The teaching of rheumatology in a graduate entry medical school.
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Curriculum↗
Biomedical subjects
M Smith
Publications and source records attributed to M Smith.
Characterization of hemoglobin variants by MALDI-TOF MS using a polyurethane membrane as the sample support.
A new method for the sampling and off-site analysis of hemoglobin variants by mass spectrometry is reported. This technique uses a nonporous polyurethane membrane as the collection device and transportation medium of a blood sample for analysis. The same membrane is then used as the MALDI-TOF MS sample support for mass spectrometric analysis. Minimal invasive sample collection is afforded by collecting less than 1 microL of blood using a common lancet device. MALDI-TOF MS is performed directly on the membrane, after washing off the interfering plasma components, followed by the addition of matrix. This reduces the time of analysis and prevents sample loss. Enzymatic digestion can be performed directly on the membrane, using in this case trypsin, allowing for further characterization of the sample. The method is much less invasive compared to drawing blood with a syringe. The sample may be transported to the laboratory by regular mail, and thus the method can serve remote locations. We demonstrate the procedure by characterizing the Hb Shepherds Bush hemoglobin variant, b74-(E18)Gly-->Asp.
Genetic Variation↗
Retrieving semen from a dead patient.
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Brain Death↗
A product of the Drosophila stoned locus regulates neurotransmitter release.
The Drosophila stoned locus encodes two novel gene products termed stonedA and stonedB, which possess sequence motifs shared by proteins involved in intracellular vesicle traffic. A specific requirement for stoned in the synaptic vesicle cycle has been suggested by synthetic genetic interactions between stoned and shibire, a gene essential for synaptic vesicle recycling (Petrovich et al., 1993). A synaptic role of stoned gene products also is suggested by altered synaptic transients in electroretinograms recorded from stoned mutant eyes (Petrovich et al., 1993). We show here that the stonedA protein is highly enriched at Drosophila nerve terminals. Mutant alleles that affect stonedA disrupt the normal regulation of synaptic vesicle exocytosis at neuromuscular synapses of Drosophila. Spontaneous neurotransmitter release is enhanced dramatically, and evoked release is reduced substantially in such stoned mutants. Ultrastructural studies reveal no evidence of major disorganization at stoned mutant nerve terminals. Thus, our data indicate a direct role for stonedA in regulating synaptic vesicle exocytosis. However, genetic and morphological observations suggest additional, subtle effects of stoned mutations on synaptic vesicle recycling. Remarkably, almost all phenotypes of stoned mutants are similar to those previously described for mutants of synaptotagmin, a protein postulated to regulate both exocytosis and the recycling of synaptic vesicles. We propose a model in which stonedA functions together with synaptotagmin to regulate synaptic vesicle cycling.
Amino Acid Substitution↗
In vitro evolution of horse heart myoglobin to increase peroxidase activity.
Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k1 (for H2O2 oxidation of metmyoglobin) of 1. 34 x 10(4) M-1 s-1 ( approximately 25-fold that of wild-type myoglobin) and k3 [for reducing the substrate (2, 2'-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 x 10(6) M-1 s-1 (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the Tm of the quadruple variant is decreased only slightly compared with wild-type (74.1 degreesC vs. 76.5 degreesC). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (kon, 22.2 x 10(-6) M-1 s-1 vs. 22.3 x 10(-6) M-1 s-1; koff, 24.3 s-1 vs. 24.2 s-1; KO2, 0.91 x 10(-6) M-1 vs. 0.92 x 10(-6) M-1). The affinity of the quadruple variant for CO is increased slightly (kon, 0.90 x 10(-6) M-1s-1 vs. 0.51 x 10(-6) M-1s-1; koff, 5.08 s-1 vs. 3.51 s-1; KCO, 1.77 x 10(-7) M-1 vs. 1.45 x 10(-7) M-1). All four substitutions are in the heme pocket and within 5 A of the heme group.
Amino Acid Substitution↗
Times they are a' changin'
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Journal Article↗
Nitric oxide donor-induced hyperpermeability of cultured intestinal epithelial monolayers: role of superoxide radical, hydroxyl radical, and peroxynitrite.
