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M D Wong

Publications and source records attributed to M D Wong.

18 recordsLinked to original sources
PubMed2001

Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC.

Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.

Allosteric Regulation↗
PubMed2001

Role of cysteinyl residues in sensing Pb(II), Cd(II), and Zn(II) by the plasmid pI258 CadC repressor.

The cadCA operon of Staphylococcus aureus plasmid pI258 confers resistance to salts of the soft metals lead, cadmium, and zinc. The operon is regulated by CadC, a member of the ArsR family of metal-responsive transcriptional repressors. In this study the role of the five cysteine residues of CadC in soft metal ion sensing was investigated. Cys-7, Cys-11, Cys-52, Cys-58, and Cys-60 were changed individually to glycine or serine residues. The effect of the cadC mutations was examined in Escherichia coli using a green fluorescent protein reporter system. None of the mutations affected the ability of CadC to repress gfp expression. Neither Cys-11 nor Cys-52 was required for in vivo response to Pb(II), Zn(II), or Cd(II). Cys-7, Cys-58, or Cys-60 mutations each reduced or eliminated soft metal sensing. Wild-type and mutant CadC proteins were purified, and the effect of the substitutions on DNA binding was determined using a restriction enzyme protection assay. Binding of wild-type CadC protected cad operator DNA from digestion at the single SspI site, and the addition of Pb(II), Zn(II), or Cd(II) resulted in deprotection. Chemical modification of the cysteine residues in CadC had no effect on protection but eliminated deprotection. C11G and C52G proteins exhibited wild-type properties in vitro. C7G, C58S, and C60G proteins were able to be protected from SspI digestion but had reduced responses to soft metal ions. The results indicate that Cys-7, Cys-58, and Cys-60 are involved in sensing those soft metals and suggest that they are ligands to Pb(II), Zn(II), and Cd(II).

Amino Acid Sequence↗
PubMed2001

Effects of cost sharing on care seeking and health status: results from the Medical Outcomes Study.

OBJECTIVES: This study sought to determine the effect of cost sharing on medical care use for acute symptoms and on health status among chronically ill adults. METHODS: Data from the Medical Outcomes Study were used to compare (1) rates of physician care use for minor and serious symptoms and (2) 6- and 12-month follow-up physical and mental health status among individuals at different levels of cost sharing. RESULTS: In comparison with a no-copay group, the low- and high-copay groups were less likely to have sought care for minor symptoms, but only the high-copay group had a lower rate of seeking care for serious symptoms. Follow-up physical and mental health status scores were similar among the 3 copay groups. CONCLUSIONS: In a chronically ill population, cost sharing reduced the use of care for both minor and serious symptoms. Although no differences in self-reported health status were observed, health plans featuring cost sharing need careful monitoring for potential adverse health effects because of their propensity to reduce use of care that is considered necessary and appropriate.

Adult↗
PubMed1999

A comparison of clinical performance of primary care and traditional internal medicine residents.

OBJECTIVE: To compare primary care and traditional Internal Medicine residents in their adherence to preventive medicine guidelines, performance in the management of chronic diseases, and utilization of resources. DESIGN: Prospective cohort study. SETTING: Urban Internal Medicine residency program. PARTICIPANTS: Sixteen primary care and 137 traditional Internal Medicine residents who took care of 6,307 patients (a total of 21,002 patient visits in a 1-year period). MEASUREMENTS: Adherence to preventive medicine guidelines for the screening of breast cancer, cervical cancer, hypercholesterolemia, and colon cancer; admission rates among patients with asthma, chronic pulmonary disease, and diabetes mellitus; four items in the management of diabetes; and resource utilization including the costs for laboratory and radiology tests and number of consultations. RESULTS: Primary care residents, as compared with traditional residents, adhered to preventive medicine guidelines for a greater proportion of their patients for the following: breast cancer among women aged 52 to 75 years (61% vs. 54%, respectively, P = 0.05); cholesterol screening among patients aged 20 to 64 years (39% vs. 33%, P = 0.007); colon cancer among patients older than 50 years (49% vs. 31%, P = 0.001); and cervical cancer among women aged 20 to 64 years (36% vs. 31%, P = 0.03). There were no differences in hospital admission rates for patients with diabetes or asthma. Total ambulatory care costs for tests, procedures, consults, and office visits were greater for patients of primary care residents ($1,045 vs. $899, P = 0.0001), although total costs per primary care visit were similar between the two patient groups. CONCLUSIONS: Primary care residents more closely adhered to preventive medicine guidelines but were similar to traditional residents in their management of chronic diseases. Patients of primary care residents had greater ambulatory care costs that were not entirely attributable to greater adherence to preventive medicine guidelines.

