Retiform non-blanchable purpuric plaques in a patient with systemic lupus erythematosus.

Livedoid vasculopathy (LV) is a small vessel occlusive disease that can present with a painful purpuric eruption. Predominantly affecting young women, LV has been associated with hypercoagulable states and antiphospholipid syndrome. We present an unusual case of LV occurring in the setting of acute kidney injury secondary to lupus nephritis. It is important to differentiate LV from vasculitis as the treatment recommendation centers on anticoagulation therapy rather than immunosuppression. Additionally, antiphospholipid syndrome should be ruled out in cases of systemic lupus erythematosus with LV due to risk of thrombotic events.

An Update on Canine, Feline and Bovine Papillomaviruses.

Over recent years, a growing number of papillomaviruses have been identified, which cause a wide range of lesions in domestic and wild animals. Papillomavirus-induced lesions may have a great impact on animal health, and some diseases observed in farm animals are associated with significant economic losses. This concise review brings together recent advancements on animal papillomavirus research, providing the scientific community and veterinary practitioners with an update on this rapidly evolving field. Among others, bovine, canine and feline papillomaviruses (BPV, CPV and FcaPV) are most extensively discussed, in view of the recent discovery of new viral types and their worldwide importance for animal health. Feline papillomaviruses 2 is an emerging, highly prevalent pathogen in domestic cats, associated with a subset of malignant skin lesions. Aspects related to cross-species infection by BPV and its environmental co-factors are also addressed. Animal papillomaviruses are also fascinating models for studying molecular and cell biology and have recently inspired some major breakthroughs. Overall, it is clear that additional, international and systematic efforts are needed to clarify which lesions are caused by which viral types and to develop experimental models for studying animal papillomavirus.

Solitary esophageal leiomyoma with eosinophilic infiltrate: case report and review of the literature.

Leiomyomas are common benign tumors of the esophagus representing 10% of all mesenchymal tumors of the gastrointestinal tract. Prominent numbers of eosinophils involving a leiomyoma have only rarely been described. They have never been described involving a solitary leiomyoma of the esophagus. We present an unusual case of a solitary esophageal leiomyoma with a prominent number of eosinophils and mast cells, review the previous literature regarding this topic and discuss possible causes of the eosinophil infiltrate.

Mechanisms of cell transformation by papillomavirus E5 proteins.

The papillomavirus E5 proteins are short, hydrophobic transforming proteins. The transmembrane E5 protein encoded by bovine papillomavirus transforms cells by activating the platelet-derived growth factor beta receptor tyrosine kinase in a ligand-independent fashion. The bovine papillomavirus E5 protein forms a stable complex with the receptor, thereby inducing receptor dimerization and activation, trans-phosphorylation, and recruitment of cellular signaling proteins to the receptor. The E5 proteins of the human papillomaviruses also appear to affect the activity of growth factor receptors and their signaling pathways. The interaction of papillomavirus E5 proteins with a subunit of the vacuolar ATPase may also contribute to transformation. Further analysis of these unique mechanisms of viral transformation will yield new insight into the regulation of growth factor receptor activity and cellular signal transduction pathways.

Induced senescence in HeLa cervical carcinoma cells containing elevated telomerase activity and extended telomeres.

Proliferation of normal somatic human cells in culture is limited by replicative senescence, a growth-arrested state that appears to be triggered by the erosion of telomeres. Tumor cells such as HeLa cervical carcinoma cells, which contain short telomeres, can be induced to undergo senescence by various manipulations including oncogene withdrawal. Repression of the human papillomavirus (HPV) type 18 E6/E7 genes in HeLa cells by the bovine papillomavirus E2 transcriptional regulatory protein results in reactivation of the dormant p53 and p105(Rb) tumor suppressor pathways in these cells, repression of telomerase, and profound growth arrest. Strikingly, the growth-arrested cells rapidly and synchronously acquired numerous characteristics of primary cells undergoing replicative senescence. To explore the role of telomerase and telomere length in induced senescence, we expressed an exogenous hTERT gene, which encodes the catalytic subunit of telomerase, to generate stable HeLa cell clones with elevated telomerase activity and extended telomeres. Expression of the E2 protein in these cells repressed HPV E6/E7 expression, activated tumor suppressor pathways, and induced senescence as assessed by growth arrest, morphological changes, senescence-associated beta-galactosidase expression, and increased autofluorescence. Cells carrying the hTERT gene and control cells displayed identical responses to E2 expression. Therefore, HeLa cell senescence induced by HPV repression is not triggered by short telomeres or low levels of telomerase activity.

