scholarly journals Quantitative relationships betweenlacZmutant frequency and DNA adduct frequency in Muta™Mouse tissues and cultured cells exposed to 3-nitrobenzanthrone

Mutagenesis ◽  
2017 ◽  
pp. gew067 ◽  
Author(s):  
Paul A. White ◽  
George R. Douglas ◽  
David H. Phillips ◽  
Volker M. Arlt
Keyword(s):  
Cultured Cells ◽  
Dna Adduct ◽  
Mouse Tissues

Characterization of a factor that stimulates hydrolysis of GTP bound to ras gene product p21 (GTPase-activating protein) and correlation of its activity to cell density

1988 ◽  
Vol 8 (10) ◽  
pp. 4169-4173
Author(s):  
M Hoshino ◽  
M Kawakita ◽  
S Hattori
Keyword(s):  
Gene Product ◽  
Cell Density ◽  
Cultured Cells ◽  
Specific Activity ◽  
Gtpase Activating Protein ◽  
Ras Gene ◽  
Cell Extracts ◽  
Mouse Tissues ◽  
Almost All ◽  
Hydrolysis Of

The postmicrosomal fraction of the extract from NIH 3T3 and BALB/c 3T3 cells stimulated the hydrolysis of GTP bound to H-ras gene product p21 by severalfold. The stimulation was observed with normal p21 but not with p21 with valine as the 12th residue. This specificity is similar to that of GTPase-activating protein (GAP) for N-ras p21 described by M. Trahey and F. McCormick (Science 238:542-545, 1987). Consistent with this specificity, analysis of p21-bound nucleotides in living cells revealed that almost all normal p21 bound GDP, whereas oncogenic mutant p21s bound both GTP and GDP. Similar activity was also found in various mouse tissues, with brain tissue showing the highest specific activity. When cell extracts were prepared from cultured cells, there was a linear relationship between GAP activity and cell density. These results suggest the factor is involved in the regulation of cell proliferation.

scholarly journals High-resolution visualization and quantification of nucleic acid–based therapeutics in cells and tissues using Nanoscale secondary ion mass spectrometry (NanoSIMS)

2020 ◽  
Author(s):  
Cuiwen He ◽  
Michael T Migawa ◽  
Kai Chen ◽  
Thomas A Weston ◽  
Michael Tanowitz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Nucleic Acid ◽  
Secondary Ion Mass Spectrometry ◽  
Cultured Cells ◽  
Significant Advance ◽  
Mouse Tissues ◽  
Ion Mass Spectrometry ◽  
The Impact ◽  
Secondary Ion ◽  
In Cells

Abstract Nucleic acid therapeutics (NATs) have proven useful in promoting the degradation of specific transcripts, modifying gene expression, and regulating mRNA splicing. In each situation, efficient delivery of nucleic acids to cells, tissues and intracellular compartments is crucial—both for optimizing efficacy and reducing side effects. Despite successes in NATs, our understanding of their cellular uptake and distribution in tissues is limited. Current methods have yielded insights into distribution of NATs within cells and tissues, but the sensitivity and resolution of these approaches are limited. Here, we show that nanoscale secondary ion mass spectrometry (NanoSIMS) imaging can be used to define the distribution of 5-bromo-2′-deoxythymidine (5-BrdT) modified antisense oligonucleotides (ASO) in cells and tissues with high sensitivity and spatial resolution. This approach makes it possible to define ASO uptake and distribution in different subcellular compartments and to quantify the impact of targeting ligands designed to promote ASO uptake by cells. Our studies showed that phosphorothioate ASOs are associated with filopodia and the inner nuclear membrane in cultured cells, and also revealed substantial cellular and subcellular heterogeneity of ASO uptake in mouse tissues. NanoSIMS imaging represents a significant advance in visualizing uptake and distribution of NATs; this approach will be useful in optimizing efficacy and delivery of NATs for treating human disease.

scholarly journals Interaction between DNA-damage protein GADD34 and a new member of the Hsp40 family of heat shock proteins that is induced by a DNA-damaging reagent

