Biomedical subjects

David D Roberts

Publications and source records attributed to David D Roberts.

33 records · Page 2Linked to original sources

PubMed2004

Thrombospondin-1 is a major activator of TGF-beta in fibrotic renal disease in the rat in vivo.

BACKGROUND: Transforming growth factor-beta (TGF-beta), a profibrotic cytokine involved in many scarring processes, has to be activated extracellularly before it can bind to its receptors. Thrombospondin 1 (TSP1), a multifunctional matricellular glycoprotein, has been identified as an activator of TGF-beta in in vitro systems and during mouse postnatal development in vivo. TSP1 is expressed de novo in many inflammatory disease processes, including glomerular disease. METHODS: In this study we investigated whether peptides specifically interfering with the activation process of TGF-beta by TSP1 may be able to block activation of TGF-beta in an in vivo model of mesangial proliferative glomerulonephritis. RESULTS: Continuous intravenous infusion of blocking peptide by minipumps significantly reduced expression of active TGF-beta in glomeruli on day 7 of disease as indicated by immunohistochemistry, bioassay, and activation of the TGF-beta signal transduction pathway, while total TGF-beta expression was unchanged. Inhibition of glomerular TGF-beta activation was accompanied by a decrease of glomerular extracellular matrix accumulation and proteinuria, but was without effect on mesangial cell proliferation or influx of monocytes/macrophages. CONCLUSION: TSP1 is a major endogenous activator of TGF-beta in experimental inflammatory glomerular disease. Drugs interfering with the activation of TGF-beta by locally produced TSP1 may be considered as a future specific treatment of scarring kidney disease.

Animals↗

PubMed2004

Hemoglobin regulates expression of an activator of mating-type locus alpha genes in Candida albicans.

Phenotypic switching from the white to the opaque phase is a necessary step for mating in the pathogenic fungus Candida albicans. Suppressing switching during vascular dissemination of the organism may be advantageous, because opaque cells are more susceptible to host defenses. A repressor of white-opaque switching, HBR1 (hemoglobin response gene 1), was identified based on its specific induction following growth in the presence of exogenous hemoglobin. Deletion of a single HBR1 allele allowed opaque phase switching and mating competence, accompanied by a lack of detectable MTL alpha1 and alpha2 gene expression and enhanced MTLa1 gene expression. Conversely, overexpression of Hbr1p or exposure to hemoglobin increased MTLalpha gene expression. The a1/alpha2 repressed target gene CAG1 was derepressed in the same mutant in a hemoglobin-sensitive manner. Regulation of CAG1 by hemoglobin required an intact MTLa1 gene. Several additional Mtlp targets were perturbed in HBR1 mutants in a manner consistent with commitment to an a mating phenotype, including YEL007w, MFalpha, HST6, and RAM2. Therefore, Hbr1 is part of a host factor-regulated signaling pathway that controls white-opaque switching and mating in the absence of allelic deletion at the MTL locus.

Alleles↗

PubMed2004

Differential expression of S100C in thyroid lesions.

Identification of new potential markers that may help in the diagnosis of benign and malignant thyroid lesions is needed. By comparative 2-dimensional gel electrophoresis of microdissected cells from tumors and normal thyroid tissue, we identified a new protein, S100C, which is highly expressed in papillary carcinomas. In order to validate this finding, we investigated the immunohistochemical expression and the potential role in diagnosis of these markers in 94 specimens representing the spectrum of malignant and benign thyroid lesions. Normal thyroid tissue was evaluated in 57 specimens. Galectin-3, a marker reported as specific for malignant lesions, was also evaluated in the same lesions. S100C protein was expressed in the nuclei of normal tissue, hyperplastic nodules, and follicular adenomas and carcinomas. Papillary carcinomas showed a strong, but cytoplasmic, pattern of staining. Galectin-3 immunostaining was strongly positive in papillary carcinomas, and negative in benign lesions, confirming its value in differential diagnosis. These findings suggest that immunohistochemical staining of S100C could be helpful in the pathological study of thyroid lesions, especially in cases in which follicular variants of papillary carcinoma and follicular carcinoma are considered in the differential diagnosis.