Many of the cytopathic effects of nitric oxide (NO*) are mediated by peroxynitrite (PN), a product of the reaction between NO* and superoxide radical (O2*-). In the present study, we investigated the role of PN, O2*- and hydroxyl radical (OH*) as mediators of epithelial hyperpermeability induced by the NO* donor, S-nitroso-N-acetylpenicillamine (SNAP), and the PN generator, 3-morpholinosydnonimine (SIN-1). Caco-2BBe enterocytic monolayers were grown on permeable supports in bicameral chambers. Epithelial permeability, measured as the apical-to-basolateral flux of fluorescein disulfonic acid, increased after 24 h of incubation with 5.0 mM SNAP or SIN-1. Addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO* scavenger, or Tiron, an O2*- scavenger, reduced the increase in permeability induced by both donor compounds. The SNAP-induced increase in permeability was prevented by allopurinol, an inhibitor of xanthine oxidase (a source of endogenous O2*-). Diethyldithiocarbamate, a superoxide dismutase inhibitor, and pyrogallol, an O2* generator, potentiated the increase in permeability induced by SNAP. Addition of the PN scavengers deferoxamine, urate, or glutathione, or the OH* scavenger mannitol, attenuated the increase in permeability induced by both SNAP and SIN-1. Both donor compounds decreased intracellular levels of glutathione and protein-bound sulfhydryl groups, suggesting the generation of a potent oxidant. These results support a role for PN, and possibly OH*, in the pathogenesis of NO* donor-induced intestinal epithelial hyperpermeability.
Antioxidants↗
Capillary patency after transient middle cerebral artery occlusion of 2 h duration.
Reperfusion after transient focal ischemia of 2 h duration is followed by secondary bioenergetic failure after 4 h of reperfusion. The objective of the present study was to explore whether or not this secondary deterioration is due to secondary microcirculatory compromise. Normal fasted rats were subjected to 2 h of MCA occlusion and allowed reperfusion for 2, 4, 6 and 8 h. At predetermined reperfusion times, rats were injected with Evans blue and decapitated. Capillary patency was determined using a fluorescent double-staining technique. No capillary perfusion deficits were detected in the ischemic neocortical penumbra, neocortical focus or striatal focus. We concluded that the secondary deterioration of bioenergetic state is not due to microcirculatory compromise. Since hyperglycemic animals show pan-necrotic lesions, a hyperglycemic group was added at 8 h of reperfusion to test if the adverse effect of hyperglycemia on ischemic damage is related to capillary compromise. The results showed that, in hyperglycemic rats, capillary perfusion in the striatal focus was compromised after 8 h of recirculation following 2 h of MCA occlusion. It is concluded that when normoglycemic rats are subjected to 2 h of MCA occlusion, capillary patency is not affected during the first 4-6 h of reflow. At 8 h of reflow, though, particularly in hyperglycemic rats, microcirculation is compromised in the caudoputamenal focus, probably reflecting infarction.
Animals↗
Heart period variability and depressive symptoms: gender differences.
BACKGROUND: Deceased heart period variability has been associated with cardiac events and depressive symptoms; however, the results of studies are not unequivocal. We hypothesize that gender differences in the various study samples may have contributed to the lack of uniformity of findings. METHODS: Time and frequency domain measures of heart period variability during a 5-min resting baseline were examined in 15 depressed (8 female) and 11 nondepressed (6 female) college students. RESULTS: Both time and frequency domain measures indicated an interaction between gender and depressive symptoms, such that depressed male subjects showed less heart period variability, whereas depressed female subjects showed more heart period variability compared to their nondepressed counterparts. CONCLUSIONS: These results suggest that future studies investigating the relationship between heart period variability and depressive symptoms examine gender differences.
Adult↗
The effect of apotransferrin on iron release from Caco-2 cells, an intestinal epithelial cell line.
The Caco-2 cell line grown in bicameral chambers was used to study the effect of transferrin in the basal chamber on the transepithelial transport of iron. We have shown that when iron was offered as 59Fe on the apical surface of the Caco-2 cells, transport of 59Fe into the basal chamber was stimulated by 50 micromol/L apotransferrin. Here, we examined the effect on 59Fe transport of lower concentrations of apotransferrin, as well as the effects on transport of ovo-, cobalt-, and ferri-transferrin and of iron chelators with an affinity for iron greater than that of transferrin. The stimulation of 59Fe transport was more sensitive to the presence of apotransferrin with a Km of 0.078 +/- 0.008 micromol/L compared with ferri-transferrin with a Km of 1.24 +/- 0.39 micromol/L (P < .006). 59Fe transport was less sensitive to diethylenetriaminopenta-acetic acid (DTPA) than apotransferrin with Kms of 1.52 +/- 0.70. The chelator nitrilotriacetic acid (NTA) exhibited no stimulation of 59Fe transport. Analysis of laser scanning confocal micrographs showed that apotransferrin labeled with Texas Red is internalized by Caco-2 cells from the basal side and localizes in distinct vesicles above the nucleus. The sensitivity of apotransferrin in stimulating Fe transport suggests a unique interaction of apotransferrin with the basal surface of the intestinal epithelium.
