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Biomedical subjects

Stephen Lee

Publications and source records attributed to Stephen Lee.

At least 37 records · Page 2Linked to original sources
PubMed2004

HIF activation by pH-dependent nucleolar sequestration of VHL.

Hypoxia and acidosis occur in a wide variety of physiological and pathological settings that include muscle stress, tumour development and ischaemic disorders. A central element in the adaptive response to cellular hypoxia is HIF (hypoxia-inducible factor), a transcription factor that activates an array of genes implicated in oxygen homeostasis, tumour vascularization and ischaemic preconditioning. HIF is activated by hypoxia, but undergoes degradation by the VHL (von Hippel-Lindau) tumour suppressor protein in the presence of oxygen. Here, we demonstrate that hypoxia induction or normoxic acidosis can neutralize the function of VHL by triggering its nucleolar sequestration, a regulatory mechanism of protein function that is observed rarely. VHL is confined to nucleoli until neutral pH conditions are reinstated. Nucleolar sequestration of VHL enables HIF to evade destruction in the presence of oxygen and activate its target genes. Our findings suggest that an increase in hydrogen ions elicits a transient and reversible loss of VHL function by promoting its nucleolar sequestration.

Acidosis↗
PubMed2004

Application of the trak-C HCV core assay for monitoring antiviral activity in HCV replication systems.

The Ortho trak-C immunoassay has recently established detection of the HCV core antigen as a viable indirect marker of HCV replication in clinical samples. In this study, trak-C is used to monitor HCV replication in three pre-clinical models: the cellular HCV replicon system, transient transfection of HCV genomes, and the murine Alb-uPa/SCID HCV infection model. All of these systems utilize full-length HCV genomes that direct the expression of core, facilitating its detection with monoclonal antibodies. When performed with purified protein, the assay detects HCV core with a lower limit of detection at 1.5pg, and exhibits linear detection up to 100pg. When assaying extracts prepared from Huh-7 clone 21-5 cells harboring a full-length HCV replicon, core is detectable from as few as 63 cell equivalents. The assay was used to determine the sensitivity of Huh 21-5 cells to the antiviral effects of interferon (IFN). Inhibition by IFN-alpha using core detection was comparable to that observed using branched-DNA (bDNA 3.0) detection of HCV RNA. Replication of transfected full-length HCV 1a Con1 genomes in Huh-7 cells was also detectable using the trak-C assay. Finally, in the transgenic murine HCV infection model, the course of viral amplification was detected from serum using trak-C with kinetics similar to those observed with RNA detection. Given its ease of use and the lack of requirement for RNA purification, the trak-C assay has several advantages over RNA-based methods of viral monitoring.

Animals↗
PubMed2004

A NotI-EcoRV promoter library for studies of genetic and epigenetic alterations in mouse models of human malignancies.

Aberrant promoter methylation and associated chromatin changes are primarily studied in human malignancies. Thus far, mouse models for human cancer have been rarely utilized to study the role of DNA methylation in tumor onset and progression. It would be advantageous to use mouse tumor models to a greater extent to study the role and mechanism of DNA methylation in cancer because mouse models allow manipulation of the genome, study of samples/populations with a homogeneous genetic background, the possibility of modulating gene expression in vivo, the statistical power of using large numbers of tumor samples, access to various tumor stages, and the possibility of preclinical trials. Therefore, it is likely that the mouse will emerge as an increasingly utilized model to study DNA methylation in cancer. To foster the use of mouse models, we developed an arrayed mouse NotI-EcoRV genomic library, with clones from three commonly used mouse strains (129SvIMJ, FVB/NJ, and C57BL/6J). A total of 23,040 clones representing an estimated three- to fourfold coverage of the mouse genome were arrayed in 60 x 384-well plates. We developed restriction landmark genomic scanning (RLGS) mixing gels with 32 plates to enable the cloning of methylated sequences from RLGS profiles run with NotI-EcoRV-HinfI. RLGS was used to study aberrant methylation in two mouse models that overexpressed IL-15 or c-Myc and developed either T/NK-cell leukemia or T-cell lymphomas, respectively. Careful analysis of 198 sequences showed that 188 (94.9%) identified CpG-island sequences, 132 sequences (66.7%) had homology to the 5' regions of known genes or mRNAs, and all 132 NotI-EcoRV clones were located at the same CpG islands with the predicted promoter sequences. We have also developed a modified pGL3-based luciferase vector that now contains the NotI, AscI, and EcoRV restriction sites and allows the rapid cloning of NotI-EcoRV library fragments in both orientations. Luciferase assays using NotI-EcoRV clones confirmed that the library is enriched for promoter sequences. Thus, this library will support future genetic and epigenetic studies in mouse models.

