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Biomedical subjects

D R Evans

Publications and source records attributed to D R Evans.

At least 37 records · Page 2Linked to original sources
PubMed2000

Bioavailability of iron in two prenatal multivitamin/multimineral supplements.

OBJECTIVE: To determine the iron bioavailability in two popular prenatal multivitamin/multimineral supplement tablets containing 27 mg elemental iron. STUDY DESIGN: Iron absorption during an eight-hour period following ingestion of a multivitamin/multimineral formulation, both fasting and with a standardized meal, was measured in a group of 30 pregnant women (24-32 weeks of gestation) and statistically compared. The prenatal formulations were Stuartnatal Plus and Materna (Wyeth-Ayerst Pharmaceuticals, Philadelphia, Pennsylvania), and each contains 27 mg of elemental iron. A placebo was included in the study for the control group in this crossover, single-blind study. RESULTS: The net iron bioavailability (mean +/- SE) of Stuartnatal Plus and Materna, accounting for diurnal variation, and the iron ingested with the standardized meal was 5.4 +/- 0.4 and 4.6 +/- 0.2 mg, respectively, while fasting and 2.9 +/- 0.4 and 2.7 +/- 0.4 mg, respectively, postprandially. The total amount of iron absorption in the fasting states from both prenatal formulations exceeded the 3 mg of supplemental iron absorption per day recommended by the National Academy of Sciences. CONCLUSION: The results of this study indicate that these two prenatal multivitamin/multimineral formulations provide > 3.0 mg of supplemental iron absorption (fasting) as recommended by the National Academy of Sciences and 2.7 mg of iron absorption above the levels achieved following ingestion of a standard, low-iron test meal.

Adult↗
PubMed1999

Functional linkage between the glutaminase and synthetase domains of carbamoyl-phosphate synthetase. Role of serine 44 in carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (cad).

Mammalian carbamoyl-phosphate synthetase is part of carbamoyl-phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase (CAD), a multifunctional protein that also catalyzes the second and third steps of pyrimidine biosynthesis. Carbamoyl phosphate synthesis requires the concerted action of the glutaminase (GLN) and carbamoyl-phosphate synthetase domains of CAD. There is a functional linkage between these domains such that glutamine hydrolysis on the GLN domain does not occur at a significant rate unless ATP and HCO(3)(-), the other substrates needed for carbamoyl phosphate synthesis, bind to the synthetase domain. The GLN domain consists of catalytic and attenuation subdomains. In the separately cloned GLN domain, the catalytic subdomain is down-regulated by interactions with the attenuation domain, a process thought to be part of the functional linkage. Replacement of Ser(44) in the GLN attenuation domain with alanine increases the k(cat)/K(m) for glutamine hydrolysis 680-fold. The formation of a functional hybrid between the mammalian Ser(44) GLN domain and the Escherichia coli carbamoyl-phosphate synthetase large subunit had little effect on glutamine hydrolysis. In contrast, ATP and HCO(3)(-) did not stimulate the glutaminase activity, indicating that the interdomain linkage had been disrupted. In accord with this interpretation, the rate of glutamine hydrolysis and carbamoyl phosphate synthesis were no longer coordinated. Approximately 3 times more glutamine was hydrolyzed by the Ser(44) --> Ala mutant than that needed for carbamoyl phosphate synthesis. Ser(44), the only attenuation subdomain residue that extends into the GLN active site, appears to be an integral component of the regulatory circuit that phases glutamine hydrolysis and carbamoyl phosphate synthesis.

Adenosine Triphosphate↗
PubMed1999

Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms.

The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer. Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function. However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation. Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p. Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells. The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function.

Alleles↗
PubMed1999

Channeling of carbamoyl phosphate to the pyrimidine and arginine biosynthetic pathways in the deep sea hyperthermophilic archaeon Pyrococcus abyssi.

