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Biomedical subjects

C J Campbell

Publications and source records attributed to C J Campbell.

At least 19 recordsLinked to original sources
PubMed2006

Time-resolved FRET and FLIM of four-way DNA junctions.

Conformational transitions in a 4-way DNA junction when titrated with ionic solutions are studied using time-resolved fluorescence resonance energy transfer. Parameters characterising the transition in terms of critical ion concentration (c1/2) and the Hill coefficient for ion binding are obtained by fitting a simple two-state model using steady-state spectra. Data obtained from a fluorescence lifetime plate reader and analysed by fitting a single exponential to donor fluorescence lifetime decays are shown to be in good agreement with the parameters obtained from steady-state measurements. Fluorescence lifetimes, however, offer advantages, particularly in being independent of fluorophore concentration, output intensity, inhomogeneity in the excitation source and output wavelength. We demonstrate preliminary FRET-FLIM images of DNA junction solutions obtained using a picosecond gated CCD which are in agreement with results from a fluorescence lifetime plate reader. The results suggest that time-resolved FRET-FLIM is sensitive to subtle structural changes and may be useful in assays based on 4-way DNA junctions.

DNA, Cruciform↗
PubMed2006

The stability and characteristics of a DNA Holliday junction switch.

A Holliday junction (HJ) consists of four DNA double helices, with a branch point discontinuity at the intersection of the component strands. At low ionic strength, the HJ adopts an open conformation, with four widely spaced arms, primarily due to strong electrostatic repulsion between the phosphate groups on the backbones. At high ionic strength, screening of this repulsion induces a switch to a more compact (closed) junction conformation. Fluorescent labelling with dyes placed on the HJ arms allows this conformational switch to be detected optically using fluorescence resonance energy transfer (FRET), producing a sensitive fluorescent output of the switch state. This paper presents a systematic and quantitative survey of the switch characteristics of such a labelled HJ. A short HJ (arm length 8 bp) is shown to be prone to dissociation at low switching ion concentration, whereas an HJ of arm length 12 bp is shown to be stable over all switching ion concentrations studied. The switching characteristics of this HJ have been systematically and quantitatively studied for a variety of switching ions, by measuring the required ion concentration, the sharpness of the switching transition and the fluorescent output intensity of the open and closed states. This stable HJ is shown to have favourable switch characteristics for a number of inorganic switching ions, making it a promising candidate for use in nanoscale biomolecular switch devices.

DNA, Cruciform↗
PubMed2004

Multicolour micropatterning of thin films of dry gels.

Micropatterning of surfaces with several chemicals at different spatial locations usually requires multiple stamping and registration steps. Here, we describe an experimental method based on reaction-diffusion phenomena that allows for simultaneous micropatterning of a substrate with several coloured chemicals. In this method, called wet stamping (WETS), aqueous solutions of two or more inorganic salts are delivered onto a film of dry, ionically doped gelatin from an agarose stamp patterned in bas relief. Once in conformal contact, these salts diffuse into the gelatin, where they react to give deeply coloured precipitates. Separation of colours in the plane of the surface is the consequence of the differences in the diffusion coefficients, the solubility products, and the amounts of different salts delivered from the stamp, and is faithfully reproduced by a theoretical model based on a system of reaction-diffusion partial differential equations. The multicolour micropatterns are useful as non-binary optical elements, and could potentially form the basis of new applications in microseparations and in controlled delivery.

Chlorides↗
PubMed2004

Absorption of water by thin, ionic films of gelatin.

This paper discusses absorption of water by thin, dry films of gelatin. Experiments using a wet-stamping technique were performed to characterize water uptake in terms of (i) equilibrium profiles of the water density inside the gel and (ii) the kinetics of water absorption. It was found that, in contrast to pure gelatin films, which absorb water approximately uniformly, films of gelatin doped with ionic additives have exponentially decaying equilibrium water profiles. The process of water absorption by both doped and undoped gels was described by a theoretical model based on the minimization of grand potential functional. The results of this model are in agreement with the experiment.

Gelatin↗
PubMed2001

The transcriptional corepressor NAB2 blocks Egr-1-mediated growth factor activation and angiogenesis.

Effective tissue repair results from a rapid, temporally orchestrated series of events. At the site of local tissue injury, the production of many growth factors and cytokines is, in part, stimulated by the early growth response transcription factors such as Egr-1. Egr-1 protein binds to a family of corepressor proteins called NAB which function to block or limit Egr-1 trans-activation of cognate target genes. NAB2 blocks Egr-1 activation of the tissue factor (TF) promoter, Egr-1 stimulated production of PDGF-AB, HGF, TGFbeta(1), and VEGF and the endogenous expression of PDGF-AB and TGFbeta(1). Expression of a wild-type NAB2 but not a dominant negative NAB2 mutant abrogates Egr-1 driven TF promoter activity and tubule formation in an in vitro model of angiogenesis. These findings may have importance in any tissue that is subject to scarring after acute or chronic injury.

Cells, Cultured↗
PubMed2001

Homing markers for atherosclerosis: applications for drug delivery, gene delivery and vascular imaging.

