scholarly journals Induction of Integral Membrane PAM Expression in AtT-20 Cells Alters the Storage and Trafficking of POMC and PC1

1999 ◽  
Vol 144 (3) ◽  
pp. 459-471 ◽  
Author(s):  
Giuseppe D. Ciccotosto ◽  
Martin R. Schiller ◽  
Betty A. Eipper ◽  
Richard E. Mains
Keyword(s):  
Cell Line ◽  
Transmembrane Domain ◽  
Expression System ◽  
Immunofluorescent Staining ◽  
Soluble Form ◽  
Cytoskeletal Organization ◽  
Prohormone Convertase 1 ◽  
Essential Enzyme ◽  
Inducible Cell Line ◽  
Dose Dependent Increase

Peptidylglycine α-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH2-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.

scholarly journals Determination of Three Isoforms of the Receptor Activator of Nuclear Factor-κΒ Ligand and Their Differential Expression in Bone and Thymus*

Endocrinology ◽  
2001 ◽  
Vol 142 (4) ◽  
pp. 1419-1426 ◽  
Author(s):  
Tohru Ikeda ◽  
Michiyuki Kasai ◽  
Masanori Utsuyama ◽  
Katsuiku Hirokawa
Keyword(s):  
Cell Line ◽  
Transmembrane Domain ◽  
Osteoclast Differentiation ◽  
Soluble Form ◽  
Nuclear Factor ◽  
Double Positive ◽  
Dihydroxyvitamin D3 ◽  
Nih3t3 Cells ◽  
Receptor Activator ◽  
Osteoprotegerin Ligand

Abstract The receptor activator of nuclear factor (NF)-κB ligand [RANKL; also known as tumor necrosis factor-related activation-induced cytokine, osteoprotegerin ligand, and osteoclast differentiation factor] is known to bind with the receptor activator of NF-κB (RANK) and act not only as a key factor for osteoclastogenesis but also as a regulator of lymphocyte development. In this study, we found two additional isoforms of RANKL. RANKL 2 has a shorter intracellular domain than the original RANKL (RANKL 1), and RANKL 3 lacks a transmembrane domain and was thought to act as a soluble form. In the bone marrow stromal cell line ST2 and preosteoblastic cell line MC3T3-E1, all three RANKL isoforms were detected, but the expression of RANKL 2 was preferentially suppressed by treatment with 1α,25-dihydroxyvitamin D3 and dexamethasone. In young adult thymus, CD4−CD8− double-negative cells were positive for all three isoforms, CD4+CD8+ double-positive cells were positive for RANKL 1 and RANKL 3 but negative for RANKL 2, and CD4+CD8− and CD4−CD8+ single-positive cells were positive for all three isoforms. Immunofluorescence analyses of NIH3T3 cells transfected with each RANKL isoform indicated that the three RANKL isoforms were translated, and RANKL 2 protein predominantly stayed in the endoplasmic reticulum and Golgi networks. These results indicate that there are three kinds of RANKL-RANK pathways. The presence of multiple RANKL-RANK pathways suggests a more complicated RANKL-RANK system for osteoclastogenesis or T cell differentiation than previously thought.

scholarly journals Enhancing Anti-Tumoral Potential of CD-NHF by Modulating PI3K/Akt Axis in U87 Ex Vivo Glioma Model

2021 ◽  
Vol 22 (8) ◽  
pp. 3873
Author(s):  
Gabriel Luta ◽  
Mihail Butura ◽  
Adrian Tiron ◽  
Crina E. Tiron
Keyword(s):  
Cell Line ◽  
Ex Vivo ◽  
Glioma Cells ◽  
In Vitro Study ◽  
Immunofluorescent Staining ◽  
Expression Levels ◽  
Disease Diagnostics ◽  
Phosphorylated Akt ◽  
Significant Impairment

Background: In the latest years, there has been an increased interest in nanomaterials that may provide promising novel approaches to disease diagnostics and therapeutics. Our previous results demonstrated that Carbon-dots prepared from N-hydroxyphthalimide (CD-NHF) exhibited anti-tumoral activity on several cancer cell lines such as MDA-MB-231, A375, A549, and RPMI8226, while U87 glioma tumor cells were unaffected. Gliomas represent one of the most common types of human primary brain tumors and are responsible for the majority of deaths. In the present in vitro study, we expand our previous investigation on CD-NHF in the U87 cell line by adding different drug combinations. Methods: Cell viability, migration, invasion, and immunofluorescent staining of key molecular pathways have been assessed after various treatments with CD-NHF and/or K252A and AKTVIII inhibitors in the U87 cell line. Results: Association of an inhibitor strongly potentiates the anti-tumoral properties of CD-NHF identified by significant impairment of migration, invasion, and expression levels of phosphorylated Akt, p70S6Kinase, or by decreasing expression levels of Bcl-2, IL-6, STAT3, and Slug. Conclusions: Using simultaneously reduced doses of both CD-NHF and an inhibitor in order to reduce side effects, the viability and invasiveness of U87 glioma cells were significantly impaired.

