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M Stewart

Publications and source records attributed to M Stewart.

At least 109 records · Page 6Linked to original sources
PubMed2000

Insights into the molecular mechanism of nuclear trafficking using nuclear transport factor 2 (NTF2).

Nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. The simplicity and specialization of this system, combined with the availability of crystal structures of NTF2, RanGDP and their complex, has facilitated the investigation of the molecular mechanism of its trafficking. NTF2 binds to both RanGDP and FxFG repeat-containing nucleoporins. Mutants engineered on the basis of structural information together with determination of binding constants have been used to dissect the roles of these interactions in transport. Thus, NTF2 binds to RanGDP sufficiently strongly for the complex to remain intact during transport through NPCs, but the interaction between NTF2 and FxFG nucleoporins is much more transient, which would enable NTF2 to move through the NPC by hopping from one repeat to another. An analogous nucleoporin hopping mechanism may also be used by carrier molecules of the importin-beta family to move through NPCs.

Active Transport, Cell Nucleus↗
PubMed2000

Lupus nephritis outcomes: health maintenance organizations compared to non-health maintenance organizations.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) in USA increasingly have their care centered in health maintenance organizations (HMO). We examined whether HMO and non-HMO SLE patients with renal involvement, who had the same university rheumatologist, differed in their utilization of health care or renal outcomes. METHODS: Consecutive patients with SLE with renal involvement (n = 24), 10 enrolled in an HMO, 14 not, were studied. Laboratory values were prospectively determined. RESULTS: At the first visit to the rheumatologist, there was no significant difference between HMO and non-HMO patients in laboratory values. There was no difference in the 2 groups in the final prednisone dose (HMO 12 mg vs non-HMO 15.9 mg) or use of azathioprine (20% vs. 57%; p = 0.07) or cyclophosphamide (60% vs. 57%). The serum creatinine was higher in the HMO patients (HMO 1.1 mg/dl vs. non-HMO 0.78 mg/dl; p = 0.05). No difference was found in the number of rheumatology visits. There was a significant difference in the number of communications from the rheumatologist to the HMO versus the HMO to the rheumatologist (p = 0.026). CONCLUSION: Other than the serum creatinine, there are no differences in the treatment or renal outcomes for HMO compared to non-HMO patients with SLE seeing the same rheumatologist. There is a potential barrier in physician communication, however, with the majority of communications going from the rheumatologist to the HMO provider.

Adult↗
PubMed2000

Fibrinogen, fibrin and crosslinking in aging arterial thrombi.

The assumption that fibrin and crosslinked fibrin impart irreversibility to arterial thrombi is explored with procedure developed for measuring changes in platelet function, morphology and fibrinogen metabolism in aging occlusive thrombi, in which the condition of stasis is imposed uniformly. Arterial thrombi containing autologous (111)In labeled platelets were generated in vivo by bilateral mechanical injury of porcine carotid arteries. Vessels containing the platelet-rich thrombi were harvested and incubated intact (37 degrees C) for intervals ranging from 30 min to 12 h. The isolated vessels were then bisected and agitated in culture medium containing tick anticoagulant and hirudin for 60 min. Disaggregated platelets were evaluated for yield (from (111)In radioactivity) viability (dense body ATP secretion) and morphology (electron microscopy). Western analysis of fibrin(ogen) in thrombus extracts was performed using anti-fibrinogen Bbeta- and gamma-chain monoclonal antibodies for thrombi at each time point. A stable recovery of nearly 50% of platelets was observed during 12 h of thrombus aging. As thrombi aged, viability of disaggregated platelets gradually decreased with platelet necrosis the predominant feature beyond 6 h. By western analysis of thrombus extracts, nearly 50% of fibrinogen was cleaved to fibrin and extensively crosslinked within 30 min of injury with no evidence of fibrinolysis. With the exception of a declining proportion of gamma-monomer, these features remain relatively constant during 12 h of thrombus maturation. It is concluded that neither fibrin nor crosslinked fibrin are dominant factors imparting cohesion within platelet thrombi. Furthermore, under conditions of complete arterial occlusion imposed by this experimental design, there is no evidence of endogenous fibrinolysis.

