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Biomedical subjects

David R Brown

Publications and source records attributed to David R Brown.

At least 73 records · Page 4Linked to original sources
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

Pharmacological characterization of a 7-benzylidenenaltrexone-preferring opioid receptor in porcine ileal submucosa.

In the intestine, opioids produce antidiarrhoeal and constipating actions that are mediated by enteric neurones. Through interactions with opioid receptors (ORs) on submucosal neurones, opioids suppress active ion transport evoked by transmural electrical stimulation (TES) in mucosa-submucosa sheets from the porcine ileum. In this study, we examined the pharmacological characteristics of the previously described OR, which is sensitive to the delta1-OR antagonist 7-benzylidenenaltrexone and modulates neurogenic transepithelial ion transport in this tissue preparation. Increases in short-circuit current (Isc, a measure of active anion transport) evoked by TES in ileal mucosa-submucosa sheets were inhibited by opioid agonists possessing high selectivity for either delta- or micro-ORs including [d-Pen2,5]enkephalin (DPDPE), [d-Ala2, Glu4]deltorphin II, and [d-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO). As determined by the Schild analysis, the actions of these agonists were competitively inhibited by 7-benzylidenenaltrexone. The nonequilibrium micro-OR antagonist beta-funaltrexamine inhibited the actions of DAMGO only at a high concentration (1 microm) but did not alter DPDPE or deltorphin II action. At concentrations up to 10 microm, the nonequilibrium delta-OR antagonist naltrindole 5'-isothiocyanate did not alter the actions of delta- or micro-OR agonists. Radioligand binding analyses of neuronal homogenates from the ileal submucosa revealed that the nonselective OR ligand [3H]diprenorphine bound to two populations of specific binding sites. One of these sites possessed binding characteristics similar to the delta-OR. In summary, neurogenic ion transport in the porcine intestine is modulated by an OR which shares pharmacological characteristics of both micro- and delta-ORs and may represent a novel receptor entity.

Amphibian Proteins↗
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

Prion protein expression modulates neuronal copper content.

The prion protein is a copper (Cu)-binding protein. The abnormal isoform of this protein is associated with the transmissible spongiform encephalopathies or prion diseases. In prion diseases, the prion protein loses its Cu binding capacity. The effect of prion protein expression on the Cu content of the brain was investigated. Transgenic mice, either overexpressing the prion protein or expressing a mutant form lacking the Cu-binding region of the protein, were compared with wild-type mice and mice in which expression of the protein was knocked out. Age-dependent differences in Cu content of the brain were detected. Also, synaptosomal fractions from the brains of the mice showed different Cu content depending on the expression of the prion protein. Mice expressing prion protein, but without the Cu-binding domain showed reduced Cu content. Mice overexpressing the prion protein showed little difference in Cu in the brain compared with wild type but also the prion protein expressed by the mice showed a reduction in the level of Cu bound. These results confirm that prion protein expression modulates the Cu level found at the synapse and this effect is dependent on its Cu binding capacity. Loss of normal Cu binding by the prion protein altered age-related increases in metals in the brain. This may explain why many forms of human prion disease do not develop until late in life.

Age Factors↗
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↗
PubMed2002

Relationship of satisfaction with body size and trying to lose weight in a national survey of overweight and obese women aged 40 and older, United States.

BACKGROUND: Despite the potential benefits of weight loss, the factors associated with weight loss behavior are only beginning to be identified. We examined the association between sociodemographic factors, perceived health, satisfaction with body size, and trying to lose weight. METHODS: Data were obtained from the 1996-1997 U.S. Women's Determinants Study. We included over 1,700 overweight and obese women aged 40 and older from the following four racial/ethnic groups: Hispanic, black, American Indian/Alaskan Native, and non-Hispanic white. RESULTS: About half of the women reported that they were satisfied or very satisfied with their body size. Satisfaction was associated with lower body mass index (BMI), greater age, lower educational level, and better self-rated health. Compared with non-Hispanic white women, women in the other racial/ethnic groups expressed greater body satisfaction. About 65% of women reported that they were currently trying to lose weight. The strongest predictor of trying to lose weight was satisfaction with body size; women who were not satisfied were nine times more likely to report trying to lose weight than those who were very satisfied. Other significant predictors were BMI, race/ethnicity, and age. CONCLUSIONS: Our findings should serve as the impetus for the inclusion of measures of body image in surveillance and intervention studies of weight loss and control.

