Biomedical subjects

David R Brown

Publications and source records attributed to David R Brown.

At least 19 recordsLinked to original sources

PubMed2003

Interactions within the intrinsic cardiac nervous system contribute to chronotropic regulation.

The objective of this study was to determine how neurons within the right atrial ganglionated plexus (RAGP) and posterior atrial ganglionated plexus (PAGP) interact to modulate right atrial chronotropic, dromotropic, and inotropic function, particularly with respect to their extracardiac vagal and sympathetic efferent neuronal inputs. Surgical ablation of the PAGP (PAGPx) attenuated vagally mediated bradycardia by 26%; it reduced heart rate slowing evoked by vagal stimulation superimposed on sympathetically mediated tachycardia by 36%. RAGP ablation (RAGPx) eliminated vagally mediated bradycardia, while retaining the vagally induced suppression of sympathetic-mediated tachycardia (-83%). After combined RAGPx and PAGPx, vagal stimulation still reduced sympathetic-mediated tachycardia (-47%). After RAGPx alone and after PAGPx alone, stimulation of the vagi still produced negative dromotropic effects, although these changes were attenuated compared with the intact state. Negative dromotropic responses to vagal stimulation were further attenuated after combined ablation, but parasympathetic inhibition of atrioventricular nodal conduction was still demonstrable in most animals. Finally, neither RAGPx nor PAGPx altered autonomic regulation of right atrial inotropic function. These data indicate that multiple aggregates of neurons within the intrinsic cardiac nervous system are involved in sinoatrial nodal regulation. Whereas parasympathetic efferent neurons regulating the right atrium, including the sinoatrial node, are primarily located within the RAGP, prejunctional parasympathetic-sympathetic interactions regulating right atrial function also involve neurons within the PAGP.

Animals↗

PubMed2003

Generation of hydrogen peroxide from mutant forms of the prion protein fragment PrP121-231.

By means of electron spin resonance spectroscopy, in conjunction with the spin trapping technique, we have shown previously that Abeta and alpha-synuclein (aggregating proteins that accumulate in the brain in Alzheimer's disease, Parkinson's disease, and related disorders) both induce the formation of hydroxyl radicals following incubation in solution, upon addition of Fe(II). These hydroxyl radicals are apparently formed from hydrogen peroxide, via Fenton's reaction. An N-terminally truncated fragment of the mouse prion protein (termed PrP121-231) is toxic to cerebellar cells in culture, and certain human mutations, responsible for inherited prion disease, enhance this toxicity. Here we report that PrP121-231 containing three such mutations (E200K, D178N, and F198S) also generated hydroxyl radicals, upon addition of Fe(II). The formation of these radicals was blocked by catalase, or by metal chelators, each of which also reduced the toxicity of the PrP121-231 fragments to cultured normal mouse cerebellar cells. Wild-type PrP121-231, full-length cellular PrP, and its homologue doppel did not generate any detectable hydroxyl radicals. We conclude that the additional cytotoxic effects of the mutant forms of PrP121-231 could be due to their ability to generate hydrogen peroxide, by a metal-dependent mechanism. Thus, one effect of these (and possibly other) prion mutations could be production of a particularly toxic form of the prion protein, with an enhanced capacity to induce oxidative damage, neurodegeneration, and cell loss.

Animals↗

PubMed2003

Detection of bovine spongiform encephalopathy, ovine scrapie prion-related protein (PrPSc) and normal PrPc by monoclonal antibodies raised to copper-refolded prion protein.

