Barriers to routine gynecological cancer screening for White and African-American obese women.

BACKGROUND: Obese women are reported to be at higher risk from gynecological cancers than nonobese women, yet these women are less likely to get cancer-screening tests. The specific factors that contribute to obese women not obtaining timely cancer screening have not been identified. OBJECTIVE: To investigate the factors that contribute to lower rates of gynecological cancer screening as related to women's body size. DESIGN: A purposeful sample of 498 White and African-American women with body mass index (BMI) from 25 to 122 kg/m(2), including 60 women with BMI > 55 kg/m(2), was surveyed concerning access to gynecological cancer screening and potential barriers that could cause delay. Health care providers (N = 129) were surveyed concerning their education, practices, and attitudes about providing care and gynecological cancer-screening tests for obese women. RESULTS: Obese women reported that they delay cancer-screening tests and perceive that their weight is a barrier to obtaining appropriate health care. The percent of women reporting these statements increased significantly as the women's BMI increased. Women with BMI > 55 kg/m(2) had a significantly lower rate (68%) of Papanicolaou (Pap) tests compared to others (86%). The lower screening rate was not a result of lack of available health care since more than 90% of the women had health insurance. Women report that barriers related to their weight contribute to delay of health care. These barriers include disrespectful treatment, embarrassment at being weighed, negative attitudes of providers, unsolicited advice to lose weight, and medical equipment that was too small to be functional. The percentage of women who reported these barriers increased as the women's BMI increased. Women who delay were significantly less likely to have timely pelvic examinations, Pap tests, and mammograms than the comparison group, even though they reported that they were 'moderately' or 'very concerned' about cancer symptoms. The women who delay care were also more likely to have been on weight-loss programs five or more times. Many health care providers reported that they had little specific education concerning care of obese women, found that examining and providing care for large patients was more difficult than for other patients, and were not satisfied with the resources and referrals available to provide care for them. CONCLUSION: Since the goal of preventive cancer screening is to improve health outcomes for all women and since obese women are at greater risk, strategies must be designed to reduce the weight barriers to these tests and improve the quality of the health care experience. Providers should receive specific training related to care of large women.

Molybdate binding by ModA, the periplasmic component of the Escherichia coli mod molybdate transport system.

ModA, the periplasmic-binding protein of the Escherichia coli mod transport system was overexpressed and purified. Binding of molybdate and tungstate to ModA was found to modify the UV absorption and fluorescence emission spectra of the protein. Titration of these changes showed that ModA binds molybdate and tungstate in a 1:1 molar ratio. ModA showed an intrinsic fluorescence emission spectrum attributable to its three tryptophanyl residues. Molybdate binding caused a conformational change in the protein characterized by: (i) a shift of tryptophanyl groups to a more hydrophobic environment; (ii) a quenching (at pH 5.0) or enhancement (at pH 7.8) of fluorescence; and (iii) a higher availability of tryptophanyl groups to the polar quencher acrylamide. The tight binding of molybdate did not allow an accurate estimation of the binding constants by these indirect methods. An isotopic binding method with 99MoO42- was used for accurate determination of KD (20 nM) and stoichiometry (1:1 molar ratio). ModA bound tungstate with approximately the same affinity, but did not bind sulfate or phosphate. These KDs are 150- to 250-fold lower than those previously reported, and compatible with the high molybdate transport affinity of the mod system. The affinity of ModA for molybdate was also determined in vivo and found to be similar to that determined in vitro.

Three-dimensional solution structure of the N-terminal receiver domain of NTRC.