Animals↗
The knowledge industry
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Journal Article↗
Early glycoxidation damage in brains from Down's syndrome.
In Down's syndrome, the presence of three copies of chromosome 21 is associated with premature aging and progressive mental retardation sharing the pathological features of Alzheimer disease. Early cortical dysgenesis and late neuronal degeneration are probably caused by an overproduction of amyloid beta-peptide, followed by an increased cellular oxidation. Interestingly, chromosome 21 codes for superoxide-dismutase and amyloid beta precursor resulting, in Down's syndrome, in an overflow of these gene products and metabolites. We studied Down's fetal brain cortex to evaluate the presence and amount of lipid and protein oxidation markers; moreover, we quantified two forms of glycation end products that are known to be involved in the process of cellular oxidation. All these parameters are significantly increased in Down's fetal brains in comparison to controls, providing the evidence that accelerated brain glycoxidation occurs very early in the life of Down's syndrome subjects.
Aldehydes↗
Intracerebroventricular prostaglandin administration increases the neural damage evoked by global hemispheric hypoxic ischemia.
This study was designed to determine if central (intracerebroventricular, i.c.v.) administration of prostaglandin E2 (PGE2, mediator of core temperature elevation following exogenous or endogenous pyrogen administration) worsens the neural damage of anesthetized rats to global hemispheric hypoxic-ischemia (GHHI) from damage seen in normothermic, i.c.v. saline control groups. The first study (no GHHI) showed that 10 or 50 ng PGE2 given i.c.v. to groups of anesthetized Long-Evans rats evoked dose-related increases in colonic (systemic core) temperature but no neural damage. In the second study anesthetized rats were given an i.c.v. injection of sterile saline or PGE2 plus GHHI (ligation of the right common carotid artery plus 35 min of 12% O2) at the peak of the temperature response. Thermal response indices (TRI, degrees C x min), determined from brain (temporalis muscle, ipsilateral and contralateral to ligation) and core (colonic) temperatures, showed significant increases in the 50-ng PGE2 group compared to the TRIs of the 10-ng PGE2 or saline control group. The 50-ng PGE2, GHHI group had a higher mortality rate and showed greater ipsilateral hemispheric neural damage than the saline-treated group given the same insult, especially due to increased damage to the cortex. The results show that i.c.v. PGE2 administration significantly increases the neural damage caused by GHHI, possibly due to the associated rise in core temperature.
Animals↗
Herbal remedies in psychiatric practice.
Patients' use of alternative and complementary health services has created a need for physicians to become informed about the current literature regarding these treatments. Herbal remedies may be encountered in psychiatric practice when they are used to treat psychiatric symptoms; produce changes in mood, thinking, or behavior as a side effect; or interact with psychiatric medications. English-language articles and translated abstracts or articles (where available) found on MEDLINE and sources from the alternative/complementary health field were reviewed. Each herb was assessed for its safety, side effects, drug interactions, and efficacy in treating target symptoms or diagnoses. A synopsis of the information available for each herb is presented. In many cases the quantity and quality of data were insufficient to make definitive conclusions about efficacy or safety. However, there was good evidence for the efficacy of St John's wort for the treatment of depression and for ginkgo in the treatment of memory impairment caused by dementia. More research is required for most of the herbs reviewed, but the information published to date is still of clinical interest in diagnosing, counseling, and treating patients who may be taking botanical remedies.
Anti-Anxiety Agents↗
Genetic monitoring of malathion-exposed agricultural workers.