The kinetics of the coupled reactions between carbamoyl-phosphate synthetase (CPSase) and both aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of carbamoyl phosphate channeling in both the pyrimidine and arginine biosynthetic pathways. Isotopic dilution experiments and coupled reaction kinetics analyzed within the context of the formalism proposed by Ovádi et al. (Ovádi, J., Tompa, P., Vertessy, B., Orosz, F., Keleti, T., and Welch, G. R. (1989) Biochem. J. 257, 187-190) are consistent with a partial channeling of the intermediate at 37 degrees C, but channeling efficiency increases dramatically at elevated temperatures. There is no preferential partitioning of carbamoyl phosphate between the arginine and pyrimidine biosynthetic pathways. Gel filtration chromatography at high and low temperature and in the presence and absence of substrates did not reveal stable complexes between P. abyssi CPSase and either ATCase or OTCase. Thus, channeling must occur during the dynamic association of coupled enzymes pairs. The interaction of CPSase-ATCase was further demonstrated by the unexpectedly weak inhibition of the coupled reaction by the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA). The anomalous effect of PALA suggests that, in the coupled reaction, the effective concentration of carbamoyl phosphate in the vicinity of the ATCase active site is 96-fold higher than the concentration in the bulk phase. Channeling probably plays an essential role in protecting this very unstable intermediate of metabolic pathways performing at extreme temperatures.

Adaptation, Biological↗
PubMed1999

The effect of ascorbic acid supplementation on the nicotine metabolism of smokers.

BACKGROUND: The relationship of serum ascorbic acid (AA) levels and excretion of nicotine metabolites was determined in 75 men who smoked at least one pack of cigarettes per day. METHODS: The subjects were randomly divided into three groups of 25 each who received a placebo, 200 mg of supplementation, or 1000 mg of supplementation of AA per day for 1 month. Baseline and weekly serum AA levels were determined and simultaneous estimates of urinary excretion of nicotine metabolites as cotinine equivalents (CE). RESULTS: The group mean serum AA levels in the placebo group decreased 13% after 2 weeks; the group mean serum levels of the supplemented groups increased significantly after 1 week (P </= 0. 001) and remained elevated over 100% per week from baseline throughout the study. There was a progressive increase in urinary CE excretion in the placebo group. In the supplemented groups, group mean CE excretion decreased an average of 5% weekly in the 200-mg group and an average of 33% weekly in the 1000-mg group. Pearson's correlation between serum AA and urine CE excretion was significant (r = -0.7980, P </= 0.0003). CONCLUSIONS: Body levels of AA affect the level of urinary nicotine metabolites. The results of this study indicate that dietary levels of AA are inversely correlated to urinary excretion of nicotine metabolites and may be due to suppressed: (1) nicotine intake by smokers who smoked fewer cigarettes or inhale less deeply, (2) nicotine metabolism, or (3) both.

Adult↗
PubMed1999

Amniotic fluid B12, calcium, and lead levels associated with neural tube defects.

The group mean differences and relationships between folate, B12, calcium, lead, and methionine levels in amniotic fluid from 29 non-NTD and 11 NTD pregnancies (15-20 weeks' gestation age) were determined. The study population was predominantly Hispanic in both groups (48% in non-NTD and 36% in NTD group) with an average maternal age of 29 years in the non-NTD group and 24 years in the NTD group; and, an average gestation age of 18.5 weeks in the non-NTD and 17.5 weeks in the NTD group. The folate, B12, lead, calcium, and methionine levels of the two groups were compared by Student's t-test and by Pearson's correlation. The NTD levels were lower for calcium (p< or =0.0001), B12 (p< or =0.001), and methionine (P< or =0.001); but, the lead level was higher (p< or =0.0001). A negative correlation was observed between lead and both B12 (p< or =0.007) and methionine (p< or =0.02).

Adult↗
PubMed1999

The effect of ascorbic acid supplementation on the blood lead levels of smokers.

BACKGROUND: The study subjects were 75 adult men (20 to 30 years of age), who smoked one pack of cigarettes per day (minimum) and had no clinical signs of ascorbic acid deficiency or lead toxicity. None had a history of industrial exposure to lead, and the blood-lead levels were anticipated to be below 1.45 micromol/L, the minimum blood level associated with toxicity symptoms. METHODS: The men were randomly assigned to three study groups of 25, and each group was provided a four-week supply of one level of daily ascorbic acid supplements (placebo, 200 mg or 1000 mg of ascorbic acid). We measured baseline and weekly serum and urine ascorbic-acid levels as well as blood and urine lead levels. The weekly group means and variations of the measured data were statistically compared by means of ANOVA and Pearson's correlation. RESULTS: The serum ascorbic-acid levels of the groups receiving ascorbic acid increased significantly after one week (p< or =.001). There was no effect of placebo or 200 mg ascorbic-acid supplementation on the blood or urine lead levels. However, there was a 81% decrease in blood-lead levels in the 1000 mg ascorbic acid group after one week of supplementation (p< or =.001). CONCLUSIONS: Daily supplementation with 1000 mg of ascorbic acid results in a significant decrease of blood-lead levels associated with the general population. Ascorbic acid supplementation may provide an economical and convenient method of reducing blood-lead levels, possibly by reducing the intestinal absorption of lead.

