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Biomedical subjects

M A Becker

Publications and source records attributed to M A Becker.

At least 19 recordsLinked to original sources
PubMed2006

Magnetic resonance imaging in the quantitative assessment of gouty tophi.

Measurements of tophus size can be important in monitoring the course of gout therapy, as tophus resolution is proposed as one measure of success of treatment. This multicentre study assessed the intra- and interreader reproducibility of quantitative tophus volume measurements from magnetic resonance images (MRI) in subjects with palpable gouty tophi. Subjects first underwent radiographic imaging of a selected tophus followed by MRI before and at <or=5, 10 and 20-min after gadolinium administration. After choosing optimal parameters, subjects underwent pre- and postgadolinium-enhanced MRIs of a selected tophus on two occasions separated by 5-10 days. Unenhanced spin-echo images provided satisfactory tophi images and were less subject to interfering artefacts than gadolinium-enhanced gradient-echo images. Intrareader reproducibility was excellent, with no statistically significant difference in mean tophus volume between visits (mean difference - 0.05 +/- 0.97 cm3). A small but statistically significant difference in interreader mean tophus volume was detected (mean difference 0.89 +/- 2.05 cm3; p < 0.05). MRI can quantify tophus size in gout and deserves further comparison with other techniques for tophus size monitoring in assessing effects of gout therapy.

Adolescent↗
PubMed2004

Febuxostat (TMX-67), a novel, non-purine, selective inhibitor of xanthine oxidase, is safe and decreases serum urate in healthy volunteers.

In order to evaluate the safety, pharmacological properties, and urate-lowering efficacy of febuxostat, a non-purine, selective inhibitor of xanthine oxidase, a Phase 1, 2-week, multiple-dose, placebo-controlled, dose-escalation study was conducted in 154 healthy adults of both sexes. Daily febuxostat doses in the range 10 mg to 120 mg resulted in proportional mean serum urate reductions ranging from 25% to 70% and in proportional increases in maximum febuxostat plasma concentrations and area under plasma concentration versus time curves. Accompanying the hypouricemic effect were increases in serum xanthine concentrations, decreases in urinary uric acid excretion, and increases in urinary xanthine and hypoxanthine excretion, confirming inhibition of xanthine oxidase activity by febuxostat. Hepatic conjugation and oxidative metabolism were the major pathways of elimination of febuxostat from the body, and renal elimination did not appear to play a significant role. Although not uncommon, adverse events were mild and self-limited, and no deaths or serious adverse events were observed. Febuxostat is a safe and potent hypouricemic agent in healthy humans.

Adult↗
PubMed2003

Attitudes towards mental health care in younger and older adults: similarities and differences.

It is commonly assumed that older and younger adults have very different attitudes about seeking mental health services and that this is a major factor in reducing the use of mental health services by the elderly. However, little evidence exists to illustrate how elders actually perceive mental health care. Responses from a survey of 474 older adults age 65 and over were compared with data from a national survey of 1001 persons age 21-65. Results indicate similarities in many attitudes including likelihood of seeking treatment for severe mental disorders, importance of mental health care, and concerns about cost and coverage as barriers to care. Differences included use of services, perceptions about less severe disorders, referral sources, and preferred providers. Clinical, policy and public education implications are discussed.

Adult↗
PubMed2001

Phosphoribosylpyrophosphate synthetase and the regulation of phosphoribosylpyrophosphate production in human cells.

between purine nucleoside diphosphate inhibition and inorganic phosphate (Pi) activation; and intracellular concentration of the PRS1 isoform. The operation of additional determinants of rates of PRPP synthesis in human cells is suggested by: (1) multiple PRS isoforms with distinctive physical and kinetic properties; (2) nearly immediate activation of intracellular PRPP synthesis in response to mitogens, growth-promoters, and increased intracellular Mg2+ concentrations; (3) tissue-specific differences in PRS1 and PRS2 transcript and isoform expression; and (4) reversible association of PRS subunits with one another and/or with PRS-associated proteins (PAPs), as a result of which the catalytic and perhaps regulatory properties of PRS isoforms are modified.

Amino Acid Sequence↗
PubMed1999

Accelerated transcription of PRPS1 in X-linked overactivity of normal human phosphoribosylpyrophosphate synthetase.

