acetaminophen to a reactive intermediate by this system. Addition of methionine to the deficient diet restored hepatic glutathione content to control levels but did little to protect against the acute lethality of acetaminophen. In methionine-supplemented animals, the addition of either selenium or vitamin E increased the LD50 of acetaminophen to 167 and 200 mg/kg, respectively. Administration of a sublethal, toxic dose of acetaminophen (LD30) to the methionine-supplemented and selenium- and vitamin E-deficient mice did not produce any hepatic damage as evidenced by a lack of plasma aminotransferase elevation. In view of the known antioxidant effects of vitamin E and selenium, these data suggest the involvement of a reactive radical in the acute lethality of acetaminophen and further suggest that death from acute acetaminophen overdose in chronic selenium- and vitamin E-deficient mice may be unrelated to liver necrosis.

Two improvements in propranolol resolution were developed. Both the (+)- and (-)-di-(p-toluoyl)tartaric acids were used as the resolving agents. This procedure reduced the number of crystallizations needed to obtain a pure product. Furthermore, synthesis of the resolving agent was improved.

The initial metabolite formed by most mammalian nitroreductases is the nitro anion free radical. We, as well as others, have proposed that nitroheterocyclic anion radicals covalently bind to protein, DNA, or thiol compounds such as reduced glutathione (GSH). Our results indicate that even at 100 mM GSH does not affect the steady-state concentration of the nitro anion free radical of N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide (NFTA) in rat hepatic microsomal or xanthine oxidase incubations. The steady-state ESR amplitude of the anion radical is also unchanged by the addition of BSA or DNA. Similar results are obtained with nitrofurazone and nitrofurantoin. The reactive chemical species which binds to tissue macromolecules and GSH upon the reduction of nitrofurans remains unknown, but the anion free radical metabolite can be excluded from consideration.

In the presence of ATP hepatic microsomes sequester calcium. This sequestration is thought to be important in the modulation of free cytosolic calcium concentration. We find that on the addition of NADPH the uptake of calcium by the hepatic microsomes is inhibited 27-85%. This inhibition is reversed by the addition of 1 mM reduced glutathione (85-91% of control), incubation under a nitrogen atmosphere (112% of control), or incubation in a 80% carbon monoxide/20% oxygen atmosphere (75% of control). Superoxide dismutase had no effect on the inhibition, while catalase reversed the inhibition by 35%. The addition of 1 mM reduced glutathione at 2 and 5 min after the addition of NADPH led to uptakes of calcium which paralleled the uptake seen when the reduced glutathione was added at the beginning of the incubation. The effect of reduced glutathione showed saturation kinetics with a Km of 10 microM. Together these data suggest that cytochrome P-450 reduces the activity of the microsomal ATP-dependent calcium pump both by the production of hydrogen peroxide and by the direct oxidation of the protein thiols. The reversal of this effect by reduced glutathione appears to be enzymatically catalyzed.

The acute toxicity of nitrofurantoin was studied in the young chick deficient in selenium (Se) and/or vitamin E (E). This new and potentially valuable animal model proved to be very sensitive to the toxicity of this nitro drug. The 48-hour LD50 for nitrofurantoin decreased from 148 mg/kg in the Se- and E-supplemented chicks to 53 mg/kg in Se- and E-deficient chicks. The addition of Se (0.10 ppm as Na2SeO3) alone, but not E (100 IU/kg diet as dl-alpha-tocopheryl acetate) reduced the toxicity of nitrofurantoin, so that the LD50 for the chicks given Se alone was the same as the LD50 for the E- and Se-fed chicks. Se and E deficiency significantly decreased the Se-dependent glutathione peroxidase and the plasma tocopherol levels. Hepatic glutathione content, hepatic catalase and superoxide dismutase were unchanged by the dietary treatments. However, a toxic dose of nitrofurantoin significantly decreased hepatic glutathione content over time. These data support the concept that the toxicity of this drug may be mediated in part by an oxidative stress generated by the futile reductive metabolism of the parent compound.

The androgen receptor was purified from rat ventral prostate. The purified receptor migrated as a single band of mol. wt. 87000 on SDS-polyacrylamide gels, had a kd for R-1881 (17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one) binding as 6 nM, and sedimentation coefficient of 4.5 S. Phosphorylation of the purified receptor was studied by incubating it with [gamma-32P]ATP in the presence of several purified protein kinases including cAMP-dependent protein kinase, and four cAMP-independent protein kinases (which were active towards substrates such as phosvitin and casein). Phosphorylation of the 87000 mol. wt. androgen receptor protein occurred only in the presence of a nuclear cAMP-independent protein kinase (of the N2 type). No auto-phosphorylation of the receptor was detected. The results indicate that the androgen receptor is a phosphoprotein. Further, phosphorylation of the androgen receptor by only a specific nuclear cAMP-independent protein kinase may be important in determining the dynamics of its function.

The hepatic, microsomal, thiol:protein disulfide oxidoreductase catalyzes the glutathione (GSH) reduction of protein disulfides to sulfhydryl groups. In the presence of physiological concentrations of glucagon this activity increased from 2.3 to 6.4 fold in isolated microsomes. The stimulation had a P50 for glucagon of 7.8 X 10(-10) M which was only observed at microsomal protein concentrations of less than 100 micrograms/ml and in the presence of a GSH reducing system. This latter observation suggests that the stimulation may be inhibited by the presence of oxidized glutathione. These data support the hypothesis that glucagon may act in part by stimulating the reduction of protein disulfides by the thiol:protein disulfide oxidoreductase.