NTRC is a transcriptional enhancer binding protein whose N-terminal domain is a member of the family of receiver domains of two-component regulatory systems. Using 3D and 4D NMR spectroscopy, we have completed the 1H, 15N, and 13C assignments and determined the solution structure of the N-terminal receiver domain of the NTRC protein. Determination of the three-dimensional structure was carried out with the program X-PLOR (Brünger, 1992) using a total of 915 NMR-derived distance and dihedral angle restraints. The resultant family of structures has an average root mean square deviation of 0.81 A from the average structure for the backbone atoms involved in well-defined secondary structure. The structure is comprised of five alpha-helices and a five-stranded parallel beta-sheet, in a (beta/alpha)5 topology. Comparison of the solution structure of the NTRC receiver domain with the crystal structures of the homologous protein CheY in both the Mg(2+)-free and Mg(2+)-bound forms [Stock, A.M., Mottonen, J. M., Stock, J. B., & Schutt, C. E. (1989) Nature 337, 745-749; Volz, K., & Matsumura, P. (1991) J. Biol. Chem. 296, 15511-15519; Stock, A. M., Martinez-Hackert, E., Rasmussen, B. F., West, A. H., Stock, J. B., Ringe, D., & Petsko, G. A. (1993) Biochemistry 32, 13375-13380; Bellsolell, L., Prieto, J., Serrano, L., & Coll, M. (1994) J. Mol. Biol. 238, 489-495] reveals a very similar fold, with the only significant difference occurring in the positioning of helix 4 relative to the rest of the protein. Examination of the conformation of consensus residues of the receiver domain superfamily [Volz, K. (1993) Biochemistry 32, 11741-11753] in the structures of the NTRC receiver domain and CheY establishes the structural importance of residues whose side chains are involved in hydrogen bonding or hydrophobic core interactions. The importance of some nonconsensus residues which may be conserved for their ability to fulfill helix capping roles is also discussed.

Molybdenum accumulation in chlD mutants of Escherichia coli.

The content of molybdenum in wild-type and chlD cells was measured under a variety of growth conditions to determine if cells with a defective chlD gene were able to accumulate molybdenum. The chlD cells accumulated less molybdenum than wild-type cells did but concentrated molybdenum to a level at least 20-fold higher than the concentration in the culture medium. Molybdenum was present within spheroplasts of chlD cells and was not dialyzable. The chlD cells accumulated as much molybdenum as wild-type cells did when grown in medium containing 0.1 mM molybdate; thus, the capability of incorporation of molybdenum into cellular component(s) was equivalent to that of the wild type under these conditions.

Effect of dietary protein and methionine on sulfite oxidase activity in rats.

Sulfite oxidase catalyzes the oxidation of sulfite to sulfate. To investigate whether or not sulfite oxidase activity (EC 1.8.3.1) is regulated by the amount of sulfur from dietary protein or excess methionine, we fed rats diets containing 5, 10, 20 and 50% casein with or without excess methionine and measured sulfite oxidase activity in liver and intestinal mucosa. Hepatic sulfite oxidase activity was significantly lower in rats fed 5 or 10% casein diets and significantly higher in rats fed 50% casein than in rats fed the control diet containing 20% casein, but activity did not change in response to the addition of methionine at any level of protein. Sulfite oxidase activity in the intestinal mucosa was only 5% of that seen in liver and did not change in response to dietary protein or methionine. Activity did not change in rats fed low iron diets (5 mg Fe/kg diet) at any level of protein tested or in response to glycine. These results show that sulfite oxidase activity can adapt to different levels of dietary protein but is unaffected by the level of methionine, total amino nitrogen or iron in the diet.

Xanthine oxidase activity and immunologically detectable protein in the C57B1/6 mouse.

1. Xanthine oxidase (XO) was purified from livers of C57B1/6 mice. Antibodies generated against the purified protein were used in an immunoassay to measure total XO protein. 2. Both the specific activity and amount of XO protein were greater in the proximal small intestine than in the liver. A pool of inactive enzyme was present in the small intestine which developed after weaning. 3. Male C57B1/6 mice had the same XO specific activity as females and neither the hepatic nor the intestinal XO activity were affected by the level of dietary protein. 4. When mice were fed diets with tungsten, XO activity was lost and the amount of XO protein in the small intestine was decreased.

Effect of dietary protein and iron on the fractional turnover rate of rat liver xanthine oxidase.

