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Biomedical subjects

David R Brown

Publications and source records attributed to David R Brown.

At least 37 records · Page 2Linked to original sources

Salmonella enterica serovar Choleraesuis infection of the porcine jejunal Peyer's patch rapidly induces IL-1beta and IL-8 expression.

Salmonella enterica serovar Choleraesuis is an enteric pathogen of swine, producing septicemia, enterocolitis, pneumonia, and hepatitis. The initial molecular events at the site of Salmonella infection are hypothesized to be critical in the initiation of innate and adaptive immune responses; however, the acute immune response elicited by porcine intestinal tissues is not well understood. To address this need, we employed explants of jejunal Peyer's patch (JPP) mucosa from pigs to examine Salmonella-induced immune responses under controlled conditions as well as to overcome limitations of whole animal approaches. JPP explants mounted in Ussing chambers maintained normal histological structure for 2 h and stable short-circuit current and electrical conductance for 2.5 h. After ex vivo luminal exposure to Salmonella serovar Choleraesuis, JPP responded with an increase in mRNA expression of IL-1beta and IL-8, but not TNFalpha. Increased IL-1beta and IL-8 expression were dependent on efficient Salmonella adhesion and internalization, whereas mutant Salmonella did not induce inflammatory cytokine expression. Commensal enteric bacteria, present in some experiments, also did not induce inflammatory cytokine expression. These findings indicate that Salmonella uptake by Peyer's patch is important in the induction of an innate response involving expression of IL-1beta and IL-8, and that ex vivo intestinal immune tissue explants provide an intact tissue model that will facilitate investigation of mucosal immunity in swine.

Animals↗

Differential acquisition of specific components of a classically conditioned arterial blood pressure response in rat.

Presenting a 15-s pulsed tone, the conditional stimulus (CS(+)), followed by 0.5-s tail shock, to a well-trained rat causes a sudden, but transient, pressor response (C(1)). Blood pressure (BP) then drops before increasing again (C(2)). A steady tone of the same frequency never followed by a shock (a discriminative stimulus, or CS(-)) evokes a C(1) but not a C(2) response. Experiment 1 tested the hypothesis that this BP response pattern does not depend on the nature of the tone (i.e., pulsed vs. steady) used for CS(+) and CS(-). The tones were reversed from the traditional paradigm, above, in nine rats. The C(1) BP increase for a steady-tone CS(+) (+4.8 +/- 1.9 mmHg, mean change +/- SE) and a pulsed CS(-) (+2.9 +/- 1.3 mmHg) did not differ. Conversely, C(2) showed a clear discrimination (CS(+): +5.1 +/- 1.2 mmHg, CS(-): +0 .7 +/- 0.8 mmHg; P < 0.05). Experiment 2 tested the hypothesis that the C(1) and C(2) BP responses first appear at different times during training. On training day 1, five 15-s pulsed tones (CS(+)) were presented to each of 18 rats; the last tone was followed by a tail shock. Likewise, five steady CS(-) tones never followed by shock were given. Training continued for 2 more days, with each CS(+) followed by shock. At the end of day 2, CS(+) evoked a C(1) BP response (+3.9 +/- 0.9 mmHg) but no C(2) (+0.6 +/- 0.4 mmHg, not significant vs. pretone). By the end of day 3, CS(+) evoked a significant (vs. baseline) C(1) (+7.3 +/- 1.4 mmHg) and C(2) (+3.3 +/- 0.8 mmHg). Conversely, although CS(-) evoked a C(1) response (3.5 +/- 1.3 mmHg), there was no C(2) (+0.7 +/- 0.5 mmHg; not significant). We conclude that 1) C(1) and C(2) are acquired at different rates, 2) early in training C(1) is an orienting response evoked by both tones, and 3) C(2) is only acquired as an animal learns to associate the CS(+) tone with shock. This suggests that C(1) and C(2) are controlled by different processes in the brain.

Acoustic Stimulation↗

AT1-receptor antagonism reverses the blood pressure elevation associated with diet-induced obesity.

