scholarly journals A Conditionally Immortalized Human Podocyte Cell Line Demonstrating Nephrin and Podocin Expression

2002 ◽  
Vol 13 (3) ◽  
pp. 630-638 ◽  
Author(s):  
Moin A. Saleem ◽  
Michael J. O’Hare ◽  
Jochen Reiser ◽  
Richard J. Coward ◽  
Carol D. Inward ◽  
...  
Keyword(s):  
Cell Line ◽  
Kinase Inhibitors ◽  
Growth Arrest ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Cell Cycle Protein ◽  
Filtration Barrier ◽  
Health And Disease ◽  
Podocyte Proteins

ABSTRACT. Recent molecular insights have established the podocyte as a key component of the glomerular filtration barrier, and hence an important common pathway in proteinuric diseases. A conditionally immortalized human podocyte cell line has been developed by transfection with the temperature-sensitive SV40-T gene. These cells proliferate at the “permissive” temperature (33°C). After transfer to the “nonpermissive” temperature (37°C), they entered growth arrest and expressed markers of differentiated in vivo podocytes, including the novel podocyte proteins, nephrin, podocin, CD2AP, and synaptopodin, and known molecules of the slit diaphragm ZO-1, α-, β-, and γ-catenin and P-cadherin. The differentiation was accompanied by a growth arrest and the upregulation of cyclin-dependent kinase inhibitors, p27 and p57, as well as cyclin D1, whereas cyclin A was downregulated. These data are consistent with cell cycle protein expression during podocyte maturation in vivo. In conclusion, the development of this cell line provides a new tool in the study of podocyte biology, which will enable accurate assessment of the behavior of these complex cells in health and disease.

Phenotype–genotype relationships in peroxisome biogenesis disorders of PEX1-defective complementation group 1 are defined by Pex1p–Pex6p interaction

2001 ◽  
Vol 357 (2) ◽  
pp. 417-426 ◽  
Author(s):  
Shigehiko TAMURA ◽  
Naomi MATSUMOTO ◽  
Atsushi IMAMURA ◽  
Nobuyuki SHIMOZAWA ◽  
Yasuyuki SUZUKI ◽  
...  
Keyword(s):  
Zellweger Syndrome ◽  
Complementation Group ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Peroxisome Biogenesis ◽  
Causative Gene ◽  
Aaa Atpase ◽  
Peroxisome Biogenesis Disorders ◽  
The Stability

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleucodystrophy (NALD) and infantile Refsum disease (IRD), are fatal autosomal recessive diseases caused by impaired peroxisome biogenesis, of which 12 genotypes have been reported. ZS patients manifest the severest clinical and biochemical abnormalities, whereas those with NALD and IRD show less severity and the mildest features respectively. We have reported previously that temperature-sensitive peroxisome assembly is responsible for the mildness of the clinical features of IRD. PEX1 is the causative gene for PBDs of complementation group E (CG-E, CG1 in the U.S.A. and Europe), the PBDs of highest incidence, encoding the peroxin Pex1p of the AAA ATPase family. It has been also reported that Pex1p and Pex6p interact with each other. In the present study we investigated phenotype–genotype relationships of CG1 PBDs. Pex1p from IRD such as Pex1p with the most frequently identified mutation at G843D was largely degraded in vivo at 37°C, whereas a normal level of Pex1p was detectable at the permissive temperature. In contrast, PEX1 proteins derived from ZS patients, including proteins with a mutation at L664P or the deletion of residues 634–690, were stably present at both temperatures. Pex1p-G843D interacted with Pex6p at approx. 50% of the level of normal Pex1p, whereas Pex1p from ZS patients mostly showing non-temperature-sensitive peroxisome biogenesis hardly bound to Pex6p. Taking these results together, we consider it most likely that the stability of Pex1p reflects temperature-sensitive peroxisome assembly in IRD fibroblasts. Failure in Pex1p–Pex6p interaction gives rise to more severe abnormalities, such as those manifested by patients with ZS.