Prion-related protein (PrP) is a glycosylphosphatidylinositol-linked cell-surface protein expressed by a wide variety of cells, including those of the nervous system and the immune system. Several functions of normal cellular PrP (PrPc) have been proposed that may be associated with the capacity of this protein to bind copper. In the present study, we describe the generation of a panel of monoclonal antibodies raised to copper-refolded PrP, which may be used to analyse the normal and disease-associated forms of this protein. The anti-PrP monoclonal antibodies were reactive by Western blot and ELISA with recombinant murine PrPc refolded in the presence or absence of either copper or manganese, and with the disease-susceptible allelic form V136R154Q171 ('VRQ'; where single-letter amino-acid notation has been used) and disease-resistant allelic form A136R154R171 ('ARR') of recombinant ovine PrPc. FACS analysis of lymphoid cells using these monoclonal antibodies showed that wild-type non-activated mouse lymphocytes expressed little, if any, PrPc. These monoclonal antibodies were shown to react with the unglycosylated and monoglycosylated forms of PrPSc (abnormal disease-specific conformation of PrP) in prion-infected tissue samples from all of the different species tested by Western blot. In addition, this analysis allowed one to make a distinction between bovine spongiform encephalopathy ('BSE') and scrapie PrPSc) isolates from experimentally infected sheep on the basis of their different electrophoretic mobilities.

Animals↗

PubMed2003

Kinin-induced anion-dependent secretion in porcine ileum: characterization and involvement of opioid- and cannabinoid-sensitive enteric neural circuits.

The intestinal secretory actions of the proinflammatory peptide kallidin (lysyl-bradykinin) are mediated partially by enteric neurons. We hypothesized that kallidin produces neurogenic anion secretion through opioid- and cannabinoid-sensitive enteric neural pathways. Changes in short-circuit current (I(sc)) across sheets of porcine ileal mucosa-submucosa mounted in Ussing chambers were measured in response to kallidin (1 microM) or drugs added to the contraluminal bathing medium. Kallidin transiently increased I(sc), an effect reduced after inhibition of neuronal conduction by 0.1 microM saxitoxin, cyclooxygenase inhibition by 10 microM indomethacin, or kinin B(2) receptor blockade by 1 microM d-arginyl-l-arginyl-l-prolyl-trans-4-hydroxy-l-prolylglycyl-3-(2-thienyl)-l-alanyl-l-seryl-d-1,2,3,4-tetrahydro-3-isoquinolinecarbonyl-l-(2alpha,3beta,7alphabeta)-octahydro-1H-indole-2-carbonyl-l-arginine (HOE-140). Its action was dependent upon extracellular Cl(-) or HCO(3)(-) ions, but was resistant to 10 microM bumetanide or 0.3 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and seemed to involve luminal alkalinization as measured by pH-stat titration. Kallidin-induced I(sc) elevations were sensitive to saxitoxin in tissues bathed in Cl(-)-, but not HCO(3)(-)-deficient media. Tissues pretreated with 0.1 microM [d-Pen(2,5)]-enkephalin, a selective delta-opioid agonist, displayed reduced I(sc) responses to kallidin; this effect was prevented by the delta-opioid antagonist naltrindole. At a contraluminal concentration of 1 microM, the cannabinoid receptor agonist (6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol (HU-210) also attenuated responses to kallidin. Proinflammatory kinins seem to stimulate neurogenic anion secretion in porcine ileum by activating enteric neural circuits expressing inhibitory opioid and possibly cannabinoid receptors.

Analgesics, Opioid↗

PubMed2003

Copper-dependent generation of hydrogen peroxide from the toxic prion protein fragment PrP106-126.

Oligomeric forms of many of the aggregating proteins associated with neurodegenerative diseases are toxic to cultured cells. We have shown recently that Abeta and alpha-synuclein can both induce the formation of hydroxyl radicals following incubation in solution, upon the addition of Fe(II). Thus, they appear to generate hydrogen peroxide, which is converted to hydroxyl radicals via the Fenton reaction. Here we show that the widely studied toxic peptide fragment of the prion protein, PrP106-126, has exactly the same property, but only in the presence of copper ions. Since the aggregation and toxicity of PrP106-126 have been reported to be critically dependent on copper binding, our data suggest that the published cytotoxic effects of this peptide could also be due to its ability to generate hydrogen peroxide.

Copper↗

PubMed2003

Effects of nicotine and dietary salt on a learned blood pressure response in Dahl-S rats.

