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1.  Uncertainty in dual permeability model parameters for structured soils 
Water resources research  2012;48(1):WR010500-.
Successful application of dual permeability models (DPM) to predict contaminant transport is contingent upon measured or inversely estimated soil hydraulic and solute transport parameters. The difficulty in unique identification of parameters for the additional macropore- and matrix-macropore interface regions, and knowledge about requisite experimental data for DPM has not been resolved to date. Therefore, this study quantifies uncertainty in dual permeability model parameters of experimental soil columns with different macropore distributions (single macropore, and low- and high-density multiple macropores). Uncertainty evaluation is conducted using adaptive Markov chain Monte Carlo (AMCMC) and conventional Metropolis-Hastings (MH) algorithms while assuming 10 out of 17 parameters to be uncertain or random. Results indicate that AMCMC resolves parameter correlations and exhibits fast convergence for all DPM parameters while MH displays large posterior correlations for various parameters. This study demonstrates that the choice of parameter sampling algorithms is paramount in obtaining unique DPM parameters when information on covariance structure is lacking, or else additional information on parameter correlations must be supplied to resolve the problem of equifinality of DPM parameters. This study also highlights the placement and significance of matrix-macropore interface in flow experiments of soil columns with different macropore densities. Histograms for certain soil hydraulic parameters display tri-modal characteristics implying that macropores are drained first followed by the interface region and then by pores of the matrix domain in drainage experiments. Results indicate that hydraulic properties and behavior of the matrix-macropore interface is not only a function of saturated hydraulic conductivity of the macroporematrix interface (Ksa) and macropore tortuosity (lf) but also of other parameters of the matrix and macropore domains.
PMCID: PMC3903292  PMID: 24478531
2.  Protein Markers for the Differential Diagnosis of Vascular Dementia and Alzheimer's Disease 
Alzheimer's disease (AD) is the most common form of dementia found in all human populations worldwide, while vascular dementia (VaD) is the second most common form of dementia. New biomarkers for early and specific diagnosis of AD and VaD are needed to achieve greater insight into changes occurring in the brain and direct therapeutic strategies. The objective of this explorative study was to discover candidate protein biomarkers for the differential diagnosis between VaD and AD. Surface-enhanced laser desorption/ionization (SELDI) TOF-MS was used to differentially profile proteins and peptides in CSF samples from 28 AD patients and 21 patients with VaD. A combination of univariate (Kruskal-Wallis) and multivariate (independent component analysis) statistical approaches produced a list of 27 proteins and peptides that could differentiate between VaD and AD. These markers represent various physiological processes, such as protein degradation (ubiquitin), protease inhibition (cystatin C and alpha-1-antichymoptrypsin), and inflammation (C3a and C4a) that are known to be represented in neurodegenerative diseases.
PMCID: PMC3373063  PMID: 22701795
3.  Effort discounting in human nucleus accumbens 
A great deal of behavioral and economic research suggests that the value attached to a reward stands in inverse relation to the amount of effort required to obtain it, a principle known as effort discounting. In the current report, we present the first direct evidence for a neural analogue of effort discounting. Functional magnetic resonance imaging was used to measure neural responses to monetary rewards in the human nucleus accumbens, a structure previously demonstrated to encode reference-dependent reward information. The magnitude of accumbens activation was found to vary with reward outcome, but also with the degree of mental effort demanded to obtain individual rewards. For a fixed level of reward, accumbens was less strongly activated following a high demand for effort than following a lower demand. The magnitude of this effect was noted to correlate with preceding activation in the dorsal anterior cingulate cortex, a region that has been proposed to monitor information-processing demands and to mediate in the subjective experience of effort.
PMCID: PMC2744387  PMID: 19246324
4.  The role of tyrosine phosphorylation in lipopolysaccharide- and zymosan-induced procoagulant activity and tissue factor expression in macrophages. 
Infection and Immunity  1997;65(6):2362-2370.