Identification of the transmembrane dimer interface of the bovine papillomavirus E5 protein.

We have developed a genetic method to determine the active orientation of dimeric transmembrane protein helices. The bovine papillomavirus E5 protein, a 44-amino acid homodimeric protein that appears to traverse membranes as a left-handed coiled-coil, transforms fibroblasts by binding and activating the platelet-derived growth factor (PDGF) beta receptor. A heterologous dimerization domain was used to force E5 monomers to adopt all seven possible symmetric coiled-coil registries relative to one another within the dimer. Focus formation assays demonstrated that dimerization of the E5 protein is required for transformation and identified a single preferred orientation of the monomers. The essential glutamine residue at position 17 resided in the dimer interface in this active orientation. The active chimera formed complexes with the PDGF beta receptor and induced receptor tyrosine phosphorylation. We also identified E5-like structures that underwent non-productive interactions with the receptor.

Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways.

Most cervical carcinomas express high-risk human papillomaviruses (HPVs) E6 and E7 proteins, which neutralize cellular tumor suppressor function. To determine the consequences of removing the E6 and E7 proteins from cervical cancer cells, we infected HeLa cells, a cervical carcinoma cell line that contains HPV18 DNA, with a recombinant virus that expresses the bovine papillomavirus E2 protein. Expression of the E2 protein resulted in rapid repression of HPV E6 and E7 expression, followed approximately 12 h later by profound inhibition of cellular DNA synthesis. Shortly after E6/E7 repression, there was dramatic posttranscriptional induction of p53. Two p53-responsive genes, mdm2 and p21, were induced with slightly slower kinetics than p53 and appeared to be functional, as assessed by inhibition of cyclin-dependent kinase activity and p53 destabilization. There was also dramatic posttranscriptional induction of p105(Rb) and p107 after E6/E7 repression, followed shortly thereafter by induction of p130. By 24 h after infection, only hypophosphorylated p105(Rb) was detectable and transcription of several Rb/E2F-regulated genes was dramatically repressed. Constitutive expression of the HPV16 E6/E7 genes alleviated E2-induced growth inhibition and impaired activation of the Rb pathway and repression of E2F-responsive genes. This dynamic response strongly suggests that the p53 and Rb tumor suppressor pathways are intact in HeLa cells and that repression of HPV E6 and E7 mobilizes these pathways in an orderly fashion to deliver growth inhibitory signals to the cells. Strikingly, the major alterations in the cell cycle machinery underlying cervical carcinogenesis can be reversed by repression of the endogenous HPV oncogenes.

Rapid induction of senescence in human cervical carcinoma cells.

Expression of the bovine papillomavirus E2 regulatory protein in human cervical carcinoma cell lines repressed expression of the resident human papillomavirus E6 and E7 oncogenes and within a few days caused essentially all of the cells to synchronously display numerous phenotypic markers characteristic of cells undergoing replicative senescence. This process was accompanied by marked but in some cases transient alterations in the expression of cell cycle regulatory proteins and by decreased telomerase activity. We propose that the human papillomavirus E6 and E7 proteins actively prevent senescence from occurring in cervical carcinoma cells, and that once viral oncogene expression is extinguished, the senescence program is rapidly executed. Activation of endogenous senescence pathways in cancer cells may represent an alternative approach to treat human cancers.

E2F-Rb complexes assemble and inhibit cdc25A transcription in cervical carcinoma cells following repression of human papillomavirus oncogene expression.

