Association of DNAse sensitive chromatin domains with the nuclear periphery in 3T3 cells in vitro

2000 ◽  
Vol 78 (2) ◽  
pp. 67-78 ◽  
Author(s):  
Jonathan KL Chan ◽  
Paul C Park ◽  
Umberto De Boni
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Periphery ◽  
Spatial Association ◽  
3T3 Cells ◽  
Spatial Coupling ◽  
Apical Side ◽  
Cytoplasmic Transport ◽  
In Cells ◽  
Flat Geometry

DNAse sensitive chromatin, putative transcriptionally competent sequences, exists either as pan-nuclear speckles in cells with nuclei which exhibit a flat geometry, or as a shell apposed to the nuclear envelope in cells with spheroidal nuclei. To test the hypothesis that DNAse sensitive chromatin is similarly associated with the nuclear periphery in cell types with a very flat geometry such as 3T3 fibroblasts, cells were subjected to hypotonic expansion to change their nuclei from a flat ellipsoid to a spheriod. This was based on the assumption that such a spatial association is not resolvable due to the interdigitation at the nuclear midplane of DNAse sensitive chromatin associated with the upper and lower nuclear surfaces. In situ nick translation was used to visualize the distribution of DNAse sensitive chromatin as a function of nuclear geometry. Both unexpanded and expanded cells exhibit DNAse sensitive chromatin as a dome at the apical side of the nucleus, i.e., that aspect of the cell facing the culture medium. The results argue for a polarized association of DNAse sensitive chromatin with the nuclear envelope and indicate that the nuclear periphery may function as a compartment for the spatial coupling of transcription and nucleo-cytoplasmic transport. Key words: nuclear organization, DNAse sensitive chromatin, hypotonic expansion, 3T3 cells.

scholarly journals Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells.

1995 ◽  
Vol 15 (11) ◽  
pp. 6430-6442 ◽  
Author(s):  
C A Pritchard ◽  
M L Samuels ◽  
E Bosch ◽  
M McMahon
Keyword(s):  
Protein Kinases ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Map Kinases ◽  
Prolonged Exposure ◽  
Feedback Regulation ◽  
Biochemical Properties ◽  
3T3 Cells ◽  
In Cells

The protein kinase domains of mouse A-Raf and B-Raf were expressed as fusion proteins with the hormone binding domain of the human estrogen receptor in mammalian cells. In the absence of estradiol, 3T3 and rat1a cells expressing delta A-Raf:ER and delta B-Raf:ER were nontransformed, but upon the addition of estradiol the cells became oncogenically transformed. Morphological oncogenic transformation was more rapid and distinctive in cells expressing delta B-Raf:ER compared with cells expressing delta A-Raf:ER. Biochemical analysis of cells transformed by delta A-Raf:ER and delta B-Raf:ER revealed several interesting differences. The activation of delta B-Raf:ER consistently led to the rapid and robust activation of both MEK and p42/p44 MAP kinases. By contrast, the activation of delta A-Raf:ER led to a weak activation of MEK and the p42/p44 MAP kinases. The extent of activation of MEK in cells correlated with the ability of the different Raf kinases to phosphorylate and activate MEK1 in vitro. delta B-Raf:ER phosphorylated MEK1 approximately 10 times more efficiently than delta Raf-1:ER and at least 500 times more efficiently than delta A-Raf:ER under the conditions of the immune-complex kinase assays. These results were confirmed with epitope-tagged versions of the Raf kinase domains expressed in insect cells. The activation of all three delta Raf:ER proteins in 3T3 cells led to the hyperphosphorylation of the resident p74raf-1 and mSOS1 proteins, suggesting the possibility of "cross-talk" between the different Raf kinases and feedback regulation of intracellular signaling pathways. The activation of either delta B-Raf:ER or delta Raf-1:ER in quiescent 3T3 cells was insufficient to promote the entry of the cells into DNA synthesis. By contrast, the activation of delta A-Raf:ER in quiescent 3T3 cells was sufficient to promote the entry of the cells into S phase after prolonged exposure to beta-estradiol. The delta Raf:ER system has allowed us to reveal significant differences between the biological and biochemical properties of oncogenic forms of the Raf family of protein kinases. We anticipate that cells expressing these proteins and other estradiol-regulated protein kinases will be useful tools in future attempts to unravel the complex web of interactions involved in intracellular signal transduction pathways.

scholarly journals c-Src-Mediated Phosphorylation of TRIP6 Regulates Its Function in Lysophosphatidic Acid-Induced Cell Migration