The expression of surface procoagulants by exudative macrophages represents an important mechanism underlying local fibrin deposition at sites of extravascular inflammation. The present studies investigated the contribution of tyrosine phosphorylation to the generation of macrophage procoagulant activity (PCA) and tissue factor expression in response to proinflammatory stimuli. Both lipopolysaccharide (LPS) and zymosan rapidly stimulated tyrosine phosphorylation in elicited murine peritoneal macrophages. This effect was prevented by the tyrosine kinase inhibitors genistein and herbimycin and augmented by the addition of the phosphotyrosine phosphatase inhibitor vanadate. The vanadate-mediated rise in phosphotyrosine accumulation was abrogated by the use of diphenylene iodonium, an inhibitor of the respiratory burst oxidase, suggesting a role for peroxides of vanadate as contributors to the tyrosine phosphorylation. This notion was supported by the finding that vanadyl hydroperoxide markedly increased the accumulation of phosphotyrosine residues. To define the role of tyrosine phosphorylation in the induction of macrophage PCA by LPS, the effects of tyrosine kinase inhibition by genistein and herbimycin were investigated. Both agents inhibited the expression of macrophage PCA. Further, Northern blot analysis with the cDNA probe for murine tissue factor indicated that the inhibition occurred at the mRNA level or earlier. Since vanadate augmented phosphotyrosine accumulation, it was hypothesized that it might enhance generation of macrophage products. However, vanadate reduced induction of PCA in response to LPS. By contrast, vanadate augmented basal prostaglandin E2 (PGE2) release and stimulated PGE2 release by macrophages. Indomethacin prevented the increase in PGE2 but only partially restored normal levels of PCA. The effect of vanadate on tissue factor expression appeared to be posttranscriptional. These studies thus demonstrate, by functional Western blotting and Northern blotting techniques, that tyrosine phosphorylation plays a role in the regulation of macrophage PCA and tissue factor expression in response to proinflammatory stimuli.
PMCID: PMC175327  PMID: 9169775
5.  Functional interference between hypoxia and dioxin signal transduction pathways: competition for recruitment of the Arnt transcription factor. 
Molecular and Cellular Biology  1996;16(10):5221-5231.
Hypoxia-inducible factor 1 alpha (HIF-1 alpha) and the intracellular dioxin receptor mediate hypoxia and dioxin signalling, respectively. Both proteins are conditionally regulated basic helix-loop-helix (bHLH) transcription factors that, in addition to the bHLH motif, share a Per-Arnt-Sim (PAS) region of homology and form heterodimeric complexes with the common bHLH/PAS partner factor Arnt. Here we demonstrate that HIF-1 alpha required Arnt for DNA binding in vitro and functional activity in vivo. Both the bHLH and PAS motifs of Arnt were critical for dimerization with HIF-1 alpha. Strikingly, HIF-1 alpha exhibited very high affinity for Arnt in coimmunoprecipitation assays in vitro, resulting in competition with the ligand-activated dioxin receptor for recruitment of Arnt. Consistent with these observations, activation of HIF-1 alpha function in vivo or overexpression of HIF-1 alpha inhibited ligand-dependent induction of DNA binding activity by the dioxin receptor and dioxin receptor function on minimal reporter gene constructs. However, HIF-1 alpha- and dioxin receptor-mediated signalling pathways were not mutually exclusive, since activation of dioxin receptor function did not impair HIF-1 alpha-dependent induction of target gene expression. Both HIF-1 alpha and Arnt mRNAs were expressed constitutively in a large number of human tissues and cell lines, and these steady-state expression levels were not affected by exposure to hypoxia. Thus, HIF-1 alpha may be conditionally regulated by a mechanism that is distinct from induced expression levels, the prevalent model of activation of HIF-1 alpha function. Interestingly, we observed that HIF-1 alpha was associated with the molecular chaperone hsp90. Given the critical role of hsp90 for ligand binding activity and activation of the dioxin receptor, it is therefore possible that HIF-1 alpha is regulated by a similar mechanism, possibly by binding an as yet unknown class of ligands.
PMCID: PMC231522  PMID: 8816435
6.  Prenatal diagnosis for recessive dystrophic epidermolysis bullosa in 10 families by mutation and haplotype analysis in the type VII collagen gene (COL7A1). 
Molecular Medicine  1996;2(1):59-76.
