Determination of metabolite and regulatory enzyme levels in Dirofilaria immitis and Ascaris suum: a comparative study.

Studies on metabolite levels in Dirofilaria immitis revealed similarities in several metabolites with those of Ascaris suum. The glycogen level in the filariid was however 3-4 times lower than that in A. suum. Levels of three regulatory enzymes were also determined in D. immitis and compared with those in A. suum. The activities of Hexokinase and Phosphofructokinase were similar. However, the levels of Glycogen phosphorylase b appeared to be much lower in the filariid than in A. suum. The subtle but important differences observed may reflect modifications of the parasite enzymes suggesting salient differences in the regulation of energy production from carbohydrates in the worms. The differences may also represent specialization required for the unique life style of the worms in their different locations in their hosts.

Kinetic characterization of a T-state of Ascaris suum phosphofructokinase with heterotropic negative cooperativity by ATP eliminated.

The affinity analogue, 2',3'-dialdehyde ATP has been used to chemically modify the ATP-inhibitory site of Ascaris suum phosphofructokinase, thereby locking the enzyme into a less active T-state. This enzyme form has a maximum velocity that is 10% that of the native enzyme in the direction of fructose 6-phosphate (F6P) phosphorylation. The enzyme displays sigmoid saturation for the substrate fructose 6-phosphate (S0.5 (F6P) = 19 mM and nH = 2.2) at pH 6.8 and a hyperbolic saturation curve for MgATP with a Km identical to that for the native enzyme. The allosteric effectors, fructose 2,6-bisphosphate and AMP, do not affect the S0.5 for F6P but produce a slight (1.5- and 2-fold, respectively) V-type activation with Ka values (effector concentration required for half-maximal activation) of 0.40 and 0.24 mM, respectively. Their activating effects are additive and not synergistic. The kinetic mechanism for the modified enzyme is steady-state-ordered with MgATP as the first substrate and MgADP as the last product to be released from the enzyme surface. The decrease in V and V/K values for the reactants likely results from a decrease in the equilibrium constant for the isomerization of the E:MgATP binary complex, thus favoring an unisomerized form. The V and V/KF6P are pH dependent with similar pK values of about 7 on the acid side and 9.8 on the basic side. The microenvironment of the active site appears to be affected minimally as evidenced by the similarity of the pK values for the groups involved in the binding site for F6P in the modified and native enzymes.

Infertility and early parent-infant interactions.

Approximately 50% of infertile couples will become parents through pregnancy or adoption, but they experience major difficulties while working towards this goal. Infertility treatments are associated with physical pain and psychological distress, and adoption procedures are prolonged and emotionally stressful. The extent to which these stressors alter the parenting of these couples is not known. The purpose of this study, therefore, was to examine the early parent-infant interactions in infertile couples who become parents through pregnancy or adoption. Two groups of infertile couples (30 who achieved pregnancy and 21 who adopted) and a group of 19 couples without fertility problems were observed interacting with their infants twice, 7 to 21 days after the infant's arrival and a week later, at a time when both parents were at home. Their babies were between 9 days and 5 months of age. Behaviours of the mother, father and infant were recorded every 10 seconds, beginning when the baby was picked up and ending when the baby was put down asleep or 1 1/2 hours had passed. Repeated measures ANOVAs were used to compare the three groups over the observations. There were no differences between fertile and infertile biological parents. Adopted infants showed more alertness, less sleeping, more smiles, and more looking than biological infants. Adoptive mothers spent less time as the sole interactor. Adoptive parents spent more time in playing with their infants and held and touched them less than did biological parents. Infertility, therefore, does not appear to affect early parenting. In general, the amounts of behaviours exhibited by infertile biological parents were very close to those of fertile parents. Differences in the behaviours of adoptive as compared to biological parents can best be explained as responses to the behaviours of their older infants, rather than as evidence of different parenting styles.

Expression, purification, and characterization of the recombinant NAD-malic enzyme from Ascaris suum.

