scholarly journals Processing of mutated proinsulin with tetrabasic cleavage sites to bioactive insulin in the non-endocrine cell line, COS-7

FEBS Letters ◽  
1992 ◽  
Vol 311 (1) ◽  
pp. 55-59 ◽  
Author(s):  
Masahiko Yanagita ◽  
Kazuhisa Nakayama ◽  
Toshiyuki Takeuchi
Keyword(s):  
Cell Line ◽  
Endocrine Cell ◽  
Cleavage Sites

scholarly journals Tetracycline-regulated secretion of human insulin in a transfected non-endocrine cell line

2003 ◽  
Vol 30 (3) ◽  
pp. 331-346 ◽  
Author(s):  
KT Scougall ◽  
CA Maltin ◽  
JA Shaw
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
Human Insulin ◽  
Endocrine Cell ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Host Cells ◽  
Cleavage Sites ◽  
Wild Type ◽  
Transfected Cells

Long-term constitutive secretion of insulin by implantation of ex vivo transfected cells such as fibroblasts or myoblasts or in situ by intramuscular injection of naked plasmid DNA provides a potential approach to gene therapy for diabetes mellitus. A mechanism for regulating insulin secretion will be necessary to realize the therapeutic potential of this approach. A second obstacle is the inability of non-endocrine host cells to fully process proinsulin. Therefore, alteration of the wild-type cDNA will be necessary to achieve processing of proinsulin by endogenous endoproteases within these cells. The cDNAs for beta-galactosidase (beta), human wild-type proinsulin (hppI1) and a mutated construct (hppI4), in which the dibasic PC2 and PC3 cleavage sites had been altered to form furin cleavage sites, were sub-cloned into four vectors (pCR3, pVR1012, pIRES, pTRE), including a tetracycline responsive plasmid (pTRE) that requires co-transfection with another plasmid encoding a transactivator (pTet-off) for transgene expression. Transient transfection of the COS-7 fibroblast cell line with these constructs was performed using DEAE-dextran and liposomes. Analysis of vector efficiencies revealed that pTRE/pTet-off>pIRES>pCR3>pVR1012. Further analysis demonstrated total pro/insulin secretion of 2.33 ng/10(6) cells/24 h with > or =25% processed to insulin in hppI-1.pTRE/pTet-off-transfected cells compared with 0.39 ng/10(6) cells/24 h and >70% processing in hppI-4.pTRE/pTet-off-transfected cells. In co-transfection studies with pTRE-hppI1/pTet-off and pTRE-hppI4/pTet-off constructs, pro/insulin secretion was inhibited to 65-66% and 36-38% of control (100%) in the presence of 0.01 and 0.1 microg/ml tetracycline respectively over a 24-h incubation period. Furthermore, reversal of tetracycline inhibition was demonstrated for pTRE-hppI1/pTet-off- and pTRE-hppI4/pTet-off-transfected cells. After a 48-h incubation with 1.0 microg/ml tetracycline, total pro/insulin levels were 10 and 14% compared with untreated cells respectively. On tetracycline removal, total proinsulin levels increased and were equivalent to untreated groups 72 h later. In conclusion, regulation of fully processed human insulin secretion has been achieved in a transiently transfected non-endocrine cell line.

scholarly journals Activities of Thrombin and Factor Xa Are Essential for Replication of Hepatitis E Virus and Are Possibly Implicated in ORF1 Polyprotein Processing

2018 ◽  
Vol 92 (6) ◽  
Author(s):  
Gayatri D. Kanade ◽  
Kunal D. Pingale ◽  
Yogesh A. Karpe
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Liver Cell ◽  
Serine Proteases ◽  
Hepatitis E Virus ◽  
Factor Xa ◽  
Hepatitis E ◽  
Cleavage Sites ◽  
Polyprotein Processing ◽  
Liver Cell Line

ABSTRACTHepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a nonstructural polyprotein of 1,693 amino acids. It is not clear whether the ORF1 polyprotein (pORF1) is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave various results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bears conserved cleavage sites of thrombin and factor Xa. Using a reverse genetics approach, we demonstrated that an HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstratedin vitroprocessing when we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of a liver cell line with a serine protease inhibitor as well as small interfering RNA (siRNA) knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both of these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in a liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV.IMPORTANCEHepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. A high mortality rate of about 30% is observed in HEV-infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to the variability of study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine proteases, thrombin and factor Xa, that are conserved among HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study sheds light on the presence and roles of clotting factors with respect to virus replication in the cells.

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line, CHO

1996 ◽  
Vol 21 (3) ◽  
pp. 279-288 ◽  
Author(s):  
S. M. N. Hunt ◽  
A. S. Tait ◽  
P. P. Gray ◽  
M. J. Sleigh
Keyword(s):  
Cell Line ◽  
Endocrine Cell ◽  
Human Proinsulin

Stimulation of glucagon-like peptide-1 secretion by muscarinic agonist in a murine intestinal endocrine cell line.

Endocrinology ◽  
1994 ◽  
Vol 134 (5) ◽  
pp. 2011-2017 ◽  
Author(s):  
J Abello ◽  
F Ye ◽  
A Bosshard ◽  
C Bernard ◽  
J C Cuber ◽  
...  
Keyword(s):  
Cell Line ◽  
Endocrine Cell ◽  
Muscarinic Agonist ◽  
Glucagon Like Peptide ◽  
Glucagon Like Peptide 1 ◽  
Stimulation Of

scholarly journals PKD1, PKD2, and Their Substrate Kidins220 Regulate Neurotensin Secretion in the BON Human Endocrine Cell Line

2007 ◽  
Vol 283 (5) ◽  
pp. 2614-2621 ◽  
Author(s):  
Jing Li ◽  
L. Andy Chen ◽  
Courtney M. Townsend ◽  
B. Mark Evers
Keyword(s):  
Cell Line ◽  
Endocrine Cell

384 ERK1c, a Novel Spliced Form of ERK1, Positively Regulates PGE2-Stimulated NT Secretion in the Human Endocrine Cell Line, BON

2008 ◽  
Vol 134 (4) ◽  
pp. A-51
Author(s):  
Jing Li ◽  
Tianyan Gao ◽  
Courtney M. Townsend ◽  
B.M. Evers
Keyword(s):  
Cell Line ◽  
Endocrine Cell

Intestinal-type fibroblasts selectively influence proliferation rate and peptide synthesis in the murine entero-endocrine cell line STC-1

1997 ◽  
Vol 62 (3) ◽  
pp. 139-147 ◽  
Author(s):  
Christelle Ratineau ◽  
Michelina Plateroti ◽  
Jérôme Dumortier ◽  
Martine Blanc ◽  
Michèle Kédinger ◽  
...  
Keyword(s):  
Cell Line ◽  
Peptide Synthesis ◽  
Endocrine Cell ◽  
Proliferation Rate ◽  
Intestinal Type

scholarly journals Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line.

1995 ◽  
Vol 15 (7) ◽  
pp. 3870-3881 ◽  
Author(s):  
B M Evers ◽  
X Wang ◽  
Z Zhou ◽  
C M Townsend ◽  
G P McNeil ◽  
...  
Keyword(s):  
Cell Line ◽  
Endocrine Cell ◽  
Mutational Analysis ◽  
Constitutive Expression ◽  
N Gene ◽  
Specific Expression ◽  
Response Element ◽  
High Level ◽  
Neuromedin N ◽  
Bon Cells

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP-1-like motif and other upstream control elements play an important role in the high-level constitutive expression of NT/N in the human endocrine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/N expression and to better understand the mechanisms responsible for the terminal differentiation of the N-cell lineage in the gut.

Sign in / Sign up

Export Citation Format

Share Document