Animals↗
PubMed2004

Adhesion of microfabricated particles on vascular endothelium: a parametric analysis.

"Smart" drug delivery systems should be selective and effective to ensure drug administration at the right time, at the right dosage, and anywhere in the body. Among the several administration routes and delivery systems that have been proposed, one of the most effective and promising is based on microfabricated particles injected directly into the blood stream. The shape, size, and material properties of the particles can be designed and optimized depending on the specific applications and targets (cell, tissue, or circulating virus). Since the binding affinity of particles to cells is affected by both the binding force and its growth rate with time, it is of great importance to consider the viscous response of the system. In this work, a parametric analysis is presented where the probability of adhesion of a microfabricated particle on the endothelium is expressed as a function of (i) the hemodynamic conditions, (ii) the viscoelastic properties of the particle and targeted cell, and (iii) density of ligands grafted over the particle. Criteria for the optimal design of particles are proposed.

Adsorption↗
PubMed2004

Unusual case of subcutaneous panniculitis-like T-cell lymphoma.

An unusual case of subcutaneous panniculitis-like T-cell lymphoma is presented involving multiple organ systems, which eventually culminated in rapid demise from the haemophagocytic syndrome, after an initial protracted course. A 44-year-old man presented in April 2001 with bronchiolitis obliterans organising pneumonia that initially responded well to corticosteroids. However, the condition relapsed on attempted prednisone withdrawal in January 2002 and the patient was noted to have developed truncal subcutaneous nodules. Initial skin biopsy revealed lobular panniculitis, with negative microbiological culture. In July 2002, mononeuritis multiplex was diagnosed after the patient presented with paresthesiae and was treated with pulse cyclophosphamide therapy. By November 2002 there was ulceration of the subcutaneous nodules. Repeat skin biopsy revealed subcutaneous panniculitis-like T-cell lymphoma. The clinical manifestations were supportive of an unifying diagnosis of malignancy involving pulmonary, cutaneous and nervous systems. Combination chemotherapy with fludarabine, mitoxantrone and dexamethasone was commenced. However, the patient deteriorated, with fevers, weight loss, pancytopenia and laboratory features consistent with the haemophagocytic syndrome. Despite maximal supportive therapy the patient succumbed to his disease.

Adult↗
PubMed2003

Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL(-/-) renal cell carcinoma cells.

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL(-/-) RCC). VHL(-/-) RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL(-/-) RCC cells by activating the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL(-/-) RCC cells. TGF-alpha is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL(-/-) RCC cells is a direct consequence of HIF activation. In contrast to TGF-alpha, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL(-/-) RCC cells. VHL(-/-) RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-alpha and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.

Basic Helix-Loop-Helix Proteins↗
PubMed2003

Nuclear export of the glucocorticoid receptor is accelerated by cell fusion-dependent release of calreticulin.

Nucleocytoplasmic exchange of nuclear hormone receptors is hypothesized to allow for rapid and direct interactions with cytoplasmic signaling factors. In addition to recycling between a naïve, chaperone-associated cytoplasmic complex and a liganded chaperone-free nuclear form, the glucocorticoid receptor (GR) has been observed to shuttle between nucleus and cytoplasm. Nuclear export of GR and other nuclear receptors has been proposed to depend on direct interactions with calreticulin, which is predominantly localized to the lumen of the endoplasmic reticulum. We show that rapid calreticulin-mediated nuclear export of GR is a specific response to transient disruption of the endoplasmic reticulum that occurs during polyethylene glycol-mediated cell fusion. Using live and digitonin-permeabilized cells we demonstrate that, in the absence of cell fusion, GR nuclear export occurs slowly over a period of many hours independent of direct interaction with calreticulin. Our findings temper expectations that nuclear receptors respond rapidly and directly to cytoplasmic signals in the absence of additional regulatory control. These results highlight the importance of verifying findings of nucleocytoplasmic trafficking using techniques in addition to heterokaryon cell fusion.

Active Transport, Cell Nucleus↗
PubMed2003

Stat5 synergizes with T cell receptor/antigen stimulation in the development of lymphoblastic lymphoma.

