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Biomedical subjects

G Elliott

Publications and source records attributed to G Elliott.

At least 37 records · Page 2Linked to original sources
PubMed1999

Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.

A receptor that mediates osteoprotegerin ligand (OPGL)-induced osteoclast differentiation and activation has been identified via genomic analysis of a primary osteoclast precursor cell cDNA library and is identical to the tumor necrosis factor receptor (TNFR) family member RANK. The RANK mRNA was highly expressed by isolated bone marrow-derived osteoclast progenitors and by mature osteoclasts in vivo. Recombinant OPGL binds specifically to RANK expressed by transfected cell lines and purified osteoclast progenitors. Transgenic mice expressing a soluble RANK-Fc fusion protein have severe osteopetrosis because of a reduction in osteoclasts, similar to OPG transgenic mice. Recombinant RANK-Fc binds with high affinity to OPGL in vitro and blocks osteoclast differentiation and activation in vitro and in vivo. Furthermore, polyclonal Ab against the RANK extracellular domain promotes osteoclastogenesis in bone marrow cultures suggesting that RANK activation mediates the effects of OPGL on the osteoclast pathway. These data indicate that OPGL-induced osteoclastogenesis is directly mediated through RANK on osteoclast precursor cells.

Animals↗
PubMed1999

Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation.

Activation of endothelial cells, important in processes such as angiogenesis, is regulated by cell surface receptors, including those in the tyrosine kinase (RTK) family. Receptor activity, in turn, can be modulated by phosphorylation, turnover, or proteolytic release of a soluble extracellular domain. Previously, we demonstrated that release of soluble tie-1 receptor from endothelial cells by phorbol myristate acetate (PMA) is mediated through protein kinase C and a Ca2+-dependent protease. In this study, the release of soluble tie-1 was shown to be stimulated by inflammatory cytokines and vascular endothelial growth factor (VEGF), but not by growth factors such as basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFalpha). Release of soluble tie by tumor necrosis factor alpha (TNFalpha) or VEGF occurred within 10 minutes of stimulation and reached maximal levels within 60 minutes. Specificity was shown by fluorescence-activated cell sorting (FACS) analysis; endothelial cells exhibited a significant decrease in cell surface tie-1 expression in response to TNF, whereas expression of epidermal growth factor receptor (EGF-R) and CD31 was stable. In contrast, tie-1 expression on megakaryoblastic UT-7 cells was unaffected by PMA or TNFalpha. Sequence analysis of the cleaved receptor indicated that tie-1 was proteolyzed at the E749/S750 peptide bond in the proximal transmembrane domain. Moreover, the hydroxamic acid derivative BB-24 demonstrated dose-dependent inhibition of cytokine-, PMA-, and VEGF-stimulated shedding, suggesting that the tie-1 protease was a metalloprotease. Protease activity in a tie-1 peptide cleavage assay was (1) associated with endothelial cell membranes, (2) specifically activated in TNFalpha-treated cells, and (3) inhibited by BB-24. Additionally, proliferation of endothelial cells in response to VEGF, but not bFGF, was inhibited by BB-24, suggesting that the release of soluble tie-1 receptor plays a role in VEGF-mediated proliferation. This study demonstrated that the release of soluble tie-1 from endothelial cells is stimulated by inflammatory cytokines and VEGF through the activation of an endothelial membrane-associated metalloprotease.

Amino Acid Sequence↗
PubMed1999

Intercellular delivery of thymidine kinase prodrug activating enzyme by the herpes simplex virus protein, VP22.

We demonstrate that fusion proteins consisting of the herpes simplex virus (HSV) transport protein VP22 linked in frame to HSV thymidine kinase (tk) retain the ability to be transported between cells. In vivo radiolabelling experiments and in vitro assays show that the fusion proteins also retain tk activity. When transfected COS cells, acting as a source of the VP22-tk chimera, were co-plated on to gap junction-negative neuroblastoma cells, ganciclovir treatment induced efficient cell death in the recipient neuroblastoma cell monolayer. No such effect was observed with COS cells transfected with tk alone. Tumours established in mice with neuroblastoma cell lines expressing VP22-tk regressed upon administration of ganciclovir. Furthermore tumours established from 50:50 mixtures of VP22-tk transduced and nontransduced cells also regressed while no significant effect was observed in similar experiments with cells transduced with tk alone. VP22 mediated transport may thus have application in a clinical setting to amplify delivery of the target protein in enzyme-prodrug protocols.

