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Biomedical subjects

M E Jones

Publications and source records attributed to M E Jones.

At least 19 recordsLinked to original sources
PubMed1992

Orotidylate decarboxylase: insights into the catalytic mechanism from substrate specificity studies.

Pyrimidine nucleotides were tested as substrates for pure yeast orotidylate decarboxylase in an attempt to gain insight into the nature of the catalytic mechanism of the enzyme. Substitutions of the 5-position in the pyrimidine ring of the orotidylate substrate resulted in compounds that are either excellent inhibitors or substrates of the enzyme. The 5-bromo- and 5-chloroorotidylates are potent inhibitors while the 5-fluoro derivative is a good substrate with a turnover number 30 times that observed with orotidylate. When carbon 5 of the pyrimidine ring is replaced by nitrogen in 5-azaorotidylate, the resulting compound is unstable in solution with a half-life of 25 min at pH 6. However, studies with freshly generated 5-azaorotidylate show that an enzyme-dependent reaction occurs, presumably decarboxylation. This enzyme reaction follows simple Michaelis-Menten kinetics. Because the 5-aza group is not electrophilic, an enzyme mechanism utilizing a nucleophilic addition of the enzyme at the 5-position is ruled out. We also present studies that are not compatible with a mechanism requiring the formation of a Schiff's base prior to decarboxylation. The enzyme is tolerant of modest substitution at the 4-position, for the 4-keto group can be replaced with a thioketone. However, no catalysis is observed when the same substitution is made at the 2-position. Similarities in the substrate specificity of orotate phosphoribosyltransferase and orotidylate decarboxylase led us to compare the amino acid sequences of the two enzymes; significant (20%) sequence homology was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

A unique catalytic and inhibitor-binding role for Lys93 of yeast orotidylate decarboxylase.

The presence of a proton-donating catalytic amino acid side chain in orotidylate decarboxylase (ODCase) was sought by site-directed mutagenesis. Replacement of yeast ODCase Lys93 with a cysteine resulted in a mutant protein (K93C) with no measurable activity, representing a decrease in activity by a factor of, at most, 2 x 10(-8) times the activity of the wild-type enzyme. Treatment of this mutant protein with 2-bromoethylamine, designed to append Cys93 to yield S-(2-aminoethyl)cysteine, restored activity by a factor of at least 5 x 10(5) over the untreated mutant protein. Activity could not be restored by treatment with other brominated reagents designed to replace the epsilon-amino of S-(2-aminoethyl)Cys93 with a different functional group. The overall architecture of the K93C protein was not significantly changed, as judged by the similar dimerization properties (in the absence of ligands) of the mutant enzyme compared to the wild-type enzyme. The binding affinity of the substrate orotidylate was not measurably changed by the mutation, indicating that Lys93 has an essential role in catalysis which is mechanistically distinguishable from substrate binding. Apparently the mutation removes an integral portion of the active site and does not drastically affect the structural or substrate binding properties. However, the affinities of the mutant protein for the competitive inhibitors 6-azauridylate (6-azaUMP) and UMP are significantly altered from the pattern seen with the wild-type enzyme. The K93C protein has an affinity for the neutral ligand UMP which is greater than that for the anionic 6-azaUMP, in clear contrast to the preference for 6-azaUMP displayed by the wild-type enzyme. Lys93 is apparently critical for catalysis of the substrate to product and for the binding of anionic inhibitors; the data are discussed in terms of previously existing models for transition-state analogue inhibitor binding and catalysis.

Animals↗
PubMed1992

Analysis of algebraic weighted least-squares estimators for enzyme parameters.