Academic Medical Centers↗
PubMed1996

Epithelial-matrix interactions : laminin downregulates enterocyte EGF receptor and IGF-I receptor expression.

Intestinal crypt cells continuously proliferate to yield functional villus cells which terminally differentiate prior to sloughing into the intestinal lumen. Growth factors and extracellular matrix have been shown to regulate this process. To investigate how these effectors may interact, Epidermal Growth Factor (EGF) receptor and Insulin-like Growth Factor-I (IGF-I) receptor expression was evaluated in IEC-6 cells incubated on laminin or collagen I. EGF receptor and IGF-I receptor expression were decreased in cells grown on laminin. The data suggest that the mechanism by which laminin inhibits enterocyte growth is downregulation of growth factor receptors for important enterocyte mitogens.

Animals↗
PubMed1995

Retrograde gastroesophageal intussusception complicating chronic achalasia.

We treated a patient with retrograde gastroesophageal intussusception complicating chronic achalasia. Operation consisted of diaphragmatic division in the median plane to facilitate reduction, followed by Heller myotomy and fundoplication for the achalasia. The patient was able to eat normally after recovery.

Adult↗
PubMed1990

Partial purification and properties of nicotinamide adenine dinucleotide synthetase from human erythrocytes: evidence that enzyme activity is a sensitive indicator of lead exposure.

We have examined properties of nicotinamide adenine dinucleotide (NAD) synthetase from human erythrocytes. The enzyme was found to be cold labile and extremely unstable in crude hemolysate, with complete loss of activity occurring after 24 hours at 4 degrees C. However, maintenance of crude hemolysate at 20 to 25 degrees C in the presence of EDTA and KCl increased NAD synthetase stability substantially (half-life = 10 days). Using these conditions, NAD synthetase was purified 3,100-fold with a 29% yield using DEAE-cellulose column chromatography, ammonium sulfate fractionation, and dialysis. The apparent Michaelis-Menten constants for nicotinic acid adenine dinucleotide (NAAD), adenosine triphosphate, Mg2+, glutamine, and K+ were 0.108, 0.154, 1.36, 2.17, and 8.32 mmol/L, respectively. The pH optimum ranged between 6.8 and 7.4, and the molecular weight was estimated to be 483 +/- 5 Kd. The enzyme was markedly inhibited by Pb2+ and Zn2+, with concentrations necessary for 50% inhibition of activity of 1.3 and 2.0 mumol/L, respectively. The incubation of intact red blood cells with lead followed by rigorous washing to remove lead abolished nearly all NAD synthetase activity. In contrast, glucose-6-phosphate dehydrogenase activity, which is not sensitive to lead, was unaffected, whereas pyrimidine 5'-nucleotidase activity, which is sensitive to lead, was decreased 30% to 50% under these conditions. More importantly, patients with lead overburden (34 to 72 micrograms Pb2+/dL blood) all had markedly decreased NAD synthetase activity. These data together with other results suggest that erythrocyte NAD synthetase activity is a sensitive indicator of lead exposure in humans.

Amide Synthases↗
PubMed1988

Impaired erythrocyte phosphoribosylpyrophosphate formation in hemolytic anemia due to pyruvate kinase deficiency.