2000 ◽  
Vol 352 (3) ◽  
pp. 795-800 ◽  
Author(s):  
Tadao HASEGAWA ◽  
Hengyi XIAO ◽  
Fumiyasu HAMAJIMA ◽  
Ken-ichi ISOBE
Keyword(s):  
Dna Damage ◽  
Heat Shock ◽  
Cultured Cells ◽  
Similar Region ◽  
C Terminus ◽  
Mouse Tissues ◽  
Time Courses ◽  
Shock Proteins ◽  
Two Hybrid

GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest. To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34. As bait we used the product of the partial GADD34 cDNA, including the regions rich in proline, glutamic acid, serine and threonine (PEST) and γ134.5 regions. A cDNA clone, named GAHSP40, which is a mouse DnaJ family protein with a high similarity to human HLJ1 was cloned. The interaction between GADD34 and GAHSP40 in cultured cells was confirmed by a co-immunoprecipitation experiment and in NIH 3T3 cells by two-hybrid analysis in vivo. For binding of the two proteins, the γ134.5-similar region of GADD34 was necessary; however, the PEST region was also involved and the C-terminus of GAHSP40, but not the J-domain, was important. GAHSP40 was detected in all mouse tissues examined, but a different transcript was found in the testis. Both GADD34 mRNA and GAHSP40 mRNA were significantly elevated by treatment with methyl methanesulphonate, although the time courses were different. In addition, both GAHSP40 and GADD34 mRNA were induced by heat shock.

Identification of the cells expressing cot proto-oncogene mRNA

1995 ◽  
Vol 108 (1) ◽  
pp. 97-103
Author(s):  
R. Ohara ◽  
S. Hirota ◽  
H. Onoue ◽  
S. Nomura ◽  
Y. Kitamura ◽  
...  
Keyword(s):  
Parotid Gland ◽  
Cultured Cells ◽  
Expression Patterns ◽  
Cell Types ◽  
Gene Expressions ◽  
Developmentally Regulated ◽  
Gastric Glands ◽  
Gastric Adenocarcinomas ◽  
Mouse Tissues ◽  
Glandular Cells

The cell types expressing cot proto-oncogene mRNA were identified by in situ hybridization (ISH) histochemistry. Among a variety of adult mouse tissues examined, four types of glandular cells expressing cot gene were identified: (1) granular duct cells in the submandibular and sublingual glands; (2) serous cells in the parotid gland; (3) peptic (chief) cells in gastric glands; and (4) goblet cells in colonic glands. Investigation of the developmentally regulated expression of cot mRNA using tissues of 14-day and 18-day embryos, newborn and weanling mice showed that cot gene is expressed only in morphologically differentiated and functionally activated cells of these four types. No other types of cells showing ISH signals were observed. Based on these results, cot gene expressions in cultured cells of colonic adenocarcinomas and gastric adenocarcinomas were examined. SW 480 and WiDr cells showed high expression of this gene and so should be useful for functional analysis of Cot kinase. The expression patterns of cot gene in tumor tissues of the parotid gland, and gastric and colonic glands were investigated. Two of the tissues overexpressed this gene markedly, suggesting that overproduction of Cot kinase may be one cause of their transformation.

In Vivo Analysis of Erythroid Protein 4.1 Pre-mRNA Splicing Mechanisms: Use of Antisense Morpholinos to Assay Function of Deep Intron Regulatory Elements

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 815-815
Author(s):  
Marilyn Parra ◽  
Xiuli An ◽  
Narla Mohandas ◽  
John G. Conboy
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Cultured Cells ◽  
Conflicts Of Interest ◽  
Regulatory Elements ◽  
Protein Isoforms ◽  
Start Codon ◽  
Exon 2 ◽  
Mouse Tissues