Growth Properties of Neural Cell Lines Immortalized with the Tsa58 Allele of Sv40 Large T Antigen

1997 ◽  
Vol 6 (3) ◽  
pp. 231-238 ◽  
Author(s):  
M.E. Truckenmiller ◽  
Ora Dillon-Carter ◽  
Carlo Tornatore ◽  
Henrietta Kulaga ◽  
Hidetoshi Takashima ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cell Line ◽  
Cell Lines ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Large T Antigen ◽  
Wild Type ◽  
Sv40 Large T Antigen ◽  
Growth Properties

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33° C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5°C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5°C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5°C for all three cell lines. After 3 wk at 39.5°C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.

Cyclic AMP regulates PDGF-stimulated signal transduction and differentiation of an immortalized optic-nerve-derived cell line

1999 ◽  
Vol 202 (4) ◽  
pp. 461-473
Author(s):  
R.I. Cohen ◽  
R. Mckay ◽  
G. Almazan
Keyword(s):  
Signal Transduction ◽  
Optic Nerve ◽  
Growth Factor ◽  
Cyclic Amp ◽  
Cell Line ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Permissive Temperature

To facilitate the study of the molecular events underlying the development of optic-nerve-derived oligodendrocytes and their growth-factor-related signal transduction events, we immortalized perinatal rat optic nerve cells with a temperature-sensitive simian virus 40 large T-antigen, carrying the tsA58 and U19 mutations, via a retrovirus vector. The line, tsU19-9, was selected on the basis of the expression of the neural precursor marker nestin. At the permissive temperature, 33 degreesC, tsU19-9 cells had a flat epithelial morphology. In contrast, following exposure to platelet-derived growth factor (PDGF), a factor important in the lineage progression of oligodendrocytes, or in the presence of dibutyryl cyclic AMP at 39 degreesC (the non-permissive temperature), the cells underwent morphological and antigenic differentiation to cells characteristic of the oligodendrocyte lineage. We used this cell line to investigate the binding characteristics of PDGF and related signalling cascades. Competition binding, phosphoinositide hydrolysis and intracellular Ca2+ mobilization assays all demonstrated that the three different isoforms of PDGF (AA, AB and BB) bound to and acted on the cell line. Overnight exposure to forskolin, a treatment that initiated morphological and phenotypic progression into an oligodendrocyte lineage, decreased PDGF-BB-induced intracellular Ca2+ mobilization and inhibited basal and PDGF-stimulated [3H]thymidine incorporation. Our results demonstrate that tsU19-9 may serve as a resource to study early optic-nerve oligodendrocyte development.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

An Alternatively Spliced Survivin Variant Is Positively Regulated by p53 and Sensitizes Leukemia Cells to Chemotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3497-3497
Author(s):  
Ningxi Zhu ◽  
Lubing Gu ◽  
Harry W. Findley ◽  
Fengzhi Li ◽  
Muxiang Zhou
Keyword(s):  
Cell Line ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Doxorubicin Treatment ◽  
Cryptic Exon ◽  
Apoptosis Protein ◽  
Induced Apoptosis

Abstract Survivin is a unique member of the inhibitor of apoptosis protein (IAP) family, and its expression is regulated by p53. Recent identification of several functionally divergent survivin variants augments the complexity of survivin action as well as its regulation. Here we report that survivin-2B (retaining a part of intron 2 as a cryptic exon) is positively regulated by p53, and its overexpression plays a role in sensitizing leukemia cells to chemotherapeutic drug doxorubicin. Doxorubicin treatment activated p53, downregulated survivin and survivin-DEx3 but upregulated survivin-2B in EU-3, an acute lymphocytic leukemia (ALL) cell line with wild type (wt)-p53 phenotype. In contrast, doxorubicin treatment failed to induce these alterations in EU-6 cells, a mutant-p53 ALL cell line. To specify the role of wt-p53 in regulating survivin and its variants, a temperature-sensitive p53 mutant plasmid p53–143 was transfected into EU-4, a p53-null ALL cell line, to establish a subline EU-4/p53–143. When EU-4/p53–143 cell culture was shifted from 37.5°C to the wt-p53-permissive temperature (32.5°C), the expression of survivin and survivin-DEx3 was decreased whereas survivin-2B expression was increased, confirming the distinct regulatory effect of p53 on survivin and its variants. To clarify the role of survivin-2B in the process of apoptosis, survivin-2B cDNA was cloned into pcDNA3HA vector and transfected into EU-4 cells. Enforced expression of survivin-2B in EU-4 cells inhibited cell growth and sensitized these cells to doxorubicin-induced apoptosis. These results suggest that survivin-2B variant is a pro-apoptotic factor and its expression is upregulated by p53.