RBCs from patients with hemolytic anemia due to pyruvate kinase (PK) deficiency are characterized by a decreased total adenine and pyridine nucleotide content. Because phosphoribosylpyrophosphate (PRPP) is a precursor of both adenine and pyridine nucleotides, we investigated the ability of intact PK-deficient RBCs to accumulate PRPP. The rate of PRPP formation in normal RBCs (n = 11) was 2.89 +/- 0.80 nmol/min.mL RBCs. In contrast, the rate of PRPP formation in PK-deficient RBCs (n = 4) was markedly impaired at 1.03 +/- 0.39 nmol/min.mL RBCs. Impaired PRPP formation in these cells was not due to the higher proportion of reticulocytes. To study the mechanism of impaired PRPP formation, PK deficiency was simulated by incubating normal RBCs with fluoride. In normal RBCs, fluoride inhibited PRPP formation, caused adenosine triphosphate (ATP) depletion, prevented 2,3-diphosphoglycerate (DPG) depletion, and inhibited pentose phosphate shunt (PPS) activity. These results together with other data suggest that impaired PRPP formation is mediated by changes in ATP and DPG concentration, which lead to decreased PPS and perhaps decreased hexokinase and PRPP synthetase activities. Impaired PRPP formation may be a mechanism for the decreased adenine and pyridine nucleotide content in PK-deficient RBCs.

2,3-Diphosphoglycerate↗
PubMed1976

Metabolism of natural and synthetic corticosteroids in relation to their effects on mouse fetuses.

14C-glucose uptake by mouse fetuses was reduced by doses of dexamethasone, 200 mug or more, which over 2 days caused fetal death. Uptake by strain C57B1/6J greater than A/J greater than SWV. Corticosterone or cortisol, 4 mg, caused neither reduced uptake nor fetal death. These differences occurred despite similar maternal hyperglycemia in all cases. Recovery of 3H-steroid after 15 min, total and unchanged steroid per gram fetal tissue was: corticosterone 1.7 and 0.3%; dexamethasone 0.12 and 0.06% of the dose. Rapid metabolism of corticosterone apparently prevents accumulation in fetal tissue sufficient to evoke a response under these conditions.

Animals↗
PubMed1975

Subcellular distribution of glucocorticoid receptors in mouse fibroblasts.

Mouse fibroblasts contain a macromolecular binding component (receptor) which binds glucocorticoids specifically and with high affinity. This study shows that there are three different cellular forms of bound receptor and that it is experimentally possible to markedly alter the subcellular distribution of these three forms. Cells incubated with (3H)triamcinolone acetonide were broken after hypotonic shock and a 7000g hypotonic supernatant was obtained; the pellet was extracted with 0.3 M KCl, yielding a nuclear extract; the remaining pellet was resuspended in water, sonicated, and assayed for "nuclear residual" (i.e., nonextractable) radioactivity. If whole cells are incubated at 0 degrees in a growth medium, almost all of the bound steroid is located in the hypotonic supernatant fraction. Incubation at 37 degrees produces a shift of the steroid-bound macromolecule into the nuclear extractable form, while omission of glucose and addition of KCN at 37 degrees markedly increase the nuclear residual form at the expense of both the nuclear-extractable and supernatant forms. Since DNase treatment of chromatin liberates a soluble steroid-receptor complex, we believe that the nuclear residual form may be steroid-receptor complex tightly bound to chromatin. We propose a model suggesting that an energy-requiring process is required to generate free receptor from the chromatin complex to complete the normal cellular recycling system.

Animals↗
PubMed1972

Quantitative radioautography of sugar transport in intestinal biopsies from normal humans and a patient with glucose-galactose malabsorption.

Both galactose accumulation and phlorizin binding by columnar epithelial cells have been investigated in vitro with a recently developed technique for high-resolution, plastic-section radioautography which is particularly suited to small quantities of biopsy tissue. Grain density analysis of the radioautographs provides definitive support for the view that the cellular mechanisms underlying glucose-galactose absorption in laboratory animals are fully applicable to the small intestine of man. Even the number of sugar carriers at the microvillar membrane appears similar and the major quantitative difference, lower affinity for phlorizin in man, correlates with the finding that phlorizin is also a less potent inhibitor of uphill, galactose transport at the microvilli. In addition, radioautographs of biopsies taken 2 yr apart from a patient with glucose-galactose malabsorption provide evidence that the cellular defect in this inborn error of transport is a persistent reduction in the number of functioning sugar carriers at the microvillar membrane.

Adult↗