The cDNA encoding the 65-kDa subunit of malic enzyme from Ascaris suum was cloned into the bacterial expression vector pKK223-3 and overproduced in Escherichia coli. A protein with a subunit molecular mass of 65,000 was expressed at a level of up to 3% of the total soluble protein in JM109, as judged by SDS-PAGE. The enzyme was purified using column chromatography on phenyl-Sepharose followed by orange-A agarose. The purification procedure resulted in a 32-fold purification with an overall yield of 51%. The bacterially expressed enzyme exhibits kinetic constants identical to those measured for native A. suum NAD-malic enzyme.

Expression of Ascaris suum malic enzyme in a mutant Escherichia coli allows production of succinic acid from glucose.

The malic enzyme gene of Ascaris suum, was cloned into the vector pTRC99a in two forms encoding alternative amino-termini. The resulting plasmids, pMEA1 and pMEA2, were introduced into Escherichia coli NZN111, a strain that is unable to grow fermentatively because of inactivation of the genes encoding pyruvate dissimilation. Induction of pMEA1, which encodes the native animoterminus, gave better overexpression of malic enzyme, approx 12-fold compared to uninduced cells. Under the appropriate culture conditions, expression of malic enzyme allowed the fermentative dissimilation of glucose by NZN111. The major fermentation product formed in induced cultures was succinic acid.

Methods and outcomes of breastfeeding instruction for nursing students.

Support from nurses can influence breastfeeding rates, but many nurses are not well-informed about breastfeeding topics. Surveys were used to assess the breastfeeding instruction provided in five nursing programs. Most students attended breastfeeding lectures, but only one-fourth received breastfeeding information during clinical activities. After completing their maternity rotation, less than 25 percent had as many as three clinical opportunities to teach breastfeeding techniques or counsel about lactation problems. Completion of maternity rotation did not improve student's knowledge of breastfeeding health benefits or clinical advice. Previous personal breastfeeding experience was associated with more accurate clinical advice and rating breastfeeding instruction as inadequate. We conclude that nursing education may not prepare students for effective breastfeeding promotion, and we suggest solutions for lactation consultants.

Isotope partitioning with Ascaris suum phosphofructokinase is consistent with an ordered kinetic mechanism.

Isotope partitioning and initial velocity studies have been used to study the kinetic mechanism of Ascaris suum phosphofructokinase (PFK) at pH 8.0 for the native enzyme (nPFK), and at pH 6.8 for a form of enzyme desensitized (dPFK) to hysteresis in the reaction time course, to ATP allosteric inhibition, and to F6P homotropic cooperativity. Complete trapping (P*max approximately equal to 100%) of the E:MgATP* complex as fructose (1-32P)-1, 6-bisophosphate for both enzyme forms is consistent with the previously proposed steady-state ordered mechanism [Rao, G.S.J., Harris, B.G., & Cook, P.F. (1987) J.Biol. Chem. 262, 14074-14079] with MgATP binding before fructose 6-phosphate (F6P). K'F6P values for trapping of MgATP of 0.54 +/- 0.09 mM for nPFK and 0.85 +/- 0.15 mM for dPFK were obtained. Saturating amounts of the heterotropic activator fructose 2, 6-bisphosphate (F26P2) gives no change in the trapping parameters for nPFK with a P*max of 100% and a K'F6P of 0.40 +/- 0.06 mM. For dPFK, however, F26P2 causes a decrease in both parameters, giving a P*max of 54% and a K'F6P of 0.26 +/- 0.07 mM. The partial trapping of E:MgATP* in the presence of F26P2 for dPFK suggests that the activator changes the kinetic mechanism from an ordered to a random binding of substrates. Initial velocity studies confirm the change in mechanism. Uncompetitive inhibition by arabinose 5-phosphate (Ara5P), a dead-end inhibitory analog of F6P, versus MgATP for nPFK in the absence and presence of F26P2 is consistent with an ordered mechanism with MgATP adding to enzyme prior to F6P. An uncompetitive pattern is also obtained with dPFK for Ara5P versus MgATP in the absence of F26P2, but the pattern becomes noncompetitive in the presence of F26P2, consistent with a change to a random mechanism. No trapping of the E:[14C]F6P complex could be detected, indicating either that the E:[14C]F6P complex does not form in a significant amount under the conditions used or that the off-rate for F6P from enzyme is much faster than the net rate constant for formation of the first product, FBP. The data are consistent with a predominantly ordered mechanism with MgATP binding prior to F6P. The minor pathway with MgATP dissociating from the E:F6P:MgATP ternary complex becomes apparent for the dPFK in the presence of F26P2.

