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M A Pearson

Publications and source records attributed to M A Pearson.

At least 19 recordsLinked to original sources
PubMed2000

Kinetic and structural characterization of urease active site variants.

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar approximately 10(-)(5)-fold deficiencies in rates, modest K(m) changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s.

Aspartic Acid↗
PubMed2000

Structure of the ERM protein moesin reveals the FERM domain fold masked by an extended actin binding tail domain.

The ezrin-radixin-moesin (ERM) protein family link actin filaments of cell surface structures to the plasma membrane, using a C-terminal F-actin binding segment and an N-terminal FERM domain, a common membrane binding module. ERM proteins are regulated by an intramolecular association of the FERM and C-terminal tail domains that masks their binding sites. The crystal structure of a dormant moesin FERM/tail complex reveals that the FERM domain has three compact lobes including an integrated PTB/PH/ EVH1 fold, with the C-terminal segment bound as an extended peptide masking a large surface of the FERM domain. This extended binding mode suggests a novel mechanism for how different signals could produce varying levels of activation. Sequence conservation suggests a similar regulation of the tumor suppressor merlin.

Actins↗
PubMed2000

Floundering about at cell membranes: a structural view of phospholipid signaling.

Structures are now available for the majority of the enzyme families involved in the phosphorylation, dephosphorylation and hydrolysis of signaling phospholipids. Lipid kinase and phosphatase structures recapitulate catalytic motifs involved in protein phosphorylation and dephosphorylation, whereas cytosolic phospholipase A(2) manifests novel catalytic geometry. Structures have been determined for most known intracellular phospholipid 'receptor' domains, both those that bind membrane-embedded phospholipids and those that bind lipid monomers.

Cell Membrane↗
PubMed2000

Accuracy in gamma camera measurements of point source velocities.

Mucociliary clearance is impaired in many diseases of the respiratory system. We have developed a method for measuring tracheal mucus velocity by the dynamic study of a single point source of radioactivity deposited in the trachea by cricothyroid injection. Preliminary results suggest that patients with airways disease have very low tracheal mucus velocities (<2 mm x min(-1)). The aim of this experiment was to explore the ability of current scintillation detection systems to track a single point as it moves in a dynamic study in small increments and at low velocity (movements of the order of 1 mm). Background noise was estimated to contribute an error in positioning of 0.16 mm (1 standard deviation). Overall errors in velocity were estimated at 0.2 mm x min(-1). This suggests that standard instrumentation in use in most nuclear medicine departments has the capacity to measure accurately velocities as low as 1 mm x min(-1).

Gamma Cameras↗
PubMed2000

Autonomic changes during orthostasis: a time-frequency analysis.

The transition between sitting and standing represents a period of dynamic changes in cardiac autonomic control. Previous research using heart rate (HR) has suggested a biphasic HR response from sitting to standing with an initial increase in HR followed by an HR decrease before stabilizing at a level above the sitting level. These HR changes have been interpreted as mediated by sympathetic nervous system (SNS) activity. However, more direct measures of cardiac autonomic control have not been investigated in an active orthostasis paradigm. This is due in part to the requirement of stationarity for traditional spectral analytic techniques. In the present study, time-frequency analysis was used to assess the relative contributions of sympathetic and parasympathetic nervous system activity to the cardiac autonomic control during the transition of orthostasis. We examined the heart rate time series during the transition between sitting and standing, and the 60 s prior and the 60 s following the transition. The high frequency component of the HR spectra (an index of parasympathetic activity) decreased sharply at the point of transition and remained depressed relative to the low frequency component (an index of sympathetic activity). These results suggest that time-frequency analysis may be a useful technique to examine non-stationary time series. Furthermore, the transition of orthostasis may be mediated more via parasympathetic activity than by sympathetic activity.

Adult↗
PubMed1999

Characterization of metal-substituted Klebsiella aerogenes urease.