Animals↗
PubMed1999

Intercellular trafficking of VP22-GFP fusion proteins.

The herpes simplex virus protein VP22 exhibits the unusual property of intercellular transport whereby after being synthesised in a subpopulation of cells, in which it is largely cytoplasmic, the protein is transported to adjacent cells where it accumulates mainly in the nucleus. Here we examine the transport of a fusion protein consisting of VP22 linked to the green fluorescent protein (GFP). Intercellular transport, nuclear accumulation and chromatin binding of VP22-GFP could be detected by intrinsic GFP fluorescence in fixed cells. However, while the cytoplasmic localisation of VP22-GFP could be detected in live cells actively synthesising the protein, we were unable to detect intercellular transport by intrinsic GFP fluorescence in livecells, indicating that the levels of transported protein may be below those required for live detection, or that GFP fluorescence was quenched. The use of antibody to GFP was more sensitive than intrinsic GFP fluorescence and allowed ready detection of transport and nuclear accumulation of VP22-GFP. Intercellular transport was also confirmed in coplating experiments. Consistent with previous results showing a requirement for the C-terminus of VP22 in transport of the native protein, a fusion protein consisting of GFP linked to the N-terminal 1-192 residues of VP22 failed to transport between cells. The results support the proposal that VP22 has the ability to transport cargo proteins between cells and that it has significant potential in the field of gene therapy.

Animals↗
PubMed1999

Thrombolytic therapy for venous thromboembolism.

The role of thrombolytic therapy for venous thromboembolism remains uncertain. To date, no well-designed randomized clinical trial has shown that the benefits of thrombolysis exceed the risks for a well-defined subgroup of patients with acute pulmonary embolism. Recent reports have underscored the risks of thrombolytic therapy. The largest multinational registry of patients in whom acute pulmonary embolism had been diagnosed found that 3% of 311 patients who underwent thrombolysis for acute pulmonary embolism suffered an intracranial hemorrhage. Because of the serious risk associated with systemic thrombolysis, investigators have continued to study methods that rapidly remove venous thromboemboli while minimizing the risk. Reports of catheter fragmentation combined with thrombolysis as well as catheter-directed thrombolysis are promising, but well-designed clinical trials are needed to clarify the utility of these techniques.

Humans↗
PubMed1999

Live-cell analysis of a green fluorescent protein-tagged herpes simplex virus infection.

Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here we describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show that this virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection. Moreover, we show that GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy. Consequently, we have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, we have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.

Animals↗
PubMed1999

Identification of phosphorylation sites within the herpes simplex virus tegument protein VP22.

The herpes simplex virus protein VP22 is a major phosphoprotein of infected cells. In this study, we identify two serine phosphorylation sites within VP22 and show that the N-terminal site is a substrate for casein kinase II, while the extreme C-terminal site is a substrate for another, as yet unidentified, cellular kinase. Furthermore, we show that a mutant of VP22 which has both sites altered is unable to incorporate phosphate in vivo, confirming that there are no other phosphorylation sites within VP22.

Animals↗
PubMed1999

Medical text books on the World Wide Web.

The availability of medical textbooks for physicians on the web is rapidly increasing. Internet sites that maintain a listing of such resources are briefly described, and descriptions of particularly valuable fee based and free medical textbooks web sites are categorized.

Internet↗
PubMed1998

Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.

The ligand for osteoprotegerin has been identified, and it is a TNF-related cytokine that replaces the requirement for stromal cells, vitamin D3, and glucocorticoids in the coculture model of in vitro osteoclastogenesis. OPG ligand (OPGL) binds to a unique hematopoeitic progenitor cell that is committed to the osteoclast lineage and stimulates the rapid induction of genes that typify osteoclast development. OPGL directly activates isolated mature osteoclasts in vitro, and short-term administration into normal adult mice results in osteoclast activation associated with systemic hypercalcemia. These data suggest that OPGL is an osteoclast differentiation and activation factor. The effects of OPGL are blocked in vitro and in vivo by OPG, suggesting that OPGL and OPG are key extracellular regulators of osteoclast development.

