scholarly journals Molecular forms of N-CAM and its RNA in developing and denervated skeletal muscle.

1986 ◽  
Vol 102 (3) ◽  
pp. 731-739 ◽  
Author(s):  
J Covault ◽  
J P Merlie ◽  
C Goridis ◽  
J R Sanes
Keyword(s):  
Primary Cultures ◽  
Apparent Molecular Weight ◽  
Fold Increase ◽  
Nucleic Acid Hybridization ◽  
Molecular Forms ◽  
Neuraminidase Treatment ◽  
Adult Muscle ◽  
Neural Cell Adhesion

The neural cell adhesion molecule (N-CAM) is present in both embryonic and perinatal muscle, but its distribution changes as myoblasts form myotubes and axons establish synapses (Covault, J., and J. R. Sanes, 1986, J. Cell Biol., 102:716-730). Levels of N-CAM decline postnatally but increase when adult muscle is denervated or paralyzed (Covault, J., and J. R. Sanes, 1985, Proc. Natl. Acad. Sci. USA., 82:4544-4548). To determine the molecular forms of N-CAM and N-CAM-related RNA during these different periods we used immunoblotting and nucleic acid hybridization techniques to analyze N-CAM and its RNA in developing, cultured, adult, and denervated adult muscle. As muscles develop, the extent of sialylation of muscle N-CAM decreases, and a 140-kD desialo form of N-CAM (generated by neuraminidase treatment) is replaced by a 125-kD form. This change in the apparent molecular weight of desialo N-CAM is paralleled by a change in N-CAM RNA: early embryonic muscles express a 6.7-kb RNA species which hybridizes with N-CAM cDNA, whereas in neonatal muscle this form is largely replaced by 5.2- and 2.9-kb species. Similar transitions in the desialo form of N-CAM, but not in extent of sialylation, accompany differentiation in primary cultures of embryonic muscle and in cultures of the clonal muscle cell lines C2 and BC3H-1. Both in vivo and in vitro, a 140-kD desialo form of N-CAM and a 6.7-kb N-CAM RNA are apparently associated with myoblasts, whereas a 125-kD desialo form and 5.2- and 2.9-kb RNAs are associated with myotubes and myofibers. After denervation of adult muscle, a approximately 12-15-fold increase in the levels of N-CAM is accompanied by a approximately 30-50-fold increase in N-CAM RNA, suggesting that N-CAM expression is regulated at a pretranslational level. Forms of N-CAM and its RNA in denervated muscle are similar to those seen in perinatal myofibers.

Role of IFN-γ and IL-2 in rat lung epithelial cell migration and apoptosis after oxidant injury

2004 ◽  
Vol 286 (1) ◽  
pp. L4-L14 ◽  
Author(s):  
Olivier Lesur ◽  
Marcel Brisebois ◽  
Alexandre Thibodeau ◽  
Frédéric Chagnon ◽  
Denis Lane ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Primary Cultures ◽  
Fold Increase ◽  
Protective Role ◽  
Scatchard Analysis ◽  
Membrane Expression ◽  
Extracellular Signal ◽  
Ifn Γ

In the present study, IFN-γ exposure to primary cultures of rat type II epithelial cells (TIIP) upregulated membrane expression of the common γ-chain of the IL-2 receptor (∼2.5- to 4-fold increase) and redistributed receptor affinity in TIIP, as assessed by Western blot, cell, and tissue histochemistry and Scatchard analysis. As for restitution processes of the lung epithelium, functionality of IL-2R on TIIP was conditional to IFN-γ exposure: 1) IFN-γ priming promoted a fivefold increase of IL-2-driven TIIP locomotion ( P < 0.05 vs. control at 100 U/ml) and 2) IFN-γ coincubation with IL-2 reduced bleomycin-induced TIIP apoptosis in vitro by 25% (caspase-3 activity) and by ∼70% (TdT-mediated dUTP nick end labeling/4′,6′-diamidino-2-phenylindole assay) as well as in vivo by ∼90% (caspase-3 activity; P < 0.05 vs. control). Sustained p42/44 extracellular signal-regulated kinase activity played a protective role in this process, whereas specific inhibition by PD-98059 (50 μM) significantly reversed bleomycin-induced TIIP apoptosis ( P < 0.05 vs. control). From these in vitro and in vivo data, it is proposed that combinations of IFN-γ and IL-2 can drive repair activity of TIIP by stimulating migration and preventing programmed cell death, both of which are speculated to be very fast restitution events after oxidant-induced acute lung injury.

