Administration of NF-kappaB decoy inhibits pancreatic activation of NF-kappaB and prevents diabetogenesis by alloxan in mice.
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Alloxan↗
Biomedical subjects
T Bray
Publications and source records attributed to T Bray.
Mutant PTR1 proteins from Leishmania tarentolae: comparative kinetic properties and active-site labeling.
PTR1, the gene promoting MTX resistance following gene amplification or DNA transfection in Leishmania tarentolae and selected mutants, has been cloned and heavily overexpressed (>100 mg/liter) in Escherichia coli strain BL21 (DE3). Protein has been purified, essentially to homogeneity, in two steps, via ammonium sulfate precipitation and chromatography on DEAE-Trisacryl. The active proteins are tetramers and display optimal pteridine reductase activity at pH 6.0 using biopterin as substrate and NADPH as the reduced dinucleotide cofactor. 2,4-Diaminopteridine substrate analogues are strong competitive inhibitors (K(i) approximately 38 --> 3 nM) against the pterin substrate and both NADP(+) and folate are inhibitors although somewhat weaker. Dihydropteridines are poor substrates compared to the fully oxidized pteridine. Kinetic analysis affords the usual Michaelis constants and in addition shows that inhibition by NADP(+) allows the formation of ternary nonproductive complexes with folate. The kinetic results are consistent with a sequential ordered bi-bi kinetic mechanism in which first NADPH and then pteridine bind to the free enzyme. Sequence comparisons suggest that PTR1 belongs to the short-chain dehydrogenase/reductase (SDR) family containing an amino-terminal glycine-rich dinucleotide binding site plus a catalytic Y(Xaa)(3)K motif. In accord with this observation, the mutants K16A, Y37D, and R39A and the double mutants K17A:R39A and Y37D:R39A all show a two- to threefold lower binding affinity for NADPH and exhibit low or zero activity. Two Y(Xaa)(3)K regions are present in wild-type PTR1 at 152 and 194. Only Y194F gives protein with zero activity. This observation coupled with affinity labeling of PTR1 by oNADP(+) (2', 3'-dialdehyde derivative of NADP(+)) followed by NaBH(4) reduction, V8 protease digestion, and mass spectral analysis suggests that the motif participating in catalysis is that at 194. The mutation K198Q eliminates inactivation by oNADP(+) supporting the hypothesis that K198 is associated with nucleotide orientation, as has been demonstrated for similar lysine residues in other members of the SDR family.
Animals↗
Comparative properties of three pteridine reductases.
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Amino Acid Sequence↗
Assessing the impact of neuropsychiatric symptoms in Alzheimer's disease: the Neuropsychiatric Inventory Caregiver Distress Scale.
OBJECTIVES: To develop an adjunct scale to the Neuropsychiatric Inventory (NPI) for assessing the impact of neuropsychiatric symptoms in Alzheimer's disease (AD) patients on caregiver distress. DESIGN: Cross-sectional descriptive and correlational study. SETTING: University out-patient memory disorders clinics. PARTICIPANTS: Eighty-five AD subjects and their caregivers (54 spouses, 31 children). MEASUREMENTS: The NPI and NPI Caregiver Distress Scale (NPI-D) were used to assess neuropsychiatric symptoms in AD patients and related caregiver distress, respectively. Criterion validity of the NPI-D was examined (N = 69) by comparison with an abridged version of the Relatives' Stress Scale (RSS'), a general measure of caregiver stress, using item clusters that had previously been correlated to behavioral disturbances in demented patients. Test-retest (n = 20) and inter-rater reliability (n = 16) of the NPI-D were also assessed. RESULTS: Test-retest and interrater reliability of the NPI-D were both adequate. Overall, caregiver NPI-D distress ratings were correlated significantly with the RSS' (r = .60, P < .001). RSS' ratings correlated strongly with NPI scores (r = .64, P < .001), even after controlling for degree of cognitive impairment based on the Mini-Mental State Exam (MMSE) score (r = .61). MMSE scores showed a moderate correlation to RSS' ratings (-.30, P = .02), but this association was markedly attenuated when controlling for the degree of neuropsychiatric disturbance based on the NPI score (r = -. 14). NPI-D ratings for 9 of 10 NPI symptom domains correlated most strongly with either NPI symptom severity or total (frequency x severity) scores. Agitation, dysphoria, irritability, delusions, and apathy were the symptoms most often reported to be severely distressing to caregivers. CONCLUSIONS: The NPI-D provides a reliable and valid measure of subjective caregiver distress in relation to neuropsychiatric symptoms measured by the NPI. Neuropsychiatric alterations are more strongly associated than cognitive symptoms to caregiver distress. The NPI-D may be useful in both clinical and research settings for assessing the contribution to caregiver distress of neuropsychiatric symptoms in AD patients.
Aged↗
Pterin and folate reduction by the Leishmania tarentolae H locus short-chain dehydrogenase/reductase PTR1.