Signal transducer and activator of transcription (STAT) proteins are latent transcription factors that mediate a wide range of actions induced by cytokines, interferons, and growth factors. We now report the development of thymic T cell lymphoblastic lymphomas in transgenic mice in which Stat5a or Stat5b is overexpressed within the lymphoid compartment. The rate of lymphoma induction was markedly enhanced by immunization or by the introduction of TCR transgenes. Remarkably, the Stat5 transgene potently induced development of CD8+ T cells, even in mice expressing a class II-restricted TCR transgene, with resulting CD8+ T cell lymphomas. These data demonstrate the oncogenic potential of dysregulated expression of a STAT protein that is not constitutively activated, and that TCR stimulation can contribute to this process.

Animals↗
PubMed2003

A new quasi-one-dimensional ternary chalcogenide: synthesis, crystal structure, and electronic structure of Nb1+xV1-xS5 (x = 0.18).

The new low-dimensional ternary chalcogenide, Nb(1+x)V(1-x)S(5) (x = 0.18), has been prepared and characterized. This compound crystallizes in the monoclinic space group, C2(2h)-P2(1)/m with two formula units in a cell with dimensions a = 9.881(4) A, b = 3.329(1) A, c = 8.775(3) A, and beta = 114.82(3) degrees. The layer is composed of two unique chains of face-sharing Nb-centered bicapped trigonal prisms and edge-sharing M-centered octahedra (M = Nb or V). The electronic structures of the monomeric basic building units, NbS(8) and VS(6), and hypothetical and real one-, two-, and three-dimensional structures making up the compound are examined to understand the nature of inter- and intrachain interactions and orbital overlapping among metals and sulfur atoms. The electronic structure of Nb(1+x)V(1-x)S(5) is essentially given by superimposing those of the individual chains. V d orbitals are found to be crucial for the one-dimensional metallic conductivity along the chain axis.

Journal Article↗
PubMed2003

A role for Stat5 in CD8+ T cell homeostasis.

Cytokine signals are known to contribute to CD8+ memory T cell homeostasis, but an exact understanding of the mechanism(s) has remained elusive. We have now investigated the role of Stat5 proteins in this process. Whereas Stat5a and Stat5b KO mice have decreased numbers of CD8+ T cells, Stat5-transgenic mice have an increased number of these cells. Stat5b-transgenic mice exhibit increased Ag-induced cell death of CD4+ T cells and augmented proliferation and Bcl-2 expression in CD8+ T cells, providing a basis for this finding. Moreover, CD8+ memory T cells are substantially affected by Stat5 levels. These findings identify Stat5 proteins as critical signaling mediators used by cytokines to regulate CD8+ T cell homeostasis.

Animals↗
PubMed2003

Cutaneous Wegener's granulomatosis: a variant or atypical localized form?

A 74-year-old woman presented with an antineutrophil cytoplasmic antibody titre-negative, treatment-responsive Wegener's granulomatosis confined to the integument. She initially presented with a painful left postauricular nodulo-ulcerative lesion with chronically discharging sinuses. This lesion was effectively treated with a short, 3-month course of cyclophosphamide and 24 months of oral prednisone. After 5 months in remission, she developed further similar ulcers, in addition to painless nodules on her ankles and feet bilaterally. These lesions resolved with an extra 32 months of high-dose oral prednisone therapy before complete remission. At most recent review, there was no evidence of disease recurrence 21 months after ceasing all active treatment. Histology demonstrated a granulomatous inflammation. No systemic disease progression to the upper respiratory tract, lung or kidney was detected. This case highlights the importance of being aware of atypical or partial presentations of Wegener's granulomatosis. This diagnosis needs to be considered with patients presenting with a culture-negative chronic ulcer, where malignancy and trauma have been excluded. This will avoid unnecessary surgery and ensure early diagnosis and effective treatment of a disease that is disfiguring and usually fatal if inappropriately treated.

Aged↗
PubMed2002

Central regulation of cerebellar climbing fibre input during motor learning.