Adenosine Triphosphate↗
PubMed2000

The impact of patient-centered care on outcomes.

BACKGROUND: We designed this observational cohort study to assess the association between patient-centered communication in primary care visits and subsequent health and medical care utilization. METHODS: We selected 39 family physicians at random, and 315 of their patients participated. Office visits were audiotaped and scored for patient-centered communication. In addition, patients were asked for their perceptions of the patient-centeredness of the visit. The outcomes were: (1) patients' health, assessed by a visual analogue scale on symptom discomfort and concern; (2) self-report of health, using the Medical Outcomes Study Short Form-36; and (3) medical care utilization variables of diagnostic tests, referrals, and visits to the family physician, assessed by chart review. The 2 measures of patient-centeredness were correlated with the outcomes of visits, adjusting for the clustering of patients by physician and controlling for confounding variables. RESULTS: Patient-centered communication was correlated with the patients' perceptions of finding common ground. In addition, positive perceptions (both the total score and the subscore on finding common ground) were associated with better recovery from their discomfort and concern, better emotional health 2 months later, and fewer diagnostic tests and referrals. CONCLUSIONS: Patient-centered communication influences patients' health through perceptions that their visit was patient centered, and especially through perceptions that common ground was achieved with the physician. Patient-centered practice improved health status and increased the efficiency of care by reducing diagnostic tests and referrals.

Adult↗
PubMed2000

Exploring information technology adoption by family physicians: survey instrument valuation.

As the information needs of family physicians become more complex, there will be a greater need to successfully implement the technologies needed to manage that information. The ability to stratify primary care physicians can enable the implementation process to be more efficient. This research tested a new instrument on 101 family physicians, and was able to stratify physicians into high, intermediate, and low information technology (IT) usage groups. It is expected that this stratification would allow managers of IT implementation to target specific adoption strategies for each group. The instrument is available from ddixon@julian.uwo.ca.

Analysis of Variance↗
PubMed2000

Patients' attitudes to comforting touch in family practice.

OBJECTIVE: To examine patients' attitudes to comforting touch in family practice. DESIGN: A survey was designed with statements and responses to proposed scenarios. SETTING: Twenty family practices throughout Ontario. PARTICIPANTS: Family practice patients; of 400 surveys distributed, 376 were completed (94% response rate). MAIN OUTCOME MEASURES: Patients responded to scenarios on a five-point Likert scale, ranging from strongly disagree to strongly agree. Results were analyzed using SPSS for DOS. RESULTS: Most patients in this population believed that touch can be comforting (66.3%) and healing (57.9%). Women were more accepting of comforting touch than men in all scenarios. Acceptance of comforting touch declined for both sexes as touch became proximal and more intimate. Men and women were more accepting of comforting touch from female doctors. Acceptance of all comforting touch declined markedly if a physician was unfamiliar to a patient, regardless of the physician's sex. CONCLUSION: Most patients surveyed believed touch is comforting and healing and viewed distal touches (on the hand and shoulder) as comforting.

Adolescent↗
PubMed1999

Re-entrant activity in a presubiculum-subiculum circuit generates epileptiform activity in vitro.

