Biomedical subjects

David R Brown

Publications and source records attributed to David R Brown.

At least 19 recordsLinked to original sources

PubMed2007

NMR characterization of the pH 4 beta-intermediate of the prion protein: the N-terminal half of the protein remains unstructured and retains a high degree of flexibility.

Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely alpha-helical isoform to a beta-sheet-rich oligomer. CD has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an alpha-helical protein into one that contains a high proportion of beta-sheet-like conformation. In the present study, we characterize this soluble pH 4 folding intermediate using NMR. (15)N-HSQC (heteronuclear single-quantum correlation) studies with mPrP (mouse PrP)-(23-231) show that a total of 150 dispersed amide signals are resolved in the native form, whereas only 65 amide signals with little chemical shift dispersion are observable in the pH 4 form. Three-dimensional (15)N-HSQC-TOCSY and NOESY spectra indicate that the observable residues are all assigned to amino acids in the N-terminus: residues 23-118. (15)N transverse relaxation measurements indicate that these N-terminal residues are highly flexible with additional fast motions. These observations are confirmed via the use of truncated mPrP-(112-231), which shows only 16 (15)N-HSQC amide peaks at pH 4. The loss of signals from the C-terminus can be attributed to line broadening due to an increase in the molecular size of the oligomer or exchange broadening in a molten-globule state.

Circular Dichroism↗

PubMed2006

Characterization of Salmonella enterica serovar Typhimurium DT104 invasion in an epithelial cell line (IPEC J2) from porcine small intestine.

Salmonella Typhimurium DT104 is an emerging enteric pathogen in swine of increasing medical importance. In this study, the time course and the actin-dependent host signaling processes necessary for invasion of a S. Typhimurium DT104 field isolate were investigated in IPEC J2 epithelial cells derived from porcine small intestine. Internalized bacteria were quantified by a gentamicin resistance assay. DT104 internalization into epithelial monolayers increased steadily between 15 and 120min after apical inoculation. Internalization was reduced by the Rho GTPase inhibitor mevastatin, the N-WASP inhibitor wiskostatin and the actin-disrupting agent cytochalasin D, but not the Rac1 GTPase inhibitor NSC-23766. Early DT104 invasion of porcine enterocytes appears to be mediated by Rac1 GTPase-independent changes in epithelial actin assembly.

Animals↗

PubMed2006

Essential bacterial functions encoded by gene pairs.

To address the need for new antibacterials, a number of bacterial genomes have been systematically disrupted to identify essential genes. Such programs have focused on the disruption of single genes and may have missed functions encoded by gene pairs or multiple genes. In this work, we hypothesized that we could predict the identity of pairs of proteins within one organism that have the same function. We identified 135 putative protein pairs in Bacillus subtilis and attempted to disrupt the genes forming these, singly and then in pairs. The single gene disruptions revealed new genes that could not be disrupted individually and other genes required for growth in minimal medium or for sporulation. The pairwise disruptions revealed seven pairs of proteins that are likely to have the same function, as the presence of one protein can compensate for the absence of the other. Six of these pairs are essential for bacterial viability and in four cases show a pattern of species conservation appropriate for potential antibacterial development. This work highlights the importance of combinatorial studies in understanding gene duplication and identifying functional redundancy.

Amino Acid Sequence↗

PubMed2006

Prion protein does not redox-silence Cu2+, but is a sacrificial quencher of hydroxyl radicals.