Urease possesses a dinuclear Ni active site with the protein providing a bridging carbamylated lysine residue as well as an aspartyl and four histidyl ligands. The apoprotein can be activated in vitro by incubation with bicarbonate/CO2 and Ni(II); however, only approximately 15% forms active enzyme (Ni-CO2-ureaseA), with the remainder forming inactive carbamylated Ni-containing protein (Ni-CO2-ureaseB). In the absence of CO2, apoprotein plus Ni(II) forms a distinct inactive Ni-containing species (Ni-urease). The studies described here were carried out to better define the metal-binding sites for the inactive Ni-urease and Ni-CO2-ureaseB species, and to examine the properties of various forms of Co-, Mn-, and Cu-substituted ureases. Xray absorption spectroscopy (XAS) indicated that the two Ni atoms present in the Ni-urease metallocenter are coordinated by an average of two histidines and 3-4 N/O ligands, consistent with binding to the usual enzyme ligands with the lysine carbamate replaced by solvent. Neither XAS nor electronic spectroscopy provided evidence for thiolate ligation in the inactive Ni-containing species. By contrast, comparative studies of Co-CO2-urease and its C319A variant by electronic spectroscopy were consistent with a portion of the two Co being coordinated by Cys319. Whereas the inactive Co-CO2-urease possesses a single histidyl ligand per metal, the species formed using C319A apoprotein more nearly resembles the native metallocenter and exhibits low levels of activity. Activity is also associated with one of two species of Mn-CO2-urease. A crystal structure of the inactive Mn-CO2-urease species shows a metallocenter very similar in structure to that of native urease, but with a disordering of the Asp360 ligand and movement in the Mn-coordinated solvent molecules. Cu(II) was bound to many sites on the protein in addition to the usual metallocenter, but most of the adventitious metal was removed by treatment with EDTA. Cu-treated urease was irreversibly inactivated, even in the C319A variant, and was not further characterized. Metal speciation between Ni, Co, and Mn most affected the higher of two pKa values for urease activity, consistent with this pKa being associated with the metal-bound hydrolytic water molecule. Our results highlight the importance of precisely positioned protein ligands and solvent structure for urease activity.

Amino Acid Substitution↗
PubMed1998

Chemical rescue of Klebsiella aerogenes urease variants lacking the carbamylated-lysine nickel ligand.

Klebsiella aerogenes urease possesses a dinuclear metallocenter in which two nickel atoms are bridged by carbamylated Lys217. To assess whether carbamate-specific chemistry is required for urease activity, site-directed mutagenesis and chemical rescue strategies were combined in efforts to place a carboxylate group at the location of this metal ligand. Urease variants with Lys217 replaced by Glu, Cys, and Ala (K217E, K217C/C319A, and K217A proteins) were purified, shown to be activated by incubation with small organic acids plus Ni(II), and structurally characterized. K217C/C319A urease possessed a second change in which Cys319 was replaced by Ala in order to facilitate efforts to chemically modify Cys217; however, this covalent modification approach did not produce active urease. Chemical rescue of the K217E, K217C/C319A, and K217A variants required 2, 2, and 10 h, respectively, to reach maximal activity levels. The highest activity generated [224 micromol of urea degraded.min-1.(mg of protein)-1, for K217C/C319A urease incubated with 500 mM formic acid and 10 mM Ni at pH 6.5] corresponded to 56% of that measured for in vitro activation of the wild-type apoprotein. While the K217E apoprotein showed minimal structural perturbations, the K217C/C319A apoprotein showed a disordering of some active site residues, and the K217A apoprotein revealed a repositioning of His219 to allow the formation of a hydrogen bond with Thr169, thus replacing the hydrogen bond between the amino group of Lys217 and Thr169 in the native enzyme. Importantly, these structures allow rationalization of the relative rates and yields of chemical rescue experiments. The crystal structures of chemically rescued K217A and K217C/C319A ureases revealed a return of the active site residues to their wild-type positions. In both cases, noncovalently bound formate was structurally equivalent to the Lys-carbamate as the bridging metallocenter ligand. We conclude that carbamate-specific chemistry is not required for urease catalysis.