A forelimb-withdrawal classical conditioning paradigm was used in awake cats (n = 4) to investigate changes in transmission in climbing fibre (CF) pathways during motor learning. The conditioned stimulus was an auditory tone, while the unconditioned stimulus was a low-intensity, single or double (0.1 ms) electrical pulse applied to the ipsilateral superficial radial nerve. Microwires chronically implanted into the paravermal cerebellar cortex (lobule V) were used to record the CF field potentials evoked by nerve stimulation, and fields at 22 recording sites (9 C1, 7 C2 and 6 C3 zone sites) were monitored during the complete sequence of at least one training run (i.e. over a period of about 2-3 weeks of conditioning). At 19 sites (86 %) conditioning led to a significant reduction in mean size of field. Similar reductions occurred at four sites studied over two successive training runs. At 10 sites (45 %) there was a statistically significant increase prior to the reduction. The three sites that failed to exhibit a significant reduction were all located in the C1 zone. Controls showed that the changes in CF transmission were dependent on the animal being conditioned. The mean size of CF field for trials in which a conditioned EMG response was present (in either the cleidobrachialis or biceps muscle) was not significantly different from the mean size for trials in which a conditioned response was absent. Similarly, on a trial-by-trial basis, CF pathway excitability was not correlated with the conditioned EMG activity in the flexor muscles under study. Overall, the results demonstrate that (1) the capacity of spino-olivocerebellar pathways (SOCPs) to forward information to the cerebellar cortex can be altered by recent experience, (2) establishment of a conditioned forelimb flexor reflex to a tone reduces SOCP excitability at most but not all sites within a forelimb-related region of the cerebellar cortex, (3) the extent of reductions differ at different sites and some are preceded by transient increases, and (4) the changes in transmission may not be related to the conditioned movement. The implications of these findings for some key theories of cerebellar cortical function are discussed.

Acoustic Stimulation↗
PubMed2002

Bcc and Fcc transition metals and alloys: a central role for the Jahn-Teller effect in explaining their ideal and distorted structures.

Transition metal elements, alloys, and intermetallic compounds often adopt the body centered cubic (bcc) and face centered cubic (fcc) structures. By comparing quantitative density functional with qualitative tight-binding calculations, we analyze the electronic factors which make the bcc and fcc structures energetically favorable. To do so, we develop a tight-binding function, DeltaE(star), a function that measures the energetic effects of transferring electrons within wave vector stars. This function allows one to connect distortions in solids to the Jahn-Teller effect in molecules and to provide an orbital perspective on structure determining deformations in alloys. We illustrate its use by considering first a two-dimensional square net. We then turn to three-dimensional fcc and bcc structures, and distortions of these. Using DeltaE(star), we rationalize the differences in energy of these structures. We are able to deduce which orbitals are responsible for instabilities in seven to nine valence electron per atom (e(-)/a) bcc systems and five and six e(-)/a fcc structures. Finally we demonstrate that these results account for the bcc and fcc type structures found in both the elements and binary intermetallic compounds of group 4 through 9 transition metal atoms. The outline of a theory of metal structure deformations based on loss of point group operation rather than translational symmetry is presented.

Journal Article↗
PubMed2002

Expression of C/EBPbeta from the C/ebpalpha gene locus is sufficient for normal hematopoiesis in vivo.

CCAAT/enhancer-binding proteins (C/EBPs) are critical transcriptional regulators of differentiation of hematopoietic cells. Previous studies have shown that targeted disruption of the C/ebpalpha gene results in a lack of granulocytic differentiation with an arrest at the stage of immature myeloblasts. By using a gene replacement strategy in which C/EBPbeta was expressed from the C/ebpalpha gene locus of C/EBPalpha-null mice, we have evaluated the ability of C/EBPbeta to function for C/EBPalpha in directing differentiation along the granulocytic pathway. We show that the morphology and the differential cell counts of the bone marrow and peripheral blood cells from C/EBPbeta knockin mice are indistinguishable from those of their wild-type littermates, indicating that hematopoiesis occurs normally in these animals. Additionally, we analyzed expression of 21 myeloid-specific genes, including markers for distinct stages of granulocytic differentiation, and found no significant differences in their levels of expression in the bone marrow of C/EBPbeta knockin and wild-type mice. These results imply that C/EBPbeta can substitute for C/EBPalpha during hematopoiesis when expressed from the C/ebpalpha gene locus.

Animals↗
PubMed2002

Structure rationalization and topology prediction of two-distinct-component organic crystals: the role of volume fraction and interface topology.