An algorithm for the least-squares estimation of enzyme parameters Km and Vmax. is proposed and its performance analysed. The problem is non-linear, but the algorithm is algebraic and does not require initial parameter estimates. On a spreadsheet program such as MINITAB, it may be coded in as few as ten instructions. The algorithm derives an intermediate estimate of Km and Vmax. appropriate to data with a constant coefficient of variation and then applies a single reweighting. Its performance using simulated data with a variety of error structures is compared with that of the classical reciprocal transforms and to both appropriately and inappropriately weighted direct least-squares estimators. Three approaches to estimating the standard errors of the parameter estimates are discussed, and one suitable for spreadsheet implementation is illustrated.

Algorithms↗
PubMed1992

Interstate differences in incidence and mortality from melanoma. A re-examination of the latitudinal gradient.

OBJECTIVE: To investigate the patterns of cutaneous malignant melanoma (CMM) mortality in Australia. DESIGN: A descriptive analysis of melanoma incidence and mortality in Australia supplemented by a case series analysis of melanoma survival. Melanoma mortality rates were based on tabulations supplied by the Australian Bureau of Statistics for the years 1969-1989. Melanoma incidence rates were based on State cancer registry records for the years 1977-1990. The case series survival analysis was based on detailed individual records from the population-based cancer registries in Tasmania and South Australia. MAIN OUTCOME MEASURES: The level of and rise in melanoma mortality rates during 1969-1989 in Australia; the five-year survival rates for Tasmanian and South Australian cases; and male:female incidence ratios related to latitude. RESULTS: We found annual increases in melanoma mortality rates of 2.5% in men (P < 0.0001) and 1.1% in women (P < 0.0001) for all Australia. The five-year survival rates (with 95% confidence intervals [CI]) were: 67% (59%-75%) for Tasmanian men; 79% (76%-83%) for South Australian men; 80% (74%-86%) for Tasmanian women and 88% (86%-91%) for South Australian women. A change in the male:female incidence ratio with latitude was also found--women have significantly higher incidence rates at higher latitudes, but similar rates to men at lower latitudes. CONCLUSIONS: The age standardised mortality from CMM for the period 1969 to 1989 shows little variation by State for women, despite a considerable range in latitude. CMM mortality in men is increasing at a faster rate than that in women. Between 1982 and 1987 the male:female incidence ratio in high latitudes in the Southern Hemisphere showed an excess of cases in women, a finding which we believe has not been reported before.

Aged↗
PubMed1992

Inhibition kinetics of hepatic microsomal long chain fatty acid-CoA ligase by 2-arylpropionic acid non-steroidal anti-inflammatory drugs.

Microsomal long chain fatty acid CoA ligase (EC 6.2.1.3) has been implicated in the formation of CoA thioesters of xenobiotics containing a carboxylic acid moiety. In this study we have demonstrated that the microsomal enzyme from rat liver exhibits biphasic kinetics for the formation of palmitoyl-CoA, i.e. there are high affinity low capacity Kmhigh, 1.6 microM, Vmaxhigh, 12.9 nmol/mg/min) and low affinity high capacity (Kmlow, 506 microM, Vmaxlow, 58.3 nmol/mg/min) components. Inhibition of the high affinity isoform was studied using the R and S enantiomers of ibuprofen, fenoprofen, ketoprofen and naproxen. The high affinity component of palmitoyl-CoA formation was competitively inhibited by R-fenoprofen (Ki 15.4 microM) while R-ibuprofen exhibited mixed inhibition kinetics. In contrast the R and S enantiomers of ketoprofen and naproxen were non-competitive inhibitors. This diversity of inhibition kinetics observed argues in favour of a binding site in addition to the catalytic site. A competitive interaction with the high affinity form correlated with literature evidence of enantiospecific chiral inversion and "hybrid" triglyceride formation for the R enantiomers of fenoprofen and ibuprofen. Paradoxically, R-ketoprofen which is extensively inverted in rats was a non-competitive inhibitor of palmitoyl-CoA formation by the high affinity isoform suggesting that it may not act as an alternate substrate. The results of this study clearly indicate that formation of R-2-arylpropionate-CoAs is not fully explained by interaction with the high affinity isoform of a microsomal long chain (palmitoyl) CoA ligase and therefore the involvement of other isoforms cannot be discounted.