In Vitro Culture of Human Thyroid Cells: Preliminary Findings on the Role of the Pathological Condition of the Donors

1997 ◽  
Vol 25 (2) ◽  
pp. 153-160
Author(s):  
Francesca Mattioli ◽  
Marianna Angiola ◽  
Laura Fazzuoli ◽  
Francesco Razzetta ◽  
Antonietta Martelli
Keyword(s):  
Pathological Condition ◽  
Primary Cultures ◽  
S Phase ◽  
Human Thyroid ◽  
Thyroid Cells ◽  
Toxicological Studies ◽  
Predictive Indicator

Although primary cultures of human thyroid cells are used for endocrinological and toxicological studies, until now no attention has been paid toward verifying whether the hormonal conditions to which the gland was exposed in vivo prior to surgery could influence in vitro responses. Our findings suggest that the hormonal situation in vivo cannot be used as a predictive indicator of triiodothyronine and thyroxine release and/or S-phase frequency in vitro, either with or without the addition of bovine thyrotropin.

scholarly journals Methylation of rRNA as a host defense against rampant group II intron retrotransposition

Mobile DNA ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Justin M. Waldern ◽  
Dorie Smith ◽  
Carol Lyn Piazza ◽  
E. Jake Bailey ◽  
Nicholas J. Schiraldi ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Fold Increase ◽  
Group Ii Intron ◽  
In Vivo Experiments ◽  
Cellular Factors ◽  
Group Ii ◽  
Self Splicing ◽  
Native Host

Abstract Background Group II introns are mobile retroelements, capable of invading new sites in DNA. They are self-splicing ribozymes that complex with an intron-encoded protein to form a ribonucleoprotein that targets DNA after splicing. These molecules can invade DNA site-specifically, through a process known as retrohoming, or can invade ectopic sites through retrotransposition. Retrotransposition, in particular, can be strongly influenced by both environmental and cellular factors. Results To investigate host factors that influence retrotransposition, we performed random insertional mutagenesis using the ISS1 transposon to generate a library of over 1000 mutants in Lactococcus lactis, the native host of the Ll.LtrB group II intron. By screening this library, we identified 92 mutants with increased retrotransposition frequencies (RTP-ups). We found that mutations in amino acid transport and metabolism tended to have increased retrotransposition frequencies. We further explored a subset of these RTP-up mutants, the most striking of which is a mutant in the ribosomal RNA methyltransferase rlmH, which exhibited a reproducible 20-fold increase in retrotransposition frequency. In vitro and in vivo experiments revealed that ribosomes in the rlmH mutant were defective in the m3Ψ modification and exhibited reduced binding to the intron RNA. Conclusions Taken together, our results reinforce the importance of the native host organism in regulating group II intron retrotransposition. In particular, the evidence from the rlmH mutant suggests a role for ribosome modification in limiting rampant retrotransposition.

scholarly journals Expression of laminin γ 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter

1996 ◽  
Vol 313 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Françoise LEVAVASSEUR ◽  
Jocelyne LIÉTARD ◽  
Kohei OGAWA ◽  
Nathalie THÉRET ◽  
Peter D. BURBELO ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Hepatoma Cell ◽  
Primary Cultures ◽  
Regulatory Elements ◽  
Hepatoma Cells ◽  
Basement Membranes ◽  
Major Protein ◽  
Cultured Hepatocytes

Laminin γ1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin γ1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin γ1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5´ flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin γ1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a Mr 60000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.

Abstract 41: Explant-Derived Cells from Chronic Heart Failure Activated Endothelial-Mesenchymal Transition and Attenuated Pluripotency Genes Expression

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Liudmila Zakharova ◽  
Hikmet Nural ◽  
Mohamed A Gaballa
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Smooth Muscle Actin ◽  
Fold Increase ◽  
Gene Expressions ◽  
Mesenchymal Transition ◽  
Cell Outgrowth ◽  
Pluripotency Genes

Cardiac progenitor cells are generated from atria explants; however the cellular origin and the mechanisms of cell outgrowth are unclear. Using transgenic tamoxifen-induced Willms tumor 1 (Wt1)-Cre/ERT and Cre-activated GFP reporter mice, we found approximately 40% of explant-derived cells and 74% of explant-derived c-Kit+ cells originated from the epicardium. In atria from sham hearts, Wt1+ cells were located in a thin epicardial layer, while c-Kit+ cells were primarily found within both the sub-epicardium and the myocardium, albeit at low frequency. No overlap between c-Kit+ and Wt1+ cells was observed, suggesting that epicardial Wt1+ cells do not express c-Kit marker in vivo, but more likely the c-Kit marker was acquired in culture. Compared with 4 days in culture, at day 21 we observed 7 folds increase in Snail gene expression; 32% increase in α-smooth muscle actin (SMA) marker, and 30% decrease in E-cadherin marker, suggesting that the explant-derived cells underwent epithelial to mesenchymal transition (EMT) in vitro. Cell outgrowths released TGF-β (1036.4 ± 1.18 pm/ml) and exhibited active TGF-β signaling, which might triggered the EMT. Compared to shams, CHF cell outgrowths exhibited elevated levels of EMT markers, SMA (49% vs. 34%) and Snail (2 folds), and reduced level of Wt1 (11% vs. 22%). In addition, CHF cell outgrowths had two folds increase in Pai1 gene expression, a direct target of TGF-β signaling. In c-Kit+ cells derived from CHF explants, Nanog gene expression was 4 folds lower and Sox 2 was 2 folds lower compared with cells from shams. Suppression of EMT in cell outgrowth increased the percentage of c-Kit+ and Wt1+ cells by 17%, and 15%, respectively. Also suppression of EMT in c-Kit+ cells resulted in 4 folds increase in Nanog and 3 fold increase in Sox2 gene expressions. Our results showed that CHF may further exuberates EMT while diminishes the re-activation of pluripotency genes. Thus, EMT modulation in CHF is a possible strategy to regulate both the yield and the pluripotency of cardiac-explant-derived progenitor cells.