Overproduction of the short-chain dehydrogenase/reductase PTR1 confers resistance to the dihydrofolate reductase inhibitor methotrexate in the protozoan parasite Leishmania. Genetic analysis has previously implicated PTR1 in pterin and folate metabolism. PTR1 was purified from a fusion protein expressed in Escherichia coli. Purified PTR1 exhibits NADPH-dependent biopterin, dihydrobiopterin, folate, and dihydrofolate reductase activities. The highest activity was found with the most oxidized pterins. The active protein was found to be a tetramer as demonstrated by gel-filtration chromatography. Kinetic constants (K(m)), as determined by double-reciprocal plots, were calculated for NADPH and for several of PTR1's substrates. The PTR1 of Leishmania tarentolae had a K(m) of 16.9 microM for the cofactor NADPH and K(m) values ranging from 3.5 to 85 microM for the various substrates. The dissociation constant (KD), as determined by fluorescence titration, for NADPH was estimated to be 130 microM. The biochemical characterization of this important and novel enzyme involved in folate and pterin metabolism of Leishmania should be useful for structure-function analysis and for developing specific inhibitors against this putative important chemotherapeutic target.
Animals↗
SGML as a message interchange format in healthcare.
INTRODUCTION: In 1993, The European Committee for Standardization (CEN) studied several syntaxes for interchange formats in healthcare, but excluded SGML due to resource constraints. We sought to extend the CEN report and formally evaluate the use of SGML as a message interchange format. METHODS: We followed the methodology set forth by CEN, using their example scenarios and healthcare data model. General message descriptions based on this model set the functional requirements for the interchange format. These general requirements are then mapped into SGML to see how well they can be supported. RESULTS: Results follow the CEN format, enabling a direct comparison of SGML with ASN.1, ASTM E1238, EDIFACT, EUCLIDES, and ODA (those syntaxes studied by CEN). CONCLUSION: SGML compares favorably with other syntaxes investigated by CEN. None of the interchange formats support all functional requirements. Optimal and standard mechanisms of combining different formats through a modular approach to achieve greater overall functionality requires further study.
Computer Communication Networks↗
Altered structural and mechanistic properties of mutant dihydropteridine reductases.
Nine single genetic mutants of rat dihydropteridine reductase (EC 1.6.99.7), D37I, W86I, Y146F, Y146H, K150Q, K150I, K150M, N186A, and A133S and one double mutant, Y146F/K150Q, have been engineered, overexpressed in Escherichia coli and their proteins purified. Of these, five, W86I, Y146F, Y146H, Y146F/K150Q, and A133S, have been crystallized and structurally characterized. Kinetic constants for each of the mutant enzyme forms, except N186A, which was too unstable to isolate in a homogeneous form, have been derived and in the five instances where structures are available the altered activities have been interpreted by correlation with these structures. It is readily apparent that specific interactions of the apoenzyme with the cofactor, NADH, are vital to the integrity of the total protein tertiary structure and that the generation of the active site requires bound cofactor in addition to a suitably placed W86. Thus when the three major centers for hydrogen bonding to the cofactor are mutated, i.e. 37, 150, and 186, an unstable partially active enzyme is formed. It is also apparent that tyrosine 146 is vital to the activity of the enzyme, as the Y146F mutant is almost inactive having only 1.1% of wild-type activity. However, when an additional mutation, K150Q, is made, the rearrangement of water molecules in the vicinity of Lys150 is accompanied by the recovery of 50% of the wild-type activity. It is suggested that the involvement of a water molecule compensates for the loss of the tyrosyl hydroxyl group. The difference between tyrosine and histidine groups at 146 is seen in the comparably unfavorable geometry of hydrogen bonds exhibited by the latter to the substrate, reducing the activity to 15% of the wild type. The mutant A133S shows little alteration in activity; however, its hydroxyl substituent contributes to the active site by providing a possible additional proton sink. This is of little value to dihydropteridine reductase but may be significant in the sequentially analogous short chain dehydrogenases/reductases, where a serine is the amino acid of choice for this position.
Amino Acid Sequence↗
Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement.