Adenocarcinoma, Follicular↗

PubMed2003

Alpha4beta1 integrin mediates selective endothelial cell responses to thrombospondins 1 and 2 in vitro and modulates angiogenesis in vivo.

We examined the function of alpha4beta1 integrin in angiogenesis and in mediating endothelial cell responses to the angiogenesis modulators, thrombospondin-1 and thrombospondin-2. Alpha4beta1 supports adhesion of venous endothelial cells but not of microvascular endothelial cells on immobilized thrombospondin-1, vascular cell adhesion molecule-1, or recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. Chemotactic activities of this region of thrombospondin-1 and thrombospondin-2 are also mediated by alpha4beta1, whereas antagonism of fibroblast growth factor-2-stimulated chemotaxis is not mediated by this region. Immobilized N-terminal regions of thrombospondin-1 and thrombospondin-2 promote endothelial cell survival and proliferation in an alpha4beta1-dependent manner. Soluble alpha4beta1 antagonists inhibit angiogenesis in the chick chorioallantoic membrane and neovascularization of mouse muscle explants. The latter inhibition is thrombospondin-1-dependent and not observed in explants from thrombospondin-1-/- mice. Antagonizing alpha4beta1 may in part block proangiogenic activities of thrombospondin-1 and thrombospondin-2, because N-terminal regions of thrombospondin-1 and thrombospondin-2 containing the alpha4beta1 binding sequence stimulate angiogenesis in vivo. Therefore, alpha4beta1 is an important endothelial cell receptor for mediating motility and proliferative responses to thrombospondins and for modulation of angiogenesis.

Animals↗

PubMed2003

Heme oxygenase in Candida albicans is regulated by hemoglobin and is necessary for metabolism of exogenous heme and hemoglobin to alpha-biliverdin.

Candida albicans is an opportunistic pathogen that has adapted uniquely to life in mammalian hosts. One of the host factors recognized by this yeast is hemoglobin, which binds to a specific cell surface receptor. In addition to its regulating the expression of adhesion receptors on the yeast, we have found that hemoglobin induces the expression of a C. albicans heme oxygenase (CaHmx1p). Hemoglobin transcriptionally induces the CaHMX1 gene independent of the presence of inorganic iron in the medium. A Renilla luciferase reporter driven by the CaHMX1 promoter demonstrated rapid activation of transcription by hemoglobin and (cobalt protoporphyrin IX) globin but not by apoglobin or other proteins. In contrast, iron deficiency or exogenous hemin did not activate the reporter until after 3 h, suggesting that induction of the promoter by hemoglobin is mediated by receptor signaling rather than heme or iron flux into the cell. As observed following disruption of the Saccharomyces cerevisiae ortholog, HMX1, a CaHMX1 null mutant was unable to grow under iron restriction. This suggests a role for CaHmx1p in inorganic iron acquisition. CaHMX1 encodes a functional heme oxygenase. Exogenous heme or hemoglobin is exclusively metabolized to alpha-biliverdin. CaHMX1 is required for utilization of these exogenous substrates, indicating that C. albicans heme oxygenase confers a nutritional advantage for growth in mammalian hosts.

Amino Acid Sequence↗

PubMed2003

Angiogenesis inhibitors target the endothelial cell cytoskeleton through altered regulation of heat shock protein 27 and cofilin.

Inhibition of angiogenesis has emerged as a key focus for the treatment of cancer, necessitating a better understanding of the downstream molecular targets of angiogenesis inhibitors. Endostatin, thrombospondin-1, fumagillin, and its synthetic derivative, TNP-470, are potent inhibitors of endothelial cell proliferation and migration in culture and of angiogenesis in vivo. To identify targets that mediate the effects of these inhibitors, we compared two-dimensional gel electrophoresis patterns from lysates of treated and untreated human endothelial cells. Among the proteins identified were cofilin and hsp27, two proteins involved in actin dynamics. Western blotting and immunofluorescence experiments confirmed that the phosphorylation states and subcellular localization of these two proteins were affected by all of the inhibitors tested and that treated cells had a more extensive network of actin stress fibers and more numerous focal adhesion plaques compared with untreated cells. Endothelial monocyte activating polypeptide II, another angiogenesis inhibitor, elicited the same response in the actin cytoskeleton and focal adhesions of endothelial cells. This more adherent phenotype may explain the shared ability of these inhibitors to block endothelial migratory signals. Starting with a proteomics approach, we have identified common effector molecules used by a panel of angiogenesis inhibitors that perturb the cytoskeleton to prevent endothelial migration.

