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Large T Antigen (large t + antigen)
Kinds of Large T Antigen Selected AbstractsA transient expression vector for recombinant protein production in Chinese hamster ovary cellsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 1 2006Mimi ML Liao Abstract An expression vector was specifically designed for use in Chinese hamster ovary (CHO) cells to enhance the level of protein production in a transient expression system. Two key components that can increase protein production transiently are the promoter used to drive recombinant gene expression and the template copy number. In this study the modified and metal-inducible metallothionein (M2.6) promoter was shown to be superior to the human cytomegalovirus (CMV) and to the simian virus SV40 promoters. Plasmid replication was achieved using the Polyoma (Py) virus origin of replication (PyOri) and the Py Large T antigen (PyLT). An expression vector containing Py elements was shown to replicate extensively in CHO cells. The combination of the metal-inducible M2.6 promoter and episomal replication of the expression vector, named pPyOriLT resulted in elevated levels of transgene expression following transient transfection of CHO cells Copyright © 2005 Society of Chemical Industry [source] Presence and expression of JCV early gene large T Antigen in the brains of immunocompromised and immunocompetent individualsJOURNAL OF MEDICAL VIROLOGY, Issue 12 2008Serena Delbue Abstract JC virus (JCV) is a polyomavirus that asymptomatically infects up to 80% of the worldwide human population and establishes latency in the kidney. In the case of host immunodeficiency, it can cause progressive multifocal leukoencephalopathy (PML), which is a fatal demyelinating disease of the central nervous system. In an attempt to understand better PML pathogenesis and JCV infection, the presence of the JCV genome and expression of the early viral protein in the brain of deceased individuals, with and without HIV infection, was investigated. Sixty autopsy samples of brain tissues were collected from 15 HIV-positive PML patients, 15 HIV-positive patients with other neurological diseases, 15 HIV-positive patients without neurological disorders, and 15 HIV-negative individuals who died from diseases unrelated to the central nervous system. By means of specific Real Time Polymerase Chain Reaction, the JCV genome was detected in 14 of 15 PML brains, three of 15 HIV-positive brains (with and without neurological diseases), and 1 of 15 HIV-negative brains. JCV genotyping was also performed. Expression of the early JCV protein T Antigen was verified by a specific immunohistochemistry assay, and it was found in the brain tissues from 12 PML cases and one case with other neurological disease. The data obtained demonstrate that infection of the brain with JCV can also be observed in the brains of HIV-negative individuals, without neurological disorders. However, viral protein expression was limited to PML brains and to one brain from a patient with other neurological disease, suggesting that JCV can also be present in the brains of patients without PML. J. Med. Virol. 80:2147,2152, 2008. © 2008 Wiley-Liss, Inc. [source] Resistance to experimental tumorigenesis in cells of a long-lived mammal, the naked mole-rat (Heterocephalus glaber)AGING CELL, Issue 4 2010Sitai Liang Summary The naked mole-rat (NMR, Heterocephalus glaber) is a long-lived mammal in which spontaneous cancer has not been observed. To investigate possible mechanisms for cancer resistance in this species, we studied the properties of skin fibroblasts from the NMR following transduction with oncogenes that cause cells of other mammalian species to form malignant tumors. Naked mole-rat fibroblasts were transduced with a retrovirus encoding SV40 large T antigen and oncogenic RasG12V. Following transplantation of transduced cells into immunodeficient mice, cells rapidly entered crisis, as evidenced by the presence of anaphase bridges, giant cells with enlarged nuclei, multinucleated cells, and cells with large number of chromosomes or abnormal chromatin material. In contrast, similarly transduced mouse and rat fibroblasts formed tumors that grew rapidly without crisis. Crisis was also observed after > 40 population doublings in SV40 TAg/Ras-expressing NMR cells in culture. Crisis in culture was prevented by additional infection of the cells with a retrovirus encoding hTERT (telomerase reverse transcriptase). SV40 TAg/Ras/hTERT-expressing NMR cells formed tumors that grew rapidly in immunodeficient mice without evidence of crisis. Crisis could also be induced in SV40 TAg/Ras-expressing NMR cells by loss of anchorage, but after hTERT transduction, cells were able to proliferate normally following loss of anchorage. Thus, rapid crisis is a response of oncogene-expressing NMR cells to growth in an in vivo environment, which requires anchorage independence, and hTERT permits cells to avoid crisis and to achieve malignant tumor growth. The unique reaction of NMR cells to oncogene expression may form part of the cancer resistance of this species. [source] Temporary amelioration of bilirubin conjugation defect in Gunn rats by transplanting conditionally immortalized hepatocytesJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 6 2002BYUNG-HO KIM Abstract Background: Conditionally immortalized hepatocytes (CIH) have been used in hepatocyte transplantation as an alternative to primary hepatocytes to cope with the shortage of donor organs. However, CIH are known to undergo apoptosis at body temperature and survive in vivo for a short period. In the present study, we investigated whether CIH function or not and how long their function is maintained in vivo. Methods: Various CIH cell lines that were established with temperature-sensitive Simian virus 40 large T antigen were transplanted into the spleen of Gunn rats, which are defective in bilirubin uridine diphosphate glucuronoside transferase (BUGT). Then, we measured biological changes over 3 months. Results: Serum bilirubin of the syngeneic CIH recipients decreased by 30%, which was maintained for 8 weeks. Thereafter, it began to rise to basal