scholarly journals Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

2021 ◽  
Vol 22 (12) ◽  
pp. 6361
Author(s):  
Eunyoung Lee ◽  
Michelle Novais de Paula ◽  
Sangki Baek ◽  
Huynh Kim Khanh Ta ◽  
Minh Tan Nguyen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ion Exchange ◽  
Stem Cell Factor ◽  
Transmembrane Domain ◽  
Coli Strain ◽  
Exchange Chromatography ◽  
Soluble Form ◽  
Human Stem Cell ◽  
E Coli ◽  
Human Stem Cell Factor

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

scholarly journals Expression of phosphoenolpyruvate carboxykinase (PEPCK) chimeras in renal epithelial cells. Retention of appropriate physiological responsiveness using enhancerless retroviral vectors

1992 ◽  
Vol 284 (3) ◽  
pp. 725-732 ◽  
Author(s):  
A S Pollock ◽  
D H Lovett
Keyword(s):  
Epithelial Cell ◽  
Cyclic Amp ◽  
Cell Line ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Large Fraction ◽  
Expression System ◽  
Retroviral Vectors ◽  
Epithelial Cell Line ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cell Line

We used an enhancerless U3 mutant retroviral vector to deliver chimeras of the phosphoenolpyruvate carboxykinase (PEPCK) promoter region to a renal epithelial cell line capable of expressing PEPCK mRNA. Chimeras consisting of the PEPCK promoter and chloramphenicol acetyltransferase, neomycin phosphotransferase or human growth hormone genes were expressed after viral infection of the NRK52E renal epithelial cell line. Virus-delivered sequences in which the direction of PEPCK promoter transcription was antegrade to the normal direction of the long terminal repeat (LTR)-initiated transcription correctly upon stimulation with dexamethasone or 8-bromo cyclic AMP and upon lowering of the extracellular pH. Fluorescent primer extension in situ using primers specific for virus-delivered sequences of antegrade constructs indicated that a large fraction of NRK52E cells could be infected by co-cultivation with virus-producing psi-2 cells without G418 selection. Virus-delivered constructs whose orientation was opposite to that of the LTRs were expressed at very low levels, with transcripts detectable by PCR only in RNA from cyclic AMP-treated cells. Using reverse transcription/PCR, we demonstrated that the chimeric transcripts were from the internal PEPCK promoter rather than a functional or reconstituted Moloney LTR. PEPCK-reporter chimeras delivered by retroviral vectors demonstrated a level of expression more consistent with the level of expression of the native PEPCK gene than did transfected chimeras. This expression system should prove useful for studies of the physiological modulation of gene expression in renal tissues.

An endogenous carbohydrate-binding protein of baby hamster kidney (BHK21 C13) cells. Temporal changes in cellular expression in the developing kidney

1990 ◽  
Vol 97 (1) ◽  
pp. 139-148
Author(s):  
L. Foddy ◽  
S.C. Stamatoglou ◽  
R.C. Hughes
Keyword(s):  
Cell Line ◽  
Kidney Development ◽  
Immunofluorescent Staining ◽  
Carbohydrate Binding ◽  
Baby Hamster Kidney ◽  
Cellular Expression ◽  
Established Cell ◽  
Developing Kidney ◽  
Triton X ◽  
Newborn Hamster

Asialofetuin (ASF) coupled to Sepharose has been used to isolate a Mr 30,000 protein from Triton X-100 extracts of the baby hamster kidney cell line BHK21 C13. Binding to ASF-Sepharose was specific for terminal beta-galactosyl residues. The lectin requires detergent for optimal solubilization and binding is independent of Ca2+ or reducing reagents. The lectin was labelled in a lactoperoxidase-catalysed iodination of intact BHK21 C13 cells, suggesting that it is associated with the cell surface. Antibodies to the lectin identify in Western blotting cross-reactive components in established cell lines of kidney (MDCK, NRK) and non-kidney (L, CHO, 3T3) origin. In young adult hamsters, the lectin is expressed in colon and duodenum and in lesser amounts in ileum, stomach, lung, liver and testes but is absent in kidney. The lectin is expressed in late embryonic and newborn hamster kidney but expression declines during 14 days after birth. Immunofluorescent staining of cryostat sections of newborn hamster kidney and intestine show that the lectin is expressed at apical epithelial surfaces. The presence of the lectin at the luminal surface of kidney tubules suggests a tubular origin for the BHK21 C13 cell line. Possible functions of the Mr 30,000 lectin in kidney development are discussed.

scholarly journals Phosphoproteomic Analysis of Gossypol-Induced Apoptosis in Ovarian Cancer Cell Line, HOC1a