We examined the effects of chronic nicotine exposure and dietary salt on the arterial blood pressure (BP) changes learned in response to an acute behavioral stress in the Dahl salt-sensitive rat. Four groups were tested: low salt + vehicle; low salt + nicotine; high salt + vehicle; and high salt + nicotine. Rats were fed a low-salt (0.08% NaCl) or a high-salt (8% NaCl) diet for 4 wk; 2.4 mg. kg(-1). day(-1) nicotine or vehicle was given via an implanted osmotic minipump for the last 2 wk. All rats were conditioned by following one tone (CS+) with a 0.5-s tail shock; another tone (CS-) was never followed by shock. CS+ in low salt + vehicle and high salt + vehicle-treated rats evoked an initial arterial BP increase (C(1)), a component of the startle response, and an ensuing, smaller, but more sustained, pressor response (C(2)), which is acquired with training. In these rats, both C(1) and C(2) evoked by CS- were significantly smaller than those to CS+, demonstrating that these groups discriminated between the two tests. Conversely, although the low salt + nicotine-treated rats had both the C(1) and C(2) components of the conditional arterial pressure response, they did not discriminate between CS+ and CS-. Finally, the high salt + nicotine group failed to both discriminate between tones and acquire (i.e., learn) the C(2) response. The unconditional response to shock did not differ between groups. We conclude that combined exposure to high salt and to nicotine inhibits the salt-sensitive animal's acquisition of a learned conditional BP response, perhaps because nicotine acts preferentially on those central processes required for associative learning versus those involved in orientating to external stimuli.

Animals↗

PubMed2003

Physical activity and body mass index among US adolescents: youth risk behavior survey, 1999.

OBJECTIVE: To investigate associations of underweight and overweight with physical activity among high school students in the United States. METHODS: A nationally representative sample of 15 349 US high school students participated in the 1999 Youth Risk Behavior Survey; 13 295 were included in these analyses. Five measures of physical activity were examined as dichotomous variables: (1) vigorous-intensity physical activity (>/=3 vs <3 sessions lasting at least 20 minutes each per week); (2) moderate-intensity physical activity (>/=5 vs <5 sessions lasting at least 30 minutes each per week); (3) strength training (>/=3 vs <3 sessions per week); (4) enrollment in physical education (yes or no); and (5) sports participation (yes or no). Using body mass indexes, students were categorized by percentiles as underweight (</=5th percentile), at risk for underweight (>5th to </=15th percentiles), normal weight (>15th to <85th percentiles), at risk for overweight (>/=85th to <95th percentiles), or overweight (>/=95th percentile). Potential associations between physical activity and body mass index were examined using logistic regression. RESULTS: On several measures, adolescent boys who were underweight or overweight were less likely to be physically active than boys of normal weight (eg, odds ratio [OR], 0.23; 95% confidence interval [CI], 0.12-0.45; and OR, 0.75; 95% CI, 0.61-0.93; for boys who were underweight and overweight, respectively, for strength training). Adolescent girls who were overweight or at risk for overweight were less likely (OR, 0.62; 95% CI, 0.50-0.78; and OR, 0.63; 95% CI, 0.46-0.85; respectively) to be involved with sports than girls of normal weight; and girls who were underweight were less likely (OR, 0.44; 95% CI, 0.22-0.91) to be enrolled in physical education. CONCLUSIONS: Weight status among high school students is correlated with selected physical activity behavior, especially among adolescent boys. Interventions to increase physical activity for high school students should target adolescents of all shapes and sizes, and may best be achieved by school policies requiring physical education or after-school sports.

Adolescent↗

PubMed2003

Neuromodulation of enteropathogen internalization in Peyer's patches from porcine jejunum.

Jejunal Peyer's patches (JPP) are innervated sites of immune induction and enteropathogen infection. We investigated the role of enteric nerves in modulating pathogen entry into porcine JPP. Presumptive norepinephrine (NE)-containing nerve fibers were localized in JPP domes and follicle-associated villi by secondary immunofluorescence histochemistry. NE or the neuronal conduction blocker saxitoxin increased intracellular internalization of pathogenic Salmonella choleraesuis and Escherichia coli O157:H7, but not nonpathogenic E. coli, into isolated JPP mucosa. NE action was prevented by the alpha-adrenergic antagonist phentolamine. Withdrawal of enteric neural activity or NE administration appears to modulate JPP interactions with pathogenic bacteria.