levels. The recipients of allogeneic CIH showed a minor reduction of bilirubin, although this was not statistically significant. However, there was no significant change in the bilirubin level in recipients of BUGT-defective congeneic CIH throughout the study period. Bilirubin monoglucuronides in the bile were not detected in the recipients of BUGT-defective CIH. However, they appeared in recipients of non-defective CIH and made up approximately 41% of total bile pigments. Conclusions: Conditionally immortalized hepatocytes expressed hepatocyte function in vivo as well as in vitro, but the function lasted for a couple of months. According to our previous study, the limited functional duration may be related to the inevitable occurrence of apoptosis of these cells at body temperature. These data suggest that CIH can be used in hepatocyte transplantation only for temporary hepatic support. © 2002 Blackwell Publishing Asia Pty Ltd [source] Lack of protein expression of the simian virus 40 large T antigen in human lymphomas,,JOURNAL OF MEDICAL VIROLOGY, Issue 6 2008Philip Went Abstract Several studies have detected Simian virus 40 (SV40) deoxyribonucleic acid sequences in human tumor tissues, including lymphomas, mainly by the polymerase chain reaction, but these data were not confirmed by subsequent investigations. Regional differences in the distribution of the SV40 and/or technical difficulties have been taken into account to explain these divergent results, but because only a few such studies dealt with the expression of SV40 proteins in tumor tissues, we investigated the expression of the SV40 large T antigen in human lymphomas by immunohistochemistry. Tissue microarrays containing Non-Hodgkin's-lymphomas and Hodgkin's-lymphomas were constructed utilizing archival samples encompassing the years 1974,2001 from Italian, Swiss and Austrian patients. Expression of the SV40 large T antigen was analysed by highly specific and sensitive immunohistochemistry using a mouse monoclonal antibody. Protein expression of the large T antigen was not detected in 655 Non-Hodgkin's-lymphomas or in 337 Hodgkin's- lymphomas. The results suggest the absence of an association between SV40 large T antigen and human lymphomas. J. Med. Virol. 80:1112,1115, 2008. © 2008 Wiley-Liss, Inc. [source] Tumorigenic study on hepatocytes coexpressing SV40 with RasMOLECULAR CARCINOGENESIS, Issue 4 2006Beicheng Sun Abstract A model of neoplastic transformation by the combination of SV40 large T antigen (LT), SV40 small T antigen (ST), oncogenic Ras, and human telomerase reverse trasncriptase subunit (hTERT) has become established and replicated in primary human fibroblasts, however, there is no report on human hepatocytes. Here we use cell transplantation model, and show that transplantation of human hepatocytes of HL-7702 and HL-7703 expressing Ha-RasV12 and SV40 LT into subrenal capsule of immunodeficient mice results in fully malignant tumors, in contrast to conventional subcutaneous injections where tumors fail to develop. In GM-847 cell study, we have found that hTERT is not required for tumorigenic growth in subrenal capsule transplantation, however, it is required in subcutaneous injection assay. These results demonstrate that Human hepatocytes can be transformed under kidney capsule by coexpressing SV40 LT and Ha-RasV12, neither hTERT nor protein phosphatase 2A (PP2A) inhibition are required for malignant transformation, a gene which increases cell survival in the subcutaneous injection model is not required for tumorigenic growth in subrenal capsule. © 2005 Wiley-Liss, Inc. [source] Functional expression of the hyperpolarization-activated, non-selective cation current If in immortalized HL-1 cardiomyocytesTHE JOURNAL OF PHYSIOLOGY, Issue 1 2002Laura Sartiani HL-1 cells are adult mouse atrial myocytes induced to proliferate indefinitely by SV40 large T antigen. These cells beat spontaneously when confluent and express several adult cardiac cell markers including the outward delayed rectifier K+ channel. Here, we examined the presence of a hyperpolarization-activated If current in HL-1 cells using the whole-cell patch-clamp technique on isolated cells enzymatically dissociated from the culture at confluence. Cell membrane capacitance (Cm) ranged from 5 to 53 pF. If was detected in about 30 % of the cells and its occurrence was independent of the stage of the culture. If maximal slope conductance was 89.7 ± 0.4 pS pF,1 (n= 10). If current in HL-1 cells showed typical characteristics of native cardiac If current: activation threshold between ,50 and ,60 mV, half-maximal activation potential of ,83.1 ± 0.7 mV (n= 50), reversal potential at ,20.8 ± 1.5 mV (n= 10), time-dependent activation by hyperpolarization and blockade by 4 mm Cs+. In half of the cells tested, activation of adenylyl cyclase by the forskolin analogue L858051 (20 ,m) induced both a ,6 mV positive shift of the half-activation potential and a ,37 % increase in the fully activated If current. RT-PCR analysis of the hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) expressed in HL-1 cells demonstrated major contributions of HCN1 and HCN2 channel isoforms to If current. Cytosolic Ca2+ oscillations in spontaneously beating HL-1 cells were measured in Fluo-3 AM-loaded cells using a fast-scanning confocal microscope. The oscillation frequency ranged from 1.3 to 5 Hz and the spontaneous activity was stopped in the presence of 4 mm Cs+. Action potentials from HL-1 cells had a triangular shape, with an overshoot at +15 mV and a maximal diastolic potential of ,69 mV, i.e. more negative than the threshold potential for If activation. In conclusion, HL-1 cells display a hyperpolarization-activated If current which might contribute to the spontaneous contractile activity of these cells. [source] Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase ,-primaseFEBS JOURNAL, Issue 9 2003Richard W. P. Smith Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase ,-primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA replication of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase ,-primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex. [source] | ||||||||||||||||