Alanine↗
PubMed1998

Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A.

The Tyr92-Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfide-intact wild-type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92-Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C[40, 95]A variant, which is an analog of the major rate-determining three-disulfide intermediate in the oxidative folding of RNase A, missing the 40-95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.

Animals↗
PubMed1998

Investigation of the molecular mechanisms underlying growth factor synergy: the role of ERK 2 activation in synergy.

Stem Cell Factor (SCF), the ligand for the c-kit proto-oncogene, has been shown to synergistically interact with other cytokines, enhancing the responsiveness of haemopoietic precursors. In this study we have examined the effects of SCF, in combination with interleukin-3 (IL-3), on FDCP-Mix A4 cells, a murine, multipotent, haemopoietic progenitor cell line. Low concentration of IL-3 act to enhance cell survival but do not stimulate proliferation in A4 cells. Similarly, SCF when added alone, acts as a good survival stimulus, but is a poor proliferative stimulus. However, in combination with low concentrations of IL-3, SCF stimulates a synergistic enhancement of proliferation leading to a large increase in cell number after seven days. The synergy observed was not due to SCF stimulated alterations in the mRNA, protein levels or affinity of the IL-3 receptors. Therefore, interactions between cytokines at the level of cytoplasmic signalling pathways were investigated. IL-3 stimulated the rapid and transient tyrosine phosphorylation of several proteins (including those of molecular weights 130, 110, 100, 95, 80, 65, 50 and 45 kDa). Some of these proteins were identified as the Src Homology Collagen (SHC) protein, Janus kinase (JAK-2) and the Mitogen Activated Protein Kinase isoforms ERK 1 and ERK 2. IL-3 also stimulated a transient increase in the activity of both ERK 1 and 2. SCF stimulated a rapid and transient increase in the tyrosine phosphorylation of ERK 1 and ERK 2 although, coaddition of SCF with IL-3, caused no gross differences in the phosphorylation of SHC, JAK-2 or ERKs compared to those observed with IL-3 alone. Coaddition of SCF and low concentration of IL-3 stimulated a reproducible synergistic increase in the activity of ERK 2, whereas only an additive increase in the activity of ERK 1 was observed. These results suggest that ERK 2 activation represents a point at which the two pathways, stimulated by IL-3 and SCF, interact synergistically.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Structures of Cys319 variants and acetohydroxamate-inhibited Klebsiella aerogenes urease.

Cys319 is located on a mobile flap covering the active site of Klebsiella aerogenes urease but does not play an essential role in catalysis. Four urease variants altered at position C319 range from having high activity (C319A) to no measurable activity (C319Y), indicating Cys is not required at this position, but its presence is highly influential [Martin, P. R., & Hausinger, R. P. (1992) J. Biol. Chem. 267, 20024-20027]. Here, we present 2.0 A resolution crystal structures of C319A, C319S, C319D, and C319Y proteins and the C319A variant inhibited by acetohydroxamic acid. These structures show changes in the hydration of the active site nickel ions and in the position and flexibility of the active site flap. The C319Y protein exhibits an alternate conformation of the flap, explaining its lack of activity. The changes in hydration and conformation suggest that there are suboptimal protein-solvent and protein-protein interactions in the empty urease active site which contribute to urease catalysis. Specifically, we hypothesize that the suboptimal interactions may provide a significant source of substrate binding energy, and such hidden energy may be a common phenomenon for enzymes that contain mobile active site loops and undergo an induced fit. The acetohydroxamic acid-bound structure reveals a chelate interaction similar to those seen in other metalloenzymes and in a small molecule nickel complex. The inhibitor binding mode supports the proposed mode of urea binding. We complement these structural studies with extended functional studies of C319A urease to show that it has enhanced stability and resistance to inhibition by buffers containing nickel ions. The near wild-type activity and enhanced stability of the C319A variant make it useful for further studies of urease structure-function relationships.

Amino Acid Sequence↗
PubMed1997

Heart rate variability during exercise in the horse.