Oxidative stress is believed to play a central role in the pathogenesis of prion diseases, a group of fatal neurodegenerative disorders associated with a conformational change in the prion protein (PrP(C)). The precise physiological function of PrP(C) remains uncertain; however, Cu(2+) binds to PrP(C) in vivo, suggesting a role for PrP(C) in copper homeostasis. Here we examine the oxidative processes associated with PrP(C) and Cu(2+). (1)H NMR was used to monitor chemical modifications of PrP fragments. Incubation of PrP fragments with ascorbate and CuCl(2) showed specific metal-catalyzed oxidation of histidine residues, His(96/111), and the methionine residues, Met(109/112). The octarepeat region protects His(96/111) and Met(109/112) from oxidation, suggesting that PrP(90-231) might be more prone to chemical modification. We show that Cu(2+/+) redox cycling is not 'silenced' by Cu(2+) binding to PrP, as indicated by H(2)O(2) production for full-length PrP. Surprisingly, although detection of Cu(+) indicates that the octarepeat region of PrP is capable of reducing Cu(2+) even in the absence of ascorbate, H(2)O(2) is not generated unless ascorbate is present. Full-length PrP and fragments cause a dramatic reduction in detectable hydroxyl radicals in an ascorbate/Cu(2+)/O(2) system; however, levels of H(2)O(2) production are unaffected. This suggests that PrP does not affect levels of hydroxyl radical production via Fentons cycling, but the radicals cause highly localized chemical modification of PrP(C).

Animals↗

PubMed2006

Empirical and theoretical analysis of the extremely low frequency arterial blood pressure power spectrum in unanesthetized rat.

The slope of the log of power versus the log of frequency in the arterial blood pressure (BP) power spectrum is classically considered constant over the low-frequency range (i.e., "fractal" behavior), and is quantified by beta in the relationship "1/f(beta)." In practice, the fractal range cannot extend to indefinitely low frequencies, but factor(s) that terminate this behavior, and determine beta, are unclear. We present 1) data in rats (n = 8) that reveal an extremely low frequency spectral region (0.083-1 cycle/h), where beta approaches 0 (i.e., the "shoulder"); and 2) a model that 1) predicts realistic values of beta within that range of the spectrum that conforms to fractal dynamics (approximately 1-60 cycles/h), 2) offers an explanation for the shoulder, and 3) predicts that the "successive difference" in mean BP (mBP) is an important parameter of circulatory function. We recorded BP for up to 16 days. The absolute difference between successive mBP samples at 0.1 Hz (the successive difference, or Delta) was 1.87 +/- 0.21 mmHg (means +/- SD). We calculated beta for three frequency ranges: 1) 0.083-1; 2) 1-6; and 3) 6-60 cycles/h. The beta for all three regions differed (P < 0.01). For the two higher frequency ranges, beta indicated a fractal relationship (beta(6-60/h) = 1.27 +/- 0.01; beta(1-6/h) = 1.80 +/- 0.16). Conversely, the slope of the lowest frequency region (i.e., the shoulder) was nearly flat (beta(0.083-1 /h) = 0.32 +/- 0.28). We simulated the BP time series as a random walk about 100 mmHg with ranges above and below of 10, 30, and 50 mmHg and with Delta from 0.5 to 2.5. The spectrum for the conditions mimicking actual BP time series (i.e., range, 85-115 mmHg; Delta, 2.00) resembled the observed spectra, with beta in the lowest frequency range = 0.207 and fractal-like behavior in the two higher frequency ranges (beta = 1.707 and 2.057). We suggest that the combined actions of mechanisms limiting the excursion of arterial BP produce the shoulder in the spectrum and that Delta contributes to determining beta.

Animals↗

PubMed2006

Prion protein reduces both oxidative and non-oxidative copper toxicity.

The prion protein is a membrane tethered glycoprotein that binds copper. Conversion to an abnormal isoform is associated with neurodegenerative diseases known as prion diseases. Expression of the prion protein has been suggested to prevent cell death caused by oxidative stress. Using cell based models we investigated the potential of the prion protein to protect against copper toxicity. Although prion protein expression effectively protected neurones from copper toxicity, this protection was not necessarily associated with reduction in oxidative damage. We also showed that glycine and the prion protein could both protect neuronal cells from oxidative stress. Only the prion protein could protect these cells from the toxicity of copper. In contrast glycine increased copper toxicity without any apparent oxidative stress or lipid peroxidation. Mutational analysis showed that protection by the prion protein was dependent upon the copper binding octameric repeat region. Our findings demonstrate that copper toxicity can be independent of measured oxidative stress and that prion protein expression primarily protects against copper toxicity independently of the mechanism of cell death.