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Lixu Jin ◽  
Yuling Chen ◽  
Xinlin Mu ◽  
Qingquan Lian ◽  
Haiyun Deng ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Line ◽  
Cancer Cell ◽  
Ovarian Cancer Cell ◽  
Gynecological Cancer ◽  
Ovarian Cancer Cell Line ◽  
Cancer Cell Line ◽  
Actin Binding ◽  
Ovarian Cancer Cells ◽  
Cytoskeletal Organization

Ovarian cancer is a major cause for death of gynecological cancer patients. The efficacy of traditional surgery and chemotherapy is rather compromised and platinum-resistant cancer recurs. Finding new therapeutic targets is urgently needed to increase the survival rate and to improve life quality of patients with ovarian cancer. In the present work, phosphoproteomic analysis was carried out on untreated and gossypol-treated ovarian cancer cell line, HOC1a. We identified approximately 9750 phosphopeptides from 3030 phosphoproteins, which are involved in diverse cellular processes including cytoskeletal organization, RNA and nucleotide binding, and cell cycle regulation. Upon gossypol treatment, changes in phosphorylation of twenty-nine proteins including YAP1 and AKAP12 were characterized. Western blotting and qPCR analysis were used to determine expression levels of proteins in YAP1-related Hippo pathway showing that gossypol induced upregulation of LATS1, which phosphorylates YAP1 at Ser 61. Furthermore, our data showed that gossypol targets the actin cytoskeletal organization through mediating phosphorylation states of actin-binding proteins. Taken together, our data provide valuable information to understand effects of gossypol on protein phosphorylation and apoptosis of ovarian cancer cells.

scholarly journals RB459 and RB462 antibodies recognize mouse Pannexin1 protein by immunofluorescent staining

2019 ◽  
Vol 2 (2) ◽  
pp. e39
Author(s):  
Olga M Rusiecka ◽  
Christel L Roth ◽  
Brenda R Kwak ◽  
Filippo Molica
Keyword(s):  
Endothelial Cell ◽  
Cell Line ◽  
Recombinant Antibodies ◽  
Immunofluorescent Staining ◽  
Mouse Tissue ◽  
Murine Macrophage ◽  
Macrophage Cell Line ◽  
Macrophage Cell ◽  
Endothelial Cell Line ◽  
Murine Macrophage Cell Line

The recombinant antibodies RB459 and RB462 recognize mouse Pannexin1 (Panx1) by immunofluorescent staining of a mouse cardiac endothelial cell line, a murine macrophage cell line and cryosections of mouse tissue.

scholarly journals A single amino acid substitution (N297A) in the conserved NPXXY sequence of the human N-formyl peptide receptor results in inhibition of desensitization and endocytosis, and a dose-dependent shift in p42/44 mitogen-activated protein kinase activation and chemotaxis

2000 ◽  
Vol 352 (2) ◽  
pp. 399-407 ◽  
Author(s):  
Jeannie M. GRIPENTROG ◽  
Algirdas J. JESAITIS ◽  
Heini M. MIETTINEN
Keyword(s):  
Protein Kinase ◽  
Transmembrane Domain ◽  
Mitogen Activated Protein Kinase ◽  
Formyl Peptide Receptor ◽  
Wild Type ◽  
Peptide Receptor ◽  
Formyl Peptide ◽  
Mitogen Activated Protein ◽  
Dose Dependent Increase ◽  
Dose Dependent

The formyl peptide receptor (FPR) is a G-protein-coupled receptor (GPCR) that mediates chemotaxis and stimulates the mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase pathway. We have examined the functional effects of substitutions of a conserved aspartic acid residue in the second transmembrane domain (D71A) and of residues in the conserved NPXXY motif in the seventh transmembrane domain (N297A and Y301A). These mutated receptors, expressed in Chinese hamster ovary (CHO) cells, bind ligand with affinities similar to wild-type FPR, but the D71A mutant is uncoupled from G-protein [Miettinen, Mills, Gripentrog, Dratz, Granger and Jesaitis (1997) J. Immunol 159, 4045–4054]. In the present study, we show that both the D71A and N297A mutations resulted in defective endocytosis. The N297A substitution also prevented desensitization, as determined by intracellular calcium mobilization by sequential stimulation with ligand. In chemotaxis assays, the N297A mutation resulted in cell migration towards gradients of up to 100nM N-formyl-methionyl-leucyl-phenylalanine (fMLF), whereas cells expressing the wild-type FPR and the Y301A mutant were no longer chemotactically responsive at 10–100nM fMLF. Maximal activation of p42/44 MAPK occurred in CHO cells expressing wild-type FPR at 10nM–100nM fMLF, whereas cells expressing the N297A mutant showed a dose-dependent increase in the amount of phosphorylated p42/44 MAPK up to 1–10µM fMLF. Since the MAPK kinase inhibitor PD98059 blocked fMLF-induced chemotaxis, our results suggest that the dose-dependent increase in p42/44 MAPK activation may correlate with the increased chemotactic migration of N297A transfectants at 10nM–100nM fMLF.

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