Similarities and differences between the E5 oncoproteins of bovine papillomaviruses type 1 and type 4: cytoskeleton, motility and invasiveness in E5-transformed bovine and mouse cells

Bovine papillomaviruses (BPVs) are oncogenic viruses. In cattle, BPV-1/2 is associated with urinary bladder cancer and BPV-4 with upper GI tract cancer. BPV E5 is a small hydrophobic protein localised in the endoplasmic reticulum (ER) and Golgi apparatus (GA). E5 is the major transforming protein of BPVs, capable of inducing cell transformation in cultured mouse fibroblasts and, in cooperation with E7, in primary bovine cells. E5-induced cell transformation is accompanied by activation of several cellular protein kinases, including growth factor receptors, and alkalinisation of endosomes and GA. We have reported that BPV E5 causes swelling and fragmentation of the GA and extensive vacuolisation of the cytoplasm. We now show that E5 from both BPV-1 and BPV-4 disturbs the actin cytoskeleton and focal adhesions in transformed bovine cells, where these morphological and behavioural characteristics are accompanied by hyperphosphorylation of the cellular phosphotyrosine kinase c-src. Both BPV-1 and BPV-4 E5 increase the motility of transformed mouse cells, but only BPV-1 E5 causes transformed mouse cells to penetrate a matrigel matrix. BPV-1 transformed mouse cells, but not BPV-4 transformed mouse cells, have hyperhpsphorylated c-src.     

Stable expression of a neuronal dopaminergic progenitor phenotype in cell lines derived from human amniotic fluid cells

Cells from human amniotic fluid derived from the fetus are considered a source of multipotent cells. Their properties have not been fully exploited, partially because unlike other embryonic sources such as embryonic stem (ES) cells, cell lines from amniocentesis samples have not been generated. We have established and characterized the properties of eight individual cell lines. Flow cytometry using several cell surface markers showed that all cell lines generated consisted of homogeneous populations that lack HLAII antigenicity. Using a combination of immunocytochemistry, Western blotting, and RT-PCR, we found weak expression of Oct4 and nestin and strong expression of tubulin-betaIII, MAP2, and tau. Specific markers for cholinergic, (nor)adrenergic, and GABAergic neurons or glia were weakly expressed or absent, whereas expression of factors implicated in early induction of dopaminergic neurons, TGF-beta3 and beta-catenin were present. Further analysis showed strong expression of EN-1, c-RET, PTX3, and NURR1 essential for induction and survival of midbrain dopaminergic neurons, TH, AADC, and VMAT2 components of dopamine synthesis and secretion, and syntaxin1A and SNAP-25 necessary for neurotransmitter exocytosis. This phenotype was retained throughout passages and up to the current passage 36. Expression of neuronal and dopaminergic markers in individual AF cell lines was comparable to expression in neurons induced from ES cells and in IMR-32 and SH-SY5Y neuroblastomas. Our data show that cell lines can be derived from subcultures of amniocentesis, and are primarily composed of a population of progenitors with a phenotype similar to that of committed mesencephalic dopaminergic neurons.     

Right ventricular function in ischemic or idiopathic dilated cardiomyopathy.

BACKGROUND: Differentiation between ischemic (ICM) and dilated cardiomyopathy (DCM) has important therapeutic implications because the former may benefit from coronary revascularization. The aim of this study was to investigate right ventricular (RV) function using tissue Doppler echocardiography (TDE) and compare the TDE parameters of the RV among patients with ICM and DCM. METHODS AND RESULTS: Forty-two patients with ICM and 40 patients with DCM were studied with conventional echocardiography and TDE. The 2 groups did not differ in terms of New York Heart Association class, left ventricular ejection fraction and pharmacological treatment. Patients with ICM had higher pulmonary artery systolic pressure (44.4 mmHg vs 34.7 mmHg, p=0.006) and lower tricuspid annular motion systolic (RV Sa 0.06 m/s vs 0.09 m/s, p<0.0001), and diastolic velocities (RV Ea 0.05 m/s vs 0.07 m/s, p=0.0003, RV Aa 0.075 m/s vs 0.11 m/s, p=0.0016). They also exhibited a higher ratio of early transtricuspid filling velocity to early diastolic velocity of the tricuspid annulus (RV E/Ea 8.2 vs 5.7, p=0.0008). Age, pulmonary artery systolic pressure and tricuspid Sa were significant independent predictors of the diagnosis of ICM. CONCLUSIONS: RV dysfunction is more pronounced in patients with ICM than in patients with DCM. The RV TDE parameters can be used to complement clinical and conventional echocardiographic findings in the assessment of patients with ICM and DCM.     

Down-regulation of MHC class I by bovine papillomavirus E5 oncoproteins.

The papillomavirus E5 protein is localized in the endoplasmic reticulum (ER) and Golgi apparatus (GA) of the host cell. Transformed bovine fibroblasts expressing bovine papillomavirus (BPV) E5 are highly vacuolated and have a much enlarged, distorted and fragmented GA. Major histocompatibility complex class I (MHC I) is processed and transported to the cell surface through the GA. Given the cellular localization of E5 in the GA and the morphologically abnormal GA, we investigated the expression of MHC I in cells transformed by E5 from BPV-1 and BPV-4. Two cell lines were used: bovine cells that also express E6, E7 and activated ras, and NIH3T3 cells that express only E5. In addition, PalF cells acutely infected with a recombinant retrovirus expressing E5 were also examined. In contrast to non-transformed normal cells, or transformed cells expressing other papillomavirus proteins, cells expressing E5 do not express MHC I on their surface, but retain it intracellularly, independently of the presence of other viral or cellular oncogenes, or of whether the cells are long-term transformants or acutely infected. We conclude that expression of E5 prevents expression of MHC I to the cell surface and causes its retention within the cell. In addition, lower amounts of total MHC I heavy chain and of heavy chain RNA are detected in E5-transformed cells than in control cells. As surface expression of another glycosylated membrane protein, the transferrin receptor, is not affected, it appears that E5 targets MHC I with at least a degree of specificity. In papillomavirus lesions this effect would have important implications for antigen presentation by, and immunosurveillance of, virally infected cells.     

First report of Hb A2-NYU (HBD:c.39T>A) in the Hellenic population

We report the first heterozygous case of Hb A(2)-NYU (HBD:c.39T>A) in the Hellenic population. The proband, an adult female from the island of Crete, Greece, was identified during routine family screening. DEAE chromatography of the index case revealed a minor hemoglobin (Hb) fraction preceding the elution of the wild-type Hb A(2). DNA sequencing of the entire HBD gene coding regions indicated that the index case was heterozygous for the rare variant Hb A(2)-NYU. Family studies indicated that this Hb variant was inherited from the mother. This finding underlines the vast genetic heterogeneity of the HBD gene in the Hellenic population.     

Characterization of polymorphic microsatellite markers in Valencia letourneuxi (Valenciidae) and cross-amplification in two other cyprinodontiform species

Fourteen microsatellite loci were isolated from the critically endangered Corfu toothcarp Valencia letourneuxi using a microsatellite enrichment protocol and selective hybridization with a biotinylated (AC) probe. Nine of these loci were polymorphic in a V. letourneuxi population of 20 individuals (Vlychos stream) with mean number of alleles per locus = 2.55 and mean observed heterozygosity = 0.3447. Moreover, some of these 9 microsatellite markers and the remaining five loci, monomorphic in V. letourneuxi, were found to be polymorphic in the Spanish samaruc Valencia hispanica and the Mediterranean toothcarp Aphanius fasciatus. These microsatellite loci are expected to be used for population genetic analysis as well as studying the genetic diversity of this endangered species.