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Biomedical subjects

M J Brown

Publications and source records attributed to M J Brown.

At least 37 records · Page 2Linked to original sources
PubMed2001

Rigidity of circulating lymphocytes is primarily conferred by vimentin intermediate filaments.

Lymphocytes need rigidity while in circulation, but must abruptly become deformable to undergo transmigration into tissue. Previously, the control of leukocyte deformability has been attributed to microfilaments or microtubules, but the present studies demonstrate the greater importance of vimentin intermediate filaments (IFs). In circulating T lymphocytes, IFs form a distinctive spherical cage that undergoes a rapid condensation into a juxtanuclear aggregate during chemokine-induced polarization. Measurements of the resistance of peripheral blood T lymphocytes to global deformation demonstrate that their rigidity is primarily dependent on intact vimentin filaments. Microtubules, in contrast, are not sufficient to maintain rigidity. Thus, vimentin IFs are a primary source of structural support in circulating human lymphocytes, and their regulated collapse is likely to be an essential element in chemokine-induced transendothelial migration.

Animals↗
PubMed2001

Acute axonal neuropathy in maple syrup urine disease.

A 25-year-old woman with maple syrup urine disease (MSUD) developed generalized weakness over 1 week. She had severe leg and moderate arm weakness, areflexia, and distal sensory loss. Plasma branched-chain amino acid concentrations were elevated, reflecting an acute exacerbation of the disease. Electrodiagnostic studies indicated an acute axonal polyneuropathy and sural nerve biopsy revealed acute wallerian degeneration without inflammation. Peripheral neuropathy, although not identified previously as a clinical feature of MSUD, may become more common as chronic dietary restrictions and improved management of the disease allow survival into adulthood.

Acute Disease↗
PubMed2001

The structure of phosphorylated GSK-3beta complexed with a peptide, FRATtide, that inhibits beta-catenin phosphorylation.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) sequentially phosphorylates four serine residues on glycogen synthase (GS), in the sequence SxxxSxxxSxxx-SxxxS(p), by recognizing and phosphorylating the first serine in the sequence motif SxxxS(P) (where S(p) represents a phosphoserine). FRATtide (a peptide derived from a GSK-3 binding protein) binds to GSK-3 and blocks GSK-3 from interacting with Axin. This inhibits the Axin-dependent phosphorylation of beta-catenin by GSK-3. RESULTS: Structures of uncomplexed Tyr216 phosphorylated GSK-3beta and of its complex with a peptide and a sulfate ion both show the activation loop adopting a conformation similar to that in the phosphorylated and active forms of the related kinases CDK2 and ERK2. The sulfate ion, adjacent to Val214 on the activation loop, represents the binding site for the phosphoserine residue on 'primed' substrates. The peptide FRATtide forms a helix-turn-helix motif in binding to the C-terminal lobe of the kinase domain; the FRATtide binding site is close to, but does not obstruct, the substrate binding channel of GSK-3. FRATtide (and FRAT1) does not inhibit the activity of GSK-3 toward GS. CONCLUSIONS: The Axin binding site on GSK-3 presumably overlaps with that for FRATtide; its proximity to the active site explains how Axin may act as a scaffold protein promoting beta-catenin phosphorylation. Tyrosine 216 phosphorylation can induce an active conformation in the activation loop. Pre-phosphorylated substrate peptides can be modeled into the active site of the enzyme, with the P1 residue occupying a pocket partially formed by phosphotyrosine 216 and the P4 phosphoserine occupying the 'primed' binding site.

Amino Acid Motifs↗
PubMed2001

Dietary obesity in the rat induces endothelial dysfunction without causing insulin resistance: a possible role for triacylglycerols.