BACKGROUND: Epidermolysis bullosa (EB) is a group of heritable diseases that manifest as blistering and erosions of the skin and mucous membranes. In the dystrophic forms of EB (DEB), the diagnostic hallmark is abnormalities in the anchoring fibrils, attachment structures beneath the cutaneous basement membrane zone. The major component of anchoring fibrils is type VII collagen, and DEB has been linked to the type VII collagen gene (COL7A1) at 3p21, with no evidence for locus heterogeneity. Due to life-threatening complications and significant long-term morbidity associated with the severe, mutilating form of recessive dystrophic EB (RDEB), there has been a demand for prenatal diagnosis from families with affected offspring. MATERIALS AND METHODS: Intragenic polymorphisms in COL7A1 and flanking microsatellite markers on chromosome 3p21, as well as detection of pathogenetic mutations in families, were used to perform PCR-based prenatal diagnosis from DNA obtained by chorionic villus sampling at 10-15 weeks or amniocentesis at 12-15 weeks gestation in 10 families at risk for recurrence of RDEB. RESULTS: In nine cases, the fetus was predicted to be normal or a clinically unaffected carrier of a mutation in one allele. These predictions have been validated in nine cases by the birth of a healthy child. In one case, an affected fetus was predicted, and the diagnosis was confirmed by fetal skin biopsy. CONCLUSIONS: DNA-based prenatal diagnosis of RDEB offers an early, expedient method of testing which will largely replace the previously available invasive fetal skin biopsy at 18-20 weeks gestation.
PMCID: PMC2230038  PMID: 8900535
7.  Adhesion of Listeria monocytogenes to silica surfaces after sequential and competitive adsorption of bovine serum albumin and beta-lactoglobulin. 
Adsorbed bovine serum albumin was resistant to exchange with beta-lactoglobulin, and when albumin was adsorbed from a mixture, its surface concentration increased with time. The passivating character of adsorbed albumin and its resistance to desorption were consistent with the level of Listeria monocytogenes adhesion evoked by albumin-containing protein films.
PMCID: PMC167467  PMID: 7646040
8.  Suppression of Listeria monocytogenes colonization following adsorption of nisin onto silica surfaces. 
Nisin is an antimicrobial peptide proven to be an effective inhibitor of gram-positive bacteria. It is known that nisin can adsorb to various surfaces and still retain much of its original activity (M. A. Daeschel, J. McGuire, and H. Al-Makhlafi, J. Food Prot. 55:731-735, 1992). In this study, nisin films were allowed to form on silanized silica surfaces and then exposed to medium containing Listeria monocytogenes. Representative areas were selected from each surface, and images of resident listeriae were obtained at 4-h intervals for 12 h. During this time, cells on surfaces that had been in contact with a high concentration of nisin (1.0 mg/ml) exhibited no signs of growth and many displayed evidence of cellular deterioration. Surfaces treated with a lower concentration of nisin (0.1 mg/ml) had a smaller degree of inhibition. In contrast, both protein-free surfaces and those with films of heat-inactivated nisin allowed attached L. monocytogenes cells to grow and reproduce. These studies, when repeated with a nisin-resistant strain of L. monocytogenes, resulted in no inhibition of growth on surfaces with adsorbed nisin. The bactericidal effect of adsorbed nisin was also studied with iodonitrotetrazolium violet, a tetrazolium salt, which is reduced to a red formazan crystal by viable bacteria. Crystals were visible in 95% of the cells adhered to control surfaces but were present in less than 20% of the cells on surfaces with adsorbed nisin. These data indicate that adsorbed nisin may have potential for use as a food grade antimicrobial agent on food contact surfaces.
PMCID: PMC167359  PMID: 7793927
9.  Distinct roles of the molecular chaperone hsp90 in modulating dioxin receptor function via the basic helix-loop-helix and PAS domains. 
Molecular and Cellular Biology  1995;15(2):756-765.
The intracellular dioxin receptor mediates signal transduction by dioxin and functions as a ligand-activated transcription factor. It contains a basic helix-loop-helix (bHLH) motif contiguous with a Per-Arnt-Sim (PAS) homology region. In extracts from nonstimulated cells the receptor is recovered in an inducible cytoplasmic form associated with the 90-kDa heat shock protein (hsp90), a molecular chaperone. We have reconstituted ligand-dependent activation of the receptor to a DNA-binding form by using the dioxin receptor and its bHLH-PAS partner factor Arnt expressed by in vitro translation in reticulocyte lysate. Deletion of the PAS domain of the receptor resulted in constitutive dimerization with Arnt. In contrast, this receptor mutant showed low levels of xenobiotic response element-binding activity, indicating that the PAS domain may be important for DNA-binding affinity and/or specificity of the receptor. It was not possible to reconstitute dioxin receptor function with proteins expressed in wheat germ lysate. In line with these observations, reticulocyte lysate but not wheat germ lysate promoted the association of de novo synthesized dioxin receptor with hsp90. At least two distinct domains of the receptor mediated interaction with hsp90: the ligand-binding domain located within the PAS region and, surprisingly, the bHLH domain. Whereas ligand-binding activity correlated with association with hsp90, bHLH-hsp90 interaction appeared to be important for DNA-binding activity but not for dimerization of the receptor. Several distinct roles for hsp90 in modulating dioxin receptor function are therefore likely: correct folding of the ligand-binding domain, interference with Arnt heterodimerization, and folding of a DNA-binding conformation of the bHLH domain. Thus, the dioxin receptor system provides a complex and interesting model of the regulation of transcription factors by hsp90.