Animals↗

PubMed2003

Analysis of doppel protein toxicity.

The recently described doppel protein (Dpl) is a homologue of the prion protein (PrP(c)). This protein, expressed in the brains of mice that lack the expression of PrP(c), causes neuronal death as the mice age. Previous studies have suggested this neuronal damage is caused by oxidative assault and changes in the activity of NOS proteins. We investigated the toxicity of Dpl in cell culture models and showed that Dpl was toxic to neurons. This toxicity was inhibited by the expression of PrP(c) and possibly involved direct interaction between the two proteins. The mechanism of toxicity involved stimulation of nitric oxide production via activation of the nitric oxide synthases, nNOS and iNOS. This mechanism of toxicity is quite different from that of PrP(Sc) and does not require the protein to change conformation. These results provide the first evidence for the mechanism of Dpl toxicity.

Amino Acid Sequence↗

PubMed2003

Mapping the functional domain of the prion protein.

Prion diseases such as Creutzfeldt-Jakob disease are possibly caused by the conversion of a normal cellular glycoprotein, the prion protein (PrPc) into an abnormal isoform (PrPSc). The process that causes this conversion is unknown, but to understand it requires a detailed insight into the normal activity of PrPc. It has become accepted from results of numerous studies that PrPc is a Cu-binding protein and that its normal function requires Cu. Further work has suggested that PrPc is an antioxidant with an activity like that of a superoxide dismutase. We have shown in this investigation that this activity is optimal for the whole protein and that deletion of parts of the protein reduce or abolish this activity. The protein therefore contains an active domain requiring certain regions such as the Cu-binding octameric repeat region and the hydrophobic core. These regions show high evolutionary conservation fitting with the idea that they are important to the active domain of the protein.

Animals↗

PubMed2003

A novel method of generating neuronal cell lines from gene-knockout mice to study prion protein membrane orientation.

The technology of gene knockout and transgenic mice has allowed the study of the role of genes and their proteins in animal physiology and metabolism. However, these techniques have often been found to be limited in that some genetic manipulations of mice led either to a fatal phenotype or to compensations that mask the loss of function of the target protein. The experimentation on neurons from transgenic mice is particularly critical in the study of key proteins that may be involved in neurodegeneration. The cell fusion technique has been implemented as a novel way to generate cell lines from prion protein knockout mice. Fusion between neonatal mouse neurons and a neuroblastoma cell line have led to a Prnp degrees / degrees cell line that facilitates the study of the knockout phenotype. These cells are readily transfectable and allowed us to study the expression of prion protein mutants on a PrP-knockout background. Using this cell line we have examined the effect of PrP mutations reported to alter PrPc to a transmembrane form. Our results suggest that these mutations do not create transmembrane forms of the protein, but block normal transport of PrP to the cell membrane.

Animals↗

PubMed2003

Catecholamines modulate Escherichia coli O157:H7 adherence to murine cecal mucosa.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important food-borne pathogen. While the molecular mechanisms governing E. coli O157:H7 pathogenesis have been intensively investigated, the role of host factors has received less attention. In this study, we tested the hypothesis that the enteric catecholamines norepinephrine (NE) and dopamine (DA) modulate interactions of the cecal mucosa with E. coli O157:H7. Full-thickness sheets of murine cecum were mounted in Ussing chambers and short circuit current and tissue electrical conductance were periodically determined to assess active transepithelial ion transport and ionic permeability, respectively. Neurochemicals and stationary-phase E. coli O157:H7 were exposed respectively to the contraluminal and luminal aspects of the mucosa. Epithelial adherence of E. coli O157:H7 was quantified by a bacterial adhesion assay after 90 min of luminal E. coli O157:H7 exposure. DA and NE increased E. coli O157:H7 adherence relative to untreated control tissues at 50% effective concentrations of 3.8 microM and 4.2 microM respectively. Pretreatment of tissues with either the alpha-adrenergic antagonist phentolamine or the beta-adrenergic antagonist propranolol prevented the action of NE. The effect of DA was prevented by the dopamine antagonist haloperidol. The drugs did not impair tissue viability or transepithelial conductance. The present findings suggest that enteric catecholamines modulate E. coli O157:H7 adherence to the cecal epithelium. Conditions associated with elevated catecholamine release, such as stress exposure, may influence host susceptibility to E. coli O157:H7 infection.