scholarly journals Trapping of a Spiral-Like Intermediate of the Bacterial Cytokinetic Protein FtsZ

2006 ◽  
Vol 188 (5) ◽  
pp. 1680-1690 ◽  
Author(s):  
Katherine A. Michie ◽  
Leigh G. Monahan ◽  
Peter L. Beech ◽  
Elizabeth J. Harry
Keyword(s):  
Ring Formation ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Bacterial Cell Division ◽  
Temporal Regulation ◽  
Division Site ◽  
Spiral Form ◽  
Z Ring

ABSTRACT The earliest stage in bacterial cell division is the formation of a ring, composed of the tubulin-like protein FtsZ, at the division site. Tight spatial and temporal regulation of Z-ring formation is required to ensure that division occurs precisely at midcell between two replicated chromosomes. However, the mechanism of Z-ring formation and its regulation in vivo remain unresolved. Here we identify the defect of an interesting temperature-sensitive ftsZ mutant (ts1) of Bacillus subtilis. At the nonpermissive temperature, the mutant protein, FtsZ(Ts1), assembles into spiral-like structures between chromosomes. When shifted back down to the permissive temperature, functional Z rings form and division resumes. Our observations support a model in which Z-ring formation at the division site arises from reorganization of a long cytoskeletal spiral form of FtsZ and suggest that the FtsZ(Ts1) protein is captured as a shorter spiral-forming intermediate that is unable to complete this reorganization step. The ts1 mutant is likely to be very valuable in revealing how FtsZ assembles into a ring and how this occurs precisely at the division site.

scholarly journals Yeast 18S rRNA Dimethylase Dim1p: a Quality Control Mechanism in Ribosome Synthesis?

1998 ◽  
Vol 18 (4) ◽  
pp. 2360-2370 ◽  
Author(s):  
Denis L. J. Lafontaine ◽  
Thomas Preiss ◽  
David Tollervey
Keyword(s):  
18S Rrna ◽  
Small Subunit ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rrna Processing ◽  
Small Subunit Rrna ◽  
Enzymatic Function ◽  
Processing Steps

ABSTRACT One of the few rRNA modifications conserved between bacteria and eukaryotes is the base dimethylation present at the 3′ end of the small subunit rRNA. In the yeast Saccharomyces cerevisiae, this modification is carried out by Dim1p. We previously reported that genetic depletion of Dim1p not only blocked this modification but also strongly inhibited the pre-rRNA processing steps that lead to the synthesis of 18S rRNA. This prevented the formation of mature but unmodified 18S rRNA. The processing steps inhibited were nucleolar, and consistent with this, Dim1p was shown to localize mostly to this cellular compartment. dim1-2 was isolated from a library of conditionally lethal alleles of DIM1. In dim1-2strains, pre-rRNA processing was not affected at the permissive temperature for growth, but dimethylation was blocked, leading to strong accumulation of nondimethylated 18S rRNA. This demonstrates that the enzymatic function of Dim1p in dimethylation can be separated from its involvement in pre-rRNA processing. The growth rate ofdim1-2 strains was not affected, showing the dimethylation to be dispensable in vivo. Extracts of dim1-2 strains, however, were incompetent for translation in vitro. This suggests that dimethylation is required under the suboptimal in vitro conditions but only fine-tunes ribosomal function in vivo. Unexpectedly, when transcription of pre-rRNA was driven by a polymerase II PGKpromoter, its processing became insensitive to temperature-sensitive mutations in DIM1 or to depletion of Dim1p. This observation, which demonstrates that Dim1p is not directly required for pre-rRNA processing reactions, is consistent with the inhibition of pre-rRNA processing by an active repression system in the absence of Dim1p.

scholarly journals A Tick-Borne Langat Virus Mutant That Is Temperature Sensitive and Host Range Restricted in Neuroblastoma Cells and Lacks Neuroinvasiveness for Immunodeficient Mice