Animals↗
PubMed1992

Climatic temperature and variation in the incidence of sudden infant death syndrome between the Australian states.

OBJECTIVE: To describe the relationship between climatic temperature and the incidence of sudden infant death syndrome (SIDS) for the Australian States and examine the extent to which differences in climatic temperature might explain the regional variation of SIDS in Australia. DESIGN: Case series study. A generalised linear model was used to model the association between monthly average temperature and the incidence of SIDS. SETTING: The report is population based. Data are available from all Australian States. SUBJECTS: Cases of SIDS from birth to less than 12 months of age occurring in Queensland (1981-1987), New South Wales (1981-1987), Victoria (1984-1987), Tasmania (1975-1989), South Australia (1980-1989), and Western Australia (1980-1988). RESULTS: Every one degree Celsius decrease in average monthly temperature within the range 9 degrees C to 25 degrees C is associated with a 10.6% (95% confidence interval, 9.6%-11.7%) increase in the incidence of SIDS. Climatic temperature accounts for 84% of the interstate variation in the rate of SIDS. After controlling for the effect of temperature, a significant overall difference in SIDS incidence remains (P less than 0.0001) for the Australian States. CONCLUSION: Climatic temperature accounts for most but not all of the regional variation of SIDS incidence in the Australian States. The remaining variation may reflect differences in the maternal and infant characteristics of the State populations.

Australia↗
PubMed1992

Sudden infant death syndrome: factors contributing to the difference in incidence between Victoria and Tasmania.

OBJECTIVE: To examine how much of the difference in incidence of sudden infant death syndrome (SIDS) between Tasmania and Victoria could be accounted for by the effect of differing climatic temperature and the effect of the differing prevalence of maternal and infant characteristics in the two State populations. DESIGN: A two population ecological comparison. Two previously published predictive models were applied to quantify the contribution of several factors to the higher incidence of SIDS in Tasmania compared with Victoria. SETTING: A population based study involving the two Australian States of Tasmania and Victoria. PATIENTS: The characteristics of the 1985 to 1987 live birth cohorts of Tasmania and Victoria were examined. Cases were defined as all infants dying in 1985 to 1987 whose cause of death was stated as SIDS. RESULTS: The rate of SIDS for Tasmania and Victoria 1985 to 1987 was 3.76 per 1000 live births and 2.18 per 1000 live births respectively. Adjustment of the Tasmanian rate for the effect of the interstate difference in climatic temperature resulted in a lower Tasmanian rate of 2.92 per 1000 live births. Adjustment for the effect of interstate differences in maternal age, birthweight, infant sex, month of birth and intention to breast-feed at hospital discharge decreased the Tasmanian rate to 2.47 per 1000 live births. CONCLUSION: Approximately 82% of the interstate difference in SIDS incidence between Tasmania and Victoria from 1985 to 1987 can be accounted for by differences in climatic temperature, maternal age, birth-weight, infant sex, month of birth and feeding intention at hospital discharge.

Adult↗
PubMed1992

Thermal environment and sudden infant death syndrome: case-control study.