Abstract Abstract 815 Erythroid stage-specific alternative splicing plays an essential role in the expression of protein 4.1R isoforms that interact with other skeletal proteins to strengthen the membrane. In late erythroblasts, 4.1R mRNA is processed from pre-mRNA that initiates transcription at alternative first exon 1A (E1A) and splices exclusively to the more distal of two alternative 3' splice sites at exon 2 (E2dis), ~100kb downstream. This splicing event is important because it is required to generate the shorter N-terminal domain characteristic of 80kDa isoforms of 4.1R protein in red cells. We have reported that E1A splicing to E2dis requires two nested intrasplicing events mediated by an essential deep intron element originally annotated as exon 1B. However, these studies employed small minigenes transfected into cultured cells, an artificial system that may not correctly reflect in vivo mechanisms. Here we used an antisense RNA strategy to explore splicing of endogenous full length 4.1R pre-mRNA in tissues of live mice and in primary erythroblasts. Chemically modified oligonucleotides known as vivo-morpholinos (vMOs), introduced via tail vein injection and internalized into selected organs, can base pair with complementary cellular RNA sequences and block function of candidate regulatory motifs. Importantly, two independent vMOs directed against the 4.1R intraexon regulatory element both substantially abrogated intrasplicing in several mouse tissues, robustly switching E1A splicing from E2dis to the proximal 3' splice site in E2 (E2prox). This switch results in inclusion of start codon AUG1 in mature 4.1R mRNA and synthesis of larger isoforms of 4.1R protein. These results were highly sequence-specific, since negative control vMOs directed against other genes did not alter E1A splicing to E2dis. Interestingly, we have recently used vMOs to confirm the existence of a similar deep intron element required for analogous E1A-E2dis splicing in the paralogous 4.1B gene. Together these findings strongly support the in vivo physiological function of deep intron elements in the control of intrasplicing in both 4.1R and 4.1B pre-mRNAs. To test whether the 4.1R intrasplicing mechanism is also active in erythroid cells, we incubated mouse splenic erythroblasts isolated from FVA-treated animals with morpholinos directed against the intraexon. Two independent morpholinos against its 5' splice site and branch point both induced a concentration-dependent switch in E1A splicing from E2dis to E2prox. Control morpholinos had no effect on E1A splicing. Because the splicing switch results in inclusion of alternative translation initiation codon AUG1, it was predicted to induce synthesis of larger isoforms of 4.1R including the N-terminal headpiece known to influence 4.1R binding affinities for other skeletal proteins. Western blot analysis of erythroblast proteins confirmed a switch to expression of larger 4.1R protein isoforms that are not present in normal late stage erythroblasts. Intrasplicing is mediated by deep intron elements, and is essential for accurate physiological splicing of natural 4.1R pre-mRNA in erythroid and other cells. Antisense morpholinos represent a new tool for alternative splicing studies in vivo or in cultured erythroblasts. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of Palladin, a Novel Protein Localized to Stress Fibers and Cell Adhesions

2000 ◽  
Vol 150 (3) ◽  
pp. 643-656 ◽  
Author(s):  
Mana M. Parast ◽  
Carol A. Otey
Keyword(s):  
Cultured Cells ◽  
Focal Adhesions ◽  
Cell Types ◽  
Cell Junctions ◽  
Stress Fibers ◽  
Western Blots ◽  
Embryonic Tissues ◽  
Mouse Tissues ◽  
Antisense Constructs

Here, we describe the identification of a novel phosphoprotein named palladin, which colocalizes with α-actinin in the stress fibers, focal adhesions, cell–cell junctions, and embryonic Z-lines. Palladin is expressed as a 90–92-kD doublet in fibroblasts and coimmunoprecipitates in a complex with α-actinin in fibroblast lysates. A cDNA encoding palladin was isolated by screening a mouse embryo library with mAbs. Palladin has a proline-rich region in the NH2-terminal half of the molecule and three tandem Ig C2 domains in the COOH-terminal half. In Northern and Western blots of chick and mouse tissues, multiple isoforms of palladin were detected. Palladin expression is ubiquitous in embryonic tissues, and is downregulated in certain adult tissues in the mouse. To probe the function of palladin in cultured cells, the Rcho-1 trophoblast model was used. Palladin expression was observed to increase in Rcho-1 cells when they began to assemble stress fibers. Antisense constructs were used to attenuate expression of palladin in Rcho-1 cells and fibroblasts, and disruption of the cytoskeleton was observed in both cell types. At longer times after antisense treatment, fibroblasts became fully rounded. These results suggest that palladin is required for the normal organization of the actin cytoskeleton and focal adhesions.