Previous studies in our laboratory demonstrated that rats exhibiting obesity in response to a moderately high-fat (MHF) diet developed hypertension associated with activation of the local and systemic renin-angiotensin system. In this study, we examined the effect of the angiotensin type 1 (AT(1))-receptor antagonist, losartan, on blood pressure in obesity-prone (OP) and obesity-resistant (OR) rats fed a MHF diet. Using telemetry monitoring, we characterized the evolution of blood pressure elevations during the development of obesity. Male Sprague-Dawley rats were implanted with telemetry transducers for chronic monitoring of blood pressure, and baseline measurements were obtained. Rats were then switched to the MHF diet (32% kcal as fat) and were segregated into OP and OR groups at week 5. At week 9 on the MHF diet, OP rats exhibited significantly greater 24-h mean arterial blood pressure compared with OR rats (OP: 105 +/- 4 mmHg, OR: 96 +/- 2 mmHg; P < 0.05). Elevations in blood pressure in OP rats were manifest as an increase in systolic pressure. Administration of losartan to all rats at week 9 resulted in a reduction in blood pressure; however, losartan had the greatest effect in OP rats (percent decrease in mean arterial pressure by losartan; OP: 19 +/- 4, OR: 10 +/- 2%; P < 0.05). These results demonstrate that elevations in blood pressure occur subsequent to established obesity in rats fed a high-fat diet. Moreover, these results demonstrate the ability of losartan to reverse the blood pressure increase from diet-induced obesity, supporting a primary role for the renin-angiotensin system in obesity-associated hypertension.

Angiotensin II Type 1 Receptor Blockers↗

Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations.

The prion protein (PrP) is a Cu2+ binding cell surface glyco-protein. Misfolding of PrP into a beta-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni2+ as a diamagnetic probe to further understand Cu2+ binding to PrP. Like Cu2+, Ni2+ preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni2+ ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni2+ binding sites form square-planar diamagnetic complexes. We have previously shown that Cu2+ forms a paramagnetic square-planar complex in this region, suggesting that Ni2+ could be used as a probe for Cu2+ binding. In addition, competition studies show that two Cu2+ ions can displace Ni2+ from these sites. Upon Ni2+ addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni2+ binding sites. Analysis of Cu2+ loaded visible-CD spectra show that as with Ni2+, PrP(90-115) binds two Cu2+ ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Delta51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni2+ to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease.

Animals↗

Copper binding is the governing determinant of prion protein turnover.

The cellular isoform of the prion protein (PrP(c)) is located at the cell membrane, anchored externally by a glycosylphosphatidylinositol (GPI) anchor. It is a copper (Cu) binding glycoprotein with a rapid basal turnover. Previous studies have shown that exposure of cells to Cu causes internalisation of PrP(c) in vitro. In this study, we show that physiological levels of Cu promote internalisation of PrP(c). Interaction between PrP(c) and Cu was found to be the overriding factor in stimulating the internalisation response with other metals showing no effect. Deletion mutation studies have shown that two domains are essential for copper-induced internalisation to occur. These two domains are the octameric repeat region, encompassing amino acids 51-89, and the palindromic region, amino acids 112-119 with the sequence AGAAAAGA. The decrease in detectable levels of PrP(c) at the cell surface following Cu treatment was found to be the result of rapid internalisation rather than loss into the surrounding environment. These results have implications for both normal metabolism of PrP(c) and the possible mechanism of conversion of PrP(c) to PrP(sc).

Amino Acid Sequence↗

Adrenocorticotrophic hormone modulates Escherichia coli O157:H7 adherence to porcine colonic mucosa.

Exposure to stress is associated with susceptibility to disease and one stress mediator, norepinephrine, has been reported to enhance the adherence of enterohemorrhagic Escherichia coli O157:H7 (EHEC) to the colonic mucosa. We tested the hypothesis that adrenocorticotropic hormone (ACTH) and other stress-related hormones may act in a similar fashion. Explants of distal colonic mucosa from young pigs were mounted in Ussing chambers and their luminal aspect was exposed to EHEC strain 700728 for 30-90 min. When added to the contraluminal, but not luminal bathing medium, ACTH increased EHEC adherence within 90 min in a concentration-dependent manner (EC50 = 1.2 nM), but did not alter tissue electrical conductance. ACTH had no effect on the adherence of a pig-adapted non-O157 E. coli strain. The effect of 0.1 microM ACTH on luminal EHEC adherence was prevented in tissues pretreated contraluminally with the type 2 melanocortin receptor antagonist ACTH7-38, the neuronal conduction blocker saxitoxin, or the muscarinic cholinergic antagonist atropine. Moreover, ACTH7-38 decreased EHEC adherence in the absence of ACTH. These results suggest that ACTH acts via melanocortin receptors located on enteric nerves to enhance mucosal adherence of EHEC.

Adhesins, Bacterial↗

An aggregation-specific enzyme-linked immunosorbent assay: detection of conformational differences between recombinant PrP protein dimers and PrP(Sc) aggregates.

The conversion of the normal cellular prion protein, PrP(C), into the protease-resistant, scrapie PrP(Sc) aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrP(Sc) aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrP(Sc) aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease.