Animals↗

PubMed2002

Coupling of sympathetic nerve traffic and BP at very low frequencies is mediated by large-amplitude events.

This study explores the functional association between renal sympathetic nerve traffic (NT) and arterial blood pressure (BP) in the very-low-frequency range (i.e., <0.1 Hz). NT and BP (n = 6) or BP alone (n = 17) was recorded in unanesthetized rats (n = 6). Data were collected for 2-5 h, and wavelet transforms were calculated from data epochs of up to 1 h. From these transforms, we obtained probability distributions for fluctuation amplitudes over a range of time scales. We also computed the cross-wavelet power spectrum between NT and BP to detect the occurrence in time of large-amplitude transient events that may be important in the autonomic regulation of BP. Finally, we computed a time sequence of cross correlations between NT and BP to follow the relationship between NT and BP in time. We found that NT and BP follow comparable self-similar scaling relationships (i.e., NT and BP fluctuations exhibit a certain type of power law behavior). Scaling of this nature 1) points to underlying dynamics over a wide range of scales and 2) is related to large-amplitude events that contribute to the very-low-frequency variability of NT and BP. There is a strong correlation between NT and BP during many of these transient events. These strong correlations and the uniformity in scaling imply a functional connection between these two signals at frequencies where we previously found no connection using spectral coherence.

Animals↗

PubMed2002

Active bicarbonate-dependent secretion evoked by 5-hydroxytryptamine in porcine ileal mucosa is mediated by opioid-sensitive enteric neurons.

5-Hydroxytryptamine (5-HT) mediates intestinal hypersecretion associated with infection and inflammation. We tested the hypothesis that 5-HT-induced anion secretion is mediated by an opioid-sensitive enteric neural circuit. 5-HT, at a contraluminal concentration of 10 microM, increased short-circuit current by 58 +/- 7 microA/cm(2) in sheets of porcine ileal mucosa with attached inner submucosal plexus. Responses to 5-HT were inhibited by saxitoxin or indomethacin, and reduced in tissues bathed in Cl(-)- or HCO(3)(-)-deficient media. 5-HT action was attenuated by saxitoxin in tissues bathed in Cl(-)-free media, but not HCO(3)-free media. The delta-opioid receptor agonist [D-Pen(2,5)]enkephalin (0.1 microM) blunted the 5-HT change in short-circuit current by a mechanism sensitive to the delta-opioid receptor antagonist naltrindole. The inhibitory actions of [D-Pen(2,5)]enkephalin and saxitoxin were not additive. These results suggest that 5-HT stimulates HCO(3)(-)-dependent ion transport through a mechanism involving prostanoids and an enteric neural pathway modulated by opioids.

Analgesics, Opioid↗

PubMed2002

Plasminogen activation is stimulated by prion protein and regulated in a copper-dependent manner.