2005 ◽  
Vol 25 (14) ◽  
pp. 5859-5868 ◽  
Author(s):  
Yun-Ju Lai ◽  
Chen-Shan Chen ◽  
Weei-Chin Lin ◽  
Fang-Tsyr Lin
Keyword(s):  
Cell Migration ◽  
Lysophosphatidic Acid ◽  
Morphological Changes ◽  
Focal Adhesions ◽  
Dependent Manner ◽  
3T3 Cells ◽  
Erk Activation ◽  
Nih 3T3 ◽  
In Cells

ABSTRACT TRIP6 (thyroid receptor-interacting protein 6), also known as ZRP-1 (zyxin-related protein 1), is a member of the zyxin family that has been implicated in cell motility. Previously we have shown that TRIP6 binds to the LPA2 receptor and associates with several components of focal complexes in an agonist-dependent manner and, thus, enhances lysophosphatidic acid (LPA)-induced cell migration. Here we further report that the function of TRIP6 in LPA signaling is regulated by c-Src-mediated phosphorylation of TRIP6 at the Tyr-55 residue. LPA stimulation induces tyrosine phosphorylation of endogenous TRIP6 in NIH 3T3 cells and c-Src-expressing fibroblasts, which is virtually eliminated in Src-null fibroblasts. Strikingly, both phosphotyrosine-55 and proline-58 residues of TRIP6 are required for Crk binding in vitro and in cells. Mutation of Tyr-55 to Phe does not alter the ability of TRIP6 to localize at focal adhesions or associate with actin. However, it abolishes the association of TRIP6 with Crk and p130cas in cells and significantly reduces the function of TRIP6 to promote LPA-induced ERK activation. Ultimately, these signaling events control TRIP6 function in promoting LPA-induced morphological changes and cell migration.

Apical and basolateral uptake and intracellular fate of dopamine precursor l-dopa in LLC-PK1 cells

1998 ◽  
Vol 274 (2) ◽  
pp. F243-F251 ◽  
Author(s):  
P. Soares-Da-Silva ◽  
M. P. Serrão ◽  
M. A. Vieira-Coelho
Keyword(s):  
Nonlinear Analysis ◽  
Basal Cell ◽  
Acid Decarboxylase ◽  
Maximal Velocity ◽  
Gbr 12909 ◽  
Apical Side ◽  
Wide Range ◽  
Cells Cultured ◽  
In Cells

The present study was aimed at the uptake ofl-3,4-dihydroxyphenylalanine (l-dopa) and its intracellular decarboxylation to dopamine. The accumulation ofl-dopa from the apical side in cells cultured in collagen-treated plastic was found to be a saturable process with a Michaelis constant ( K m) of 123 ± 17 μM and a maximal velocity ( V max) of 6.0 ± 0.2 nmol ⋅ mg protein−1 ⋅ 6 min−1. The uptake ofl-dopa applied from either the apical or basal cell borders in cells cultured in polycarbonate filters was also found to be saturable; nonlinear analysis of saturation curves for apical and basal application revealed K m values of 63.8 ± 17.0 and 42.5 ± 9.6 μM and V maxvalues of 32.0 ± 5.8 and 26.2 ± 3.4 nmol ⋅ mg protein−1 ⋅ 6 min−1, respectively. Cell monolayers incubated withl-dopa, applied from either the apical or the basal side, in the absence of benserazide, led to the accumulation of newly formed dopamine. The intracellular accumulation of newly formed dopamine was a saturable process with apparent K m values of 20.5 ± 8.2 and 247.3 ± 76.8 μM when the substrate was applied from the apical and basal side, respectively. Some of the newly formed dopamine escaped to the extracellular milieu. The basal outward transfer of dopamine was five- to sevenfold of that occurring at the apical side and was uniform over a wide range of concentrations of intracellular dopamine; the apical outward transfer of the amine depended on the intracellular concentration of dopamine and was a nonsaturable process. The apical and basal outward transfers of dopamine were insensitive to cocaine (10 and 30 μM) and GBR-12909 (1 and 3 μM). The accumulation of exogenous dopamine in LLC-PK1 cells was found to be saturable; nonlinear analysis of the saturation curves revealed for the apical and basal application of dopamine a K m of 17.7 ± 4.3 and 96.0 ± 28.1 μM and a V max of 2.0 ± 0.1 and 2.2 ± 0.3 nmol ⋅ mg protein−1 ⋅ 6 min−1, respectively. However, both cocaine (10, 30, or 100 μM) and GBR-12909 (1 or 3 μM) were found not to affect the uptake of 100 μM dopamine applied from either the apical or the basal cell border. In conclusion, the data presented here show that LLC-PK1cells are endowed with considerable aromaticl-amino acid decarboxylase (AADC) activity and transportl-dopa quite efficiently through both the apical and basal cell borders. On the other hand, our observations support the possibility of a basal-to-apical gradient of AADC activity and the possibility that LLC-PK1 cells might constitute an interesting in vitro model for the study of the renal dopaminergic physiology.

scholarly journals Rapid disappearance of statin, a nonproliferating and senescent cell-specific protein, upon reentering the process of cell cycling.