Expression of the bovine papillomavirus E2 protein in cervical carcinoma cells represses expression of integrated human papillomavirus (HPV) E6/E7 oncogenes, followed by repression of the cdc25A gene and other cellular genes required for cell cycle progression, resulting in dramatic growth arrest. To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene linked to the cdc25A promoter in HT-3 cervical carcinoma cells. Mutation of the distal E2F binding site in the cdc25A promoter abolished E2-induced repression, whereas mutation of the proximal E2F site or the E2 site had no effect. None of these mutations affected the activity of the promoter in the absence of E2 expression. Expression of the E2 protein also led to posttranscriptional increase in the level of E2F4, p105(Rb), and p130 and induced the formation of nuclear E2F4-p130 and E2F4-p105(Rb) complexes. This resulted in marked rearrangement of the protein complexes that formed at the distal E2F site in the cdc25A promoter, including the replacement of free E2F complexes with E2F4-p105(Rb) complexes. These experiments indicated that repression of E2F-responsive promoters following HPV E6/E7 repression was mediated by activation of the Rb tumor suppressor pathway and the assembly of repressing E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression.

The platelet-derived growth factor beta receptor as a target of the bovine papillomavirus E5 protein.

The 44-amino acid E5 protein of bovine papillomavirus is a homo-dimeric, transmembrane protein that transforms cells by activating the platelet-derived growth factor ss receptor in a ligand-independent fashion. The E5 protein induces receptor activation by forming a stable complex with the receptor, thereby inducing receptor dimerization, trans-phosphorylation of tyrosine residues in the cytoplasmic domain of the receptor, and recruitment of cellular SH2 domain-containing proteins into a signal transduction complex. Direct interactions between specific transmembrane and juxtamembrane amino acids in the E5 protein and the PDGF ss receptor appear to drive complex formation and dimerization of the receptor. Further analysis of this unique mechanism of viral transformation promises to yield new insight into the regulation of growth factor receptor activity and cellular signal transduction pathways.

Metastatic ocular melanoma to the left ventricle inducing near-syncope attacks in an 84-year-old woman.

Cardiac tumors may represent mechanical causes for syncope by limiting left ventricular filling and/or by obstructing the left ventricular outflow tract. Malignant melanoma is known to metastasize to the myocardium or pericardium, but there are only a very limited number of reports describing endocardial involvement by the tumor. We describe herein an 84-year-old woman who presented with daily near-syncope episodes, 9 years after treatment for a choroidal melanoma. The echocardiography and the pathologic examination revealed a metastatic melanoma. This is the first reported case of an ocular melanoma metastasizing to the heart and presenting as a left ventricular intracavitary pedunculated mass.

Bovine papillomavirus E5 protein induces the formation of signal transduction complexes containing dimeric activated platelet-derived growth factor beta receptor and associated signaling proteins.

The bovine papillomavirus E5 protein binds to the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in constitutive activation of the receptor and cell growth transformation. By subjecting extracts from E5-transformed or PDGF-treated cells to velocity sedimentation in sucrose gradients, activated PDGF beta receptor complexes were separated from monomeric, inactive receptor. Rapidly sedimenting activated complexes contained oligomeric (apparently dimeric), tyrosine-phosphorylated PDGF beta receptor, the E5 protein, and associated cellular signaling proteins including the p85 subunit of phosphoinositol 3'-kinase, phospholipase Cgamma, and Ras-GTPase activating protein. These signaling proteins made the major contribution to the increased sedimentation rate of the activated receptor complexes. Pairwise analysis of components of these complexes indicated that multiple signaling proteins and the E5 protein were simultaneously present in the activated complexes. Our results also showed that the E5 protein and PDGF activated only a small fraction of the total PDGF beta receptor, that not all receptor molecules associated with the E5 protein were tyrosine-phosphorylated, and that signaling proteins could bind to hemiphosphorylated receptor dimers. On the basis of these results, we propose a model for the assembly of multiprotein, activated PDGF beta receptor complexes in response to the E5 protein.

Minocycline pigmentation of heart valves.

Minocycline, a derivative of tetracycline, is a broad spectrum antibiotic used in the treatment of gram-positive and gram-negative infections. Benitz et al. (1) were the first to report black discoloration of the thyroid gland in rats, dogs, and monkeys given minocycline. Since that time, there have been numerous reports in the literature describing minocycline related black pigmentation of the skin, thyroid gland, and other sites. We report an unusual case of minocycline induced pigmentation of the cardiac valves and coronary vessels. The pigment stained with Fontana-Masson and was reduced with bleaching. The exact nature of the pigment is unclear; however, various theories have been advocated. Ochronosis is another cause of black pigmentation of the heart valves; the clinical history should allow distinction between the two.