Prion diseases are associated with the conversion of the normal prion protein, PrP(C), to the infectious disease form PrP(Sc). Discrimination between these isoforms would significantly enhance diagnosis of these diseases, and it has recently been reported that PrP(Sc) is specifically recognized by the serine protease zymogen plasminogen (Fischer et al. (2000) Nature 408, 479). Here we have tested the hypothesis that PrP is a regulator of the plasminogen activation system. The effect of recombinant PrP, either containing copper (holo-PrP) or devoid of it (apo-PrP), on plasminogen activation by both uPA and tPA was determined. PrP had no effect on plasminogen activation by uPA. By contrast, the activity of tPA was stimulated by up to 280-fold. This was observed only with the apo-PrP isoforms. The copper-binding octapeptide repeat region of PrP was involved in the effects, as a mutant lacking this region failed to stimulate plasminogen activation, although a synthetic peptide corresponding to this region was unable to stimulate tPA activity. Competition experiments demonstrated that, in addition to plasminogen binding, the stimulation required a high-affinity interaction between tPA and PrP (K(d) < 2.5 nM). Kinetic analysis revealed a template mechanism for the stimulation, suggesting independent binding sites for tPA and plasminogen. Lack of copper-binding may be an early event in the conversion of PrP(C) to PrP(Sc), and our data therefore suggest that tPA-catalyzed plasminogen activation may provide the basis for a sensitive detection system for the early stages of prion diseases and also play a role in the pathogenesis of these diseases.

Animals↗

PubMed2002

Metal imbalance and compromised antioxidant function are early changes in prion disease.

The prion protein (PrP) has been shown to bind copper. In the present study we have investigated whether prion disease in a mouse scrapie model resulted in modification of metal concentrations. We found changes in the levels of copper and manganese in the brains of scrapie-infected mice prior to the onset of clinical symptoms. Interestingly, we noted a major increase in blood manganese in the early stages of disease. Analysis of purified PrP from the brains of scrapie-infected mice also showed a reduction in copper binding to the protein and a proportional decrease in antioxidant activity between 30 and 60 days post-inoculation. We postulate that alterations in trace-element metabolism as a result of changes in metal binding to PrP are central to the pathological modifications in prion disease.

Animals↗

PubMed2002

Lack of prion protein expression results in a neuronal phenotype sensitive to stress.

The prion protein is a highly conserved glycoprotein expressed most highly in the synapse. Evidence has recently been put forward to suggest that the prion protein is an antioxidant. However, the functional importance of the prion protein has been disputed; it is claimed that mice genetically ablated to lack prion protein expression are normal and have no specific phenotype. We have reexamined the phenotype of prion protein knockout mice and found that there are multiple biochemical changes in the mice, including increased levels of nuclear factor NF-kappaB and Mn superoxide dismutase, COX-IV decreased levels of Cu/Zn superoxide dismutase activity, decreased p53, and altered melatonin levels. Additionally, cultured cells from these mice are more sensitive to a range of insults, all linked to increased neuronal sensitivity to oxidative stress. These results imply that prion protein knockout mice are more sensitive to oxidative stress and have an altered phenotype that must be taken into account when considering the additional effects of increased levels of proteins such as Doppel. The implication of these results is that the consequence of genetic ablation of genes must include biochemical analysis as well as analyses of possible developmental and behavioral changes.

Animals↗

PubMed2002

High extracellular potassium protects against the toxicity of cytosine arabinoside but is not required for the survival of cerebellar granule cells in vitro.

Depolarization of cerebellar granule cells with elevated potassium has been described as essential to maintain their survival in culture. There are several reports that this is only specific for rat cerebellar granule cells and not those of mouse. We reinvestigated this issue and found that although high potassium enhanced the survival of cerebellar granule cells from both rat and mouse it was not essential for the survival of those cultures. Further analysis of the culture system indicated that high potassium offered protection against the toxicity of glutamate and cytosine arabinose (Ara C), a standard antimitotic additive to cultures of granule cells. Ara C was found to be toxic to cerebellar cells after potassium withdrawal at concentrations standardly used in culturing these cells (10 microM). High potassium was found to diminish the expression of p53. Ara C toxicity is known to utilize the p53-dependent signaling pathway to initiate apoptosis. Another depolarizing agent, veratridine, offers no protection against Ara C but we provide evidence that the protective effect of high potassium against Ara C is mediated through calcium balance within the cells. We suggest that there is no requirement for high potassium in terms of cerebellar granule cell survival. The previously proposed role for high potassium in the survival cerebellar granule cells is rather a protective effect against toxic substances in serum such as glutamate or against agents such as Ara C.

Animals↗