Animals↗

Physical activity among adults >or=50 yr with and without disabilities, BRFSS 2001.

PURPOSE: Before 2001, the Behavioral Risk Factor Surveillance System (BRFSS), a national survey of health behaviors, assessed only leisure-time physical activity. In 2001, the BRFSS used newly developed physical activity items to evaluate combined leisure-time, household, and transportation activities. Using BRFSS 2001 data, this cross-sectional study describes the prevalence of inactivity and insufficient and recommended physical activity for older adults (i.e., aged >or=50 yr). METHODS: BRFSS 2001 data were analyzed using prevalence estimates and logistic regression to assess physical activity patterns among older adults (N=74,960) stratified by disability status, and select sociodemographic and health status characteristics. RESULTS: A total of 43.4, 39.1, and 17.5% of respondents without disabilities were active at a recommended level, insufficiently active, and inactive, respectively, taking into account nonoccupational physical activities. A total of 28.8% of older adults with disabilities were active at a recommended level, 35.7% insufficiently active, and 35.5% inactive. Among persons with and without disabilities, groups with the highest odds of inactivity and insufficient activity were women, persons aged >or=75 yr, blacks, persons with lower education levels and low incomes, and those who were obese. CONCLUSIONS: Not all persons with disabilities can be active at recommended levels, but it is possible for the vast majority to do some types of physical activity, even if at insufficient levels. Thus, it may be possible for the prevalence of inactivity among persons with and without disabilities to be similar. This was not found. At the start of the new millennium, almost 60% of older adults without disabilities and 70% with disabilities were not obtaining a recommended amount of combined leisure-time, transportation, and household physical activity.

Activities of Daily Living↗

Neurodegeneration and oxidative stress: prion disease results from loss of antioxidant defence.

Prion diseases or transmissible spongiform encephalopathies (TSEs) are rare neurodegenerative disorders that can be acquired either by direct transmission, inherited through dominant mutations in the prion protein gene or via an unknown sporadic cause. This latter group constitutes the vast majority of cases. Like many neurodegenerative diseases the hallmarks of oxidative damage can be readily detected throughout the brain of the affected individual. However, unlike most other neurodegenerative diseases, prion diseases are connected with a dramatic loss of antioxidant defence. As abnormal protein accumulates in the diseased brain there is both an increase of oxidative substances and a loss of the defences that keep them in check. In particular the normal cellular prion protein has been shown to be an antioxidant. Conversion of this protein to the protease resistant isoform is accompanied by a loss of this antioxidant activity. This change creates a paradox as the loss of activity is not accompanied by a loss of protein expression. It is likely that this prevents other cellular defences from responding sufficiently to protect neurons from the heightened oxidative burden. Recent experiments with transgenic mice have shown that when prion protein expression is switched off during the course of prion disease, cell death is dramatically halted and the mouse recovers from the disease. This result clearly illustrates that the continued expression of non-function prion protein is essential for disease progression. This implies that the presence of this abnormal protein during prion disease causes a failure of cellular antioxidant defence. This failed defence is the fundamental cause of the massive neurodegeneration that results in the fatal nature of TSEs. The role of oxidative stress in TSEs and other neurodegenerative disorders are discussed in this review.

Animals↗

Prion protein fate governed by metal binding.

The conversion of the normal cellular prion protein to an abnormal isoform is considered to be causal to the prion diseases or transmissible spongiform encephalopathies. The prion protein is a copper binding protein but under some conditions may bind other metals. In particular, the binding of manganese has been suggested to convert the prion protein (PrP) to a protease resistant isoform. Therefore, the differences in the way the protein binds copper and manganese might be revealing in terms of the mechanism of conversion of the protein or its normal cellular activity. We report the use of near-infrared spectroscopy for studies on aqueous solutions of prion protein binding Cu or Mn. These alloforms of the protein were analyzed by spectral data acquisition and multivariate analysis. Our results indicate that PrP binds both Mn and Cu differently. Analyses of Cu binding suggest that the PrP-Cu complex protected Cu from the water increasing protein stability. PrP-Mn does not protect Mn from water interactions. A real-time study of the protein alloforms showed that PrP-Cu remains stable in solution, but that PrP-Mn underwent highly different changes that led to fibril formation.

Binding Sites↗

Synthesis of analogues of Congo red and evaluation of their anti-prion activity.