The retrohippocampal cortices form the transition between neocortex and the hippocampus. Area CA3 of the hippocampus and the entorhinal cortex (EC) of the retrohippocampal region are established as brain regions that generate epileptiform activity. Interictal activity generated in EC consists of a primary population burst followed by multiple afterdischarges. The presubiculum is similar to EC in its six-layered structure, but lacks a columnar circuitry that the EC possesses. Isolated presubicular tissue cannot generate afterdischarges and isolated subicular tissue generates no spontaneous activity under some conditions. We report epileptiform activity in combined presubiculum-subiculum slices that consists of synchronous population bursts and multiple afterdischarges. Intracellular and field potential recordings reveal two re-entrant paths for interaction of presubicular and subicular neurons. We demonstrate a deep presubicular input to subiculum and separate return paths from subicular bursting neurons onto deep and superficial layer pre-/parasubicular neurons. Recordings from subicular cell apical dendrites showed repetitive burst firing during sustained depolarizing current injection. We conclude that re-entrant activity in a presubiculum-subiculum circuit generates epileptiform activity in both regions. Presubicular inputs to subiculum depolarize apical dendrites which can then burst repetitively. These bursts are transmitted back to the presubiculum. We suggest that iterations on this circuit act to prolong the dendritic depolarization of subicular bursting neurons and to entrain the activity across subicular cells resulting in multiple afterdischarges.

Animals↗
PubMed1999

A full-length Cbfa1 gene product perturbs T-cell development and promotes lymphomagenesis in synergy with myc.

The Cbfa1/PEBP2 alpha A/AML3 gene plays an essential role in osteogenesis but is also expressed in the T-cell lineage where it has been implicated in lymphoma development as a target for retroviral insertional mutagenesis. As lymphoma cells with til-1 insertion express at least five distinct Cbfa1 isoforms, it is important to establish which, if any, have intrinsic oncogenic potential. We have generated transgenic mice in which the most abundant lymphoma isoform (G1/p57) is expressed under the control of the CD2 locus control region. Co-precipitation analysis of transgenic thymus revealed high levels of Cbfa1 protein in an abundant complex containing the binding cofactor Cbfb. CD2-Cbfa1-G1 mice displayed abnormal T-cell development, with a pronounced skew towards CD8 SP cells in the thymus and developed a low incidence of spontaneous lymphomas (6% at 12 months) with cells of similar phenotype. Strongly synergistic tumour development was seen when CD2-Cbfa1-G1 mice were crossed with lines carrying myc transgenes (CD2-myc or tamoxifen-regulatable CD2-mycER) and Cbfa1 was found to rescue expression of the CD2-myc transgene in pre-leukaemic mice. However, synergy did not appear to be due to a dominant block of myc-induced apoptosis by Cbfa1 as explanted primary tumours and cell lines from CD2-Cbfa1-G1/CD2-mycER mice showed accelerated death on induction with tamoxifen at similar rates to CD2-mycER controls. Moreover, thymocytes from preleukaemic CD2-Cbfa1-G1 mice showed reduced survival in vitro and increased sensitivity to the inhibitory effects of TGF-beta. This study demonstrates that a full-length Cbf alpha-chain gene can act as an oncogene without fusion to a heterologous protein.

Animals↗
PubMed1999

Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP.

Nuclear transport factor 2 (NTF2) is a small, homodimeric protein that binds to both RanGDP and xFxFG repeat-containing nucleoporins, such as yeast Nsp1p and vertebrate p62. NTF2 is required for efficient nuclear protein import and has been shown to mediate the nuclear import of RanGDP. We have used the crystal structures of rat NTF2 and its complex with RanGDP to design a mutant, W7A-NTF2, in which the affinity for xFxFG-repeat nucleoporins is reduced while wild-type binding to RanGDP is retained. The 2.5 A resolution crystal structure of W7A-NTF2 is virtually superimposable upon the wild-type protein structure, indicating that the mutation had not introduced a more general conformational change. Therefore, our data suggest that the exposed side-chain of residue 7 is crucial to the interaction between NTF2 and xFxFG repeat-containing nucleoporins. Consistent with its reduced affinity for xFxFG nucleoporins, fluorescently labelled W7A-NTF2 binds less strongly to the nuclear envelope of permeabilized cultured cells than wild-type NTF2 and, when microinjected into Xenopus oocytes, colloidal gold coated with W7A-NTF2 binds less strongly to the central channel of nuclear pore complexes than wild-type NTF2-coated gold. Significantly, W7A-NTF2 only weakly stimulated the nuclear import of fluorescein-labelled RanGDP, providing direct evidence that an interaction between NTF2 and xFxFG repeat-containing nucleoporins is required to mediate the nuclear import of RanGDP.