Bovine papillomavirus E2 protein activates a complex growth-inhibitory program in p53-negative HT-3 cervical carcinoma cells that includes repression of cyclin A and cdc25A phosphatase genes and accumulation of hypophosphorylated retinoblastoma protein.

The bovine papillomavirus E2 protein can inhibit the proliferation of HT-3 cells, a p53-negative cervical carcinoma cell line containing integrated human papillomavirus type 30 DNA. Here, we analyzed HT-3 cells to explore the mechanism of p53-independent E2-mediated growth inhibition. Expression of the E2 protein repressed expression of the endogenous human papillomavirus type 30 E6/E7 genes. This was accompanied by hypophosphorylation and increased accumulation of p105Rb and repression of E2F1 expression. The E2 protein also caused reduced cyclin-dependent kinase (cdk) 2 activity, but this did not appear to be due to increased expression of cdk inhibitors. Rather, expression of cyclin A, which regulates cdk2 activity, and the cdc25A and cdc25B phosphatases, which are thought to activate cdk2, was significantly reduced at both the RNA and protein levels in response to E2 expression. The E2 protein reduced expression of cdc25A and cdc25B in both HT-3 and HeLa cells, but not in cells that were not growth-inhibited by the E2 protein. E2 point mutants unable to inhibit cell growth did not repress cdc25A and cdc25B expression, nor did the cell cycle inhibitors hydroxyurea and mimosine. Based on these results and the known properties of cell cycle components, we propose a model to account for E2-induced growth inhibition of cervical carcinoma cell lines.

The bovine papillomavirus E5 protein requires a juxtamembrane negative charge for activation of the platelet-derived growth factor beta receptor and transformation of C127 cells.

The bovine papillomavirus E5 gene encodes a 44-amino-acid, homodimeric transmembrane protein that is the smallest known transforming protein. The E5 protein transforms cultured fibroblasts by forming a stable complex with the endogenous platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, leading to sustained receptor activation. Aspartic acid 33 in the extracellular juxtamembrane region of the E5 protein is important for cell transformation and interaction with the PDGF beta receptor. A. N. Meyer et al. (Proc. Natl. Acad. Sci USA 91:4634-4638, 1994) speculated that this residue interacted with lysine 499 on the receptor. We constructed E5 mutants containing all possible substitutions at position 33, as well as several double mutants containing substitutions at aspartic acid 33 and at glutamic acid 36, and we examined the ability of these mutants to transform C127 mouse fibroblasts and to bind to and induce activation of the PDGF beta receptor. There was an excellent correlation between the transformation activities of the various mutants and their ability to bind to and activate the PDGF beta receptor. Analysis of the mutants demonstrated that a juxtamembrane negative charge on the E5 protein was required for cell transformation and for productive interaction with the PDGF beta receptor and indicated that aspartic acid 33 was more important for these activities than was glutamic acid 36. These results are consistent with the existence of an essential juxtamembrane salt bridge between lysine 499 on the PDGF beta receptor and an acidic residue in the C terminus of the E5 protein and lend support to our proposed model for the complex between the E5 dimer and the PDGF beta receptor.

Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor beta receptor.

The bovine papillomavirus E5 protein is a 44-aa transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor and induces constitutive tyrosine phosphorylation and activation of the receptor, resulting in cell transformation. The E5 protein does not resemble PDGF, but rather activates the receptor in a ligand-independent fashion, thus providing a unique system to examine activation of receptor tyrosine kinases. Here, we used a variety of approaches to explore the mechanism of receptor activation by the E5 protein. Chemical cross-linking experiments revealed that the E5 protein activated only a small fraction of the endogenous PDGF beta receptor in transformed fibroblasts and suggested that this fraction was constitutively dimerized. Coimmunoprecipitation experiments using extracts of cells engineered to coexpress full-length and truncated PDGF beta receptors confirmed that the E5 protein induced oligomerization of the receptor. Furthermore, in cells expressing the E5 protein, a kinase-active receptor was able to trans-phosphorylate a kinase-negative mutant receptor but was unable to catalyze intramolecular autophosphorylation. These results indicated that the E5 protein induced PDGF beta receptor activation by forming a stable complex with the receptor, resulting in receptor dimerization and trans-phosphorylation.

