In vitro and in vivo characterisation of glial cells immortalised with a temperature sensitive SV40 T antigen-containing retrovirus

1994 ◽  
Vol 37 (2) ◽  
pp. 182-196 ◽  
Author(s):  
M. Jung ◽  
A. J. Crang ◽  
W. F. Blackemore ◽  
D. Hoppe ◽  
H. Kettenmann ◽  
...  
Keyword(s):  
Glial Cells ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Sv40 T Antigen

Establishment and characterization of conditionally immortalized cells from the mouse urogenital ridge

1996 ◽  
Vol 109 (5) ◽  
pp. 899-909 ◽  
Author(s):  
B. Capel ◽  
J.R. Hawkins ◽  
E. Hirst ◽  
D. Kioussis ◽  
R. Lovell-Badge
Keyword(s):  
Primordial Germ Cells ◽  
Cell Types ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Sv40 Large T Antigen ◽  
In Vitro Morphogenesis ◽  
Immortalized Cells ◽  
Clonal Lines

Cell cultures from the urogenital ridge have been established to facilitate the study of the regulation and downstream interactions of Sry in mammalian sex determination. Cells have been explanted from transgenic mice carrying a temperature sensitive SV40 large T-antigen, and established in ongoing cultures. Analysis of the cells in these cultures at the electron microscope level reveals multiple cell types that compare to the cell types found in vivo during this period of development. Primordial germ cells, that are simultaneously explanted in the course of these experiments, also survive in culture. The explants undergo a morphogenetic organization into branching cord-like structures when cells are trypsinized and plated in extracellular matrix (Matrigel). We analyzed the expression of a number of molecular markers of the fetal gonad during monolayer culture, during in vitro morphogenesis in Matrigel, and in clonal lines derived from the complex explants. This analysis included Sry which is found to be expressed in some cultures from XY urogenital ridges that have been maintained for as long as 8 months.

The yeast nucleoporin Nsp1 binds nuclear localization sequences in vitro

1996 ◽  
Vol 74 (3) ◽  
pp. 363-372 ◽  
Author(s):  
Werner Barth ◽  
Ursula Stochaj
Keyword(s):  
Nuclear Localization ◽  
Specific Binding ◽  
T Antigen ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Sv40 T Antigen ◽  
Nuclear Targeting ◽  
Nuclear Localization Sequences

Facilitated transport of proteins into the nucleus requires nuclear localization sequences (NLSs) be present in the protein destined for the nucleus. The specific binding of NLSs by components of the nuclear transport apparatus is essential for these targeting reactions. We now report that the yeast nucleoporin Nsp1 binds specifically nuclear localization sequences in vitro. This nucleoporin recognizes several NLSs that are functional for nuclear targeting in vivo, including the NLS of SV40 T-antigen and of the yeast transcription factor Gal4. Nsp1 is organized into three domains, and we have located NLS binding sites to the N-terminal portion and the middle repetitive region of the protein. For the interaction between the NLS of SV40 T-antigen and Nsp1, we obtained association constants of 1.2 × 107 M−1 and 5 × 107 M−1. An association constant of 5 × 107 M−1 was determined for NLS binding to the repetitive domain of Nsp1. We analyzed binding of Nsp1 and its domains to a mutant version of the NLS derived from SV40 T-antigen, which poorly functions for nuclear targeting in vivo. The affinity for the mutant signal was about two orders of magnitude lower than for the wild-type NLS.Key words: Nsp1, nuclear pore complex, nucleoporin, nuclear localization sequence, protein targeting, yeast.

Tumorigenicity of cells transformed by Simian virus 40 and of hybrids between such cells and normal diploid cells

1979 ◽  
Vol 36 (1) ◽  
pp. 223-240
Author(s):  
C.J. Gee ◽  
H. Harris
Keyword(s):  
Mouse Embryo ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Transformed Cells ◽  
Sv40 T Antigen ◽  
Malignant Phenotype ◽  
Embryo Cells ◽  
Diploid Cells

A number of newly isolated clonal cell lines derived from diploid mouse embryo cells transformed by SV40 were examined in vitro and in vivo. Although these lines showed the properties that define transformation in vitro, they were not tumorigenic for many passages after their initial isolation. Cells from tumours eventually produced by the SV40-transformed cells were fused with diploid mouse embryo cells. The hybrids formed were initially non-tumorigenic. This indicates that a normal diploid cell can suppress the malignant phenotype of a tumorigenic SV40-transformed cell. The hybrid cells did, however, express the SV40 T antigen and they nad a clearly transformed phenotype in vitro. It thus appears that neither the transformed phenotype nor the expression of the SV40 T antigen are enough to endow a cell with the ability to grow progressively in vivo. The relationship between the transformed phenotype and tumorigenicity was further studied by fusing malignant mouse melanoma cells with non-tumorigenic SV40-transformed cells. The hybrids expressed the transformed phenotype in vitro but unable to form tumours in vivo. The changes that occur in cells after transformation by SV40 do not apparently affect the ability of these cells to suppress the malignant phenotype of tumour cells.