1985 ◽  
Vol 101 (5) ◽  
pp. 1695-1701 ◽  
Author(s):  
E Wang
Keyword(s):  
Nuclear Envelope ◽  
Human Fibroblasts ◽  
Healing Process ◽  
Close Correlation ◽  
Specific Protein ◽  
Wound Healing Process ◽  
Fluorescence Staining ◽  
Immunoblotting Analysis ◽  
In Cells

Statin, a 57,000-D protein characteristically found in nonreplicating cells, was identified by a monoclonal antibody produced by hybridomas established from mice injected with extracts of in vitro aged human fibroblasts (Wang, E., 1985, J. Cell Biol., 100:545-551). Fluorescence staining with the antibody shows that the expression of statin disappears upon reinitiation of the process for cell replication. The rapid de-expression is observed in fibroblasts involved in the in vitro wound-healing process, as well as in cells that have been subcultured after trypsinization and replated from a confluent culture. Kinetic analysis shows that 50% of the cell population lose their statin expression at 12 h after replating, before the actual events of mitosis. Immunogold labeling with highly purified antibodies localizes the protein at the nuclear envelope in nonreplicating cells, but not in their replicating counterparts. Immunoblotting analysis confirms the disappearance of statin in cells that have reentered the cycling process. Using the technique of flow cytometry to examine the large number of nonreplicating fibroblasts in confluent cultures, we have found that statin is mostly expressed in those cells showing the least amount of DNA content, whose growth is blocked at the G0/G1 stage of the cell cycle. This close correlation is rapidly altered once the cells are released from the confluent state. These results suggest that the expression of statin may be regulated by a fine mechanism controlling the transition from the nonreplicating to the replicating state, and that the protein is structurally associated with the nuclear envelope.

scholarly journals Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts.

1995 ◽  
Vol 131 (5) ◽  
pp. 1275-1290 ◽  
Author(s):  
G Gurland ◽  
G G Gundersen
Keyword(s):  
Intermediate Filaments ◽  
Perinuclear Region ◽  
3T3 Cells ◽  
Noticeable Effect ◽  
3T3 Fibroblasts ◽  
Rapid Regrowth ◽  
In Cells

Separate populations of microtubules (MTs) distinguishable by their level of posttranslationally modified tubulin subunits and by their stability in vivo have been described. In polarized 3T3 cells at the edge of an in vitro wound, we have found a striking preferential coalignment of vimentin intermediate filaments (IFs) with detyrosinated MTs (Glu MTs) rather than with the bulk of the MTs, which were tyrosinated MTs (Tyr MTs). Vimentin IFs were not stabilizing the Glu MTs since collapse of the IF network to a perinuclear location, induced by microinjection of monoclonal anti-IF antibody, had no noticeable effect on the array of Glu MTs. To test whether Glu MTs may affect the organization of IFs we regrew MTs in cells that had been treated with nocodazole to depolymerize all the MTs and to collapse IFs; the reextension of IFs into the lamella lagged behind the rapid regrowth of Tyr MTs, but was correlated with the slower reformation of Glu MTs. Similar realignment of IFs with newly formed Glu MTs was observed in serum-starved cells treated with either serum or taxol to induce the formation of Glu MTs. Next, we microinjected affinity purified antibodies specific for Glu tubulin (polyclonal SG and monoclonal 4B8) and specific for Tyr tubulin (polyclonal W2 and monoclonal YL1/2) into 3T3 cells. Both injected SG and 4B8 antibodies labeled the subset of endogenous Glu MTs; W2 and YL1/2 antibodies labeled virtually all of the cytoplasmic MTs. Injection of SG or 4B8 resulted in the collapse of IFs to a perinuclear region. This collapse was comparable to that observed after complete MT depolymerization by nocodazole. Injection of W2, YL1/2, or nonspecific control IgGs did not result in collapse of the IFs. Taken together, these results show that Glu MTs localize IFs in migrating 3T3 fibroblasts and suggest that detyrosination of tubulin acts as a signal for the recruitment of vimentin IFs to MTs.

scholarly journals Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development.