Actin Cytoskeleton↗

PubMed2003

Recognition of the N-terminal modules of thrombospondin-1 and thrombospondin-2 by alpha6beta1 integrin.

In addition to its recognition by alpha3beta1 and alpha4beta1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for alpha6beta1 integrin. alpha6beta1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. alpha6beta1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an alpha6beta1-binding sequence in thrombospondin-1. This peptide inhibited alpha6beta1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by alpha6beta1, but not by alpha4beta1. alpha6beta1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of alpha6beta1 integrin in microvascular endothelial cells by the anti-beta1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of alpha4beta1 and alpha6beta1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.

Amino Acid Sequence↗

PubMed2002

Regulation of integrin function by CD47 ligands. Differential effects on alpha vbeta 3 and alpha 4beta1 integrin-mediated adhesion.

We examined the regulation of alpha4beta1 integrin function in melanoma cells and T cells by ligands of CD47. A CD47 antibody (B6H12) that inhibited alphavbeta3-mediated adhesion of melanoma cells induced by CD47-binding peptides from thrombospondin-1 directly stimulated alpha4beta1-mediated adhesion of the same cells to vascular cell adhesion molecule-1 and N-terminal regions of thrombospondin-1 or thrombospondin-2. B6H12 also stimulated alpha4beta1- as well as alpha2beta1- and alpha5beta1-mediated adhesion of CD47-expressing T cells but not of CD47-deficient T cells. alpha4beta1 and CD47 co-purified as a detergent-stable complex on a CD47 antibody affinity column. CD47-binding peptides based on C-terminal sequences of thrombospondin-1 also specifically enhanced adhesion of melanoma cells and T cells to alpha4beta1 ligands. Unexpectedly, activation of alpha4beta1 function by the thrombospondin-1 CD47-binding peptides also occurred in CD47-deficient T cells. CD47-independent activation of alpha4beta1 required the Val-Val-Met (VVM) motif of the peptides and was sensitive to inhibition by pertussis toxin. These results indicate that activation of alpha4beta1 by the CD47 antibody B6H12 and by VVM peptides occurs by different mechanisms. The antibody directly activates a CD47-alpha4beta1 complex, whereas VVM peptides may target an unidentified Gi-linked receptor that regulates alpha4beta1.

Amino Acid Sequence↗

PubMed2002

Interactions of thrombospondins with alpha4beta1 integrin and CD47 differentially modulate T cell behavior.

Thrombospondin (TSP)-1 has been reported to modulate T cell behavior both positively and negatively. We found that these opposing responses arise from interactions of TSP1 with two different T cell receptors. The integrin alpha4beta1 recognizes an LDVP sequence in the NH2-terminal domain of TSP1 and was required for stimulation of T cell adhesion, chemotaxis, and matrix metalloproteinase gene expression by TSP1. Recognition of TSP1 by T cells depended on the activation state of alpha4beta1 integrin, and TSP1 inhibited interaction of activated alpha4beta1 integrin on T cells with its counter receptor vascular cell adhesion molecule-1. The alpha4beta1 integrin recognition site is conserved in TSP2. A recombinant piece of TSP2 containing this sequence replicated the alpha4beta1 integrin-dependent activities of TSP1. The beta1 integrin recognition sites in TSP1, however, were neither necessary nor sufficient for inhibition of T cell proliferation and T cell antigen receptor signaling by TSP1. A second TSP1 receptor, CD47, was not required for some stimulatory responses to TSP1 but played a significant role in its T cell antigen receptor antagonist and antiproliferative activities. Modulating the relative expression or function of these two TSP receptors could therefore alter the direction or magnitude of T cell responses to TSPs.

Antigens, CD↗

PubMed2002

Identification of heat shock protein 60 as a molecular mediator of alpha 3 beta 1 integrin activation.