Mass spectroscopic analysis of phosphatidylinositol synthesis using 6-deuteriated-myo-inositol: comparison of the molecular specificities and acyl remodelling mechanisms in mouse tissues and cultured cells

2004 ◽  
Vol 32 (6) ◽  
pp. 1057-1059 ◽  
Author(s):  
A.D. Postle ◽  
H. Dombrowsky ◽  
H. Clarke ◽  
C.J. Pynn ◽  
G. Koster ◽  
...  
Keyword(s):  
Molecular Species ◽  
Cultured Cells ◽  
Ionization Mass ◽  
Membrane Composition ◽  
Mouse Tissues ◽  
Wide Range ◽  
Kinetics Of ◽  
Hydrolysis Of

Mammalian cell PtdIns (phosphatidylinositol) in vivo is enriched in the sn-1-stearoyl 2-arachidonoyl species, the physiological precursor of phosphatidylinositol 4,5-bisphosphate. Mechanisms regulating this specificity are unclear but are typically lost for cells in culture. We used ESI-MS (tandem electrospray ionization-mass spectrometry) to determine the molecular species of PtdIns synthesized by mouse tissues in vivo compared with cultured cells in vitro. After incorporation of deuteriated myo-d6-inositol over 3 h, endogenous and newly synthesized PtdIns and lysoPtdIns species were quantified from precursor scans of m/z 241− and m/z 247− respectively. PtdIns was synthesized as a wide range of species irrespective of the final membrane composition. Analyses of isotope enrichments argued against acyl remodelling as the major regulatory mechanism: composition of the lysoPtdIns pool under all conditions reflected that of either endogenous or newly synthesized PtdIns and was always at equilibrium. The kinetics of PtdIns synthesis, together with the prolonged time scale required for achieving final equilibrium compositions suggest that selective transport between membranes and/or hydrolysis of selected molecular species are the most probable mechanisms regulating compositions of PtdIns and, ultimately, phosphatidylinositol 4,5-bisphosphate.

scholarly journals Endogenous p53 protein generated from wild-type alternatively spliced p53 RNA in mouse epidermal cells

1994 ◽  
Vol 14 (3) ◽  
pp. 1698-1708
Author(s):  
M F Kulesz-Martin ◽  
B Lisafeld ◽  
H Huang ◽  
N D Kisiel ◽  
L Lee
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Transcriptional Activation ◽  
Cultured Cells ◽  
P53 Protein ◽  
Epidermal Cells ◽  
Wild Type ◽  
Mouse Tissues ◽  
Alternatively Spliced ◽  
In Cells

We previously demonstrated that a wild-type alternatively spliced p53 (p53as) RNA exists in mouse cultured cells and normal mouse tissues at approximately 25 to 33% of the level of the major p53 RNA form. The alternative RNA transcript is 96 nucleotides longer than the major transcript as a result of alternative splicing of intron 10 sequences. The protein expected to be generated from the p53as transcript is 9 amino acids shorter than the major p53 protein and has 17 different amino acids at the carboxyl terminus. We report here that p53as protein exists in nontransformed and malignant epidermal cells and is localized to the nucleus. In addition, p53as protein is preferentially expressed during the G2 phase of the cell cycle and in cells with greater than G2 DNA content compared with the major p53 protein, which is preferentially expressed in G1. The p53as immunoreactivity is elevated and shifted to the G1 phase of the cell cycle following actinomycin D treatment of nontransformed cells but not malignant cells. In view of the dimerization and tetramerization of p53 protein which may be necessary for its DNA binding and transcriptional activation activities, the presence of p53as protein in cells has important implications for understanding the physiological function(s) of the p53 gene.

scholarly journals Degradation intermediates as an indicator of mRNA and cell metabolism