Adult↗
PubMed1999

Amniotic fluid amino and nucleic acid in normal and neural tube defect pregnancies. A comparison.

OBJECTIVE: To explore the relationship between fetal amino acids (AAs), which are the components of structural proteins and nucleic acids, and the changes associated with neural tube defects (NTD). STUDY DESIGN: Ten weekly amniotic fluid (AF) samples from normal pregnancies (non-NTD) and 10 from NTD pregnancies between 16 and 20 weeks' gestational age were analyzed for AAs, and the weekly average for the non-NTD and NTD groups was determined. The group mean levels were compared by ANOVA. RESULTS: The weekly group mean levels of methionine, serine and aspartic acid were significantly lower than the weekly group mean levels of the other AAs. The group mean levels of methionine were significantly lower for NTD than for non-NTD pregnancies. CONCLUSION: The mean methionine level in NTD pregnancies was the only AA alteration in NTD. Although the AF levels of the non-NTD pregnancies showed that the levels of serine, aspartic acid and methionine were 3- to 19-fold lower than those of the other AAs, only methionine is essential. Consequently, methionine is rate limiting with respect to the role of structural proteins and nucleic acids in the fetus.

Amino Acids↗
PubMed1998

Regulation of an Escherichia coli/mammalian chimeric carbamoyl-phosphate synthetase.

Carbamoyl-phosphate synthetase (CPSase) consists of a 120-kDa synthetase domain (CPS) that makes carbamoyl phosphate from ATP, bicarbonate, and ammonia usually produced by a separate glutaminase domain. CPS is composed of two subdomains, CPS.A and CPS.B. Although CPS.A and CPS.B have specialized functions in intact CPSase, the separately cloned subdomains can catalyze carbamoyl phosphate synthesis. This report describes the construction of a 58-kDa chimeric CPSase composed of Escherichia coli CPS.A catalytic subdomains and the mammalian regulatory subdomain. The catalytic parameters are similar to those of the E. coli enzyme, but the activity is regulated by the mammalian effectors and protein kinase A phosphorylation. The chimera has a single site that binds phosphoribosyl 5'-pyrophosphate (PRPP) with a dissociation constant of 25 microM. The dissociation constant for UTP of 0.23 mM was inferred from its effect on PRPP binding. Thus, the regulatory subdomain is an exchangeable ligand binding module that can control both CPS.A and CPS.B domains, and the pathway for allosteric signal transmission is identical in E. coli and mammalian CPSase. A deletion mutant that truncates the polypeptide within a postulated regulatory sequence is as active as the parent chimera but is insensitive to effectors. PRPP and UTP bind to the mutant, suggesting that the carboxyl half of the subdomain is essential for transmitting the allosteric signal but not for ligand binding.

Adenosine Triphosphate↗
PubMed1998

The function of Glu338 in the catalytic triad of the carbamoyl phosphate synthetase amidotransferase domain.

The synthesis of carbamoyl phosphate by the mammalian multifunctional protein, CAD, involves the concerted action of the 40 kDa amidotransferase domain (GLN), that hydrolyzes glutamine and the 120 kDa synthetase (CPS) domain that uses the ammonia, thus produced, ATP and bicarbonate to make carbamoyl phosphate. The separately cloned GLN domain has very low activity due to a reduction in kcat and an increase in Km but forms a hybrid complex with the isolated Escherichia coli CPS subunit. The hybrid has full glutamine-dependent catalytic activity and a functional interdomain linkage. The mammalian-E. coli hybrid was used to investigate the functional consequence of replacing His336 and Glu338, two residues postulated to participate in catalysis as part of a catalytic triad. The mutant mammalian GLN domains formed stable complexes with the E. coli CPS subunit, but the catalytic activity was severely impaired. While the His336Asn mutant does not form measurable amounts of the gamma-glutamyl thioester, the steady state concentration of the intermediate with the Glu338Gly mutant was comparable to the wild type hybrid because both the rate of formation and breakdown of the thioester are reduced. This result is consistent with the postulated role of Glu338 in maintaining His336 in the optimal orientation for catalysis and suggests a mechanism for the GLN CPS functional linkage.