1997 ◽  
Vol 17 (7) ◽  
pp. 4114-4123 ◽  
Author(s):  
R Ashery-Padan ◽  
N Ulitzur ◽  
A Arbel ◽  
M Goldberg ◽  
A M Weiss ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Nuclear Envelope ◽  
Posttranslational Modifications ◽  
Nuclear Periphery ◽  
Membrane Vesicles ◽  
Cdc2 Kinase ◽  
Nuclear Assembly ◽  
Egg Chamber

Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope.

scholarly journals Nuclear pore complex clustering and nuclear accumulation of poly(A)+ RNA associated with mutation of the Saccharomyces cerevisiae RAT2/NUP120 gene.

1995 ◽  
Vol 131 (6) ◽  
pp. 1677-1697 ◽  
Author(s):  
C V Heath ◽  
C S Copeland ◽  
D C Amberg ◽  
V Del Priore ◽  
M Snyder ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Envelope ◽  
Messenger Rna ◽  
Nuclear Periphery ◽  
Nuclear Accumulation ◽  
Growth Defect ◽  
Nuclear Pore ◽  
Permissive Temperature ◽  
Mutant Cells ◽  
In Cells

To identify genes involved in the export of messenger RNA from the nucleus to the cytoplasm, we used an in situ hybridization assay to screen temperature-sensitive strains of Saccharomyces cerevisiae. This identified those which accumulated poly(A)+ RNA in their nuclei when shifted to the non-permissive temperature of 37 degrees C. We describe here the properties of yeast strains carrying mutations in the RAT2 gene (RAT - ribonucleic acid trafficking) and the cloning of the RAT2 gene. Only a low percentage of cells carrying the rat2-1 allele showed nuclear accumulation of poly(A)+ RNA when cultured at 15 degrees or 23 degrees C, but within 4 h of a shift to the nonpermissive temperature of 37 degrees C, poly(A)+ RNA accumulated within the nuclei of approximately 80% of cells. No defect was seen in the nuclear import of a reporter protein bearing a nuclear localization signal. Nuclear pore complexes (NPCs) are distributed relatively evenly around the nuclear envelope in wild-type cells. In cells carrying either the rat2-1 or rat2-2 allele, NPCs were clustered together into one or a few regions of the nuclear envelope. This clustering was a constitutive property of mutant cells. NPCs remained clustered in crude nuclei isolated from mutant cells, indicating that these clusters are not able to redistribute around the nuclear envelope when nuclei are separated from cytoplasmic components. Electron microscopy revealed that these clusters were frequently found in a protuberance of the nuclear envelope and were often located close to the spindle pole body. The RAT2 gene encodes a 120-kD protein without similarity to other known proteins. It was essential for growth only at 37 degrees C, but the growth defect at high temperature could be suppressed by growth of mutant cells in the presence of high osmolarity media containing 1.0 M sorbitol or 0.9 M NaCl. The phenotypes seen in cells carrying a disruption of the RAT2 gene were very similar to those seen with the rat2-1 and rat2-2 alleles. Epitope tagging was used to show that Rat2p is located at the nuclear periphery and co-localizes with yeast NPC proteins recognized by the RL1 monoclonal antibody. The rat2-1 allele was synthetically lethal with both the rat3-1/nup133-1 and rat7-1/nup159-1 alleles. These results indicate that the product of this gene is a nucleoporin which we refer to as Rat2p/Nup120p.

MiR-485-5p Promotes Neuron Survival through Mediating Rac1/Notch2 Signaling Pathway after Cerebral Ischemia/Reperfusion

2020 ◽  
Vol 17 (3) ◽  
pp. 259-266 ◽  
Author(s):  
Xuan Chen ◽  
Sumei Zhang ◽  
Peipei Shi ◽  
Yangli Su ◽  
Dong Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Viability ◽  
Therapeutic Option ◽  
Ischemia Reperfusion ◽  
Glucose Deprivation ◽  
Small Gtpase ◽  
Luciferase Reporter ◽  
Pathological Feature ◽  
In Cells