No cure as of yet exists for any of the transmissible spongiform encephalopathies. In this paper, we describe the synthesis of analogues of Congo red and evaluation against a cellular model of infection, the SMB (scrapie mouse brain) persistently infected cell line, for their ability to inhibit the infectivity of the abnormal form of prion protein (PrP-res). The compounds have also been tested for their ability to inhibit the polymerization of PrPC by PrP-res. A number of analogues showed inhibition of PrP-res infectivity at nanomolar concentrations. Several analogues show promise; the most active compound, 2a, inhibits the formation of PrP-res in SMB cells with an EC50 of 25-50 nM.

Animals↗

Blood pressure power within frequency range approximately 0.4 Hz in rat conforms to self-similar scaling following spinal cord transection.

This study quantified the effect of interrupting the descending input to the sympathetic preganglionic neurons on the dynamic behavior of arterial blood pressure (BP) in the unanesthetized rat. BP was recorded for approximately 4-h intervals in six rats in the neurally intact state and in the same animals after complete spinal cord transection (SCT) between T(4) and T(5). In the intact state, power within the frequency range of 0.35-0.45 Hz was 1.53 +/- 0.38 mmHg(2)/Hz (mean +/- SD by fast Fourier transform). One week after SCT, power within this range decreased significantly (P < 0.05) to 0.43 +/- 0.62 mmHg(2)/Hz. To test for self-similarity before and after SCT, we analyzed data using a wavelet (i.e., functionally, a digital bandpass filter) tuned to be maximally sensitive to fluctuations with periods of approximately 2, 4, 8, 16, 32, or 64 s. In the control state, all fluctuations with periods of >/=4 s conformed to a "self-similar" (i.e., fractal) distribution. In marked contrast, the oscillations with a period of approximately 2 s (i.e., approximately 0.4 Hz) were significantly set apart from those at lower frequencies. One day and seven days after the complete SCT, however, the BP fluctuations at approximately 0.4 Hz now also conformed to the same self-similar behavior characteristic of the lower frequencies. We conclude that 1) an intact sympathetic nervous system endows that portion of the power spectrum centered around approximately 0.4 Hz with properties (e.g., a periodicity) that differ significantly from the self-similar behavior that characterizes the lower frequencies and 2) even within the relatively high frequency range at 0.4 Hz self-similarity is the "default" condition after sympathetic influences have been eliminated.

Animals↗

Activation of the systemic and adipose renin-angiotensin system in rats with diet-induced obesity and hypertension.

In obesity-related hypertension, activation of the renin-angiotensin system (RAS) has been reported despite marked fluid volume expansion. Adipose tissue expresses components of the RAS and is markedly expanded in obesity. This study evaluated changes in components of the adipose and systemic RAS in diet-induced obese hypertensive rats. RAS was quantified in adipose tissue and compared with primary sources for the circulating RAS. Male Sprague-Dawley rats were fed either a low-fat (LF; 11% kcal as fat) or moderately high-fat (32% kcal as fat) diet for 11 wk. After 8 wk, rats fed the moderately high-fat diet segregated into obesity-prone (OP) and obesity-resistant (OR) groups based on their body weight gain (body weight: OR, 566 +/- 10; OP, 702 +/- 20 g; P < 0.05). Mean arterial blood pressure was increased in OP rats (LF: 97 +/- 2; OR: 97 +/- 2; OP: 105 +/- 1 mmHg; P < 0.05). Quantification of mRNA expression by real-time PCR demonstrated a selective increase (2-fold) in angiotensinogen gene expression in retroperitoneal adipose tissue from OP vs. OR and LF rats. Similarly, plasma angiotensinogen concentration was increased in OP rats (LF: 390 +/- 48; OR: 355 +/- 24; OP: 530 +/- 22 ng/ml; P < 0.05). In contrast, other components of the RAS were not altered in OP rats. Marked increases in the plasma concentrations of angiotensin peptides were observed in OP rats (angiotensin II: LF: 95 +/- 31; OR: 59 +/- 20; OP: 295 +/- 118 pg/ml; P < 0.05). These results demonstrate increased activity of the adipose and systemic RAS in obesity-related hypertension.

Adipose Tissue↗

Preferential Cu2+ coordination by His96 and His111 induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein.

The prion protein (PrP) is a Cu(2+) binding cell surface glycoprotein that can misfold into a beta-sheet-rich conformation to cause prion diseases. The majority of copper binding studies have concentrated on the octarepeat region of PrP. However, using a range of spectroscopic techniques, we show that copper binds preferentially to an unstructured region of PrP between residues 90 and 115, outside of the octarepeat domain. Comparison of recombinant PrP with PrP-(91-115) indicates that this prion fragment is a good model for Cu(2+) binding to the full-length protein. In contrast to previous reports we show that Cu(2+) binds to this region of PrP with a nanomolar dissociation constant. NMR and EPR spectroscopy indicate a square-planar or square-pyramidal Cu(2+) coordination utilizing histidine residues. Studies with PrP analogues show that the high affinity site requires both His(96) and His(111) as Cu(2+) ligands, rather than a complex centered on His(96) as has been previously suggested. Our circular dichroism studies indicate a loss of irregular structure on copper coordination with an increase in beta-sheet conformation. It has been shown that this unstructured region, between residues 90 and 120, is vital for prion propagation and different strains of prion disease have been linked with copper binding. The role of Cu(2+) in prion misfolding and disease must now be re-evaluated in the light of these findings.