Endothelial dysfunction plays a major role in the pathogenesis of atherosclerosis. Pro-inflammatory cytokines such as interleukin-1 beta and tumour necrosis factor alpha activate endothelial cells changing their resting phenotype to become pro-adhesive, pro-thrombotic and pro-atherogenic. Phage display in vivo biopanning has been used to identify peptide sequences that home to diseased regions of the vessel wall in low density lipoprotein receptor (LDLr) knockout mice. In LDLr knockout mice, peptide sequence determinants exhibiting organ specificity have been isolated. These sequences have applications for gene delivery, drug delivery and for improving contrast agents for vascular imaging.

Animals↗
PubMed2001

Fecal analysis of ovarian cycles in female black-handed spider monkeys (Ateles geoffroyi).

An enzyme immunoassay (EIA) was applied to characterize the reproductive endocrinology of adult female black-handed spider monkeys (Ateles geoffroyi). Analysis of paired urine and fecal samples, collected from two females housed at San Diego Zoo, confirmed that the EIAs employed provided quantitative measurements of ovarian sex steroid hormones. Fecal metabolite levels were significantly correlated with those in urine, confirming that feces are a valid source of steroid metabolites in this species. The excretion of these metabolites in feces lagged urinary excretion by 1-2 days. The ovarian cycle profiles of the two captive females and five free-ranging females are comparable, with an average length of approximately 20-23 days. Cyclical bleeding, as previously reported, was observed in one of the two captive females. Pregnancy was detected in four free-ranging females, and early fetal loss for one female was indicated by hormonal data.

Animals↗
PubMed2001

Particle diameter influences adhesion under flow.

The diameter of circulating cells that may adhere to the vascular endothelium spans an order of magnitude from approximately 2 microm (e.g., platelets) to approximately 20 microm (e.g., a metastatic cell). Although mathematical models indicate that the adhesion exhibited by a cell will be a function of cell diameter, there have been few experimental investigations into the role of cell diameter in adhesion. Thus, in this study, we coated 5-, 10-, 15-, and 20-microm-diameter microspheres with the recombinant P-selectin glycoprotein ligand-1 construct 19.ek.Fc. We compared the adhesion of the 19.ek.Fc microspheres to P-selectin under in vitro flow conditions. We found that 1) at relatively high shear, the rate of attachment of the 19.ek.Fc microspheres decreased with increasing microsphere diameter whereas, at a lower shear, the rate of attachment was not affected by the microsphere diameter; 2) the shear stress required to set in motion a firmly adherent 19.ek.Fc microsphere decreased with increasing microsphere diameter; and 3) the rolling velocity of the 19.ek.Fc microspheres increased with increasing microsphere diameter. These results suggest that attachment, rolling, and firm adhesion are functions of particle diameter and provide experimental proof for theoretical models that indicate a role for cell diameter in adhesion.

Animals↗
PubMed2000

Tissue repair with a therapeutic transcription factor.

The healing of tissue involves a wide range of molecular, cellular, and physiological events that are coordinated in a temporally specific manner. The cellular transcription factor early growth response factor 1 (Egr-1) is expressed minutes after acute injury and serves to stimulate the production of a class of growth factors whose role is to promote tissue repair. We have studied the effects of Egr-1 expression at the site of dermal wounding in rodents. We find that Egr-1 promotes angiogenesis in vitro and in vivo, increases collagen production, and accelerates wound closure. These results show that Egr-1 gene therapy accelerates the normal healing process and raises the potential use of this therapeutic transcription factor for any aspect of tissue repair.

Animals↗
PubMed2000

Craniometric variation in a population of mantled howler monkeys (Alouatta palliata): evidence of size selection in females and growth in dentally mature males.

A large body of work on monkey cranial metrics (involving conclusions about interspecific variation, sexual dimorphism, and ontogeny) depends on the assumptions that growth effectively ceases with dental maturity and that intraspecific variation is negligible. We test these assumptions by examining variation in 39 measurements of 166 dentally mature Alouatta palliata skulls from animals found dead on Barro Colorado Island (BCI), Panama. We also investigate whether this population is under size-based selection, since our found-dead sample reflects the natural mortality in this population. The sample was divided into three age stages by occlusal wear (A-C, least to most wear). Female stage A means are significantly smaller than female stage B means for three cranial measures. Female stage B means are significantly smaller than female stage C means for five cranial measures. Male stage A means are significantly smaller than male stage B means for 21 cranial measures. Multivariate analyses confirm this trend of expansion between adult age stages. The dental metric and suture closure data suggest that the cranial expansion in females is due to size-based selection, while the cranial expansion in males is due to significant growth after dental maturity. Sexual dimorphism ratios are highly variable across different samples of A. palliata, indicating that dimorphism varies between populations of this species. These results provide insight into the selective forces operating on the BCI howlers and challenge the validity of the many studies which pool subspecies and assume growth ceases with maturity.

Aging↗
PubMed2000

Fur rubbing behavior in free-ranging black-handed spider monkeys (Ateles geoffroyi) in Panama.