Comparison of the substrate specificities of cAMP-dependent protein kinase from bovine heart and Ascaris suum muscle.

The catalytic subunits of cAMP-dependent protein kinases (protein kinase A) from bovine heart and Ascaris suum muscle exhibit only 48% sequence identity and show quantitative differences in substrate specificity. These differences were not obvious at the level of short synthetic substrate peptides but were distinct for some protein substrates. Phosphofructokinase from Ascaris, a physiological substrate, was a better substrate for the protein kinase from the nematode in comparison to the mammalian protein kinase due to a 10-fold lower Michaelis constant. Selective phosphorylation by the two kinases was also observed with some in vitro substrates. In addition, quantitative differences in the interactions between R- and C-subunits from Ascaris and bovine heart were observed. However, several synthetic peptides whose sequence reflected the phosphorylation site of Ascaris suum phosphofructokinase (AKGRSDS*IV), or variations of it, were phosphorylated with the same efficiency by both protein kinases. Based on the data the following are concluded: (1) In agreement with the conservation of structure in the catalytic cleft, the recognition of substrates by protein kinases from phylogenetically distant organisms exhibits similarity. (2) Non-conserved parts of the surface of the protein kinase molecule may contribute to binding of protein substrates and thus to selective recognition.

Fertility status and symptoms in childbearing couples.

The symptoms of 58 pregnant couples--37 with a history of infertility and 21 without a history of infertility--were compared. The Symptomatology Inventory, a checklist of 42 common physical and psychological symptoms of pregnancy, was completed by each spouse from months 4 to 9 of pregnancy. For purposes of analysis, the individual symptoms were grouped into three categories: physical symptoms, negative affective symptoms, and positive affective symptoms. Although the infertile pregnant couples did not experience more symptoms than fertile couples, their pattern of reporting pregnancy-related symptoms was quite different. In terms of both number and type of symptoms, infertile spouses' symptoms tended to be positively related. Compared to fertile couples, the infertile couples experienced symptoms globally and were more consistent in the number of symptoms reported by each spouse. Additional research is needed to confirm these findings and to determine the implications of these differences for childbearing and the martial relationship.

A pH-dependent allosteric transition in Ascaris suum phosphofructokinase distinct from that observed with fructose 2,6-bisphosphate.

Ascaris suum phosphofructokinase exhibits dramatic shifts in its circular dichroic spectra in the pH range 6 to 8. These shifts are quite distinct from those induced by the activators AMP and fructose 2,6-bisphosphate. Concomitant with these pH-induced spectral shifts, the enzyme also displays changes in its allosteric behavior. Inorganic ions such as K+, NH+4, SO4(2-), and PO4(3-) also cause CD-spectral shifts similar to those produced by a change in pH. Based on the evidence derived from gel filtration and sedimentation equilibrium studies, the observed CD-spectral shifts are interpreted as due to conformational changes in the enzyme tetramer rather than due to a change in its aggregation state. Further, since the pK value of 6.4 obtained from pH dependence of increase in ellipticity at 210 nm agrees very well with the pK value of 6.8 for the loss of ATP inhibition due to modification of a histidine residue (G. S. J. Rao, B. A. Wariso, P. F. Cook, and B. G. Harris (1987) J. Biol. Chem. 262, 14068-14073), it is concluded that a single histidine residue in the ATP-inhibitory site acts as a trigger for the structural changes accompanying ATP inhibition of the enzyme. This view is strongly supported by the observation that the enzyme desensitized to ATP inhibition by chemical modification of a histidine residue in the ATP-inhibitory site shows absolutely no change in its CD spectrum in the pH range 6 to 8. This study demonstrates that the mechanism of activation of phosphofructokinase at higher pH and by inorganic ions involves conformational transitions that are quite distinct from those induced by AMP and fructose 2,6-bisphosphate. A scheme is presented that incorporates all of the different states of the enzyme dependent upon effectors and pH.

Comparison of pregnancy symptoms of infertile and fertile couples.