Animals↗

PubMed2006

Food supply controls the body condition of a migrant bird wintering in the tropics.

The hypothesis that migratory bird populations are limited during the non-breeding season is increasingly supported by empirical studies that also suggest consequences that carry-over into subsequent seasons. Although variation in food supply is the best supported explanation for non-breeding season limitation, the ecological mechanisms and physiological consequences are not well understood. We both supplemented and reduced Ovenbird (Seiurus aurocapilla) food availability on replicate plots in Jamaica in each of 3 years to determine the direct role of food in limiting the physical condition of Ovenbirds. Annual variation in rainfall and food supply created a natural experiment in parallel with manipulations. Sex and age-classes of Ovenbirds did not respond differently in terms of body condition to either food manipulation or natural variation in environmental conditions, suggesting that this population is not structured by strong dominance relationships. Ovenbird body mass, fat, and pectoralis muscle shape were positively and predictably related to manipulated food availability. Feather regrowth rate also responded positively to food supplementation and negatively to food reduction in the drier of 2 years. Prior to manipulation, annual variation in body mass corresponded to annual variation in food supply and rainfall, providing additional, correlational evidence of food limitation. Since multiple intercorrelated body condition indices of Ovenbirds responded directly to food supply, and since food supply influenced body condition independently of other habitat features, we argue that food is a primary driver of non-breeding season population limitation. Moreover, since these effects were observed during the late non-breeding period, when individuals are preparing to migrate, we infer that food availability likely initiates carry-over effects.

Age Factors↗

PubMed2006

Mucosally-directed adrenergic nerves and sympathomimetic drugs enhance non-intimate adherence of Escherichia coli O157:H7 to porcine cecum and colon.

The sympathetic neurotransmitter norepinephrine has been found to increase mucosal adherence of enterohemorrhagic Escherichia coli O157:H7 in explants of murine cecum and porcine distal colon. In the present study, we tested the hypothesis that norepinephrine augments the initial, loose adherence of this important pathogen to the intestinal mucosa. In mucosal sheets of porcine cecum or proximal, spiral and distal colon mounted in Ussing chambers, norepinephrine (10 microM, contraluminal addition) increased mucosal adherence of wild-type E. coli O157:H7 strain 85-170; in the cecal mucosa, this effect occurred within 30-90 min after bacterial inoculation. In addition, norepinephrine transiently increased short-circuit current in cecal and colonic mucosal sheets, a measure of active anion transport. Norepinephrine was effective in promoting cecal adherence of a non-O157 E. coli strain as well as E. coli O157:H7 eae or espA mutant strains that are incapable of intimate mucosal attachment. Nerve fibers immunoreactive for the norepinephrine synthetic enzyme dopamine beta-hydroxylase appeared in close proximity to the cecal epithelium, and the norepinephrine reuptake blocker cocaine, like norepinephrine and the selective alpha2-adrenoceptor agonist UK-14,304, increased E. coli O157:H7 adherence. These results suggest that norepinephrine, acting upon the large bowel mucosa, modulates early, non-intimate adherence of E. coli O157:H7 and probably other mucosa-associated bacteria. Sympathetic nerves innervating the cecocolonic mucosa may link acute stress exposure or psychostimulant abuse with an increased microbial colonization of the intestinal surface. This in turn may alter host susceptibility to enteric infections.

Adhesins, Bacterial↗

PubMed2006

Genetic manipulation of intraspinal plasticity after spinal cord injury alters the severity of autonomic dysreflexia.