Members of the population of black-handed spider monkeys (Ateles geoffroyi) on Barro Colorado Island, Panama, use the leaves of three Rutaceae species in a behavior that resembles fur rubbing in the white faced capuchin (Cebus capucinus). This behavior has not been reported from other sites where Ateles has been studied. During more than 1,200 hours of observation, 30 episodes of this behavior were recorded ad libitum. Adult males engage in this behavior more than adult females. Season did not impact the frequency of the behavior. The behavior described here differs in many respects from that reported for Cebus capucinus, and does not fit the hypotheses that the behavior functions in repelling insects or other antiseptic purposes. It is proposed that fur rubbing in this group of spider monkeys is a modification of a behavior previously recorded in Ateles and may function in scent marking.

Animal Communication↗
PubMed2000

CD11b/CD18-coated microspheres attach to E-selectin under flow.

Neutrophils can attach to E-selectin under flow. Proposed ligands for E-selectin carry SLe(x)-type glycans. The leukocyte beta2 integrins are glycosylated with SLe(x). Thus, we speculated that beta2 integrins could support attachment to E-selectin. To test this hypothesis, we coated 10-microm-diameter microspheres with purified CD11b/CD18 (alphaMbeta2) and investigated the adhesion of the resulting alphaMbeta2 microspheres to E-selectin. Under in vitro flow conditions, the alphaMbeta2 microspheres attached to Chinese hamster ovary cells expressing E-selectin (CHO-E) and 4-h interleukin-1beta-activated human umbilical vein endothelial cells (HUVEC). At a shear stress of 1.8 dynes/cm2, the attachment events were eliminated by pretreatment of the cellular monolayers with a mAb to E-selectin. alphaMbeta2 microspheres did not attach to untransfected CHO cells or unactivated HUVEC at 1.8 dynes/cm2. Taken together, the results strongly suggest that the CD11b/CD18-E-selectin bond has sufficient biophysical properties to mediate attachment of neutrophil-sized particles to E-selectin under flow.

Animals↗
PubMed2000

Full promoter sequence of human early growth response factor-1 (Egr-1): demonstration of a fifth functional serum response element.

The early growth response factor-1 (Egr-1) gene encodes a zinc finger transcription factor which is critical for cell proliferation and differentiation. The human Egr-1 promoter comprises regulatory elements including two Sp1 sites, an AP1 site, two cAMP response elements and an Egr-1 binding site. In addition to these transcription factor binding sites, the promoter harbours five serum response elements (SREs) and associated binding sites for the Ets transcription factor family, previously identified from partial sequence data (Sakamoto et al, Oncogene 6; 867-871, 1991). We now report the full sequence of the human Egr-1 promoter and confirm the presence of a fifth serum response element. This element is functionally active in a minimal promoter vector in response to the MAP kinase kinase MEK1.

Base Sequence↗
PubMed1999

Delivery and expression of fluid shear stress-inducible promoters to the vessel wall: applications for cardiovascular gene therapy.

In atherosclerosis, endothelial cells at sites of stenosis experience elevated levels of shear stress. We have constructed a series of shear stress-inducible transcription units (SITUs) expressing the luciferase reporter gene and determined their activation by fluid shear stress in transfected endothelial cells. Chimeric promoters were constructed that comprised basal transcription factor-binding sites coupled to a shear stress response element (SSRE). We have used consensus binding sites for transcription factors NF-kappaB, Ap1, Sp1, Oct1, and Egr-1/Sp1 in either the presence or absence of the previously defined "GAGACC" SSRE. The response of the promoters to shear stress was determined after transfection into human umbilical vein endothelial cells (HUVECs). After transient transfection into HUVECs, fluid shear stress activated the promoters by between two- and eightfold. The most responsive SITUs comprised an overlapping Sp1/Egr-1-binding site linked to a TATA box with (SP5) or without (SP7) the GAGACC SSRE. Instillation of SP5 DNA in vivo into the left carotid artery of rabbit and subsequent generation of a stenosis using a mechanical wire occluder caused a 10-fold upregulation of luciferase reporter gene expression at the site of vessel occlusion. These vectors show promise for therapeutic gene expression at sites of occlusive vascular disease.

Animals↗
PubMed1999

Evaluation of a multidimensional measure of dyspepsia-related health for use in a randomized clinical trial.

In previous work, we developed a multidimensional measure of dyspepsia-related health. To evaluate the adequacy of this instrument as an outcome measure for a large-scale, multicenter, randomized clinical trial, we used Rasch analysis to address three questions: (1) Are the scales interval-level? (2) Do the scales measure precisely across the entire range of dyspepsia outcomes? (3) Do the scales' items have an optimal number of response categories? We found that the scales were not interval-level and that they did not measure effectively at low or high levels of the dyspepsia-related outcomes. Our results also suggest that patients were capable of discriminating among only four- to seven-item response categories. Further studies are needed to identify items that effectively measure high and low levels of dyspepsia-related outcomes and to validate that decreasing the number of response categories improves the psychometric properties of these scales.

Adult↗