Impaired arterial vasorelaxation, due primarily to endothelial dysfunction, is associated with obesity. To clarify the relationship with insulin resistance and other metabolic disturbances, we studied endothelial-dependent and -independent vascular responses in rats with dietary-induced obesity. Dietary-obese rats had significantly higher body weights (10-32%; P<0.001) and fat-pad masses (220-280%; P<0.001) than lean controls, together with raised plasma levels of triacylglycerols (15-80%; P<0.001), non-esterified fatty acids (13-38%; P<0.05) and leptin (85-180%; P<0.001). However, measures of insulin sensitivity (including the hyperinsulinaemic-euglycaemic clamp in a parallel experiment) were comparable with those in controls. Contractions induced in mesenteric arteries by noradrenaline (0.5-8 micromol/l) were comparable in lean and obese groups, but vasorelaxation in noradrenaline-preconstricted arteries was markedly reduced in dietary-obese rats of both sexes. Concentration-response curves to endothelium-dependent vasorelaxants (acetylcholine, A23187 and insulin) showed significant reductions in maximal relaxation (20-95% less than in leans; P<0.001) and significant rightward shifts in EC(40) (concentration giving 40% of maximal response) (P<0.01). Relaxation in response to the direct NO donor, sodium nitroprusside, showed a lesser impairment (12%; P<0.01) in dietary-obese rats. Maximal relaxation to acetylcholine was correlated inversely in both sexes with fat-pad mass (r(2)=0.37, P<0.05) and plasma triacylglycerols (r(2)=0.51, P<0.01), and with leptin in males only (r(2)=0.35, P<0.05). Independent determinants of acetylcholine-induced relaxation were fat mass and plasma triacylglycerols; plasma insulin and insulin sensitivity had no effect. Dietary-induced obesity severely impaired arterial relaxation in both sexes, particularly at the endothelial level. This is not attributable to insulin resistance, but may be related to moderate hypertriglyceridaemia.

Analysis of Variance↗
PubMed2001

Once-daily oral gatifloxacin versus oral levofloxacin in treatment of uncomplicated skin and soft tissue infections: double-blind, multicenter, randomized study.

This was a double-blind, multicenter study in which 410 adults (> or =18 years of age) with uncomplicated skin and soft tissue infections (SSTIs) were randomized to receive either 400 mg of gatifloxacin orally once daily or 500 mg of levofloxacin orally once daily for 7 to 10 days. The study protocol called for four assessments-before and during treatment, at the end of treatment, and posttreatment. Efficacy evaluations included clinical response and bacterial eradication rates. Of 407 treated patients, 202 (108 women, 94 men) received gatifloxacin and 205 (111 women, 94 men) received levofloxacin. For clinically evaluable patients, the cure rates were 91% for gatifloxacin and 84% for levofloxacin (95% confidence interval [CI] for the difference, -2.0 to 15.2%). Clinical cure rates for microbiologically evaluable patients were 93% for gatifloxacin and 88% for levofloxacin (95% CI for the difference, -6.5 to 16.8%). The bacterial eradication rate was 92% for each group, with gatifloxacin eradicating 93% of the methicillin-susceptible Staphylococcus aureus isolates and levofloxacin eradicating 91% of them. Both drugs were well tolerated. Most of the adverse events were mild to moderate, and nausea was the most common adverse event in each treatment arm. Once-daily oral gatifloxacin (400 mg) is clinically efficacious and well tolerated compared with once-daily levofloxacin (500 mg) for the treatment of patients with uncomplicated SSTIs.

Administration, Oral↗
PubMed2001

Beta(2) adrenergic receptors mediate important electrophysiological effects in human ventricular myocardium.

OBJECTIVE: To define the effects of beta(2) adrenergic receptor stimulation on ventricular repolarisation in vivo. DESIGN: Prospective study. SETTING: Tertiary referral centre. PATIENTS: 85 patients with coronary artery disease and 22 normal controls. INTERVENTIONS: Intravenous and intracoronary salbutamol (a beta(2) adrenergic receptor selective agonist; 10-30 microg/min and 1-10 microg/min), and intravenous isoprenaline (a mixed beta(1)/beta(2) adrenergic receptor agonist; 1-5 microg/min), infused during fixed atrial pacing. MAIN OUTCOME MEASURES: QT intervals, QT dispersion, monophasic action potential duration. RESULTS: In patients with coronary artery disease, salbutamol decreased QT(onset) and QT(peak) but increased QT(end) duration; QT(onset)-QT(peak) and QT(peak)-QT(end) intervals increased, resulting in T wave prolongation (mean (SEM): 201 (2) ms to 233 (2) ms; p < 0.01). There was a large increase in dispersion of QT(onset), QT(peak), and QT(end) which was more pronounced in patients with coronary artery disease-for example, QT(end) dispersion: 50 (2) ms baseline v 98 (4) ms salbutamol (controls), and 70 (1) ms baseline v 108 (3) ms salbutamol (coronary artery disease); p < 0.001. Similar responses were obtained with isoprenaline. Monophasic action potential duration at 90% repolarisation shortened during intracoronary infusion of salbutamol, from 278 (4.1) ms to 257 (3.8) ms (p < 0.05). CONCLUSIONS: beta(2) adrenergic receptors mediate important electrophysiological effects in human ventricular myocardium. The increase in dispersion of repolarisation provides a mechanism whereby catecholamines acting through this receptor subtype may trigger ventricular arrhythmias.