Animals↗
PubMed1999

Localized depolymerization of the major sperm protein cytoskeleton correlates with the forward movement of the cell body in the amoeboid movement of nematode sperm.

The major sperm protein (MSP)-based amoeboid motility of Ascaris suum sperm requires coordinated lamellipodial protrusion and cell body retraction. In these cells, protrusion and retraction are tightly coupled to the assembly and disassembly of the cytoskeleton at opposite ends of the lamellipodium. Although polymerization along the leading edge appears to drive protrusion, the behavior of sperm tethered to the substrate showed that an additional force is required to pull the cell body forward. To examine the mechanism of cell body movement, we used pH to uncouple cytoskeletal polymerization and depolymerization. In sperm treated with pH 6.75 buffer, protrusion of the leading edge slowed dramatically while both cytoskeletal disassembly at the base of the lamellipodium and cell body retraction continued. At pH 6.35, the cytoskeleton pulled away from the leading edge and receded through the lamellipodium as its disassembly at the cell body continued. The cytoskeleton disassembled rapidly and completely in cells treated at pH 5.5, but reformed when the cells were washed with physiological buffer. Cytoskeletal reassembly occurred at the lamellipodial margin and caused membrane protrusion, but the cell body did not move until the cytoskeleton was rebuilt and depolymerization resumed. These results indicate that cell body retraction is mediated by tension in the cytoskeleton, correlated with MSP depolymerization at the base of the lamellipodium.

Actin Cytoskeleton↗
PubMed1999

Molecular architecture of the rod domain of the Dictyostelium gelation factor (ABP120).

The Dictyostelium discoideum gelation factor is a two-chain actin-cross-linking protein that, in addition to an N-terminal actin-binding domain, has a rod domain constructed from six tandem repeats of a 100-residue motif that has an immunoglobulin fold. To define the architecture of the rod domain of gelation factor, we have expressed in E. coli a series of constructs corresponding to different numbers of gelation factor rod repeats and have characterised them by chemical crosslinking, ultracentrifugation, column chromatography, matrix-assisted laser desorption ionisation (MALDI) mass spectrometry and NMR spectroscopy. Fragments corresponding to repeats 1-6 and 5-6 dimerise, whereas repeats 1-5 and single repeats 3 and 4 are monomeric. Repeat 6 interacts weakly and was present as monomer and dimer when analysed by analytical ultracentrifugation. Proteolytic digestion of rod5-6 resulted in the generation of two polypeptides that roughly corresponded to rod5 and part of rod6. None of these polypeptides formed dimers after chemical crosslinking. Stable dimerisation therefore appears to require repeats 5 and 6. Based on these data a model of gelation factor architecture is presented. We suggest an arrangement of the chains where only the carboxy-terminal repeats interact as was observed for filamin/ABP280, the mammalian homologue of gelation factor.

Animals↗
PubMed1999

Engineered mutants in the switch II loop of Ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import.

Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction. X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran. The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2. Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p.

Animals↗
PubMed1999

Columnar activity supports propagation of population bursts in slices of rat entorhinal cortex.