Ammonia↗
PubMed1998

Pressure-induced dissociation of carbamoyl-phosphate synthetase domains. The catalytically active form is dimeric.

Carbamoyl-phosphate synthetase consists of an amidotransferase domain or subunit (GLN) that hydrolyzes glutamine and transfers the ammonia to the synthetase component (CPS) where the biosynthetic reaction occurs. The CPS domain is composed of two homologous subdomains, CPS.A and CPS.B, that catalyze different ATP-dependent reactions involved in carbamoyl phosphate synthesis. When the individual CPS.A and CPS.B subdomains were individually cloned and expressed in Escherichia coli (Guy, H. I., and Evans, D. R. (1996) J. Biol. Chem. 271, 13762-13769), they were found to be functionally equivalent and could each independently catalyze carbamoyl phosphate synthesis. The proposal was advanced that, although the monomers could catalyze the individual partial reactions, overall synthesis of carbamoyl phosphate required a homodimer of CPS.A or CPS.B. To test this hypothesis, the GLN-CPS.B dimer was reversibly dissociated at 1500 bar in a high pressure cell. Dissociation was accompanied by a loss of both glutamine- and ammonia-dependent CPSase activity. Activity was recovered once the protein was returned to atmospheric pressure. If the sample was cross-linked before exposure to high pressure, there was no dissociation and no loss of biosynthetic activity. In contrast, the bicarbonate-dependent ATPase and the carbamoyl phosphate-dependent ATP synthetase activities were largely unaffected by pressure-induced dissociation. These experiments confirmed the hypothesis that the synthesis of carbamoyl phosphate requires the concerted action of the two active sites within the homodimer.

Adenosine Triphosphate↗
PubMed1998

Comparison of sperm viability with seminal plasma metal levels.

This study compares the semen levels of lead (Pb), cadmium (Cd), and aluminum (Al) in relation to live sperm in semen samples from 64 apparently healthy men. The measured levels were separated into live sperm count tertiles (<25% [18 subjects], 25-50% [26 subjects], and >50% [20 subjects]). The mean +/- SD for each group was calculated, and the difference between the means of the high and low tertiles were compared by ANOVA. Significant differences were observed between the high and low live sperm groups for Pb (p < 0.01) and Al (p < 0.05), but not Cd. Spearman's rank correlation between sperm viability and the semen plasma metal levels showed a direct relation to Mg (p < 0.05). However, there was an inverse relation to lead (p < 0.001), cadmium (p < 0.01), and aluminum (p < 0.01). There was no significant correlation between Ca and Zn. Linear regression between the live sperm counts and semen level of the three metals show that metal levels were inversely correlated with the percentage of live sperm (p < 0.001, < 0.01). Apparently, the presence of these metals in the environment and in seminal plasma exerts a toxic effect on sperm.

Adult↗
PubMed1998

Amniotic fluid B12 and folate levels associated with neural tube defects.

Amniotic fluid levels of B12 and folate in neural tube defect (NTD) affected pregnancies were compared with the weekly group mean +/-SD changes in amniotic fluid B12 and folate levels of 10 unaffected pregnancies each week between 15 and 20 weeks' gestation age. Comparison was by analysis of variance (ANOVA) and Pearson's correlation to B12 and folate levels and to the NTD samples of corresponding gestation age. Amniotic fluid B12 and folate decreased 67 and 62%, respectively, between 15 and 20 weeks' gestation in the unaffected pregnancies, associated with an increase in amniotic fluid volume. The mean +/-SD B12 and folate of the NTD affected pregnancies (308+/-156 pg/mL and 3.1+/-1.6 ng/mL) were below the mean +/-SD B12 and folate of the total population of unaffected pregnancies (453+/-155 pg/mL and 3.9+/-1.2 ng/mL). The correlation between gestation age and amniotic fluid B12 was -0.9914 (p< or =.0001) and -0.9599 (p< or =.002) for amniotic fluid folate. The B12 levels of the affected pregnancies were below the range of unaffected pregnancies in four of the nine affected pregnancies, and folate levels in two of the nine affected pregnancies.

Amniotic Fluid↗
PubMed1998

The yeast protein complex containing cdc68 and pob3 mediates core-promoter repression through the cdc68 N-terminal domain.