Objective: Ischemia-reperfusion (I/R) injury is a pathological feature of ischemic stroke. This study investigated the regulatory role of miR-485-5p in I/R injury. Methods: SH-SY5Y cells were induced with oxygen and glucose deprivation/reoxygenation (OGD/R) to mimic I/R injury in vitro. Cells were transfected with designated constructs (miR-485- 5p mimics, miR-485-5p inhibitor, lentiviral vectors overexpressing Rac1 or their corresponding controls). Cell viability was evaluated using the MTT assay. The concentrations of lactate dehydrogenase, malondialdehyde, and reactive oxygen species were detected to indicate the degree of oxidative stress. Flow cytometry and caspase-3 activity assay were used for apoptosis assessment. Dual-luciferase reporter assay was performed to confirm that Rac family small GTPase 1 (Rac1) was a downstream gene of miR-485-5p. Results: OGD/R resulted in decreased cell viability, elevated oxidative stress, increased apoptosis, and downregulated miR-485-5p expression in SH-SY5Y cells. MiR-485-5p upregulation alleviated I/R injury, evidenced by improved cell viability, decreased oxidative markers, and reduced apoptotic rate. OGD/R increased the levels of Rac1 and neurogenic locus notch homolog protein 2 (Notch2) signaling-related proteins in cells with normal miR-485-5p expression, whereas miR- 485-5p overexpression successfully suppressed OGD/R-induced upregulation of these proteins. Furthermore, the delivery of vectors overexpressing Rac1 in miR-485-5p mimics-transfected cells reversed the protective effect of miR-485-5p in cells with OGD/R-induced injury. Conclusion: This study showed that miR-485-5p protected cells following I/R injury via targeting Rac1/Notch2 signaling suggest that targeted upregulation of miR-485-5p might be a promising therapeutic option for the protection against I/R injury.

Biologic Aspects of Expression of Stably Integrated Transgenes in Cells of the Skin In Vitro and In Vivo

1999 ◽  
Vol 111 (3) ◽  
pp. 198-205 ◽  
Author(s):  
Gerald G. Krueger ◽  
Jeffery R. Morgan ◽  
Marta J. Petersen
Keyword(s):  
In Cells

scholarly journals 1592U89, a novel carbocyclic nucleoside analog with potent, selective anti-human immunodeficiency virus activity.

1997 ◽  
Vol 41 (5) ◽  
pp. 1082-1093 ◽  
Author(s):  
S M Daluge ◽  
S S Good ◽  
M B Faletto ◽  
W H Miller ◽  
M H St Clair ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Oral Bioavailability ◽  
Human Peripheral Blood ◽  
Cross Resistance ◽  
Immunodeficiency Virus ◽  
Carbocyclic Nucleoside ◽  
Hiv 1 ◽  
In Cells

1592U89, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclo pentene-1-methanol, is a carbocyclic nucleoside with a unique biological profile giving potent, selective anti-human immunodeficiency virus (HIV) activity. 1592U89 was selected after evaluation of a wide variety of analogs containing a cyclopentene substitution for the 2'-deoxyriboside of natural deoxynucleosides, optimizing in vitro anti-HIV potency, oral bioavailability, and central nervous system (CNS) penetration. 1592U89 was equivalent in potency to 3'-azido-3'-deoxythymidine (AZT) in human peripheral blood lymphocyte (PBL) cultures against clinical isolates of HIV type 1 (HIV-1) from antiretroviral drug-naive patients (average 50% inhibitory concentration [IC50], 0.26 microM for 1592U89 and 0.23 microM for AZT). 1592U89 showed minimal cross-resistance (approximately twofold) with AZT and other approved HIV reverse transcriptase (RT) inhibitors. 1592U89 was synergistic in combination with AZT, the nonnucleoside RT inhibitor nevirapine, and the protease inhibitor 141W94 in MT4 cells against HIV-1 (IIIB). 1592U89 was anabolized intracellularly to its 5'-monophosphate in CD4+ CEM cells and in PBLs, but the di- and triphosphates of 1592U89 were not detected. The only triphosphate found in cells incubated with 1592U89 was that of the guanine analog (-)-carbovir (CBV). However, the in vivo pharmacokinetic, distribution, and toxicological profiles of 1592U89 were distinct from and improved over those of CBV, probably because CBV itself was not appreciably formed from 1592U89 in cells or animals (<2%). The 5'-triphosphate of CBV was a potent, selective inhibitor of HIV-1 RT, with Ki values for DNA polymerases (alpha, beta, gamma, and epsilon which were 90-, 2,900-, 1,200-, and 1,900-fold greater, respectively, than for RT (Ki, 21 nM). 1592U89 was relatively nontoxic to human bone marrow progenitors erythroid burst-forming unit and granulocyte-macrophage CFU (IC50s, 110 microM) and human leukemic and liver tumor cell lines. 1592U89 had excellent oral bioavailability (105% in the rat) and penetrated the CNS (rat brain and monkey cerebrospinal fluid) as well as AZT. Having demonstrated an excellent preclinical profile, 1592U89 has progressed to clinical evaluation in HIV-infected patients.

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