Kinetic constants for the interaction of NADH and NADPH with native rat dihydropteridine reductase (DHPR) and an Escherichia coli expressed mutant (D-37-I) have been determined. Comparison of kcat and Km values measured employing quinonoid 6,7-dimethyldihydropteridine (q-PtH2) as substrate indicate that the native enzyme has a considerable preference for NADH with an optimum kcat/Km of 12 microM-1 s-1 compared with a figure of 0.25 microM-1 s-1 for NADPH. Although the mutant enzyme still displays an apparent preference for NADH (kcat/Km = 1.2 microM-1 s-1) compared with NADPH (kcat/Km = 0.6 microM-1 s-1), kinetic analysis indicates that NADH and NADPH have comparable stickiness in the D-37-I mutant. The dihydropteridine site is less affected, since the Km for q-PtH2 and K(is) for aminopterin are unchanged and the 14-26-fold synergy seen for aminopterin binding to E.NAD(P)H versus free E is decreased by less than 2-fold in the D-37-I mutant. No significant changes in log kcat and log kcat/Km versus pH profiles for NADH and NADPH were seen for the D-37-I mutant enzyme. However, the mutant enzyme is less stable to proteolytic degradation, to elevated temperature, and to increasing concentrations of urea and salt than the wild type. NADPH provides maximal protection against inactivation in all cases for both the native and D-37-I mutant enzymes. Examination of the rat DHPR sequence shows a typical dinucleotide binding fold with Asp-37 located precisely in the position predicted for the acidic residue that participates in hydrogen bond formation with the 2'-hydroxyl moiety of all known NAD-dependent dehydrogenases. This assignment is consistent with x-ray crystallographic results that localize the aspartate 37 carboxyl within ideal hydrogen bonding distance of the 2'- and 3'-hydroxyl moieties of adenosine ribose in the binary E.NADH complex.
Amino Acid Sequence↗
Role of aspartate-37 in determining cofactor specificity and binding in rat liver dihydropteridine reductase.
Full-length rat dihydropteridine reductase (DHPR) cDNAs have been combined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express dihydropteridine reductase immunoreactive proteins and demonstrate conversion of quinonoid dihydropteridines to their tetrahydro forms. Several recombinant enzymes have been purified to homogeneity and biochemical studies have been carried out comparing their properties with those exhibited by the rat liver enzyme. The optimal reaction conditions, kinetic constants, and stability are similar for the recombinant and naturally occurring enzyme. The results indicate that the nonmutant recombinant rat DHPR is an authentic replica of the natural protein and that the characteristics of DHPR activity are determined by a single gene product and do not require specific modification via the eukaryotic cell. In addition to the wild type, three specific mutagenic forms of the reductase, A-6-V, W-104-F, and D-37-I, and an additional abbreviated structure have also been formed. Each of the products exhibits reductase activity, although they show varied affinities for their cofactor, NADH, and less stability to chromatography, dialysis, and concentration than the wild-type enzyme. The N-terminal sequence contains a classic NADH binding region between amino acids 9 and 36, and Asp 37 is essential for binding the cofactor as is shown by the approximately 20-fold increase in dissociation constant for the D-37-I mutant and diminished kcat (approximately 43 s-1 compared to 156 s-1 for the wild-type enzyme). The results indicate that the DHPR cofactor binding site is similar to typical dinucleotide requiring dehydrogenases such as lactic acid and liver alcohol dehydrogenase.
Amino Acid Sequence↗
Open theatre. Interview by Tim Rice.
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Afghanistan↗
Cost of orthopedic injuries sustained in motorcycle accidents.
Fifty-one serial admissions to the orthopedic services at the University of California, Davis, Medical Center, Sacramento, for motorcycle accident trauma with open fractures were reviewed. Fifty-five percent of those tested were alcohol intoxicated at the time of admission. Seventy-five percent carried no insurance of any kind, and for the total group, 72% of the cost of acute hospitalization (+17,704 per patient) was paid by the state of California, with an additional 10% paid by other tax-based sources. Care of motorcycle trauma consumes a substantial portion of public health care funds in California. This could be reduced by legislative action concerning helmet use, licensing, and rigid enforcement of compulsory insurance.
Accidents, Traffic↗
Operative treatment of displaced talus fractures.
The talus is a bone with unique biomechanical features and vascular supply. Displaced fractures of the talus, therefore, frequently create problems of proper management. Forty-one severe talar fractures were treated operatively. The incidence of avascular necrosis was relatively low in this series (16%), and all of these were of Type III and IV fractures of the Marti-Weber classification. Type IV fractures were successfully treated by arthrodesis per primam, and suggested that fusion may be the indicated method of treatment in these severe injuries. Fusion of the tibiotalar joint has been used to encourage revascularization and to preserve the important function of the subtalar joint. In all other fracture types with dislocation, anatomic reduction is performed to restore joint congruity and encourage maintenance of talar dome viability. Painstaking postoperative management is important for the complete restoration of function.
Adolescent↗
Correlation among the changes in the levels of thyroid hormones, thyrotropin and thyrotropin-releasing hormone during the development of Xenopus laevis.
The influence of thyroid hormones on neurogenesis suggested that these hormones might affect the maturation and development of neural centers which ultimately regulate thyroid function. Changes in the levels of the thyroid hormones, thyrotropin and the thyrotropin-releasing hormone in 'brain' and 'thyroid' fractions were, therefore, correlated at several developmental stages. All hormones showed increases from their initial detection through metamorphosis. The thyroid hormones were observed earliest in both fractions. These data are consistent with the notion that thyroid hormones influence the maturation of the particular neural regions whose later function is the regulation of the hypophyseal-thyroid axis.
Animals↗