Rat liver xanthine oxidase activity is regulated in response to dietary protein and iron. To investigate whether the change in activity was mediated by a change in the rate of protein degradation, we measured the fractional turnover rate using the double-isotope technique with [3H]- and [14C]leucine and calculated the apparent half-life of xanthine oxidase in rats fed diets containing either 20 or 5% casein with either 35 or 5 mg iron/kg diet. Under control conditions, xanthine oxidase had an apparent half-life of 4.8 d and approximately 65% of the enzyme subunits were active. Rats fed diets with low dietary protein had lower xanthine oxidase activity, but the enzyme had a slower fractional turnover rate, resulting in an apparent half-life of 6.4 d, and only 15-20% of the enzyme was active. The apparent half-life of xanthine oxidase increased to 7.5 d in rats fed diets with low dietary iron, but dietary iron did not affect the specific activity of the enzyme or the percentage of active subunits. These results suggest that the loss of enzyme activity is not due to loss of enzyme protein by increased degradation, but rather to inactivation of the enzyme.

Regulation of xanthine oxidase activity and immunologically detectable protein in rats in response to dietary protein and iron.

To investigate the mechanism for changes in xanthine oxidase activity in response to dietary protein and iron, we fed rats diets containing 50, 20 or 5% casein with either normal iron (35 mg Fe/kg diet) or low iron (5 mg Fe/kg diet). Xanthine oxidase activity changed in liver and intestinal mucosa in response to protein and iron, but immunologically detectable xanthine oxidase protein did not change. When total liver RNA isolated from these rats was translated by a rabbit reticulocyte lysate, we found no difference in the amount of xanthine oxidase that was translated. These results demonstrated that the changes in xanthine oxidase activity were not accompanied by changes in the amount of protein. Since xanthine oxidase can exist in an inactive desulfo form, we asked if xanthine oxidase activity was changed by the content of sulfur-containing amino acids in the diet. Xanthine oxidase activity in intestinal mucosa of the rats fed the 5% casein + methionine diet was significantly greater than that of the rats fed the 5% casein diet alone. These findings suggest that xanthine oxidase activity may be regulated by interconversion of active and inactive desulfo enzyme.

Molybdenum-sensitive transcriptional regulation of the chlD locus of Escherichia coli.

The chlD gene in Escherichia coli is required for the incorporation and utilization of molybdenum when the cells are grown with low concentrations of molybdate. We constructed chlD-lac operon fusions and measured expression of the fusion, Mo cofactor, and nitrate reductase activities under a variety of growth conditions. The chlD-lac fusion was highly expressed when cells were grown with less than 10 nm molybdate. Increasing concentrations of molybdate caused loss of activity, with less than 5% of the activity remaining at 500 nM molybdate; when tungstate replaced molybdate, it had an identical affect on chlD expression. Expression of chlD-lac was increased in cells grown with nitrate. Strains with chlD-lac plus an additional mutation in a chl or nar gene were constructed to test whether the regulation of chlD-lac required the concerted action of gene products involved with Mo cofactor or nitrate reductase synthesis. Mutations in narL prevented the increase in activity in response to nitrate; mutations in chlB, narC, or narI resulted in partial constitutive expression of the chlD-lac fusion: the fusion was regulated by molybdate, but it no longer required the presence of nitrate for maximal activity. Mutations in chlA, chlE, or chlG which affect Mo cofactor metabolism, did not affect the expression of chlD-lac.

Effect of molybdenum-deficient and low iron diets on xanthine oxidase activity and iron status in rats.

We tested the hypothesis proposed by Topham, Woodruff and Walker that intestinal xanthine oxidase is important for iron absorption. We made weanling rats xanthine oxidase-deficient and measured their growth and iron status. There were no significant differences between control and xanthine oxidase-depleted rats in growth or iron absorption or a variety of measures of iron metabolism, except that xanthine oxidase-depleted rats accumulated nonheme iron in the liver. Iron deficiency caused a loss in intestinal xanthine oxidase activity, but also caused an increase in hepatic xanthine oxidase activity. This result may be important for understanding changes in purine and protein metabolism during iron deficiency.