Amino Acid Sequence↗
PubMed1998

Natural killer and B-lymphoid potential in CD34+ cells derived from embryonic stem cells differentiated in the presence of vascular endothelial growth factor.

Differentiation of totipotent mouse embryonic stem (ES) cells to various lymphohematopoietic cells is an in vitro model of the hematopoietic cell development during embryogenesis. To understand this process at cellular levels, differentiation intermediates were investigated. ES cells generated progeny expressing CD34, which was significantly enhanced by vascular endothelial growth factor (VEGF). The isolated CD34+ cells were enriched for myeloid colony-forming cells but not significantly for erythroid colony-forming cells. When cultured on OP9 stroma cells in the presence of interleukin-2 and interleukin-7, the CD34+ cells developed two types of B220+ CD34- lymphocytes: CD3- cytotoxic lymphocytes and CD19+ pre-B cells, and such lymphoid potential was highly enriched in the CD34+ population. Interestingly, the cytotoxic cells expressed the natural killer (NK) cell markers, such as NKR-P1, perforin, and granzymes, classified into two types, one of which showed target specificity of NK cells. Thus, ES cells have potential to generate NK-type cytotoxic lymphocytes in vitro in addition to erythro-myeloid cells and pre-B cells, and both myeloid and lymphoid cells seem to be derived from the CD34+ intermediate, on which VEGF may play an important role.

Animals↗
PubMed1998

Intercellular delivery of functional p53 by the herpesvirus protein VP22.

The herpes simplex virus type 1 (HSV-1) virion protein VP22 exhibits the remarkable property of intercellular trafficking whereby the protein spreads from the cell in which it is synthesized to many surrounding cells. In addition to having implications for protein trafficking mechanisms, this function of VP22 might be exploited to overcome a major hurdle in gene therapy, i.e., efficient delivery of genes and gene products. We show that chimeric polypeptides, consisting of VP22 linked to the entire p53 protein, retain their ability to spread between cells and accumulate in recipient cell nuclei. Furthermore the p53-VP22 chimeric protein efficiently induces apoptosis in p53 negative human osteosarcoma cells resulting in a widespread cytotoxic effect. The intercellular delivery of functional p53-VP22 fusion protein is likely to prove beneficial in therapeutic strategies based on restoration of p53 function. These results, demonstrating intracellular transport of large functional proteins, indicate that VP22 delivery may have applications in gene therapy.

Animals↗
PubMed1998

Examination of determinants for intranuclear localization and transactivation within the RING finger of herpes simplex virus type 1 IE110k protein.

The herpesvirus regulatory protein IE110k possesses a cysteine-rich, RING finger motif required for its role in transactivation and virus replication. IE110k also localizes to subnuclear compartments termed PODs (PML oncogenic domains). Localization to PODs induces redistribution of the proteins associated with this nuclear compartment, including the cellular RING finger protein, PML. Here we construct a series of deletions, RING domain swaps and point mutations to analyse specific requirements within the IE110k RING finger for subnuclear localization, redistribution of PML and transactivation and we examine the relationship between these activities. We find that IE110k localizes to distinct nuclear subdomains that are more numerous than the cellular PODs and that mutation of two residues within a predicted loop of the RING finger, or replacing the IE110k RING finger with a RING finger from a cellular gene abrogates the ability of IE110k to localize to these extra compartments and traps IE110k in the original PODs. We further demonstrate that RING fingers from the cellular genes mdm-2 and Bmi I, when placed within IE110k, alter the nuclear distribution of IE110k, do not transactivate, and do not redistribute PML. We also demonstrate that the majority of wild-type IE110k, like PML, is associated with the nuclear matrix. Although substitutions and deletions within the RING finger abolish transactivation, these mutant proteins remain tightly associated with the matrix. These results further dissect the determinants involved in different aspects of nuclear compartmentalization of IE110k and are discussed in relation to PML, PODs and the IE110k RING finger.