The alpha 3 beta 1 integrin is involved in the adhesion of metastatic breast cancer cells to the lymph nodes and to osteoblasts in the bone. Regulation of the affinity or avidity of integrins for their ligands may result from conformational changes induced by changes in the microenvironment of the integrin. Two surface proteins, 55 and 32 kDa, coimmunoprecipitated with the alpha 3 beta 1 integrin from breast carcinoma cells. The 55-kDa protein preferentially associated with the active form of the alpha 3 beta 1 integrin. The protein was identified as HSP60 using two-dimensional electrophoresis and mass spectrometry and confirmed by reimmunoprecipitation of the integrin immune complex with an anti-HSP60 antibody. In cell spreading assays on a thrombospondin-1 substrate, addition of exogenous-recombinant HSP60 was sufficient to specifically activate alpha 3 beta 1 integrin but not to activate function of alpha 2 beta 1, alpha v beta 3, alpha 4 beta 1, or alpha 5 beta 1 integrins. Furthermore, mizoribine, an HSP60-binding drug, blocked activation of the alpha 3 beta 1 integrin induced by insulin-like growth factor 1 (IGF1) or exogenous recombinant HSP60 and inhibited the association of HSP60 with the integrin. Additionally, inhibiting the surface expression of endogenous HSP60 by nonactin inhibited activation of the alpha 3 beta 1 integrin by IGF1. These data demonstrate that HSP60 binding is sufficient to activate alpha 3 beta 1 integrin function and suggest that association of endogenous HSP60 with alpha 3 beta 1 integrin is necessary for IGF1-induced activation.

Chaperonin 60↗

PubMed2002

Enhanced gene expression in breast cancer cells in vitro and tumors in vivo.

Gene therapy clinical trials for cancer frequently produce inconsistent results. Some of this variability could result from differences in transcriptional regulation that limit expression of therapeutic genes in specific cancers. Systemic liposomal delivery of a nonviral plasmid DNA showed efficacy in animal models for several cancers. However, we observed large differences in the levels of gene expression from a CMV promoter-enhancer between lung and breast cancers. To optimize gene expression in breast cancer cells in vitro and in vivo, we created a new promoter-enhancer chimera to regulate gene expression. Serial analyses of gene expression data from a panel of breast carcinomas and normal breast cells predicted that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter is highly active in breast cancers. Furthermore, GAPDH is up-regulated by hypoxia, which is common in tumors. We added the GAPDH promoter, including the hypoxia enhancer sequences, to our in vivo gene expression plasmid. The novel CMV-GAPDH promoter-enhancer showed up to 70-fold increased gene expression in breast tumors compared to the optimized CMV promoter-enhancer alone. No significant increase in gene expression was observed in other tissues. These data demonstrate tissue-specific effects on gene expression after nonviral delivery and suggest that gene delivery systems may require plasmid modifications for the treatment of different tumor types. Furthermore, expression profiling can facilitate the design of optimal expression plasmids for use in specific cancers.

Animals↗

PubMedDate not supplied

Fibronectin-based masking molecule blocks platelet adhesion.

Vessel wall extracellular matrix, which underlies the endothelium, is a potent stimulator of platelet adhesion and activation. Exposure of this matrix can result from damage incurred by vascular interventions, such as saphenous vein bypass grafting and angioplasty. Fibrillar collagens are an important component of the thrombogenic extracellular matrix. Herein we describe a means of targeting poly(ethylene glycol) (PEG)-mediated blockade directly to platelet-binding ECM molecules, such as type I collagen, thereby selectively blocking platelet adhesion to vascular matrix. Purified fibronectin (FN), a matrix protein that interacts with fibrillar collagens and platelets, was selectively pegylated to generate a targeted molecular shielding reagent that masked ECM ligands from platelet recognition and adhesion. This approach protects the functions of other vascular proteins, including surface proteins on intact endothelium. To mask the platelet-binding site of FN, PEG-propyl moieties (5000 Da) were covalently appended to lysine residues on the surface of FN, generating FNPEG-5K. To preserve the collagen-binding function of FN, it was pegylated while bound to a gelatin agarose matrix. We demonstrate that FNPEG-5K blocks platelet adhesion to purified type I collagen. Moreover, the same preparation blocks platelet adhesion to vascular wall components, including collagens.