Population bursts including epileptiform spikes and sharp waves can be generated in and propagate through the retrohippocampal cortices. The propagation of these events within the entorhinal cortex was studied with field potential recordings in horizontal slices from rat brain. Population bursts were elicited by repetitive extracellular stimuli in normal media. Transections of particular laminae and microknife cuts parallel to the pial surface were used to interrupt propagation or to isolate potential pathways. Population spikes were not found to propagate more than 0.5 mm in either superficial (layers I-III) or deep (layers V-VI) strips of tissue formed by cuts along layer IV. Events propagated over 2 mm when 'columnar' connectivity was intact. Population spikes propagated past microknife cuts which started at the pial surface and sectioned layers I-IV. Population spikes also propagated past microknife cuts which sectioned the angular bundle and layers V-VI. When deep layer cuts included layer IV, horizontal propagation of population spikes was blocked. Cuts running approximately in layer IV diminished the amplitude and duration of population events recorded in layers II and V (above and below the cut, respectively) and eliminated a high frequency oscillation which occurred during the burst event. It is concluded that superficial and deep layer neurons interact during propagation of population events in entorhinal cortex and that this 'columnar' activity: (a) intensifies the excitatory activity of superficial and deep layer neurons; and (b) provides multiple paths for the spread of activity from column to column within the entorhinal cortex.

Action Potentials↗
PubMed1999

Quantifying GPIIb/IIIa receptor binding using 2 monoclonal antibodies: discriminating abciximab and small molecular weight antagonists.

BACKGROUND: Dosing of glycoprotein (GP) IIb/IIIa receptor antagonists is frequently based on the inhibition of platelet aggregation, which may be influenced by the agonist used or concurrent medications. Here we describe a monoclonal antibody-based technique to quantify total and ligand-occupied GPIIb/IIIa receptors. METHODS AND RESULTS: In vitro binding of monoclonal antibodies, LYP18 (Mab1) and 4F8 (Mab2), to the GPIIb/IIIa complex, was characterized using purified receptor and to platelets by flow cytometry. Patients undergoing coronary angioplasty received a single 20 mg dose of the oral GPIIb/IIIa antagonist, xemilofiban, or matching placebo, and antibody binding was compared with inhibition of platelet aggregation. Mab1 and Mab2 were bound to purified GPIIb/IIIa and to unoccupied, inactivated receptor on platelets. Mab2 identified the beta3 subunit, whereas Mab1 was complex-specific. Neither antibody interfered with the other's binding, suggesting that they identified distinct sites. Mab1 identified 53 300+/-5423 GPIIb/IIIa sites per platelet, whereas Mab2 identified 50 120+/-5066 sites per platelet. Mab1 binding was inhibited by abciximab in a dose dependent manner (IC50, 0.85+/-0.1 microg/mL), whereas Mab2 binding was unaffected. In contrast, the 2 small molecular weight antagonists, SC-57101A (IC50, 0.22+/-0.06 micromol/L) and eptifibatide (IC50, 0.35+/-0.14 micromol/L) inhibited Mab2 but not Mab1 binding. In patients treated with xemilofiban, Mab1 binding was unaltered but Mab2 binding decreased from 37 930+/-2061 sites per platelet at baseline to 8318+/-870 sites per platelet 6 hours after dosing (P<0.0001). Platelet aggregation to adenosine diphosphate (20 micromol/L) fell to 3+/-3% of baseline in line with the inhibition of Mab2 binding (correlation coefficient 0.8, P<0.0001). CONCLUSIONS: Mab1 and Mab2 bind to GPIIb/IIIa and are differentially displaced by abciximab and small molecular weight antagonists. These antibodies may be used to monitor receptor number and occupancy during administration of a GPIIb/IIIa antagonist.

Abciximab↗
PubMed1999

Characterisation of the 5-HT receptor binding profile of eletriptan and kinetics of [3H]eletriptan binding at human 5-HT1B and 5-HT1D receptors.