This study was undertaken to describe the most common symptoms experienced during pregnancy by couples with a history of infertility and to compare them with symptoms of expectant couples without a history of reproductive problems. The Symptomatology Inventory, a 42-item checklist of common pregnancy symptoms, was used. The 10 most frequent symptoms reported and their rank order were very similar for the women from both groups. Men from the two groups frequently reported similar symptoms, but differed on their rank order. This research provides evidence that in terms of pregnancy symptoms infertile and fertile couples are more alike than they are different.

Acid-base catalytic mechanism and pH dependence of fructose 2,6-bisphosphate activation of the Ascaris suum phosphofructokinase.

A form of phosphofructokinase (PFK) from Ascaris suum desensitized to hysteresis in the reaction time course and ATP allosteric inhibition has been used to study the activation by fructose 2,6-bisphosphate (F26P2) at varied pH in both reaction directions. In the direction of phosphorylation of F6P, V and V/KMgATP are constant over the pH range 6-9, while V/KF6P decreases at low pH, giving a pK value of 7.0, and at high pH, giving a pK of 8.9. V and V/KMgATP are insensitive to the presence of F26P2, but V/KF6P is increased by a constant amount in the presence of saturating F26P2 over the entire pH range studied. The concentration of F26P2 that gives half the change in V/KF6P, Kact, increases as the pH decreases, giving a pK of 7.4, reflecting an enzyme group that must be unprotonated for optimum binding of F26P2. In the direction of phosphorylation of MgADP, V and V/KMgADP are pH-independent, and both are insensitive to the presence of F26P2. V/KFBP decreases at high pH, giving a pK of about 7.3, and is increased by a constant amount in the presence of F26P2 over the entire pH range studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Beyond couvade: pregnancy symptoms in couples with a history of infertility.

Thirty-six couples with a history of infertility reported their physical, positive emotional, and negative emotional symptoms during pregnancy. The women experienced more physical symptoms than did the men. Both the men and women experienced second-trimester decreases in negative emotional symptoms and third-trimester increases in negative emotional symptoms. The women's physical symptoms also showed a second-trimester dip. Each symptom type was correlated for husbands and wives, but only 6 husbands showed evidence of couvade syndrome by exhibiting exact correspondence with their wives' symptoms. Symptom attunement appears to be a better term than couvade for most infertile men's experiences of pregnancy.

Pre-steady-state kinetics reveal a slow isomerization of the enzyme-NAD complex in the NAD-malic enzyme reaction.

Stopped-flow experiments obtained in the pre-steady-state time scale of the NAD-malic enzyme reaction exhibit a lag prior to the attainment of steady state. Previous results from isotope effect studies in which the deuterium isotope effect on Vmax decreases to a value of 1 at low pH have been interpreted as suggesting a slow release of NADH [Kiick, D. M., Harris, B. G., & Cook, P. F. (1986) Biochemistry 25, 227-236]. The latter, however, requires a burst in the pre-steady-state time course, and thus the previous data have been reinterpreted in view of the observed lag. Preincubation with NAD and/or Mg increases the lag rate, with the latter having the greater effect, while preincubation with Mg and malate (or a malate analog) eliminates the lag. Data suggest a slow isomerization of E:NAD that is increased by addition of malate prior to NAD in the presence of Mg. The lag is also eliminated at low pH as a result of the overall rate being limited by the isomerization; that is, the isomerization is pH-dependent. Fumarate, an activator of the NAD-malic enzyme, when preincubated with enzyme also eliminates the lag, suggesting that the activator preferentially binds the isomerized form of the enzyme or increases the isomerization rate, or both. Stopped-flow data are corroborated by circular dichroism experiments. The unliganded enzyme is approximately 50% alpha-helix on the basis of secondary structural analysis. Binding of NAD and Mg exhibits a substantial change, with a further change observed upon binding the malate analog tartronate.

Cloning and nucleotide sequence of a full-length cDNA encoding Ascaris suum malic enzyme.