Transcription of nuclear genes usually involves trans-activators, whereas repression is exerted by chromatin. For several genes the transcription mediated by trans-activators and the repression mediated by chromatin depend on the CP complex, a recently described abundant yeast nuclear complex of the Pob3 and Cdc68/Spt16 proteins. We report that the N-terminal third of the Saccharomyces cerevisiae Cdc68 protein is dispensable for gene activation but necessary for the maintenance of chromatin repression. The absence of this 300-residue N-terminal domain also decreases the need for the Swi/Snf chromatin-remodeling complex in transcription and confers an Spt- effect characteristic of chromatin alterations. The repression domain, and indeed the entire Cdc68 protein, is highly conserved, as shown by the sequence of the Cdc68 functional homolog from the yeast Kluyveromyces lactis and by database searches. The repression-defective (truncated) form of Cdc68 is stable but less active at high temperatures, whereas the known point-mutant form of Cdc68, encoded by three independent mutant alleles, alters the N-terminal repression domain and destabilizes the mutant protein.

Adenosine Triphosphatases↗
PubMed1997

The smallest carbamoyl-phosphate synthetase. A single catalytic subdomain catalyzes all three partial reactions.

Escherichia coli carbamoyl-phosphate synthetase (CPSase) is comprised of a 40-kDa glutaminase (GLN) and a 120-kDa synthetase (CPS) subunit. The CPS subunit consists of two homologous domains, CPS.A and CPS.B, which catalyze the two different ATP-dependent partial reactions involved in carbamoyl phosphate synthesis. Sequence similarities and controlled proteolysis experiments suggest that the CPS subdomains consist, in turn, of three subdomains, designated A1, A2, A3 and B1, B2, B3 for CPS.A and CPS.B, respectively. Previous studies of individually cloned CPS.A and CPS. B from E. coli and mammalian CPSase have shown that homologous dimers of either of these "half-molecules" could catalyze all three reactions involved in ammonia-dependent carbamoyl phosphate synthesis. Four smaller recombinant proteins were made for this study as follows: 1) A1-A2 in which the A3 subdomain was deleted from CPS.A, 2) B1-B2 lacking subdomain B3 of CPS.B, 3) the A2 subdomain of CPS.A, and 4) the B2 subdomain of CPS.B. When associated with the GLN subunit, A1-A2 and B1-B2 had both glutamine- and ammonia-dependent CPSase activities comparable to the wild-type protein. In contrast, the 27-kDa A2 and B2 recombinant proteins, which represent only 17% of the mass of the parent protein, were unable to use glutamine as a nitrogen donor, but the ammonia-dependent activity was enhanced 14-16-fold. The hyperactivity suggests that A2 and B2 are the catalytic subdomains and that A1 and B1 are attenuation domains which suppress the intrinsically high activity and are required for the physical association with the GLN subunit.

Animals↗
PubMed1997

Trapping an activated conformation of mammalian carbamyl-phosphate synthetase.

The amidotransferase or glutaminase domain (GLN domain) of mammalian carbamyl-phosphate synthetase II (CPSase II) catalyzes glutamine hydrolysis and transfers ammonia to the synthetase domain (CPS domain), where carbamyl phosphate formation is catalyzed in three consecutive reactions. The GLN and CPS domains are part of a single polypeptide and are connected via a 29-amino acid chain segment (GC linker). In contrast, the two comparable domains of Escherichia coli CPSase are not fused, but are separate, noncovalently associated subunits. To establish the function of the GC linker in mammalian CPSase, it was deleted, and the two domains were directly fused. The deletion mutant not only catalyzed glutamine-dependent carbamyl phosphate synthesis, but was activated 10-fold relative to its wild-type counterpart. However, ammonia-dependent synthesis of carbamyl phosphate was abolished, indicating that ammonia no longer had access to the active site on the CPS domain. The mutant was still sensitive to inhibition by the allosteric effector UTP, but was no longer activated by the allosteric effector phosphoribosyl pyrophosphate, although evidence indicated that the latter could bind to the enzyme. The linker appears to serve as a spacer that allows the complex to cycle between two conformations, an open low activity form in which the ammonia site on the CPS domain is accessible and an activated conformation in which the ammonia generated in situ from glutamine is directly channeled to the CPS active site and access to exogenous ammonia is blocked.

Amino Acid Sequence↗