Phosphoribosylpyrophosphate (PRPP) synthetase (PRS) superactivity is an X-linked disorder characterized by gout with overproduction of purine nucleotides and uric acid. Study of the two X-linked PRS isoforms (PRS1 and PRS2) in cells from certain affected individuals has shown selectively increased concentrations of structurally normal PRS1 transcript and isoform, suggesting that this form of the disorder involves pretranslational dysregulation of PRPS1 expression and might be more appropriately termed overactivity of normal PRS. We applied Southern and Northern blot analyses and slot blotting of nuclear runoffs to delineate the process underlying aberrant PRPS1 expression in fibroblasts and lymphoblasts from patients with overactivity of normal PRS. Neither PRPS1 amplification nor altered stability or processing of PRS1 mRNA was identified, but PRPS1 transcription was increased relative to GAPDH (3- to 4-fold normal in fibroblasts; 1.9- to 2.4-fold in lymphoblasts) and PRPS2. Nearly coordinate relative increases in each process mediating transfer of genetic information from PRPS1 transcription to maximal PRS1 isoform expression in patient fibroblasts further supported the idea that accelerated PRPS1 transcription is the major aberration leading to PRS1 overexpression. In addition, modulated relative increases in PRS activities at suboptimal Pi concentration and in rates of PRPP and purine nucleotide synthesis in intact patient fibroblasts indicate that despite an intact allosteric mechanism of regulation of PRS activity, PRPS1 transcription is a major determinant of PRPP and purine synthesis. The genetic basis of disordered PRPS1 transcription remains unresolved; normal- and patient-derived PRPS1s share nucleotide sequence identity at least 850 base pairs 5' to the consensus transcription initiation site.

Allosteric Regulation↗
PubMed1997

Melatonin production in an aerobic photosynthetic bacterium: an evolutionarily early association with darkness.

Melatonin was measured in a species of aerobic photosynthetic bacteria, Erythrobacter longus, grown in either constant light or constant dark. A radioimmunoassay was used to quantify melatonin levels and thin-layer chromatography to confirm the identity of melatonin immunoactivity. Melatonin levels were significantly higher (nearly 2.3-fold) in the dark-grown than in the light-grown samples. Also, the homogenates of the dark-grown bacteria retained melatonin-producing enzymatic activity, whereas the light-grown homogenates did not; melatonin levels extracted from the dark-grown homogenates increased with increasing extraction time, reaching as high as 29.2 ng.mg-1 protein at 120 min. Removal of membrane fragments from homogenates did not influence melatonin levels in light-grown homogenate, but this procedure increased melatonin levels in dark-grown homogenate, indicating that at least some of the enzymes in the pathway of melatonin production are not membrane-bound. This study is the second to demonstrate the presence of melatonin at the prokaryotic level, supporting the evidence that melatonin appeared very early in evolution. Its function in prokaryotes has not been determined, but may relate to its antioxidative actions.

Bacteria, Aerobic↗
PubMed1996

Overexpression of the normal phosphoribosylpyrophosphate synthetase 1 isoform underlies catalytic superactivity of human phosphoribosylpyrophosphate synthetase.

To define the enzymatic and genetic basis of X-linked phosphoribosylpyrophosphate synthetase (PRS) catalytic superactivity, we measured concentrations of X-linked PRS1 and PRS2 isoforms in cultured fibroblasts and lymphoblasts by immunoblotting after separation by polyacrylamide-urea isoelectric focusing. PRS1 comprised >80% of measurable PRS isoforms in all fibroblast strains, but PRS1 concentrations in cells from six affected males exceeded those in normal cells by 2-6-fold. PRS absolute specific activities (activity per mg of PRS isoforms) were comparable in all fibroblast strains and in purified recombinant normal PRS1, confirming selectively increased levels of PRS1 isoform as the enzymatic basis of PRS catalytic superactivity. Cloning, sequencing, and expression of normal subject- and patient-derived PRS cDNAs predicted normal translated region sequences for both PRS isoforms and revealed no differences in catalytic properties of recombinant PRS1. Normal and patient PRPS1 transcribed but untranslated DNA sequences were also identical. Northern blot analysis showed selective increase in relative concentrations of PRS1 transcripts in patient fibroblasts. In PRS catalytic superactivity, overexpression of the normal PRS1 isoform thus appears to result from an altered pretranslational mechanism of PRPS1 expression. In lymphoblasts, however, expression of this alteration is attenuated, explaining the absence of phenotypic expression of PRS catalytic superactivity in these cells.