We consider here small-length-scale crystal structures with two clearly different molecular components (e.g., hydrophobic and hydrophilic). Using a perspective developed by studies on large-length-scale block copolymers and liquid crystals, we focus on the crystalline interface between the two components. We examine four types of two-component crystals: aromatic ammonium carboxylates, aromatic oligo(ethylene oxides), cyclohexylammonium carboxylates, and ether-thioether compounds. Of the 111 crystal structures found in the Cambridge Structure Database (CSD), 108 adopt one of the five generic topologies found in diblock copolymers: spheres, columns, perforated layers, layers, and bicontinuous structures. As in diblock copolymers, a key factor controlling the interfacial topology is shown to be the volume ratio of the two components. When the volume fraction of one component is less than 30% of the whole, more than five-sixths of the examined crystal structures are of columnar or spherical type. For volume fractions between 40 and 50% more than three-quarters are of lamellar or bicontinuous type. We use this model to predict the topologies of small-length-scale two-component crystals. We predict the crystal topolgies of six new crystal structures: three are predicted to be columnar, and the other three, lamellar or bicontinuous. The crystal structures of these systems were then determined by single-crystal X-ray methods. Five of the structures form in topologies consistent with the predictions: three in columns and two in layers. The remaining one forms as a perforated layer instead of the predicted columnar structure. Such predictive accuracy is consistent with the statistics of the CSD investigation.

Journal Article↗
PubMed2002

Differentiation of adherent human monocytes into macrophages markedly enhances tissue factor protein expression and procoagulant activity.

Circulating monocytes as well as resident monocyte-derived macrophages (MDMs) in the atherosclerotic plaque express tissue factor. We tested the hypothesis that differentiation of monocytes into macrophages increases their tissue factor expression. This was done by isolating monocytes from human blood and culturing them for 24 h or allowing their differentiation into macrophages for 8 days before assay. Tissue factor procoagulant activity was assessed by recalcification clotting assay while Western blotting quantitated tissue factor protein. Immunocytochemistry was applied to localize antigenic tissue factor in cells. The results showed that freshly-isolated monocytes expressed low baseline procoagulant activity (0.0004+/-0.0003 ng rhTF/10(6) cells) which increased 100-fold in 24-h cultured monocytes (0.04+/-0.01 ng/10(6) cells, P<0.0001) and was associated with detectable tissue factor protein by Western blotting. Furthermore, MDMs expressed 25-fold higher procoagulant activity (1.0+/-0.5 ng/10(6) cells, P<0.04) and increased tissue factor protein content compared with 24-h monocytes as corroborated by immunocytochemistry. LPS stimulation increased baseline binding activity of transcription factor kappaB three-fold (P<0.03) and 2.6-fold (P<0.09) in monocytes and in macrophages, respectively. The results demonstrate that differentiation of human monocytes into macrophages in culture enhances their tissue factor expression. The observed tissue factor increase that can be further stimulated by LPS may contribute to thrombogenicity of macrophage-rich atheroma. Activation of the TF-kappaB transduction pathway suggests its potential role in the transcriptional regulation of TF gene in response to acute stimulation in these cells. This cell-culture model may provide the opportunity to evaluate the effect of various interventions on macrophage procoagulant activity.

Blood Coagulation Factors↗
PubMed2002

Oxygen-dependent ubiquitination and degradation of hypoxia-inducible factor requires nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein.

It is becoming increasingly evident that the degradation of nuclear proteins requires nuclear-cytoplasmic trafficking of both the substrate proteins, as well as the E3 ubiquitin-ligases. Here, we show that nuclear-cytoplasmic trafficking of the von Hippel-Lindau tumor suppressor protein (VHL) is required for oxygen-dependent ubiquitination and degradation of the alpha subunits of hypoxia-inducible factor (HIF-alpha). VHL engages in a constitutive transcription-sensitive nuclear-cytoplasmic shuttle unaffected by oxygen tension or levels of nuclear substrate HIF-alpha. Ubiquitinated forms of HIF-alpha, as well as VHL/ubiquitinated HIF-alpha complexes, are found solely in the nuclear compartment of normoxic or reoxygenated VHL-competent cells. HIF-alpha localizes exclusively in the nucleus of hypoxic cells but is exported to the cytoplasm upon reoxygenation. Oxygen-dependent nuclear ubiquitination and nuclear export of HIF-alpha can be prevented by treatment with an HIF-specific prolyl hydroxylase inhibitor. Treatment with inhibitors of RNA polymerase II activity, which interfere with the ability of VHL to engage in nuclear export, also prevents cytoplasmic accumulation of HIF-alpha in reoxygenated cells. This caused a marked increase in the HIF-alpha half-life without affecting its nuclear ubiquitination. We present a model by which VHL-mediated ubiquitination of HIF-alpha and its subsequent degradation are dependent upon dynamic nuclear-cytoplasmic trafficking of both the E3 ubiquitin-ligase and the nuclear substrate protein.

Active Transport, Cell Nucleus↗