Infiltrating Foxp3+ regulatory T cells and histopathological features in canine classical and spermatocytic seminomas.

In humans, regulatory T (T reg) cells are known to play a critical role in both the regulation of immune homoeostasis and the progression of cancer. However, there is little information about the identification, characterization and the function of T reg cells in canine tumours. We identified T reg cells in 28 canine seminoma samples using a Forkhead box P3 (Foxp3) antibody and investigated the relationship between T reg cell infiltration and histopathological features of classical and spermatocytic seminomas (SE and SS, respectively). The Foxp3 protein showed nuclear immunostaining in infiltrating lymphocytes, and Foxp3+ cells were diffused or focally distributed in seminoma tissues. Foxp3+ cells were frequently present in the SS histotype, in seminomas that showed no evidence of tumour cell invasion into the vessels and in seminomas showing a diffuse growth pattern with three cell types. Neither the SE/SS histotype nor the histopathological features of the tumour correlated with Foxp3+ cell counts. These results indicate that Foxp3+ T reg cells may be associated with a less malignant histological phenotype or may not play a critical role in the immune response of canine seminomas. Moreover, Foxp3+ T reg cells may be associated with SS seminoma, but further studies, involving a larger number of samples, are required to better understand whether these cells play a critical role in the immune response in canine seminomas. This is the first report to demonstrate the characteristics of T reg cell infiltration in canine seminoma.

P-glycoprotein expression in canine mammary gland tumours related with myoepithelial cells.

P-glycoprotein is influential in chemotherapy-resistance in numerous cancers and has been widely studied in human breast cancer research, but is less studied in canine mammary gland tumour (MGT). The study was to evaluate P-glycoprotein expression and its localisations related with prognostic factors with monoclonal antibody C219, by immunohistochemistry (IHC) of 68 cases of canine malignant (n=54) and benign (n=14) MGT. Additional immunofluorescence (IF) and reverse transcriptase-polymerase chain reaction (RT-PCR) were also performed. There was a novel finding that P-glycoprotein expression with C219 localised at two different cell types: epithelial and myoepithelial cells. Myoepithelial localised tumours were 5 benign (35.5%) and 21 malignant (63.6%), while epithelial localised tumours were 12 cases, all malignant (36.5%). Unlike conventional belief, semi-quantitative evaluation of IHC intensity scores of C219 expression in malignant MGT was related with favourable histopathological parameters.

Adeno-associated virus Rep78 binds to E2-responsive element 1 of bovine papillomavirus type 1.

Adeno-associated virus type-2 (AAV-2) is a helper-dependent parvovirus that has been implicated in the inhibition of replication and oncogenic transformation of bovine papillomavirus type-1 (BPV-1) and other transforming DNA viruses. Previous studies have suggested that the Rep78 protein of AAV-2 is a key player mediating this effect. In this report we have analyzed the effect of AAV-2 Rep78 protein on the regulation of gene expression of a reporter gene under the control of the long control region (LCR) of BPV-1. Our results show that Rep78 is capable of down-regulating the promoter activity of the LCR in vivo in tissue culture cells. Inhibition of LCR activity in vivo suggested the need for Rep78 to bind to a region of the LCR promoter spanning the E2-responsive elements of BPV-1. This observation was further confirmed in vitro with gel shift assays showing specific binding of Rep78 to DNA oligonucleotides containing E2-responsive element 1 (E2RE1) sequences of BPV-1 LCR. Our results expand the understanding of the mechanism of trans-regulation mediated by Rep78 and involving this protein and DNA sequences with complex secondary structure.

Genetic analysis of the internal ribosome entry segment of bovine viral diarrhea virus.

Pestiviruses and hepaciviruses are atypical members of the Flaviviridae due to their unique biological properties, including the utilization of internal ribosome entry for translation initiation. In contrast to internal initiation in picornaviruses, which depends on numerous canonical initiation factors, the mode of internal ribosome entry in pestiviruses and hepaciviruses resembles prokaryotic translation initiation. To identify functionally important elements within the bovine viral diarrhea virus (BVDV) internal ribosome entry segment (IRES), we carried out a mutational analysis of the 5' untranslated region (5' UTR) of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid. IRES function was assessed in a bicistronic transcript by inserting the 5' 901 nucleotides of BVDV genome, which correspond to the 385 nucleotides of the 5' UTR and 515 nucleotides of the open reading frame (ORF) encoding for Npro and 4 amino acids from the capsid protein. The resulting Npro-luciferase fusion encoded by the 3' cistron was cleaved efficiently to release the luciferase reporter. In vivo translation analyses showed that stem-loops Ia and Ib in the 5' UTR were completely dispensable for efficient translation, whereas stem-loop IIIe and the hairpin end of IIIb were only partially required. In contrast, deletions or insertions in any of other four stem-loop structures, including domains II, IIIa, IIIc, and IIId, caused nearly 10-fold reductions of in vivo IRES activity. The tolerance of structural modifications within the distal portion of domain IIIb and IIIe correlated with a low level of sequence conservation in these regions among pestiviruses. The 5' boundary of the IRES resides at the 5' end of stem-loop II near nucleotide 75. The 3' of the IRES extends into the 5' end of the polyprotein ORF because removal of the Npro coding region reduced translation by 21%.

Overexpression and simple purification of a truncated, immunologically reactive GST-HCV core (1-123) fusion protein.

A full-length and a truncated gene for the core protein of hepatitis C virus (HCV) were linked to the gene for glutathione S-transferase (GST), and the expression of each GST-HCV core fusion protein was analyzed. The truncated GST-HCV core (1-123) fusion protein was expressed as a mostly soluble and partly insoluble form comprising more than 50% of the total protein in Escherichia coli after induction by isopropylthio-beta-D-galactoside (IPTG), while the full length GST-HCV core (1-191) fusion protein was not expressed, suggesting that the hydrophobic carboxy terminal region in the core protein affects its expression. In addition, the GST-HCV core (1-123) fusion protein purified by GST-agarose chromatography reacted specifically with an anti-HCV serum from a patient.

A cDNA of Schizosaccharomyces pombe encoding a homologue of DnaJ-like protein.

A Schizosaccharomyces pombe homologue, Psi, was cloned from Schizosaccharomyces pombe cDNA library. Deduced amino acid sequence of the cDNA has 55% sequence homology with the Saccharomyces cerevisiae Sis1 protein and contains the structural features of a family of DnaJ proteins. This homology suggests Psi protein may be implicated in the initiation of translation as like Sis1 function of Saccharomyces cerevisiae.