PMCID: PMC231944  PMID: 7823943
10.  Influence of preadsorbed milk proteins on adhesion of Listeria monocytogenes to hydrophobic and hydrophilic silica surfaces. 
Applied and Environmental Microbiology  1994;60(10):3560-3565.
The adsorption of beta-lactoglobulin, bovine serum albumin, alpha-lactalbumin, and beta-casein for 8 h and beta-lactoglobulin and bovine serum albumin for 1 h at silanized silica surfaces of low and high hydrophobicity, followed by incubation in buffer and contact with Listeria monocytogenes, resulted in different numbers of cells adhered per unit of surface area. Adhesion to both surfaces was greatest when beta-lactoglobulin was present and was lowest when bovine serum albumin was present. Preadsorption of alpha-lactalbumin and beta-casein showed an intermediate effect on cell adhesion. Adsorption of beta-lactoglobulin for 1 h resulted in a generally lower number of cells adhered compared with the 8-h adsorption time, while the opposite result was observed with respect to bovine serum albumin. The adhesion data were explainable in terms of the relative rates of arrival to the surface and postadsorptive conformational change among the proteins, in addition to the extent of surface coverage in each case.
PMCID: PMC201855  PMID: 7986033
11.  A cellular factor stimulates ligand-dependent release of hsp90 from the basic helix-loop-helix dioxin receptor. 
Molecular and Cellular Biology  1994;14(4):2438-2446.
In response to dioxin, the nuclear basic helix-loop-helix (bHLH) dioxin receptor forms a complex with the bHLH partner factor Arnt that regulates target gene transcription by binding to dioxin-responsive sequence motifs. Previously, we have demonstrated that the latent form of dioxin receptor present in extracts from untreated cells is stably associated with molecular chaperone protein hsp90, and Arnt is not a component of this complex. Here, we used a coimmunoprecipitation assay to demonstrate that the in vitro-translated dioxin receptor, but not Arnt, is stably associated with hsp90. Although it showed ligand-binding activity, the in vitro-translated dioxin receptor failed to dissociate from hsp90 upon exposure to ligand. Addition of a specific fraction from wild-type hepatoma cells, however, to the in vitro-expressed receptor promoted dioxin-dependent release of hsp90. This stimulatory effect was mediated via the bHLH dimerization and DNA-binding motif of the receptor. Moreover, ligand-dependent release of hsp90 from the receptor was not promoted by fractionated cytosolic extracts from mutant hepatoma cells which are deficient in the function of bHLH dioxin receptor partner factor Arnt. Thus, our results provide a novel model for regulation of bHLH factor activity and suggest that derepression of the dioxin receptor by ligand-induced release of hsp90 may require bHLH-mediated concomitant recruitment of an additional cellular factor, possibly the structurally related bHLH dimerization partner factor Arnt. In support of this model, addition of in vitro-expressed wild-type Arnt, but not a mutated form of Arnt lacking the bHLH motif, promoted release of hsp90 from the dioxin receptor in the presence of dioxin.
PMCID: PMC358611  PMID: 8139547
12.  Pneumocystis carinii pneumonia complicating low dose methotrexate treatment for rheumatoid arthritis. 
Thorax  1991;46(3):205-207.
Low dose methotrexate has been used effectively for various rheumatic and non-rheumatic diseases. Three cases of Pneumocystis carinii pneumonia occurring during treatment of rheumatoid arthritis with low dose methotrexate are presented. Several mechanisms might contribute to impaired immunity and the rare development of opportunist lung infection with methotrexate. A high degree of suspicion may result in earlier diagnosis and treatment.
PMCID: PMC463037  PMID: 2028435
13.  Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation. 
Journal of Clinical Investigation  1987;80(2):430-436.