OBJECTIVE: To compare the thermal environment of infants who died of the sudden infant death syndrome with that of age matched control infants. DESIGN: Case-control study. Infants who died were matched with two controls, one for age and one for age and birth weight. Thermal measurements were conducted at the death scene for cases and at the scene of last sleep for control infants, who were visited unexpectedly within four weeks of the index infant's death on a day of similar climatic conditions. A follow up questionnaire was administered to parents of cases and controls. SETTING: The geographical area served by the professional Tasmanian state ambulance service, which includes 94% of the Tasmanian population. SUBJECTS: 41 infants died of the sudden infant death syndrome at home; thermal observations at death scene were available for 28 (68%), parental questionnaire data were available for 40 (96%). 38 controls matched for age and 41 matched for age and birth weight. RESULTS: Cases had more excess thermal insulation for their given room temperature (2.3 togs) than matched controls (0.6 togs) (p = 0.009). For every excess thermal insulation unit (tog) the relative risk of the sudden infant death syndrome was 1.26 (95% confidence interval 1.05 to 1.52). The average thermal bedding value calculated from parental recall was similar to that observed by attendant ambulance officers (mean difference = 0.4 tog, p = 0.39). Cases were more likely to have been found prone (odds ratio 4.58; 1.48 to 14.11). Prone sleeping position was not a confounder or effect modifier of the relation between excess thermal insulation and the syndrome. CONCLUSIONS: Overheating and the prone sleeping position are independently associated with an increased risk of the sudden infant death syndrome. Further work on infant thermal balance and sudden infant death is required and guidelines for appropriate infant thermal care need to be developed.

Bedding and Linens↗
PubMed1992

Circulating CD34+ cells: an adverse prognostic factor in the myelodysplastic syndromes.

As part of an epidemiological survey of myelodysplastic syndromes (MDS) in southern Tasmania, 62 MDS patients identified over a 2 year period were tested for the presence of CD34, the human progenitor cell antigen (HPCA), in their peripheral blood. The results were correlated with transformation to acute myeloid leukemia (AML) and patient survival, and CD34+ status was compared as a prognostic indicator with Bournemouth score, cytogenetics, and CFU-GM colony growth which were also assessed. Circulating CD34+ cells were found in 23 of the 62 MDS patients; 9 of the 23 patients with circulating CD34+ cells transformed to AML, as compared with none of the 39 CD34 negative patients (P less than 0.0001); and 11 of the 23 patients with circulating CD34+ cells were dead at the end of the 2 year period, as opposed to 6 of the 39 with no CD34+ cells (P less than 0.03). The Bournemouth score was also significantly associated with transformation to AML (P less than 0.0001) and poor survival (P less than 0.04). These were the only significant associations of the possible prognostic factors studied with either transformation or survival. In summary, the presence of circulating CD34+ cells was significantly associated with both progression to AML and poor survival and was found to be a better prognostic indicator than cytogenetics or CFU-GM colony growth.

Adult↗
PubMed1992

Orotidylate decarboxylase of yeast and man.

The mechanism for ODCase appears to involve the formation of a zwitterion of OMP and a ylid on decarboxylation. Thiamin pyrophosphate catalyzes various decarboxylation and transfer reactions involving ketone groups because the thiazolium ring with its positively charged N atom can, on the loss of a proton from the adjacent C-2, generate a ylid which adds to carbonyl groups to produce a substrate ylid. The unusual aspect, then, of the ODCase reaction is that the substrate itself becomes the ylid, presumably by gaining a proton from ODCase, which results in a positive charge on the N-1 atom of the pyrimidine ring. It is a zwitterion in the transition state which momentarily becomes a ylid on decarboxylation of OMP which then yields the product, UMP. There is no known cofactor for the ODCase reaction. It will be of interest to discover the groups on the enzyme that aid in formation of the zwitterion and the ylid. Further work on the crystal structure and on the production of altered enzymes (where specific amino acids suspected to be important for the reaction are changed) should reveal more details about this important and novel reaction.

Amino Acid Sequence↗
PubMed1992

Characteristics of the infant thermal environment in the control population of a case-control study of SIDS.