Molybdenum cofactor in chlorate-resistant and nitrate reductase-deficient insertion mutants of Escherichia coli.

We examined molybdenum cofactor activity in chlorate-resistant (chl) and nitrate reductase-deficient (nar) insertion mutants and wild-type strains of Escherichia coli K-12. The bacterial molybdenum cofactor was assayed by its ability to restore activity to the cofactor-deficient nitrate reductase found in the nit-1 strain of Neurospora crassa. In the wild-type E. coli strains, molybdenum cofactor was synthesized constitutively and found in both cytoplasmic and membrane fractions. Cofactor was found in two forms: the demolybdo form required additional molybdate in the assay mix for detection, whereas the molybdenum-containing form was active without additional molybdate. The chlA and chlE mutants had no detectable cofactor. The chlB and the narG, narI, narK, and narL (previously designated chlC) strains had cofactor levels similar to those of the wild-type strains, except the chlB strains had two to threefold more membrane-bound cofactor. Cofactor levels in the chlD and chlG strains were sensitive to molybdate. When grown in 1 microM molybdate, the chlD strains had only 15 to 20% of the wild-type levels of the demolybdo and molybdenum-containing forms of the cofactor. In contrast, the chlG strains had near wild-type levels of demolybdo cofactor when grown in 1 microM molybdate, but none of the molybdenum-containing form of the cofactor. Near wild-type levels of both forms of the cofactor were restored to the chlD and chlG strains by growth in 1 mM molybdate.

Identification of the molybdenum cofactor in chlorate-resistant mutants of Escherichia coli.

Experiments were performed to determine whether defects in molybdenum cofactor metabolism were responsible for the pleiotropic loss of the molybdoenzymes nitrate reductase and formate dehydrogenase in chl mutants of Escherichia coli. In wild-type E. coli, molybdenum cofactor activity was present in both the soluble and membrane-associated fractions when the cells were grown either aerobically or anaerobically, with and without nitrate. Molybdenum cofactor in the soluble fraction decreased when the membrane-bound nitrate reductase and formate dehydrogenase were induced. In the chl mutants, molybdenum cofactor activity was found in the soluble fraction of chlA, chlB, chlC, chlD, chlE, and chlG, but only chlB, chlC, chlD, and chlG expressed cofactor activity in the membrane fraction. The defect in the chlA mutants which prevented incorporation of the soluble cofactor into the membrane also caused the soluble cofactor to be defective in its ability to bind molybdenum. This cofactor was not active in the absence of molybdate, and it required at least threefold more molybdate than did the wild type in the Neurospora crassa nit-1 complementation assay. However, the cofactor from the chlA strain mediated the dimerization of the nit-1 subunits in the presence and absence of molybdate to yield the 7.9S dimer. Growth of chlA mutants in medium with increased molybdate did not repair the defect in the chlA cofactor nor restore the molybdoenzyme activities. Thus, molybdenum cofactor was synthesized in all the chl mutants, but additional processing steps may be missing in chlA and chlE mutants for proper insertion of cofactor in the membrane.

Repression of nitrate reductase activity and loss of antigenically detectable protein in Neurospora crassa.

Experiments were performed to determine whether conditions which cause the rapid loss of nitrate reductase activity in Neurospora crassa mycelia were accompanied by the loss of antigenically detectable nitrate reductase protein. When mycelia with nitrate reductase activity were transferred to ammonia media, there was a rapid loss in the reduced nicotinamide adenine dinucleotide-nitrate reductase activity plus the parallel loss of the reduced nicotinamide adenine dinucleotide-diaphorase and the reduced methyl viologen-nitrate reductase activities associated with the nitrate reductase. In addition, there was the loss of cross-reacting material to anti-nitrate reductase antisera that was concomitant with the loss of nitrate reductase activity. When mycelia were exposed to either ammonia plus cycloheximide, nitrate plus cycloheximide, or nitrogen-free media, or to media which lacked an assimilable carbon source, the amount of cross-reacting material declined in concert with the nitrate reductase activity. The mutant nit-6, which lacks nitrite reductase activity, was exposed to ammonia or nitrate plus cycloheximide media. The nitrate reductase and the amount of cross-reacting material declined together as in the wild-type mycelia. We conclude that the loss of nitrate reductase activity was accompanied by the specific loss of this protein and that no pool of inactivated nitrate reductase molecules existed.