The nucleotide sequence of a full-length cDNA encoding NAD(+)-malic enzyme from the parasitic nematode Ascaris suum was determined. The entire sequence of 2269 bases comprises a 5'-leader, a single open reading frame of 1851 bases, and the complete 3'-noncoding region of 340 bases. The first 12 amino acids of the translated sequence are hydrophobic, typical of mitochondrial translocation signals, and do not appear in the purified mature protein. The mature protein contains 605 amino acids and has a molecular mass of 68,478 Da. The amino acid sequences of tryptic peptides from the purified protein and also the N-terminal sequence show excellent correspondence with the translated nucleotide sequence. Comparison of the amino acid sequence of the ascarid protein with the human and rat liver NAD(+)-malic enzymes reveals highly conserved regions interrupted with long stretches of lesser homologous sequences. Structural motifs such as the putative nucleotide binding domains and also the malate binding site are clearly identified by alignment of the three protein sequences.

Mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate.

The mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate has been probed using initial velocity studies, deuterium isotope effects, and isotope partitioning of the E:Mg:malate complex. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:NAD:Mg:malate complexes. Fumarate is a positive heterotropic effector of the NAD-malic enzyme at low concentrations (K act approximately 0.05 mM) and an inhibitor competitive against malate (Ki approximately 25 mM). The activation by fumarate results in a decrease in the Ki malate and an increase in V/K malate of about 2-fold, while the maximum velocity remains constant. Isotope partitioning studies of E:Mg:[14C]malate indicate that the presence of fumarate results in a decrease in the malate off-rate constant by about 2.2-fold. The deuterium isotope effects on V and V/K malate are both 1.6 +/- 0.1 in the absence of fumarate, while in the presence of 0.5 mM fumarate DV is 1.6 +/- 0.1 and D(V/K malate) is 1.1 +/- 0.1. These data are also consistent with a decrease in the off-rate for malate from E:NAD:Mg:malate, resulting in an increase in the forward commitment factor for malate and manifested as a lower value for D(V/K malate). There is a discrimination between active and activator sites for the binding of dicarboxylic acids, with the activator site preferring the extended configuration of 4-carbon dicarboxylic acids, while the active site prefers a configuration in which the 4-carboxyl is twisted out of the C1-C3 plane. The physiologic importance and regulatory properties of fumarate in the parasite are also discussed.

Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum.

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.

Modification of the ATP inhibitory site of the Ascaris suum phosphofructokinase results in the stabilization of an inactive T state.

Treatment of the Ascaris suum phosphofructokinase (PFK) with 2',3'-dialdehyde ATP (oATP) results in an enzyme form that is inactive. The conformational integrity of the active site, however, is preserved, suggesting that oATP modification locks the PFK into an inactive T state that cannot be activated. A rapid, irreversible first-order inactivation of the PFK is observed in the presence of oATP. The rate of inactivation is saturable and gives a KoATP of 1.07 +/- 0.27 mM. Complete protection against inactivation is afforded by high concentrations of ATP, and the dependence of the inactivation rate on the concentration of ATP gives a Ki of 326 +/- 26 microM for ATP which is 22-fold higher than the Km for ATP at the catalytic site but close to the binding constant for ATP to the inhibitory site. Fructose 6-phosphate, fructose 2,6-bisphosphate, and AMP provide only partial protection against modification. The pH dependence of the inactivation rate gives a pKa of 8.4 +/- 0.1. Approximately 2 mol of [3H]oATP is incorporated into a subunit of PFK concomitant with 90% loss of activity, and ATP prevents the derivatization of 1 mol/subunit. The oATP-modified enzyme is not activated by AMP or fructose 2,6-bisphosphate. oATP has no effect on the activity of a desensitized form of PFK in which the ATP inhibitory site is modified with diethyl pyrocarbonate but with the active site intact [Rao, G.S.J., Wariso, B.A., Cook, P.F., Hofer, H.W., & Harris, B.G. (1987) J. Biol. Chem. 262, 14068-14073].(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction.

Deuterium isotope effects and 13C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the 13C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed [Hermes, J. D., Roeske, C. A., O'Leary, M. H., & Cleland, W. W. (1982) Biochemistry 21, 5106]. When dinucleotide substrates such as thio-NAD, 3-acetylpyridine adenine dinucleotide, and 3-pyridinealdehyde adenine dinucleotide that contain modified nicotinamide rings are used, the 13C effect increases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the 13C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a beta-secondary 13C isotope effect accompanies hydride transfer as a result of hyperconjugation of the beta-carboxyl of malate as the transition state for the hydride transfer step is approached.(ABSTRACT TRUNCATED AT 250 WORDS)