Interleukin 1 (IL-1) is a family of polypeptides initially found to be produced by activated monocytes and macrophages that mediate a wide variety of cellular responses to injury and infection. Epidermal epithelial cells (keratinocytes) produce "epidermal cell-derived thymocyte activating factor" or ETAF, which has been recently shown to be identical to IL-1. Human epidermis is normally exposed to significant amounts of solar ultraviolet radiation. Certain ultraviolet wavelengths (UVB, 290-320 nm) are thought to be responsible for most of the immediate and long-term pathological consequences of excessive exposure to sunlight. In this study, we asked whether exposure to UVB irradiation induced IL-1 gene expression in cultured human keratinocytes. Cultured human keratinocytes contain detectable amounts of IL-1 alpha and beta mRNA and protein in the absence of apparent stimulation; these levels could be significantly enhanced 6 h after exposure to 10 ng/ml of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Exposure to UVB irradiation with an emission spectrum comparable to that of sunlight (as opposed to that of an unfiltered artificial UV light source) significantly increased the steady state levels IL-1 alpha and beta mRNA in identical populations of human keratinocytes. This was reflected in the production of increased IL-1 activity by these cultures in vitro. In the same cell population, exposures to UVB irradiation did not alter the level of actin mRNA; therefore, the effect of UV irradiation on IL-1 represents a specific enhancement of IL-1 gene expression. Local increases of IL-1 may mediate the inflammation and vasodilation characteristic of acute UVB-injured skin, and systemic release of this epidermal IL-1 may account for fever, leukocytosis, and the acute phase response seen after excessive sun exposure.
PMCID: PMC442255  PMID: 3497177
14.  Anticandidal activities of terconazole, a broad-spectrum antimycotic. 
Terconazole is a new triazole ketal derivative with broad-spectrum in vitro and in vivo antifungal activities. This study further characterizes the effects of terconazole in vitro on yeast cell growth, viability, and morphology. Terconazole inhibited the growth of Candida albicans ATCC 44859 in a concentration-related manner, but with modest effects noted at levels from 10(-8) to 10(-5) M when the yeast was grown on media favoring the cell form. The inhibitory potency of terconazole on yeast cell viability varied with the strain and species of Candida tested. The susceptibility of C. albicans ATCC 44859 to terconazole was markedly enhanced when the yeast was grown on Eagle minimum essential medium, which favors mycelium formation. The effects of terconazole on the morphology of yeast cells (grown on Eagle minimum essential medium) were shown by phase-contrast and electron microscopy. There is a progression of changes, from loss of mycelia formation at 10(-8) M terconazole through complete necrosis at 10(-4) M.
PMCID: PMC180489  PMID: 3729366
15.  Biosynthesis of daunorubicin glycosides: role of epsilon-rhodomycinone. 
Daunorubicin (daunomycin; NSC 82151) is a fermentation-derived anthracycline antibiotic that is clinically useful in the treatment of human leukemias. Daunorubicin itself is found rarely in microbial fermentations, but is present normally in the form of glycoside derivatives that yield the free drug on simple acid hydrolysis. A major by-product of daunorubicin fermentations is usually the structurally related anthracyclinone epsilon-rhodomycinone. We have used mutants of a daunorubicin-producing Streptomyces species to study the biosynthetic relationship between epsilon-rhodomycinone and daunorubicin. We found that exogenously added epsilon-rhodomycinone can be converted to daunorubicin glycosides by a nonproducing mutant and by a mutant that produces daunorubicin glycosides but not epsilon-rhoeomycinone. Molar conversion efficiences were in the 15 to 30% range. The latter mutant was also shown to convert exogenous 14C-labeled epsilon-rhodomycinone to 14C-labeled daunorubicin glycosides, again at conversion efficiencies of about 25%. The same biotransformation was observed with daunorubicin production strain C5, which normally accumulates both epsilon-rhodomycinone and daunorubicin glycosides. A significant percentage (16 to 37%) of exogenously added epsilon-[14C]rhodomycinone was metabolized by strain C5, and 22 to 32% of the metabolized radioactivity could be recovered as daunorubicin glycosides. A mathematical model of epsilon-rhodomycinone metabolism was constructed based on plausible assumptions concerning the kinetics of epsilon-rhodomycinone accumulation and catabolsim. When analyzed according to this model, our data indicate that most (63 to 73%), but not all, of the daunorubicin glycosides accumulated in the experiments with production strain C5 derived from epsilon-rhodomycinone. A pathway network for the biosynthesis of daunorubicin glycosides is proposed that is in agreement with these data. In this proposed pathway network, epsilon-rhodomycinone is an intermediate in one of at least two pathways which yield daunorubicin glycosides.
PMCID: PMC284022  PMID: 7425613

Results 1-23 (23)