Characterization of molybdenum cofactor from Escherichia coli.

Molybdenum cofactor activity was found in the soluble fraction of cell-free extracts of Escherichia coli grown aerobically in media supplemented with molybdate. Cofactor was detected by its ability to complement the nitrate reductase-deficient mutant of Neurospora crossa, nit-1, resulting in the vitro formation of nitrate reductase activity. Acid treatment of E. coli extracts was not required for release of cofactor activity. Cofactor was able to diffuse through a membrane of nominal 2,000-molecular-weight cutoff and was insensitive to trypsin. The cofactor was associated with a carrier molecule (approximately 40,000 daltons) during gel filtration and sucrose gradient centrifugation, but was easily removed from the carrier by dialysis. The carrier molecule protected the cofactor from inactivation by heat or oxygen. E. coli grown in molybdenum-free media, without and with tungsten, synthesized a metal-free "empty" cofactor and its tungsten analog, respectively, both of which were subsequently activated by the addition of molybdate. Empty and tungsten-containing cofactor complemented the nitrate reductase subunits in the nit-1 extract, forming inactive, but intact, 7.9S nitrate reductase. Addition of molybdate to the enzyme complemented in this manner restored nitrate reductase activity.

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.

Purification and Characterization of the Nitrate Reductase from the Diatom Thalassiosira pseudonana.

THE ASSIMILATORY NITRATE REDUCTASE (NADH: nitrate oxidoreductase, E.C. 1.6.6.2.) from the marine diatom Thalassiosira pseudonana, Hasle and Heimdal, has been purified 200-fold and characterized. The regulation of nitrate reductase in response to various conditions of nitrogen nutrition has been investigated.Nitrate reductase activity is repressed by the presence of ammonium in vivo, and its synthesis is derepressed when ammonium is absent. The derepression process is sensitive to cycloheximide and apparently requires protein synthesis. Repression of enzyme activity by ammonium is neither inhibited nor delayed by the presence of cycloheximide. In vitro, ammonium does not inhibit enzyme activity.NADH is the physiological electron donor for the enzyme in a flavin-dependent reaction. Spectral studies have indicated the presence of a b-type cytochrome associated with the enzyme. It is possible to observe enzymatic oxidation-reduction reactions which represent partial functions of the over-all electron transport capacity of this enzyme. Nitrate reductase will accept electrons from artificial electron donors such as reduced methyl viologen in a flavin-independent reaction. Further, dithionitereduced flavin adenine dinucleotide can donate electrons to the enzyme to reduce nitrate to nitrite. Finally, the nitrate reductase will exhibit a diaphorase activity and reduce the artificial electron acceptor mammalian cytochrome c in flavin-adeninedinucleotide-dependent reaction.Inhibition studies with potassium cyanide, sodium azide, and o-phenanthroline have yielded indirect evidence for metal component (s) of the enzyme.The inhibition of the NADH-requiring enzyme activities by p-hydroxymercuribenzoate has shown that an essential sulfhydryl group is involved in the initial portion of the electron transport. Heat treatment exerts an effect similar to the p-hydroxymercuribenzoate inhibition; namely, the NADH-requiring activities are rapidly inactivated, whereas the terminal nitrate-reducing activities are relatively stable to heat.The T. pseudonana nitrate reductase molecule has the hydrodynamic properties of an ellipsoid with a frictional coefficient of 1.69 and a molecular weight of 330,000.