High heart rate variability (HRV) has been associated with more efficient autonomic control, allowing more responsivity and sensitivity to changing environmental demands. Previous results from spectral analysis of interbeat intervals of equine heart rate (HR) indicated increased HRV, reflecting more effective vagal control of the heart. This study focuses on characterizing equine HR and HRV during increasing then decreasing physical demand. A non-invasive ambulatory monitoring system continuously records HR and HRV as horses exercise on a high speed equine treadmill at increasing then decreasing workloads. After a 5 minute baseline, the horses walk, then trot, then canter or trot fast, return to the slower trot, then walk, and then remain quiet for a 5 minute recovery period. Results indicate a decrease of HRV and large individual differences in parasympathetic activity during exercise. Resulting patterns of HR fluctuation indicate a nonlinear dynamical approach may describe the task responses more completely than the more traditional models.

Animals↗
PubMed1996

Characterization of the mononickel metallocenter in H134A mutant urease.

A mutant form of Klebsiella aerogenes urease possessing Ala instead of His at position 134 (H134A) is inactive and binds approximately half the normal complement of nickel (Park, I.-S., and Hausinger, R. P.(1993) Protein Sci. 2, 1034-1041). The crystal structure of the H134A protein was obtained at 2.0-A resolution, and it confirms that only Ni-1 of the two nickel ions found in the native enzyme is present. In contrast to the pseudotetrahedral geometry observed for Ni-1 in native urease (where it is liganded by His-246, His-272, one oxygen atom of carbamylated Lys-217, and a water molecule at partial occupancy), the mononickel metallocenter in the H134A protein was found to possess octahedral geometry and was coordinated by the above protein ligands plus three water molecules. The nickel site of H134A urease was probed by UV-visible, variable temperature magnetic circular dichroism, and x-ray absorption spectroscopies. The spectroscopic data are consistent with the presence of Ni(II) in octahedral geometry coordinated by two histidylimidazoles and additional oxygen and/or nitrogen donors. These data underscore the requirement of Ni-2 for formation of active urease and demonstrate the important role of Ni-2 in establishing the proper Ni-1 coordination geometry.

Binding Sites↗
PubMed1996

An instructional model for primary health care education.

Primary health care (PHC) offers nurses a framework for community-based practice in a restructured health care system. This study examined a Nursing in Primary Health Care course model. Self-directed learning readiness among baccalaureate nursing students enrolled in the course was examined in relation to evaluation of course content and instructional methods. The majority of students responded positively to the course methods and significant differences in course evaluations were found to be relative to self-directed learning readiness. The results suggest the Nursing in Primary health care course model effectively introduces students to PHC.

Adult↗
PubMed1995

Macrophage inflammatory protein-1 alpha mediated growth inhibition in a haemopoietic stem cell line is associated with inositol 1,4,5 triphosphate generation.

Macrophage Inflammatory Protein-1 alpha (MIP-1 alpha) can inhibit the proliferation of multipotent haemopoietic cells. Using the FDCP-Mix A4 multipotent stem cell line, MIP-1 alpha was shown to inhibit 1L-3 stimulated cell cycling (assessed using the [3H]-thymidine "suicide" assay). Furthermore, MIP-1 alpha can inhibit 1L-3-stimulated [3H]-thymidine incorporation in FDCP-Mix cells, with half maximal inhibition observed at 3 ng/ml MIP-1 alpha. Prostaglandin E2, but not MIP-1 alpha was able to elevate cyclic AMP levels in FDCP-Mix A4 cells although both agents can cause growth inhibition. However, MIP-1 alpha addition resulted in a pertussis-toxin-insensitive increase in the level of the second messenger inositol 1,4,5 triphosphate (Ins 1,4,5P3). This response was both rapid (maximal at 5 seconds) and transient. A half maximal effect was observed at 5 ng/ml MIP-1 alpha and the dose dependency correlated with that for MIP-1 alpha mediated growth inhibition. A rapid increase in cytosolic Ca2+ levels was also observed in response to MIP-1 alpha. Inositol lipid hydrolysis and an increase in cytosolic Ca2+ (signals normally associated with proliferation) may therefore be implicated in growth inhibitory mechanisms in multipotent cells.