This report examines the thermal environment during last sleep of a control population to investigate how the thermal environment of the infant's bedroom varies by season, external temperature and by certain maternal and infant characteristics. Two age-matched control infants were chosen for each case, one of which was also matched on birthweight. The home visits were not pre-arranged and were matched on climatic conditions, time of year and time period of day for the index case. The initial response rate for controls (n = 108) was 86%. Although there was a large amount of variation in the infant thermal environment, thermal insulation correlated with room temperature (r = -0.44, P = 0.0001) and external temperature (r = -0.30, P = 0.002). The thermal environment of the infant, as defined by excess thermal insulation for room temperature, did not vary by indoor or outdoor temperature, but higher average values were observed in teenage mothers (mean difference = 2.7 tog [95% Cl = 0.3, 5.2]), infants who slept in an adult bed (mean difference = 2.6 tog [-0.1, 5.4]) and infants with an illness (mean difference = 0.8 tog [-0.3, 1.9]). There was a tendency for the thermal environment of infants to be higher and more variable during winter, supporting previous hypotheses that paradoxical overheating may occur in some infants during winter. Further work is required to provide a set of recommendations on the optimal thermal conditions for post-neonatal infants.

Bedding and Linens↗
PubMed1992

Sudden infant death syndrome: seasonality and a biphasic model of pathogenesis.

STUDY OBJECTIVE: This paper examines the relationship between season, age, and the sudden infant death syndrome (SIDS). It provides a theoretical model for the pathogenesis of SIDS and uses it as a framework to consider risk factor mechanism. DESIGN: A case series analysis was used to examine season and age in relation to SIDS and seasonal pattern and age at death distribution of perinatal risk factors. SETTING: The source population for the SIDS cases in this study was all live births in the state of Tasmania, Australia, 1975 to 1987 inclusive. SUBJECTS: Cases were all infants born 1975 to 1987 who died of SIDS on whom birth notification information was available (n = 348). The live birth cohort 1980-87 (n = 55,944) was used as the control population for risk factor identification. MEASUREMENTS AND MAIN RESULTS: The median ages of death for spring, summer, autumn, and winter born infants were 115, 103.5, 91 and 78 days. Spring and summer born infants died at a significantly older median age than winter born infants. The month of birth distribution of SIDS cases did not alter significantly from a uniform, nonseasonal distribution (p greater than 0.25) but month of death was seasonally distributed (p less than 0.01). Premature and low birthweight infants died at an older median age (p less than 0.05) than term and non-low-birthweight infants. An excess of male infant deaths and infant deaths to older mothers occurred during winter (p less than 0.05). CONCLUSIONS: The pathogenesis of SIDS can be represented as a biphasic model with three pathways of risk factor operation. In this study, season influenced the age at death of SIDS infants. We propose that risk factors with a strong seasonal distribution are likely to be operating in the postnatal period.

Female↗
PubMed1992

Algebraic least squares estimates of inhibitor constants.

When the data has a constant coefficient of variation, appropriately weighted least squares estimates of the parameters for competitive inhibition in both enzyme and binding experiments can be obtained algebraically. The algorithms are presented and justified.

Algorithms↗
PubMed1991

Purification and characterization of yeast orotidine 5'-monophosphate decarboxylase overexpressed from plasmid PGU2.

Orotidine 5'-monophosphate decarboxylase (ODCase) has been overexpressed in yeast 15C cells transformed with a plasmid carrying the URA3 gene that encodes ODCase. Twenty g of cells having ODCase activity equal to 30 mg of pure enzyme per liter of cell culture were obtained after 9 h of galactose induction. To remove yeast proteases, a 60-90% ammonium sulfate fractionation step plus the addition of EDTA as an inhibitor of metallopeptidases was necessary. The purification protocol yielded ODCase that was protease-free and stable to storage at 4 degrees C for 16 months. The pure enzyme had a specific activity of 40 units/mg in 50 mM phosphate buffer, pH 6, and could be stored at -20 degrees C in 20% glycerol with retention of full activity for more than 2 years. The enzyme had a Km for orotidine 5'-monophosphate of 0.7 microM at pH 6 and 25 degrees C. The molecular weight of the plasmid-derived ODCase monomer determined by electrophoresis on denaturing polyacrylamide gels was 29,500. ODCase sedimented through sucrose density gradients as a monomer of about 30 kDa at low protein concentration and in the absence of ligands that bind at the catalytic site. An increase in the sedimentation rate could be induced by increasing the ODCase concentration or by adding ligands that are competitive inhibitors. ODCase sedimented in a single band typical of a protein of 46 kDa at the highest protein concentration studied or in the presence of 50 mM phosphate or 933 microM substrate (orotidine 5'-monophosphate) or product (UMP). A dimer sedimenting as a protein of about 64 kDa occurred in the presence of 50 microM 6-azauridine 5'-monophosphate or 2 microM 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid, competitive inhibitors of ODCase. These results resemble the ligand-induced subunit association of the ODCase domain of bifunctional UMP synthase and support the use of yeast ODCase as a model for ODCases from other species.