Action Potentials↗
PubMed2001

Neurovascular lessons from a pair of identical twins with cerebral aneurysms.

A pair of hypertensive identical twins are reported, one of whom was non-compliant with her antihypertension medication, and after a subarachnoid haemorrhage was found to have multiple cerebral aneurysms. The other asymptomatic compliant twin was noted to have only a single small cerebral aneurysm. As both identical twins are likely to share the same cerebral vascular architecture it is suggested that good control of blood pressure contributed to the cerebral vascular integrity of the asymptomatic twin. This is the first report on the role of blood pressure in the formation of cerebral aneurysms and provides insights for an alternative approach to the controversial management of asymptomatic cerebral aneurysms.

Adult↗
PubMed2001

Simple absorbance-based assays for ultra-high throughput screening.

In order to accommodate the predicted increase in screening required of successful pharmaceutical companies, miniaturized, high-speed HTS formats are necessary. Much emphasis has been placed on sensitive fluorescence techniques, but some systems, particularly enzymes interconverting small substrates, are likely to be refractory to such approaches. We show here that simple absorbance-based assays can be miniaturized to 10-microl volumes in 1536-well microplates compatible with the requirements for ultra-high throughput screening. We demonstrate that, with low-cost hardware, assay performance is wholly predictable from the 2-fold decrease in pathlength for fully filled 1536-well plates compared to 96- and 384-well microplates. A number of enzyme systems are shown to work in this high-density format, and the inhibition parameters determined are comparable with those in standard assay formats. We also demonstrate the utility of kinetics measurements in miniaturized format with improvements in assay quality and the ability to extract detailed mechanistic information about inhibitors.

Adenosine Triphosphatases↗
PubMed2001

Real experiences of uHTS: a prototypic 1536-well fluorescence anisotropy-based uHTS screen and application of well-level quality control procedures.

This paper describes, for the first time, a true ultra-high throughput screen (uHTS) based upon fluorescence anisotropy and performed entirely in 1536-well assay plates. The assay is based upon binding and displacement of a BODIPY-FL-labeled antibiotic to a specific binding site on 70S ribosomes from Escherichia coli (Kd approximately 15 nM). The screen was performed at uHTS rates (i.e., >100,000 assay wells/24 h) using entirely commercially available equipment. In order to examine the reproducibility of detection of test compound effects, assays were performed in duplicate. Both overall assay statistics and reproducibility for individual compound results were excellent, at least equivalent to conventional HTS assays. Interference artifacts occurred mainly as a result of autofluorescence from test compounds. Well-level quality control procedures were developed to detect, eliminate, or even correct for such effects. Moreover, development of a brighter, longer wavelength probe (based upon Cy3B) markedly reduced such interferences. Overall, the data demonstrate that fluorescence anisotropy-based uHTS is now a practical reality.

Anti-Bacterial Agents↗
PubMed2001

Lead poisoning among refugee children resettled in Massachusetts, 1995 to 1999.

OBJECTIVE: Lead poisoning has been reported among immigrant and refugee populations in the United States; however, prevalences of elevated blood lead (BPb) and risk factors have not been described fully among newly arrived refugee children. This study was conducted to address this lack of data. METHODS: We performed analysis of BPb levels among a cohort of refugee children, aged <7 years, who arrived in Massachusetts between July 1, 1995, and December 31, 1999. Gender, age, birthplace, time of year of lead testing, intestinal parasitoses, anemia, and growth retardation were examined as predictors of elevated BPb. RESULTS: BPb levels >/=10 microgram/dL were found in 11.3% of 693 children shortly after their arrival in the United States. Children from developing countries had the highest prevalences, including 27% of Somalis and Vietnamese. Country of birth was the strongest predictor of elevated BPb. No association between elevated lead and age was found. Among 213 children with BPb tests >/=6 months after the initial test, 7% had newly elevated levels of >/=10 microgram/dL. CONCLUSIONS: The prevalence of elevated BPb levels in recently arrived refugee children is more than twice that of US-born children. Children who are at particular risk are those from developing countries where environmental exposures are more ubiquitous. In addition, a significant percentage of refugees acquired elevated levels after arrival, thus suggesting the importance of follow-up testing of refugee children. Refugee status should be considered a risk factor for lead poisoning.