Base Sequence↗
PubMed1995

The genetic and functional basis of purine nucleotide feedback-resistant phosphoribosylpyrophosphate synthetase superactivity.

The genetic and functional basis of phosphoribosylpyrophosphate synthetase (PRS) superactivity associated with purine nucleotide inhibitor-resistance was studied in six families with this X chromosome-linked purine metabolic and neurodevelopmental disorder. Cloning and sequencing of PRS1 and PRS2 cDNAs, derived from fibroblast total RNA of affected male patients by reverse transcription and PCR amplification, demonstrated that each PRS1 cDNA contained a distinctive single base substitution predicting a corresponding amino acid substitution in the PRS1 isoform. Overall, the array of substitutions encompassed a substantial portion of the translated sequence of PRS1 cDNA. Plasmid-mediated expression of variant PRS1 cDNAs in Escherichia coli BL21 (DE3/pLysS) yielded recombinant mutant PRS1s, which, in each case, displayed a pattern and magnitude of purine nucleoside diphosphate inhibitor-resistance comparable to that found in cells of the respective patient. Kinetic analysis of recombinant mutant PRS1s showed that widely dispersed point mutations in the X chromosome-linked PRPS1 gene encoding the PRS1 isoform result in alteration of the allosteric mechanisms regulating both enzyme inhibition by purine nucleotides and activation by inorganic phosphate. The functional consequences of these mutations provide a tenable basis for the enhanced production of phosphoribosylpyrophosphate, purine nucleotides, and uric acid that are the biochemical hallmarks of PRS superactivity.

Base Sequence↗
PubMed1995

Inhibition of human 5-phosphoribosyl-1-pyrophosphate synthetase by 4-amino-8-(beta-D-ribofuranosylamino)-pyrimido[5,4-d]pyrimidine-5'- monophosphate: evidence for interaction at the ADP allosteric site.

The kinetics of inhibition by the aminopyrimidopyrimidine nucleotide 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine[-5' -monophosphate (APP-MP) were assessed with two human isozymes of 5-phosphoribosyl-1-pyrophosphate synthetase (PRS) (PRS1 and PRS2) and a mutant enzyme, S.M. PRS1, derived from an individual with PRS hyperactivity. In the presence of 1 mM potassium phosphate, APP-MP inhibited PRS1 and PRS2 with half-maximal inhibition (IC50) at 5.2 microM and 23.8 microM, respectively. The degree of inhibition for both enzymes was highly dependent on the phosphate concentration; IC50 values were 70 times higher in the presence of 50 mM potassium phosphate. APP-MP exhibited mixed noncompetitive-uncompetitive inhibition against PRS1, with a Kii value of 6.1 microM and a Kis value of 14.6 microM, and produced parabolic secondary plots of slope or intercept versus APP-MP concentration. In comparison, inhibition of PRS1 by ADP was of a mixed noncompetitive-competitive type, with a Kii value of 9.6 microM and a Kis value of 2.8 microM. A similar kinetic analysis was completed using S.M. PRS1, a mutant enzyme with a single amino acid substitution resulting in diminished sensitivity to feedback inhibition by nucleotides. The noncompetitive component of ADP inhibition of PRS1 was absent with S.M. PRS1 and ADP inhibition was purely competitive, with a Ki of 6.4 microM, APP-MP was a very poor inhibitor of S.M. PRS1, displaying uncompetitive characteristics and a Ki of 1.6 mM. These data indicate that APP-MP inhibits PRS1 with a strong element of noncompetitive inhibition and appears to interact specifically at the allosteric site used by ADP. These results contrast with those obtained with ADP, which has a strong component of ATP competitive inhibition and binds at the ATP site as well as at a second, allosteric, site.

Adenosine Diphosphate↗
PubMed1995

Endogenous porphyrins in murine skin and transplanted PAM-212 squamous cell carcinoma tissues after injection of delta-aminolevulinic acid.