Animals↗
PubMed1994

The calpain cleavage sites in the epidermal growth factor receptor kinase domain.

The proteolysis of the human epidermal growth factor receptor cytoplasmic domain by calpain has been studied in vitro using purified recombinant cytoplasmic domain expressed in insect cells. Limited proteolysis produced kinase that was truncated at either N- or C-termini, as well as in the hinge region. We identified seven sites of calpain proteolysis by N-terminal sequencing of purified fragments. Calpain cleaved between the catalytic and autophosphorylation domains at two sites in the sequence Gln996-Asp1059, in the hinge region. Three new sites were also found in the autophosphorylation domain, preceding each of the major autophosphorylation sites. A fourth new site was located in the juxta-membrane domain, C-terminal to the regulatory Thr654. We purified an active 42-kDa fragment generated by calpain proteolysis between Leu659-Gln660 in the juxta-membrane domain, and in the hinge region. A fifth new site of calpain cleavage was found between the nucleotide binding motif Gly-Xaa-Gly-Xaa-Xaa-Gly and the essential Lys721 in the catalytic core of the kinase. Since both of these features are required for catalysis, calpain cleavage at this site may potentially provide a mechanism for down-regulation of kinase activity in vivo, under conditions of calpain activation. Thus the distribution of calpain cleavage sites along the kinase domain is consistent with a role for calpain both as a processing and as a degradative protease in epidermal growth factor receptor signalling.

Amino Acid Sequence↗
PubMed1994

Respite for caregivers: an evaluation study.

This evaluation study measured the effect of respite on family caregivers of the elderly. The specific aims were: (a) to learn the effect of respite on the evaluation variables of caregiver quality of life, mood, and response to caregiving; (b) to learn relations between the demographic variables and the evaluation variables; and (c) to examine respite to identify benefits other than those being directly measured. Subjects were 130 caregivers, with 6- and 12-month data on a small subsample of the 130. Interviews with caregivers and satisfaction surveys supported respite as an effective intervention. Quantitative analysis showed that none of the evaluation variables changed statistically. The caregiver mood variable evidenced positive changes; other variables changed negatively. Research issues, such as the short time subjects were in the respite program and the lack of sensitive measurement tools, are discussed. Clinical implications for community health nurses are also discussed.

Adult↗
PubMed1994

Toluene embryopathy: delineation of the phenotype and comparison with fetal alcohol syndrome.

OBJECTIVE: To determine if maternal toluene abuse produces any structural or developmental disabilities in the developing fetus, a cohort of toluene-exposed infants was ascertained and examined. METHODOLOGY: Eighteen infants with a history of in utero toluene exposure were examined at birth. Nine of these infants were reexamined 3 to 36 months after their initial evaluations. The clinical findings in these patients were compared with those of similarly exposed children from the literature and with patients who had the fetal alcohol syndrome. RESULTS: Thirty-nine percent of all toluene-exposed infants described in this and other studies were born prematurely, and 9% died during the perinatal period. Fifty-four percent were small for gestational age, and 52% exhibited continued postnatal growth deficiency. A 33% incidence of prenatal microcephaly, a 67% incidence of postnatal microcephaly, and an 80% incidence of developmental delay were observed. Eighty-three percent of the patients had craniofacial features similar to the fetal alcohol syndrome, and 89% of these children had other minor anomalies. CONCLUSIONS: Data from the patients herein described and the available scientific literature suggest that the mechanism of alcohol craniofacial teratogenesis may be nonspecific, with a variety of teratogens, including toluene, giving rise to phenotypic facial abnormalities similar to those of the fetal alcohol syndrome. We propose a common mechanism of craniofacial teratogenesis for toluene and alcohol, namely a deficiency of craniofacial neuroepithelium and mesodermal components due to increased embryonic cell death.

Abnormalities, Drug-Induced↗