Severe spinal cord injuries above mid-thoracic levels can lead to a potentially life-threatening hypertensive condition termed autonomic dysreflexia, which is often triggered by painful distension of pelvic viscera (bladder or bowel) and consequent sensory fiber activation, including nociceptive C-fibers. Interruption of tonically active medullo-spinal pathways after injury causes disinhibition of thoracolumbar sympathetic preganglionic neurons, and intraspinal sprouting of nerve growth factor (NGF)-responsive primary afferent fibers is thought to contribute to their hyperactivity. We investigated spinal levels that are critical for eliciting autonomic dysreflexia using a model of noxious colorectal distension (CRD) after complete spinal transection at the fourth thoracic segment in rats. Post-traumatic sprouting of calcitonin gene-related peptide (CGRP)-immunoreactive primary afferent fibers was selectively altered at specific spinal levels caudal to the injury with bilateral microinjections of adenovirus encoding the growth-promoting NGF or growth-inhibitory semaphorin 3A (Sema3a) compared with control green fluorescent protein (GFP). Two weeks later, cardio-physiological responses to CRD were assessed among treatment groups before histological analysis of afferent fiber density at the injection sites. Dysreflexic hypertension was significantly higher with NGF overexpression in lumbosacral segments compared with GFP, whereas similar overexpression of Sema3a significantly reduced noxious CRD-evoked hypertension. Quantitative analysis of CGRP immunostaining in the spinal dorsal horns showed a significant correlation between the extent of fiber sprouting into the spinal segments injected and the severity of autonomic dysreflexia. These results demonstrate that site-directed genetic manipulation of axon guidance molecules after complete spinal cord injury can alter endogenous circuitry to modulate plasticity-induced autonomic pathophysiology.

Adenoviridae↗

PubMed2006

Hydrogen peroxide cleavage of the prion protein generates a fragment able to initiate polymerisation of full length prion protein.

The prion protein is central to the disease pathogenesis of a variety of neurodegenerative diseases such as CJD. The protein is only able to initiate the disease process following post-translational modification. The main characteristic of this change is the ability of this altered isoform to polymerise. We wish to determine if altered cleavage of the protein could generate a protein fragment able to initiate polymerisation. During normal metabolic breakdown the protein is initially cleaved at a single site at around amino acid residue 111/112 in the mouse sequence. A second site before amino acid residue 90 has been postulated as an alternative cleavage point. We have provided evidence that hydrogen peroxide as low as 50 microM in the presence of copper, iron or manganese (but not nickel, magnesium or zinc) can cleave the recombinant protein near this site and requires a GXXH motif in the protein sequence. This reaction results in the production of 6 and 19 kDa fragments of the protein. This cleavage pattern occurs in prion proteins from different species (mouse, chicken and turtle) and is enhanced by modification of the octameric repeat region. The 19 kDa fragment produced by this reaction is protease sensitive. This fragment in a pure form caused the polymerisation of wild-type prion protein by a seeding mechanism. Therefore our results provide a possible mechanism by which altered cleavage of the prion protein could result in the kind of protein polymerisation associated with prion diseases.

Amino Acid Sequence↗

PubMed2006

Mouse galectin-1 inhibits the toxicity of glutamate by modifying NR1 NMDA receptor expression.

One of the major causes of neuronal death in neurodegenerative disease is excitotoxicity from the neurotransmitter glutamate. This form of cell death could arise from either excess levels of glutamate due to decreased astrocyte clearance or due to increased susceptibility. We have identified galectin-1, a galactose-binding lectin, as a potential neuroprotective factor secreted by astrocytes. Our results show that both native and recombinant galectin-1 protects mouse and rat cerebellar neurons from the toxic effects of glutamate. Galectin-1 applied to neurons increased their expression of the NMDA receptor NR1 and increased the proportion of the NR1a subunit subtype while antisense knockdown of the NR1a receptor blocked the neuroprotective effect of galectin-1. This effect of the protein was dependent upon it carbohydrate recognition domain, suggesting that the protein acts in a reduced dimerized form. In addition, galectin-1 caused a decreased expression of PKC associated with increased resistance to glutamate toxicity. These results suggest that the astrocytic lectin galectin-1 could protect neurons against the effects of excitotoxicity as seen in stroke and ischemic injury.

Animals↗

PubMed2006

Copper-dependent co-internalization of the prion protein and glypican-1.