A single amino acid substitution in a WW-like domain of diverse members of the PDGF receptor subfamily of tyrosine kinases causes constitutive receptor activation.

Platelet-derived growth factor beta receptor (PDGFbetaR) is a transmembrane receptor tyrosine kinase involved in a variety of cellular functions. We have generated a constitutively activated murine PDGFbetaR containing a valine to alanine substitution at residue 536, located in the cytoplasmic juxtamembrane domain. When this mutant receptor (PR-V536A) was expressed in Ba/F3 cells, it allowed the cells to survive and proliferate in the absence of IL-3 or PDGF, and tyrosine phosphorylation of PR-V536A was increased markedly compared with that of the wild-type PDGFbetaR in the absence of ligand and similar to that observed in ligand-activated PDGFbetaR. PR-V536A displayed increased tyrosine kinase activity in vitro toward an exogenous substrate, and the tyrosine kinase activity of the receptor was required for the constitutive activation of the mutant. This valine to alanine substitution also activated a PDGFbetaR mutant unable to bind PDGF. Alanine substitutions at positions homologous to V536 of the murine PDGFbetaR also activated other members of the PDGF receptor subfamily. The amino acid sequence of this region revealed a strong similarity to WW domains present in other signal transduction proteins. Furthermore, GST fusion proteins containing the juxtamembrane region of the PDGFR specifically associated with peptides containing the WW domain consensus recognition sequence PPXY. The results suggest that the cytoplasmic juxtamembrane domain plays a role in the regulation of receptor activity and function, perhaps by participating in protein-protein interactions.

Structural models of the bovine papillomavirus E5 protein.

The bovine papillomavirus E5 protein is thought to be a type II integral membrane protein that exists as a disulfide-linked homodimer in transformed cells. Polarized-infrared measurements show that the E5 dimer in membrane bilayers is largely alpha-helical and has a transmembrane orientation. Computational searches of helix-helix conformations reveal two possible low-energy dimer structures. Correlation of these results with previous mutagenesis studies on the E5 protein suggests how the E5 dimer may serve as a molecular scaffold for dimerization and ligand-independent activation of the PDGF-beta receptor. We propose that on each face of the E5 dimer a PDGF-beta receptor molecule interacts directly with Gln17 from one E5 monomer and with Asp33 from the other E5 monomer. This model accounts for the requirement of Gln17 and Asp33 for complex formation and explains genetic results that dimerization of the E5 protein is essential for cell transformation.

Role of glutamine 17 of the bovine papillomavirus E5 protein in platelet-derived growth factor beta receptor activation and cell transformation.

The bovine papillomavirus E5 protein is a small, homodimeric transmembrane protein that forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor through transmembrane and juxtamembrane interactions, resulting in receptor activation and cell transformation. Glutamine 17 in the transmembrane domain of the 44-amino-acid E5 protein is critical for complex formation and receptor activation, and we previously proposed that glutamine 17 forms a hydrogen bond with threonine 513 of the PDGF beta receptor. We have constructed and analyzed mutant E5 proteins containing all possible amino acids at position 17 and examined the ability of these proteins to transform C127 fibroblasts, which express endogenous PDGF beta receptor. Although several position 17 mutants were able to transform cells, mutants containing amino acids with side groups that were unable to participate in hydrogen bonding interactions did not form a stable complex with the PDGF beta receptor or transform cells, in agreement with the proposed interaction between position 17 of the E5 protein and threonine 513 of the receptor. The nature of the residue at position 17 also affected the ability of the E5 proteins to dimerize. Overall, there was an excellent correlation between the ability of the various E5 mutant proteins to bind the PDGF beta receptor, lead to receptor tyrosine phosphorylation, and transform cells. Similar results were obtained in Ba/F3 hematopoietic cells expressing exogenous PDGF beta receptor. In addition, treatment of E5-transformed cells with a specific inhibitor of the PDGF receptor tyrosine kinase reversed the transformed phenotype. These results confirm the central importance of the PDGF beta receptor in mediating E5 transformation and highlight the critical role of the residue at position 17 of the E5 protein in the productive interaction with the PDGF beta receptor. On the basis of molecular modeling analysis and the known chemical properties of the amino acids, we suggest a structural basis for the role of the residue at position 17 in E5 dimerization and in complex formation between the E5 protein and the PDGF beta receptor.