Binding Sites↗

PubMedDate not supplied

The biphasic nature of nitric oxide responses in tumor biology.

The dual or biphasic responses of cancer to nitric oxide (NO) arise from its concentration dependent ability to regulate tumor growth, migration, invasion, survival, angiogenesis, and metastasis. The outcome of these various NO-dependent processes is dictated by several factors including NO flux, the chemical redox environment, and the duration of NO exposure. Further, it was recently discovered that an NO-induced redox flux in vascular endothelial cells hypersensitizes these cells to the antiangiogenic effects of thrombospondin-1. This suggests a novel treatment paradigm for targeting tumor-driven angiogenesis that combines redox modulation with mimetic derivatives of thrombospondin-1. This article discusses the biphasic nature of NO in cancer biology and the implications of NO-driven redox flux for modulation of tumor-stimulated angiogenesis, growth, and metastasis.

Animals↗

PubMedDate not supplied

The activation of metabolites of nitric oxide synthase by metals is both redox and oxygen dependent: a new feature of nitrogen oxide signaling.

Nitrite (NO(2)-), N (G)-hydroxy-L-arginine (NOHA), and hydroxylamine (NH(2)OH) are products of nitric oxide synthase (NOS) activity and can also be formed by secondary reactions of nitric oxide (NO). These compounds are commonly considered to be rather stable and as such to be dosimeters of NO biosynthesis. However, each can be converted via metal-catalyzed reactions into either NO or other reactive nitrogen oxide species (RNOS), such as nitrogen dioxide (NO(2)) and nitroxyl (HNO), which have biologic activities distinct from those of the parent molecules. Consequently, certain aspects of tissue regulation controlled by RNOS may be dictated to a significant extent by metal-dependent reactions, thereby offering unique advantages for cellular and tissue regulation. For instance, because many metal-catalyzed reactions depend on the redox and oxygen status of the cellular environment, such reactions could serve as redox indicators. Formation of RNOS by metal-mediated pathways would confine the chemistry of these species to specific cellular sites. Additionally, such mechanisms would be independent both of NO and NOS, thus increasing the lifetime of RNOS that react with NO. Thus metal-mediated conversion of nitrite, NOHA, and NH(2)OH into biologically active agents may provide a unique signaling mechanism. In this review, we discuss the biochemistry of such reactions in the context of their pharmacologic and biologic implications.

Arginine↗

PubMedDate not supplied

Proteomic identification of new biomarkers and application in thyroid cytology.

OBJECTIVE: To validate proteins identified by proteomics as potentially usable markers in thyroid pathology. STUDY DESIGN: Frozen sections of thyroid tumors were manually micro-dissected and proteins extracted. Two-dimensional (2D) gel electrophoresis and subsequent liquid chromatography/mass spectroscopy were performed, and differentially expressed proteins were identified. Validation of candidates for tumor markers (galectin-1, galectin-3, S100C and voltage-dependent anion channel 1 [VDAC1]) was done by immunohistochemistry in 21 cell blocks from fine needle aspiration biopsies (FNAB) and corresponding histology specimens (13 cases). RESULTS: Galectin-3 was negative in benign lesions and positive in FNAB from papillary carcinoma (5 of 5), follicular variant of papillary carcinoma (1 of 4) and follicular carcinoma (1 of 2). S100C was positive in some benign lesions: hyperplasia (2 of 4), goiter (1 of 3) and follicular adenoma (1 of 3), with predominantly nuclear pattern of staining. S100C was positive in malignant lesions, showing cytoplasmic location. Galectin-1 was negative in benign lesions and positive in follicular carcinoma (1 of 2), papillary carcinoma (2 of 5) and follicular variant of papillary carcinoma (1 of 4). VDAC1 was detected in benign and malignant lesions, showing a strong positivity in follicular carcinomas. CONCLUSION: Immunohistochemical validation of potential markers is a crucial step before clinical application in diagnosis. Galectin-3, galectin-1 and S100C can be used to help in discriminating benign and malignant thyroid lesions.

Adenocarcinoma, Follicular↗