The aerial application of malathion over large urban populations in Southern California during the early 1990s raised concerns about adverse health effects, including the potential to cause genetic damage. Workers in the Mediterranean fruit fly eradication program, which involved application of malathion as ground treatment, were studied to examine micronucleus formation and mutation frequencies assessed by the glycophorin A (GPA) assay. In the 1992 pilot project the mean micronuclei level appeared higher in lymphocytes of exposed workers (n = 13) compared to controls (n = 4) (20.1 +/- 7.1 vs 14.3 +/- 7.2 respectively, P = 0.09). During the 1993 season, neither of the cohorts examined showed a higher level of micronuclei in workers exposed to malathion compared to unexposed, nor did the pooled total (n = 53; means = 17.8 +/- 7.2 vs 18.5 +/- 6.3, respectively), even after adjustment by multiple regression. The GPA variant frequency was not associated with malathion exposure in any of the cohorts. These results suggest that any potential risk of genotoxic damage from exposure to malathion is relatively low, but other assays may be more sensitive, and the sample size was small.
Adult↗
Outcomes following acute hospital care for stroke or hip fracture: how useful is an assessment of anxiety or depression for older people?
OBJECTIVE: To investigate the association between severe life events and mental health outcomes following acute hospital care for older patients with acute stroke or fractured neck of femur. DESIGN: Prospective longitudinal survey of stroke and hip fracture patients admitted to hospital from admission to 6-month follow-up. SETTING: Six district general hospitals, three in the North and three in the South of England. PARTICIPANTS: 642 patients admitted to hospital with an acute stroke (268) or hip fracture (374) resident in a private household at 6 months follow-up. MAIN OUTCOME MEASURES: Hospital Anxiety and Depression Scale, cognitive items of the Survey Psychiatric Assessment Scale, Clackmannan Disability Scale, Severe Life Events Inventory, Wenger Social Support Network Typology. RESULTS: 47% of 6-month survivors of stroke or hip fracture resident in private households had a possible psychiatric illness: dementia (13%), anxiety or depression (41%). 57% had severe or very severe disability and 48% experienced additional life events (17% two or more) after hospital admission. Severe disability was strongly associated with a higher prevalence of anxiety (p < 0.0005) or depression (p < 0.0001). Social contact was associated with a lower prevalence of anxiety (p < 0.01) or depression (p < 0.0001) and social support network type was strongly associated with depression (p < 0.001) but not anxiety (p = 0.096). Number of severe life events was associated with anxiety (p < 0.001) but not depression (p = 0.058). CONCLUSION: Disability is probably a more robust outcome measure than assessments of mental health for older people in uncontrolled studies.
Aged↗
Isotopically labeled bovine beta-lactoglobulin for NMR studies expressed in Pichia pastoris.
beta-Lactoglobulin (beta-Lg) is the major whey protein in ruminant milk and has been implicated in the irreversible denaturation of milk proteins and its associated poor processing behavior during heat treatment. In order to help understand this behavior, as well as to facilitate other studies into the relationship between the molecular structure and its behavior in solution, we have prepared and purified 15N-labeled and 13C/15N-double-labelled beta-Lg in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The overexpression of the labeled protein using the Pichia pastoris system proceeds with good yield but requires the removal of significant quantities of copurifying carbohydrate which otherwise interfere with the NMR experiments. At pH 2, the resulting material gives triple resonance NMR spectra of good quality that are consistent with a monomeric, globular protein rich in beta-sheet.
Amino Acid Sequence↗
2-year tracking of children's fruit and vegetable intake.
OBJECTIVE: To evaluate the tracking of children's fruit and vegetable intake over 2 years. DESIGN: Children in the control group from the Gimme 5 nutrition education intervention trial completed three 7-day food diaries once a year in 1994, 1995, and 1996, beginning when they were in third grade. SUBJECTS: Five hundred sixty-one students from public elementary schools. STATISTICAL ANALYSES PERFORMED: Tracking was examined through correlation of fruit and vegetable servings over the 2 years as well as the percent of subjects remaining in the same or adjacent quintiles of fruit and vegetable intake, using the kappa statistic. RESULTS: Tracking of fruit and vegetable intake was fair to moderate over the 2 years. One-year correlations for total fruit and vegetable servings were .37 for boys and .46 for girls; 2-year correlations were .48 for boys and .40 for girls. kappa values for remaining in the same quintile generally exceeded chance rates only for the top and bottom quintiles. No values exceeded .50. APPLICATION/CONCLUSION: The drift in ranking may have been a result of true changes in fruit and vegetable intake or measurement error (i.e., inability to accurately assess actual intake). If this drift reflects true change, it may be difficult for nutrition education programs targeting this age group to demonstrate long-term behavioral effects on fruit and vegetable intake. If the drift is the result of measurement limitations, improved dietary assessment may yield stronger tracking.
Child↗