Adult↗
PubMed2002

Immunization delays the onset of prion disease in mice.

The outbreak of new variant Creutzfeldt-Jakob disease has raised the specter of a potentially large population being at risk to develop this prionosis. None of the prionoses currently have an effective treatment. Recently, vaccination has been shown to be effective in mouse models of another neurodegenerative condition, namely Alzheimer's disease. Here we report that vaccination with recombinant mouse prion protein delays the onset of prion disease in mice. Vaccination was performed both before peripheral prion exposure and after exposure. A delay in disease onset was seen in both groups, but was more prolonged in animals immunized before exposure. The increase in the incubation period closely correlated with the anti-prion protein antibody titer. This promising finding suggests that a similar approach may work in humans or other mammalian species at risk for prion disease.

Animals↗
PubMed2002

Gastric antisecretory role and immunohistochemical localization of cannabinoid receptors in the rat stomach.

1. The role of cannabinoid (CB) receptors in the regulation of gastric acid secretion was investigated in the rat by means of functional experiments and by immunohistochemistry. 2. In anaesthetized rats with lumen-perfused stomach, the non selective CB-receptor agonist WIN 55,212-2 (0.30 - 4.00 micromol kg(-1), i.v.) and the selective CB(1)-receptor agonist HU-210 (0.03 - 1.50 micromol kg(-1), i.v.), dose-dependently decreased the acid secretion induced by both pentagastrin (30 nmol kg(-1) h(-1)) and 2-deoxy-D-glucose (1.25 mmol kg(-1), i.v.). By contrast, neither WIN 55,212-2 (1 - 4 micromol kg(-1), i.v.) nor HU-210 (0.03 - 1.50 micromol kg(-1), i.v.) did modify histamine-induced acid secretion (20 micromol kg(-1) h(-1)). The selective CB(2)-receptor agonist JWH-015 (3 - 10 micromol kg(-1), i.v.) was ineffective. 3. The gastric antisecretory effects of WIN 55,212-2 and HU-210 on pentagastrin-induced acid secretion were prevented by the selective CB(1)-receptor antagonist SR141716A (0.65 micromol kg(-1), i.v.) and unaffected by the selective CB(2)-receptor antagonist SR144528 (0.65 - 2 micromol kg(-1), i.v.). 4. Bilateral cervical vagotomy and ganglionic blockade with hexamethonium (10 mg kg(-1), i.v., followed by continuous infusion of 10 mg kg(-1) h(-1)) significantly reduced, but not abolished, the maximal inhibitory effect of HU-210 (0.3 micromol kg(-1), i.v.) on pentagastrin-induced acid secretion; by contrast, pretreatment with atropine (1 mg kg(-1), i.v.) did not modify the antisecretory effect of HU-210. 5. Immunoreactivity to the CB(1) receptor was co-localized with that of the cholinergic marker choline acetyltransferase in neural elements innervating smooth muscle, mucosa and submucosal blood vessels of rat stomach fundus, corpus and antrum. In contrast, CB(2) receptor-like immunoreactivity was not observed. 6. These results indicate that gastric antisecretory effects of cannabinoids in the rat are mediated by suppression of vagal drive to the stomach through activation of CB(1) receptors, located on pre- and postganglionic cholinergic pathways. However, the ineffectiveness of atropine in reducing the effect of HU-210 suggests that the release of non cholinergic excitatory neurotransmitters may be regulated by CB(1) receptors.

Animals↗