Child↗
PubMed2001

Childhood methanol ingestion treated with fomepizole and hemodialysis.

Fomepizole (4-methylpyrazole; Antizol) is used increasingly in the treatment of methanol toxicity in adults. Little experience exists with this drug in the pediatric population, however. We present a case of methanol poisoning in a child in whom the use of fomepizole averted intravenous ethanol infusion and the attendant side effects of this therapy.

Adult↗
PubMed2001

The effectiveness of housing policies in reducing children's lead exposure.

OBJECTIVES: This study evaluated the relation of housing policies to risk of subsequent lead exposure in addresses where lead-poisoned children had lived. METHODS: Addresses where children with lead poisoning lived between May 1992 and April 1993 were selected from lead screening registries in 2 northeastern states differing in their enforcement of lead poisoning prevention statutes. Blood lead levels of subsequently resident children, exterior condition, tax value, age, and census tract characteristics were collected. The odds of elevated blood lead levels in subsequently resident children were calculated with logistic regression. RESULTS: The risk of identifying 1 or more children with blood lead levels of 10 micrograms/dL or greater was 4 times higher in addresses with limited enforcement. Controlling for major confounders had little effect on the estimate. CONCLUSIONS: Enforcement of housing policies interrupts the cycle of repeated lead exposure.

Child, Preschool↗
PubMed2001

Protein kinase C theta is expressed in mast cells and is functionally involved in Fcepsilon receptor I signaling.

We investigated possible expression and function in mast cells of protein kinase C (PKC) theta, a member of the PKC family with demonstrated function in a limited range of cell types. We found that PKC theta is expressed in bone marrow-derived mast cells and in the RBL-2H3 mast cell line. PKC theta underwent translocation to the membrane in response to Fcepsilon receptor I (FcepsilonR I) activation. Receptor activation induced phosphorylation of PKC theta. The tyrosine phosphorylation of PKC theta is delayed relative to PKC delta and coincides temporally with PKC theta association with c-src family members Lyn and SRC: Studies of RBL-2H3 cells transduced with PKC theta constructs indicated a role for PKC theta in receptor-induced activation of extracellular regulated kinases, interleukin-3 gene transcription, and degranulation in response to antigen stimulation. These studies extend the known functions of PKCtheta to another important immune cell type and indicate the concurrent participation of multiple PKCs in the FcepsilonR I-mediated response of mast cells.

Animals↗
PubMed2001

Influence of beta1-adrenoceptor blockade on the gene expression of adenylate cyclase subtypes and beta-adrenoceptor kinase in human atrium.

There is accumulating evidence of cross-regulation between stimulatory G-protein (G(s))-coupled receptors in human atrium. Chronic beta(1)-adrenoceptor antagonist treatment can sensitize beta(2)-adrenoreceptors, 5-HT(4) receptors, histamine H(2) receptors and possibly beta(4)-adrenoreceptors. To investigate the mechanism of such cross-talk between G(s)-coupled receptors, we have measured the mRNA expression of the cardiac adenylate cyclases (types IV, V, VI and VII) and of beta-adrenoceptor kinase 1 in human atria using TaqMan quantitative PCR, and compared expression between patients chronically receiving a beta(1)-adrenoceptor antagonist and non-treated patients. The results showed no difference in gene expression between the two groups of patients; however, significant positive correlations of gene expression between adenylate cyclase subtypes were found. We conclude that beta(1)-adrenoceptor antagonists do not change the absolute levels of gene expression of adenylate cyclase subtypes in human atrium. The co-ordinate regulation among adenylate cyclase subtypes and the influence of beta(1)-adrenoceptor antagonists need to be investigated further.

Adenylyl Cyclases↗