Amyloid↗

Biological inorganic and bioinorganic chemistry of neurodegeneration based on prion and Alzheimer diseases.

A change of the prion protein conformation results in a class of neurodegenerative diseases called the transmissible spongiform encephalopathies (like mad cow and Creutzfeld-Jakob diseases). The function of the normal prion protein is unknown, although much of recent research demonstrates the it may be a copper binding protein selective for Cu(II). Amyloid precursor protein (APP) releases the 39-42 amino acid peptide, a major constituent of the deposit in plaques of Alzheimer disease brain. Also APP is a metal binding protein, including copper ions. The link between copper and both proteins may provide insight into the role of metals in neurodegenerative pathologies.

Alzheimer Disease↗

Prion protein is ubiquitinated after developing protease resistance in the brains of scrapie-infected mice.

Although the key event in the pathology of prion diseases is thought to be the conversion of cellular prion protein (PrP(C)) to the protease-resistant scrapie species termed PrP(Sc), the factors that contribute to neurodegeneration in scrapie-infected animals are poorly understood. One probable determinant could be when the accumulation of PrP(Sc) in infected brain overwhelms the ubiquitin-proteasome system and triggers the degenerative cascade. In the present study, it was found that in mouse brains infected with the ME7 scrapie strain, the level of ubiquitin protein conjugates increased significantly at approximately 144 days post-infection (pi) when clinical signs first become apparent. This elevation correlated with the detection of protease-resistant PrP(Sc) and a decline in two endopeptidase activities associated with proteasome function. However, ubiquitination of PrP was only detected at the terminal stage, 3 weeks after the development of clinical symptoms (approximately 165 days pi). These results suggest that ubiquitination of PrP is a late event phenomenon and this conjugation occurs after the formation of protease-resistant PrP(Sc). Whether this post-translational modification and the impairment of proteasome function are pivotal events in the pathogenesis of prion diseases remains to be determined.

Animals↗

BSE and vCJD cause disturbance to uric acid levels.

Bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) are two new members of the family of neurodegenerative conditions termed prion diseases. Oxidative damage has been shown to occur in prion diseases and is potentially responsible for the rapid neurodegeneration that is central to the pathogenesis of these diseases. An important nonenzymatic antioxidant in the brain is uric acid. Analysis of uric acid in the brain and cerebrospinal fluid (CSF) of cases of BSE and CJD showed a specific reduction in CSF levels for both BSE and variant CJD, but not sporadic CJD. Further studies based on cell culture experiments suggested that uric acid in the brain was produced by microglia. Uric acid was also shown to inhibit neurotoxicity of a prion protein peptide, production of the abnormal prion protein isoform (PrP(Sc)) by infected cells, and polymerization of recombinant prion protein. These findings suggest that changes in uric acid may aid differential diagnosis of vCJD. Uric acid could be used to inhibit cell death or PrP(Sc) formation in prion disease.

Animals↗

Copper(II) complexes of peptide fragments of the prion protein. Conformation changes induced by copper(II) and the binding motif in C-terminal protein region.

In this paper, we report the characterization of copper(II) complexes with two prion (PrP) protein peptide fragment analogues (VNITKQHTVTTTT), one with the N-terminus acetylated and the C-terminus amidated (PrP Ac180-193NH2) and the other with both the C- and N-termini free (PrP 180-193). Such peptide sequence almost entirely encompasses the PrPC's helix 2 in the C-terminal region. The stoichiometry, the binding modes and the conformational features of the copper(II) complexes with the above mentioned two peptides were investigated by electrospray ionization-mass spectrometry (ESI-MS), UV-visible (UV-Vis) spectrometry and electron paramagnetic resonance (EPR) spectrometry as well as by circular dichroism (CD) measurements. The binding site location of copper(II) in the structured region of the protein can be here suggested on the basis of our findings that show the involvement of His 187 residue. The similarity of the EPR parameters suggests that the anchoring imidazole residue drives the copper(II) coordination environment towards a common binding motif in different regions of the prion protein.

Amino Acid Motifs↗