Heparan sulfate chains have been found to be associated with amyloid deposits in a number of diseases including transmissible spongiform encephalopathies. Diverse lines of evidence have linked proteoglycans and their glycosaminoglycan chains, and especially heparan sulfate, to the metabolism of the prion protein isoforms. Glypicans are a family of glycosylphosphatidylinositol-anchored, heparan sulfate-containing, cell-associated proteoglycans. Cysteines in glypican-1 can become nitrosylated by endogenously produced nitric oxide. When glypican-1 is exposed to a reducing agent, such as ascorbate, nitric oxide is released and autocatalyses deaminative cleavage of heparan sulfate chains. These processes take place while glypican-1 recycles via a non-classical, caveolin-associated pathway. We have previously demonstrated that prion protein provides the Cu2+ ions required to nitrosylate thiol groups in the core protein of glypican-1. By using confocal immunofluorescence microscopy and immunomagnetic techniques, we now show that copper induces co-internalization of prion protein and glypican-1 from the cell surface to perinuclear compartments. We find that prion protein is controlling both the internalization of glypican-1 and its nitric oxide-dependent autoprocessing. Silencing glypican-1 expression has no effect on copper-stimulated prion protein endocytosis, but in cells expressing a prion protein construct lacking the copper binding domain internalization of glypican-1 is much reduced and autoprocessing is abrogated. We also demonstrate that heparan sulfate chains of glypican-1 are poorly degraded in prion null fibroblasts. The addition of either Cu2+ ions, nitric oxide donors, ascorbate or ectopic expression of prion protein restores heparan sulfate degradation. These results indicate that the interaction between glypican-1 and Cu2+-loaded prion protein is required both for co-internalization and glypican-1 self-pruning.

Animals↗

PubMed2006

High affinity copper binding by stefin B (cystatin B) and its role in the inhibition of amyloid fibrillation.

We show that human stefin B, a protease inhibitor from the family of cystatins, is a copper binding protein, unlike stefin A. We have used isothermal titration calorimetry to directly monitor the binding event at pH 7 and pH 5. At pH 7 stefin B shows a picomolar affinity for copper but at pH 5 the affinity is in the nanomolar range. There is no difference in the affinity of copper between the wildtype stefin B (E31 isoform) and a variant (Y31 isoform), whereas the mutant (P79S), which is tetrameric, does not bind copper. The conformation of stefin B remains unaltered by copper binding. It is known that below pH 5 stefin B undergoes a conformational change and amyloid fibril formation. We show that copper binding inhibits the amyloid fibril formation and, to a lesser degree, the initial aggregation. Similarities to and differences from other copper binding amyloidogenic proteins are discussed.

Amino Acid Sequence↗

PubMed2005

Differential effects of clathrin and actin inhibitors on internalization of Escherichia coli and Salmonella choleraesuis in porcine jejunal Peyer's patches.

Peyer's patches constitute both an inductive immune site and an enteropathogen invasion route. Peyer's patch mucosae from porcine jejunum were mounted in Ussing chambers, and either Salmonella choleraesuis vaccine strain SC-54 or non-pathogenic rodent and porcine Escherichia coli strains contacted the Peyer's patch mucosa for 90 min. Internalized bacteria were quantified by a gentamicin resistance assay. Monodansylcadaverine (300 microM, luminal addition), an inhibitor of clathrin-mediated endocytosis, significantly inhibited internalization of both E. coli strains relative to tissues untreated with the inhibitor; internalization of SC-54 was unaffected. The actin-disrupting agent cytochalasin D (10 microM, luminal addition), inhibited internalization of pig-adapted E. coli but not that of rodent-adapted E. coli or SC-54. Internalization of SC-54 and non-pathogenic E. coli in Peyer's patches appears to occur through different cellular routes.

Actins↗

PubMed2005

High affinity binding between copper and full-length prion protein identified by two different techniques.