Binding, Competitive↗
PubMed1991

Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects.

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).

Carbon Isotopes↗
PubMed1991

Iron uptake by human reticulocytes at physiologic and sub-physiologic concentrations of iron transferrin: the effect of interaction with aluminum transferrin.

We have studied the interaction, in vitro, between diferric transferrin (FeTr), aluminum transferrin (AlTr), and human reticulocytes harvested from human placental blood. In particular, we aimed to determine the extent to which the kinetics of AlTr and FeTr differed. Using transferrin labeled with either 59Fe or 125I, the association of radiotracer with reticulocytes, as a function both of time and of transferrin concentration, was examined. Under the conditions of the experiments, the data are consistent with a mechanism involving at least three processes. Two early processes acting in parallel behave as a high-affinity saturable receptor and a low-affinity non-saturable receptor, neither of which distinguish between AlTr and FeTr. In a subsequent process, AlTr and FeTr exhibit different kinetics. This third process may be saturated by FeTr but not by AlTr. Interpreted in terms of a current conventional view of metallo-transferrin uptake, we conjecture that the early parallel processes involve cell surface phenomena including classical transferrin-receptor binding, and that the subsequent process represents events, possibly intracellular, involved in metallo-transferrin dissociation or further iron transport. The extent to which AlTr influences the interaction of FeTr with reticulocytes offers insight into both the normal physiology of iron uptake and the potential for toxicity by aluminum.

Binding, Competitive↗
PubMed1991

Crystallization of yeast orotidine 5'-monophosphate decarboxylase complexed with 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid.

Using an incomplete factorial experimental design, we have identified conditions for crystallization of yeast orotidine 5'-monophosphate decarboxylase (ODCase) in an unliganded state and complexed separately to two inhibitors: 6-azauridine 5'-monophosphate (aza-UMP) and 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid (BMP). Crystals of X-ray diffraction quality have been obtained of yeast ODCase complexed with BMP, a putative transition state analog inhibitor (Ki = 8.8 x 10(-12) M). ODCase:BMP complex crystals with a hexagonal rod habit were grown from a solution initially containing 12 mg/ml ODCase (205 microM dimer) plus 450 microM BMP by microdialysis at 4 degrees C against a mother liquor which consisted of 0.1 M Na-PIPES-acetate (pH 6.4), 37.5 microM BMP, 5 mM mercaptoethanol, 1% polyethylene glycol 400, and 2.3 M ammonium sulfate. Crystals were analyzed using precession photography and were assigned to trigonal space group R32 with unit cell dimensions a = b = 115 A, c = 385 A. The crystal density is 1.245 g/cm3 indicating the presence of two ODCase: BMP complex dimers (118 kDa each) per asymmetric unit with a packing density of 2.08 A3/Da and 41% solvent content. The morphological habit of crystals of the ODCase:BMP complex changed when the initial ammonium sulfate concentration was increased in 0.05 M steps from 2.3 to 2.45 M. All of these crystals diffracted to at least 3.0 A resolution over a period of several weeks at room temperature and are isomorphous.

Ammonium Sulfate↗