2021 ◽  
Author(s):  
Yusheng Liu ◽  
Yiwei Zhang ◽  
Hu Nie ◽  
Falong Lu ◽  
Jiaqiang Wang
Keyword(s):  
Degradation Rate ◽  
Cultured Cells ◽  
Cell Metabolism ◽  
Single Cells ◽  
Mrna Degradation ◽  
Rate Analysis ◽  
Mouse Tissues ◽  
Degradation Intermediates ◽  
Mrna Degradation Rate

Traditional mRNA degradation rate measurements involves complex experimental design with RNA labeling or transcription blocking together with sampling at multiple timepoints. These experimental requirements limit the application of transcriptome-wide mRNA degradation rate analysis mainly in cultured cells, but rarely in in vivo samples. Therefore, a direct and simple strategy needs to be developed to study mRNA degradation rate. Here, we defined mRNA degradation intermediates as transcripts where decay is about to occur or has partially occurred in the 3′-untranslated regions after poly(A) tail deadenylation, and found that the proportion of mRNA degradation intermediates is a very simple and convenient indicator for evaluating the degradation rate of mRNA in mouse and human cell lines. In addition, we showed that a higher proportion of mRNA degradation intermediates is correlated with faster cell cycle and higher turnover rate of mouse tissues. Further, we validated that in mouse maturing oocytes where transcription is silent, the proportion of mRNA degradation intermediates is positively correlated with the mRNA degradation rate. Together, these results demonstrate that degradation intermediates can function as a good indicator of mRNA, cell, and tissue metabolism, and can be easily assayed by total RNA 3′-end sequencing from a single bulk cell sample without the need for drug treatment or multi-timepoint sampling. This finding is of great potential for studies on mRNA degradation rate at the molecular, cellular, or organic level, including samples or systems that cannot be assayed with previous methods. In addition, further application of the findings into single cells will likely greatly aid the identification and study of rare cells with unique cellular metabolism dynamics such as tissue stem cells and tumor cells.

scholarly journals Calcium pump of the plasma membrane is localized in caveolae.

1993 ◽  
Vol 120 (5) ◽  
pp. 1147-1157 ◽  
Author(s):  
T Fujimoto
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Smooth Muscle Cells ◽  
Cultured Cells ◽  
Muscle Cells ◽  
Epidermal Keratinocytes ◽  
Cardiac Muscle Cells ◽  
Mouse Tissues ◽  
Visceral Smooth Muscle

The Ca2+ pump in the plasma membrane plays a key role in the fine control of the cytoplasmic free Ca2+ concentration. In the present study, its subcellular localization was examined with immunocytochemical techniques using a specific antibody generated against the erythrocyte membrane Ca2+ pump ATPase. By immunofluorescence microscopy of cultured cells, the labeling with the antibody was seen as numerous small dots, often distributed in linear arrays or along cell edges. Immunogold EM of cryosections revealed that the dots correspond to caveolae, or smooth invaginations of the plasma membrane. The same technique applied to mouse tissues in vivo showed that the Ca2+ pump is similarly localized in caveolae of endothelial cells, smooth muscle cells, cardiac muscle cells, epidermal keratinocytes and mesothelial cells. By quantitative analysis of the immunogold labeling, the Ca2+ pump in capillary endothelial cells and visceral smooth muscle cells was found to be concentrated 18-25-fold in the caveolar membrane compared with the noncaveolar portion of the plasma membrane. In renal tubular and small intestinal epithelial cells, which have been known to contain the Ca2+ pump but do not have many caveolae, most of the labeling was randomly distributed in the basolateral plasma membrane, although caveolae were also positively labeled. The results demonstrate that the caveolae in various cells has the plasmalemmal Ca2+ pump as a common constituent. In conjunction with our recent finding that an inositol 1,4,5-trisphosphate receptor-like protein exists in the caveolae (Fujimoto, T., S. Nakade, A. Miyawaki, K. Mikoshiba, and K. Ogawa. 1992. J. Cell Biol. 119:1507-1513), it is inferred that the smooth plasmalemmal invagination is an apparatus specialized for Ca2+ intake and extrusion from the cytoplasm.

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