The cellular prion protein is known to be a copper-binding protein. Despite the wide range of studies on the copper binding of PrP, there have been no studies to determine the affinity of the protein on both full-length prion protein and under physiological conditions. We have used two techniques, isothermal titration calorimetry and competitive metal capture analysis, to determine the affinity of copper for wild type mouse PrP and a series of mutants. High affinity copper binding by wild type PrP has been confirmed by the independent techniques indicating the presence of specific tight copper binding sites up to femtomolar affinity. Altogether, four high affinity binding sites of between femto- and nanomolar affinities are located within the octameric repeat region of the protein at physiological pH. A fifth copper binding site of lower affinity than those of the octameric repeat region has been detected in full-length protein. Binding to this site is modulated by the histidine at residue 111. Removal of the octameric repeats leads to the enhancement of affinity of this fifth site and a second binding site outside of the repeat region undetected in the wild type protein. High affinity copper binding allows PrP to compete effectively for copper in the extracellular milieu. The copper binding affinities of PrP have been compared with those of proteins of known function and are of magnitudes compatible with an extracellular copper buffer or an enzymatic function such as superoxide dismutase like activity.

Animals↗

PubMed2005

Anterograde axonal transport of the exogenous cellular isoform of prion protein in the chick visual system.

The cellular isoform of endogenous, newly synthesized prion protein (PrPc) can be transported by axons in the anterograde direction. To determine whether a mechanism exists for secreted PrPc to be internalized and then axonally transported, we analyzed internalization and anterograde axonal transport of radiolabeled recombinant PrPc after its intraocular injection in chick embryos. Internalization and axonal transport of exogenous PrPc to the midbrain by retinal ganglion cells (RGCs) is efficient, saturable and likely receptor-mediated. Ultrastructural quantitative localization of radiolabeled PrPc within RGC soma showed significant labeling of vesicular/endosomal compartments and much less labeling present over the Golgi apparatus and lysosomes, which indicates slow degradation of exogenous PrPc in this system. These data show that a mechanism exists to internalize a secreted form of PrPc and then to axonally transport such PrPc in an anterograde direction. This may provide an additional, novel mechanism for prion protein to spread among neurons.

Animals↗

PubMed2005

Mediation of neurogenic ion transport by acetylcholine, prostanoids and 5-hydroxytryptamine in porcine ileum.

Enteric neural activity modulates active transepithelial ion transport in the intestine. We investigated the neural circuits mediating neurogenic secretion in mucosal explants from porcine ileum. Transmural electrical stimulation increased short-circuit current, a measure of active ion transport, by 35+/-2 microA/cm2. The neuronal Na+ channel blocker saxitoxin, the muscarinic cholinergic receptor antagonist atropine, the 5-hydroxytryptamine3 receptor antagonist tropisetron, and the cyclooxygenase inhibitor indomethacin inhibited this response. In addition, tropisetron inhibited the atropine-resistant portion of the response, and both atropine and indomethacin attenuated the saxitoxin-resistant component. Neurogenic secretion in porcine ileum appears to be mediated by tryptaminergic and prostanoid-sensitive cholinergic pathways.

8,11,14-Eicosatrienoic Acid↗

PubMed2005

Novel inhibitors of bacterial cytokinesis identified by a cell-based antibiotic screening assay.

The continuous emergence of antibiotic resistance demands that novel classes of antibiotics continue to be developed. The division machinery of bacteria is an attractive target because it comprises seven or more essential proteins that are conserved almost throughout the bacteria but are absent from humans. We describe the development of a cell-based assay for inhibitors of cell division and its use to isolate a new inhibitor of FtsZ protein, a key player in the division machinery. Biochemical, cytological, and genetic data are presented that demonstrate that FtsZ is the specific target for the compound. We also describe the effects of more potent analogues of the original hit compound that act on important pathogens, again at the level of cell division. The assay and the compounds have the potential to provide novel antibiotics with no pool of pre-existing resistance. They have provided new insight into cytokinesis in bacteria and offer important reagents for further studies of the cell division machinery.

Anti-Infective Agents↗