The affinity of eletriptan ((R)-3-(1-methyl-2-pyrrolidinylmethyl)-5-[2-(phenylsulphonyl )ethyl]-1H-indole) for a range of 5-HT receptors was compared to values obtained for other 5-HT1B/1D receptor agonists known to be effective in the treatment of migraine. Eletriptan, like sumatriptan, zolmitriptan, naratriptan and rizatriptan had highest affinity for the human 5-HT1B, 5-HT1D and putative 5-ht1f receptor. Kinetic studies comparing the binding of [3H]eletriptan and [3H]sumatriptan to the human recombinant 5-HT1B and 5-HT1D receptors expressed in HeLa cells revealed that both radioligands bound with high specificity (>90%) and reached equilibrium within 10-15 min. However, [3H]eletriptan had over 6-fold higher affinity than [3H]sumatriptan at the 5-HT1D receptor (K(D)): 0.92 and 6.58 nM, respectively) and over 3-fold higher affinity than [3H]sumatriptan at the 5-HT1B receptor (K(D): 3.14 and 11.07 nM, respectively). Association and dissociation rates for both radioligands could only be accurately determined at the 5-HT1D receptor and then only at 4 degrees C. At this temperature, [3H]eletriptan had a significantly (P<0.05) faster association rate (K(on) 0.249 min(-1) nM(-1)) than [3H]sumatriptan (K(on) 0.024 min(-1) nM(-1)) and a significantly (P<0.05) slower off-rate (K(off) 0.027 min(-1) compared to 0.037 min(-1) for [3H]sumatriptan). These data indicate that eletriptan is a potent ligand at the human 5-HT1B, 5-HT1D, and 5-ht1f receptors and are consistent with its potent vasoconstrictor activity and use as a drug for the acute treatment of migraine headache.

Animals↗
PubMed1999

Swallowing abnormalities after acute stroke: A case control study.

Dysphagia is a common and potentially fatal complication of acute stroke. However, the underlying pathophysiology, especially the relative importance of motor and sensory dysfunction, remains controversial. We conducted a case control study of 23 acute stroke patients (mean age = 72 yr) at a median of 6 days post-stroke and 15 healthy controls (mean age = 76 yr). We used novel methods to assess swallowing in detail, including a timed videoendoscopic swallow study and oral sensory threshold testing using electrical stimulation. Vocal cord mobility and voluntary pharyngeal motor activity were impaired in the stroke group compared with the controls (p = 0.01 and 0.03). There was a delay during swallowing in the time to onset of epliglottic tilt in the stroke group, particularly for semisolids (p = 0.02) and solids (p = 0.01), consistent with a delay in initiation of the swallow. Sensory thresholds were not increased in the stroke group compared with controls. We conclude that pharyngeal motor dysfunction and a delay in swallow initiation are common after acute stroke. Vocal cord mobility is reduced, and this may result in reduced airway protection. We found no evidence to support the hypothesis that oropharyngeal sensory dysfunction is common after acute stroke.

Acute Disease↗
PubMed1999

The fit-1 common integration locus in human and mouse is closely linked to MYB.

The fit-1 locus was originally identified as a common insertion site for feline leukemia virus (FeLV) in thymic lymphosarcomas induced by FeLV-myc recombinant viruses, suggesting that it harbors a gene that cooperates with Myc in T-cell leukemogenesis. We have previously mapped the fit-1 locus to feline Chromosome (Chr) B2. We have now identified conserved sequences that allow the mapping of the murine homolog using the European Interspecific Backcross (EUCIB). This shows that fit-1 is located on mouse Chr 10, 1cM proximal to Ahi-1, a murine retroviral integration locus that is closely linked to Myb. Moreover, the physical linkage to MYB is maintained in the human genome, as shown by cloning of the human homolog of fit-1 from a Chr 6 cosmid library and a series of overlapping PAC clones. Generation of a contig map around the human homolog of fit-1 reveals that it is approximately 100-kb upstream of MYB. In addition to fit-1 and Ahi-1, two other common insertion sites, Mis-2 and Mml-1, have also been mapped adjacent to Myb on mouse Chr 10. Previous analysis of tumors carrying insertions at fit-1, Mml-1, Mis-2 and Ahi-1 showed no obvious abnormalities in Myb expression. However, the cluster of viral insertion loci in this region suggests either the presence of a closely linked activation target or that subtle effects on Myb have been overlooked.

Animals↗