Amino Acid Sequence↗
PubMed1998

Herpes simplex virus type 1 tegument protein VP22 induces the stabilization and hyperacetylation of microtubules.

The role of the herpes simplex virus type 1 tegument protein VP22 during infection is as yet undefined. We have previously shown that VP22 has the unusual property of efficient intercellular transport, such that the protein spreads from single expressing cells into large numbers of surrounding cells. We also noted that in cells expressing VP22 by transient transfection, the protein localizes in a distinctive cytoplasmic filamentous pattern. Here we show that this pattern represents a colocalization between VP22 and cellular microtubules. Moreover, we show that VP22 reorganizes microtubules into thick bundles which are easily distinguishable from nonbundled microtubules. These bundles are highly resistant to microtubule-depolymerizing agents such as nocodazole and incubation at 4 degreesC, suggesting that VP22 has the capacity to stabilize the microtubule network. In addition, we show that the microtubules contained in these bundles are modified by acetylation, a marker for microtubule stability. Analysis of infected cells by both immunofluorescence and measurement of microtubule acetylation further showed that colocalization between VP22 and microtubules, and induction of microtubule acetylation, also occurs during infection. Taken together, these results suggest that VP22 exhibits the properties of a classical microtubule-associated protein (MAP) during both transfection and infection. This is the first demonstration of a MAP encoded by an animal virus.

Acetylation↗
PubMed1998

Useful sites on the Internet.

Internet medical sites offer the physician opportunities to improve patient care, to obtain continuing education and medical management services, and the ability to communicate with colleagues. This paper discusses how to choose the best sites, and highlights some valuable federal, university and commercial medical sites found on the Internet.

Computer Communication Networks↗
PubMed1998

Full text core medical journals on the Internet.

Peer reviewed, core medical journals have an increasing presence on the Internet. A significant number of them offer full text articles without fees. This paper indexes these resources, and discusses ways to keep the list current.

Computer Communication Networks↗
PubMed1998

Complementary or Alternative medicine and the Internet.

Complementary or Alternative Medicine (CAM) is being used with increasing frequency in the United States. This paper describes what CAM is, and catalogues mainstream medical Web sites. These online resources contain extensive information on CAM, and will provide a means to stay current with this sometimes-new yet often ancient area of medicine.

Complementary Therapies↗
PubMed1997

Regulation of tie receptor expression on human endothelial cells by protein kinase C-mediated release of soluble tie.

The expression and activity of receptor tyrosine kinases (RTK) at the cell surface can be modulated by several different pathways including the proteolytic release of the extracellular domain as a soluble receptor. We investigated the regulation of tie receptor expression, an orphan RTK restricted to cells of hematopoietic and endothelial lineages, on primary human endothelial cells and a stably transfected Chinese hamster ovary (CHO) cell line. Tie was expressed in cells as a doublet of 135 and 125 kD; the 135-kD band represented mature cell surface receptor containing sialic acid and N-linked oligosaccharide residues, whereas the 125-kD band represented an intracellular pool of immature receptor. Phorbol 12-myristate 13-acetate (PMA) had dramatic effects on tie expression at the cell surface. Within 15 minutes of PMA treatment, the 135-kD band disappeared from the cell surface and was accompanied by the appearance of a 100-kD band in cell supernatants. The 100-kD band continued to accumulate in the media throughout the duration of PMA treatment during which mature tie receptor was undetectable on the cell surface by fluorescence-activated cell sorting (FACS) or in cell lysates by immunoblot analysis. Using specific antibodies, this 100-kD species was shown to be a soluble form of the tie receptor containing the extracellular domain. PMA-dependent release of soluble tie was mediated through the activation of protein kinase C (PKC); soluble tie was not released in the presence of PKC inhibitors, an inactive PMA analog, or following the downregulation of PKC through chronic PMA treatment. These results indicate that tie receptor expression on endothelial cells is regulated by the release of a soluble extracellular fragment following activation of PKC. Parallel pathways regulating c-kit, tumor necrosis factor (TNF), and colony-stimulating factor (CSF) receptor expression suggest that the release of extracellular receptor fragments represents an alternative mechanism through which cells modulate responses to growth factors and cytokines.

Animals↗