A Molecular Approach to Immunoscintigraphy: A Study of the T-Antigen Conformation on the Surface of Tumors

1987 ◽  
Vol 26 (01) ◽  
pp. 1-6 ◽  
Author(s):  
S. Selvaraj ◽  
M. R. Suresh ◽  
G. McLean ◽  
D. Willans ◽  
C. Turner ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Tumor Cell ◽  
T Antigen ◽  
Human Carcinomas ◽  
Animal Tumor ◽  
Synthetic Antigens

The role of glycoconjugates in tumor cell differentiation has been well documented. We have examined the expression of the two anomers of the Thomsen-Friedenreich antigen on the surface of human, canine and murine tumor cell membranes both in vitro and in vivo. This has been accomplished through the synthesis of the disaccharide terminal residues in both a and ß configuration. Both entities were used to generate murine monoclonal antibodies which recognized the carbohydrate determinants. The determination of fine specificities of these antibodies was effected by means of cellular uptake, immunohistopathology and immunoscintigraphy. Examination of pathological specimens of human and canine tumor tissue indicated that the expressed antigen was in the β configuration. More than 89% of all human carcinomas tested expressed the antigen in the above anomeric form. The combination of synthetic antigens and monoclonal antibodies raised specifically against them provide us with invaluable tools for the study of tumor marker expression in humans and their respective animal tumor models.

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

scholarly journals C-Terminal Deletions Can Suppress Temperature-Sensitive Mutations and Change Dominance in the Phage Mu Repressor

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 661-672 ◽  
Author(s):  
Jodi L Vogel ◽  
Vincent Geuskens ◽  
Lucie Desmet ◽  
N Patrick Higgins ◽  
Ariane Toussaint
Keyword(s):  
Binding Activity ◽  
Temperature Sensitive ◽  
Dna Binding Activity ◽  
Heat Stable ◽  
C Terminus ◽  
Acid Domain ◽  
Mutant Forms ◽  
Amino Acid Domain

Abstract Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup° hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup° hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179–V196), binds Mu operator DNA more stably at 42° in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

Neuroprotective Effects of the FGF21 Analogue LY2405319

2021 ◽  
pp. 1-13
Author(s):  
Claire Rühlmann ◽  
David Dannehl ◽  
Marcus Brodtrück ◽  
Andrew C. Adams ◽  
Jan Stenzel ◽  
...  
Keyword(s):  
Glial Cells ◽  
Neuroprotective Effect ◽  
Amyloid Β ◽  
Fibroblast Growth Factor 21 ◽  
Neuroprotective Effects ◽  
Organotypic Brain Slice ◽  
Brain Slice Cultures ◽  
Abca1 Mrna

Background: To date, there are no effective treatments for Alzheimer’s disease (AD). Thus, a significant need for research of therapies remains. Objective: One promising pharmacological target is the hormone fibroblast growth factor 21 (FGF21), which is thought to be neuroprotective. A clinical candidate for medical use could be the FGF21 analogue LY2405319 (LY), which has a specificity and potency comparable to FGF21. Methods: The present study investigated the potential neuroprotective effect of LY via PPARγ/apoE/abca1 pathway which is known to degrade amyloid-β (Aβ) plaques by using primary glial cells and hippocampal organotypic brain slice cultures (OBSCs) from 30- and 50-week-old transgenic APPswe/PS1dE9 (tg) mice. By LY treatment of 52-week-old tg mice with advanced Aβ deposition, we further aimed to elaborate the effect of LY on AD pathology in vivo. Results: LY application to primary glial cells caused an upregulation of pparγ, apoE, and abca1 mRNA expression and significantly decreased number and area of Aβ plaques in OBSCs. LY treatment in tg mice increased cerebral [18F] FDG uptake and N-acetylaspartate/creatine ratio indicating enhanced neuronal activity and integrity. Although LY did not reduce the number of Aβ plaques in tg mice, the number of iba1-positive cells was significantly decreased indicating reduced microgliosis. Conclusion: These data identified LY in vitro as an activator of Aβ degrading genes leading to cerebral Aβ load amelioration in early and late AD pathology. Although Aβ plaque reduction by LY failed in vivo, LY may be used as therapeutic agent to treat AD-related neuroinflammation and impaired neuronal integrity.

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