Creation of a primary breast cancer culture repository from patients with a BMI >30 kg/m2: A Mexican endeavor.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. e12574-e12574
Author(s):  
Daniela Shveid Gerson ◽  
Alejandro Zentella - Dehesa ◽  
Raquel Gerson Cwilich ◽  
Benigno Rodriguez ◽  
Omar Serrano Villamayor ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Primary Breast Cancer ◽  
Medical Center ◽  
Primary Cultures ◽  
Obese Women ◽  
Cellular Analysis ◽  
Response To Chemotherapy

e12574 Background: Currently there are no primary cultures or cell lines derived from patients with breast cancer and obesity. It has been postulated that breast cancer in obese women behaves differently as it does in non-obese women, as is composed of distinct biological features, as was generated in a different metabolic environment, as well as pertains to a different prognosis and different response to chemotherapy, lower rates of overall survival and a greater probability of recurrence. By creating a primary breast cancer culture bank of breast cancer tumors from women with obesity (BMI > 30kg/m2), we will establish a cell line exclusive to obese women in Mexico, where targeted therapy may be tested and treatment may be individualized depending on the characteristics of the patient. Methods: This study recruited 32 women with breast cancer and a BMI > 30 kg/m2, matched by 6 controls with are non-obese women with breast cancer. Elegibility criteria was determined by women with breast cancer confirmed by pathology, who had not been subjected to prior treatment regarding the neoplasm. The breast cancer removing surgeries and the patients were selected from the ABC Medical Center in Mexico City and all procedures were approved by the research and ethics committee of the hospital in question. Results: Through extensive communication a cooperative protocol was established between the departments of surgery, oncology, pathology and nursing to coordinate efforts and be able to take a 2 – 5 mm sample of the breast tumor removed from the patient. To be able to distinguish cancer cells from non-cancer cells (epithelial cells, fibroblasts, adipocytes) the Hayflick limit was be utilized. Once a primary breast cancer culture was established, 12 million cells will be injected into the subscapular area of athymic, nu-nu mice to be able to monitor tumoral growth in vivo and conduct a subsequent cellular analysis, determining it still pertains to the same characteristics of the tumor from which it was obtained. Conclusions: A primary breast cancer culture repository from patients with a BMI > 30 kg/m2 was established. This is the first primary breast cancer culture for both Mexican and obese women with breast cancer, the first in vitro method of analysis of specific characteristics typical of the Mexican population. Translational research may now be conducted on these new tumoral cultures to create individualized therapy for women with the distinct, aforementioned characteristics.

FKBP51 Modulates Hippocampal Size and Function in Post-translational Regulation of Parkin

2021 ◽  
Author(s):  
Bin Qiu ◽  
Zhaohui Zhong ◽  
Shawn Righter ◽  
Yuxue Xu ◽  
Jun Wang ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Translational Regulation ◽  
Disease Onset ◽  
Fold Increase ◽  
Knock Out ◽  
Fk506 Binding Protein ◽  
Protein Levels ◽  
And Function

Abstract FK506-binding protein 51 (encoded by Fkpb51) has been associated with stress-related mental illness. To identify its function, we studied the morphological consequences of Fkbp51 deletion. Artificial Intelligence-assist morphological analysis identified that Fkbp51 knock-out (KO) mice possess more elongated CA and DG but shorter in height in coronal section when compared to WT. Primary cultured Fkbp51 KO hippocampal neurons were shown to exhibit larger dendritic outgrowth than wild-type (WT) controls, pharmacological manipulation experiments suggest that this may occur through regulation of microtubule-associated protein. Both in vitro primary culture and in vivo labeling support that FKBP51 regulates microtubule-associated protein expression. Furthermore, in the absence of differences in mRNA expression, Fkbp51 KO hippocampus exhibited decreases in βIII-tubulin, MAP2, and Tau protein levels, but a greater than 2.5-fold increase in Parkin protein. Overexpression and knock-down FKBP51 demonstrated that FKBP51 negatively regulates Parkin in a dose-dependent and ubiquitin-mediated manner. These results indicate a potential novel post-translational regulatory of Parkin by FKBP51 and significance of their interaction on disease onset.

Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast

1985 ◽  
Vol 5 (5) ◽  
pp. 1093-1099
Author(s):  
R J Schmidt ◽  
N W Gillham ◽  
J E Boynton
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Apparent Molecular Weight ◽  
Protein L ◽  
Mature Form ◽  
Chloroplast Protein Synthesis ◽  
Made In

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.

Sign in / Sign up

Export Citation Format

Share Document