2006 ◽  
Vol 80 (3) ◽  
pp. 1427-1439 ◽  
Author(s):  
Alexander A. Rumyantsev ◽  
Brian R. Murphy ◽  
Alexander G. Pletnev
Keyword(s):  
Reverse Genetics ◽  
Neuroblastoma Cells ◽  
Scid Mice ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Structural Protein ◽  
Human Neuroblastoma ◽  
Immunodeficient Mice ◽  
Langat Virus

ABSTRACT Langat virus (LGT), the naturally attenuated member of the tick-borne encephalitis virus (TBEV) complex, was tested extensively in clinical trials as a live TBEV vaccine and was found to induce a protective, durable immune response; however, it retained a low residual neuroinvasiveness in mice and humans. In order to ablate or reduce this property, LGT mutants that produced a small plaque size or temperature-sensitive (ts) phenotype in Vero cells were generated using 5-fluorouracil. One of these ts mutants, clone E5-104, exhibited a more than 103-fold reduction in replication at the permissive temperature in both mouse and human neuroblastoma cells and lacked detectable neuroinvasiveness for highly sensitive immunodeficient mice. The E5-104 mutant possessed five amino acid substitutions in the structural protein E and one change in each of the nonstructural proteins NS3 and NS5. Using reverse genetics, we demonstrated that a Lys46→Glu substitution in NS3 as well as a single Lys315→Glu change in E significantly impaired the growth of LGT in neuroblastoma cells and reduced its peripheral neurovirulence for SCID mice. This study and our previous experience with chimeric flaviviruses indicated that a decrease in viral replication in neuroblastoma cells might serve as a predictor of in vivo attenuation of the neurotropic flaviviruses. The combination of seven mutations identified in the nonneuroinvasive E5-104 mutant provided a useful foundation for further development of a live attenuated TBEV vaccine. An evaluation of the complete sequence of virus recovered from brain of SCID mice inoculated with LGT mutants identified sites in the LGT genome that promoted neurovirulence/neuroinvasiveness.

Properties of an in vitro system for studying temperature-sensitive DNA synthesis in ts A1S9 mouse L-cells

1978 ◽  
Vol 56 (6) ◽  
pp. 444-451 ◽  
Author(s):  
Jerome Humbert ◽  
Rose Sheinin
Keyword(s):  
Dna Synthesis ◽  
Buoyant Density ◽  
Velocity Sedimentation ◽  
Synthetic Activity ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Activity Restriction ◽  
L Cells

The in vitro DNA synthesis has been observed in whole cell lysates and in cytosol and nuclear fractions of wild-type (WT-4) mouse L-cells and ts A1S9 cells which exhibit temperature-sensitive (ts) DNA replication in vivo. The product, labelled with substrate 3H-labelled TTP, is resistant to alkali and has the buoyant density (1.709 g/cm3) expected for normal mouse DNA. Pulse-chase studies, in which newly made, single-stranded DNA was analyzed by velocity sedimentation in alkaline sucrose density gradients, revealed that in vitro DNA synthesis proceeds by a discontinuous mechanism. Approximately half of the DNA made in a 30-s pulse sedimented at 3–8S; the rest was very heterogeneous with S values between [Formula: see text] and 30S. After incubation for up to 300 s, a majority of the newly made DNA (>85%) sedimented as the larger, heterogeneous material, with some cosedimenting with chromosomal size DNA.The ts DNA synthesis phenotype of ts A1S9 cells is expressed in vitro. Thus, the activity of extracts of ts cells incubated at the nonpermissive (38.5 °C) temperature was commensurate with the in vivo activity. Restriction of the ts phenotype to DNA synthesis is evident in vitro since the RNA synthetic activity of lysates of temperature-inactivated ts A1S9 cells was equivalent to that of extracts obtained from cells grown at the permissive temperature (33.5 °C). The DNA synthetic activity of nuclei from WT-4 or ts A1S9 cells grown at 33.5 °C plus homologous cytosol is equivalent to that of the whole lysate. In contrast, such cytosol preparations give little, if any, enhancement of the activity of nuclei from ts A1S9 cells incubated at 38.5 °C for 16 h. The cytosol of such temperature-inactivated cells, which are almost fully effective with nuclei of control cells, produce little or no enhancement of DNA synthesis by homologous nuclei.

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