After intraperitoneal (IP) injection of delta-aminolevulinic acid (ALA), the endogenous porphyrins in murine skin and tumor tissues were determined by a method involving solvent and acid extractions. The results showed that the total amount of porphyrins in the tumor tissues after ALA injection was much higher than that in the skin from the same mice, although the amount of porphyrins in the skin from the ALA-injected mice was higher than that from the saline-injected (control) mice. The porphyrins in the tumor were mostly protoporphyrin and coproporphyrin, with only a small amount of uroporphyrin. The optimum period for porphyrin accumulation in the tumor as well as in the skin was 1 hour after the injection of ALA. As the period was extended to 3 and 6 hours, the amount of porphyrins in these tissues decreased considerably. These findings could be valuable for further application of ALA in the photodynamic therapy of skin cancer.

Aminolevulinic Acid↗
PubMed1994

A simplified method for the determination of phosphoribosylpyrophosphate synthetase activity in hemolysates.

Methods currently employed for measurement of phosphoribosylpyrophosphate synthetase (ribophosphate pyrophosphokinase; PRPPs) activity are cumbersome and expensive, requiring an auxiliary enzyme reaction, a radioactive nucleobase and multiple incubations, or do not allow a complete kinetic study. The aim of the present study is to describe a simplified, single step, non-isotopic method for determination of PRPPs in hemolysate, appropriate for a complete screening of PRPPs activity disorders. Briefly, the charcoal-treated hemolysate is incubated with saturating amounts of substrates and P1 P5 di(adenosine 5') pentaphosphate (Ap5A), an inhibitor of human adenylate kinase, to prevent conversion of AMP to ADP. AMP generated in this reaction is then measured by HPLC. Adenylate kinase activity was fully inhibited by Ap5A, allowing the accurate determination of AMP. The method was sensitive and reproducible and mean and variance values compared closely with those reported using other, more complicated, assay procedures. The hyperbolic curve relating Pi concentration to initial reaction velocity was shifted to sigmoidal by addition of 0.02 mmol/l GDP which inhibited PRPPs activities only at inorganic phosphate concentrations < 2 mmol/l. This suggests that this method should provide sensitive and accurate screening for regulatory, as well as catalytic, defects underlying PRPPs superactivity.

Adenosine Monophosphate↗
PubMed1994

Ocular timolol levels after drug withdrawal: an experimental model.

Carbon 14-labelled timolol maleate was instilled into both eyes of 12 pigmented rabbits daily for 42 days. Drug levels in the aqueous humour and ocular tissues were measured up to 42 days after drug withdrawal. The results indicate that timolol concentrates mainly in melanotic tissues, with slow release. Even 42 days after withdrawal the drug was still present in pigmented ocular tissues. Timolol was detected in the aqueous up to 5 days after withdrawal. These findings explain the long-term depressant effect of topically administered timolol on aqueous production. We conclude that lower or less frequent doses of timolol should be considered in patients with glaucoma.

Animals↗
PubMed1993

Human X-linked phosphoribosylpyrophosphate synthetase superactivity is associated with distinct point mutations in the PRPS1 gene.

Superactivity of phosphoribosylpyrophosphate synthetase (PRS) is an X chromosome-linked disorder of purine metabolism, characterized by gout with uric acid overproduction and, in some families, neurodevelopmental impairment. Two highly homologous isoforms of PRS (PRS1 and PRS2), each encoded by a distinct X chromosome-linked locus, have been identified, and PRS1 and 2 cDNAs have been cloned. The entire 954-base pair translated regions of PRS1 and 2 cDNAs derived from cultured lymphoblasts and fibroblasts from two patients in whom purine nucleotide feedback resistance of PRS is associated with enzyme superactivity and neurodevelopmental defects were examined by direct sequencing after polymerase chain reaction amplification of PRS transcripts. Nucleotide sequences of PRS2 cDNAs from the patients and normal individuals were identical. In contrast, PRS1 cDNAs from the patients differ from normal PRS1 cDNA, each by a single base substitution. PRS1 cDNA from patient N. B. showed an A to G transition at nucleotide 341, corresponding to an asparagine to serine change at amino acid residue 113 of mature PRS1. A G to C transversion at nucleotide 547, indicating an aspartic acid to histidine change at amino acid 182, was found for PRS1 cDNA from patient S. M. Point mutations at the sites identified in the PRS1 cDNAs of the two patients were confirmed by the results of RNase mapping analysis. Normal, N. B., and S. M. PRS1 cDNAs were introduced into Escherichia coli BL21 (DE3)/pLyS, and recombinant N. B. and S. M. PRS1s showed the purine nucleotide feedback resistance phenotypes characteristic of PRS from patients' cells.

Base Sequence↗