scholarly journals Phosphorylation of the Oncofetal Variant of the Human Bile Salt-dependent Lipase

2001 ◽  
Vol 276 (15) ◽  
pp. 12356-12361 ◽  
Author(s):  
Alain Verine ◽  
Josette Le Petit-Thevenin ◽  
Laurence Panicot-Dubois ◽  
Annick Valette ◽  
Dominique Lombardo
Keyword(s):  
High Pressure ◽  
Bile Salt ◽  
Cho Cells ◽  
Phosphorylation Site ◽  
Chinese Hamster ◽  
Human Bile ◽  
Site Specific ◽  
Phosphorylation Motif ◽  
Extracellular Compartment ◽  
First Time

In this paper, we report, for the first time, the localization of the phosphorylation site of the fetoacinar pancreatic protein (FAPP), which is an oncofetal variant of the pancreatic bile salt-dependent lipase. Using Chinese hamster ovary (CHO) cells transfected with the cDNA encoding FAPP, we radiolabeled the enzyme with32P, and then the protein was purified by affinity chromatography on cholate-immobilized Sepharose column and submitted to a CNBr hydrolysis. Analysis of peptides by high pressure liquid chromatography, associated with the radioactivity profile, revealed that the phosphorylation site is located at threonine 340. Site-specific mutagenesis experiments, in which the threonine was replaced by an alanine residue, were used to invalidate the phosphorylation of FAPP and to study the influence of the modification on the activity and secretion of the enzyme. These studies showed that CHO cells, transfected with the mutated cDNA of FAPP, kept all of their ability to synthesize the protein, but the loss of the phosphorylation motif prevented the release of the protein in the extracellular compartment. However, the mutated enzyme, which was sequestrated in the transfected CHO cells, remains active on bile salt-dependent lipase substrates.

Glycosylation of GD4 and Thy-1

1993 ◽  
Vol 342 (1299) ◽  
pp. 43-50 ◽  
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Truncated Form ◽  
Site Specific ◽  
Glycosylation Sites ◽  
Oligosaccharide Processing ◽  
Domain 3 ◽  
Protein Cell ◽  
Oligosaccharide Structures

The site-specific glycosylation of soluble recombinant variants of human and rat CD4 (sCD4) expressed in Chinese hamster ovary (CHO) cells has been characterized. The presence of identical oligosaccharides at the conserved glycosylation site in domain 3 of rat and human sCD4 and the greater abundance of oligomannose and hybrid type glycans at the non-conserved glycosylation site of rat sCD4 clearly indicate that the protein structure influences oligosaccharide processing. Comparisons of rat sCD4 glycopeptides with m utant molecules with only single glycosylation sites and with a truncated form containing only the two NH 2 -terminal domains, indicate that independent processing occurs at each glycosylation site and that dom ain interactions can also affect oligosaccharide processing. These and other analyses of sCD2 expressed in CHO cells and Thy-1 purified from various tissues suggest that the diversity of oligosaccharide structures on a protein is regulated by the location of the glycosylation sites and the nature of the target protein, cell and tissue. The functional significance of this control remains to be determined.

Effects of High Pressure and Metal Salts on Cell Growth

1991 ◽  
Vol 19 (2) ◽  
pp. 245-249
Author(s):  
Jon Jenssen ◽  
Tore Syversen
Keyword(s):  
High Pressure ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Cho Cells ◽  
Metal Salt ◽  
Chinese Hamster ◽  
Metal Salts ◽  
Control Growth ◽  
Copper Manganese ◽  
Helium Gas

The effects of chromate, vanadate, nickel, manganese, copper and barium, both separately and in combination with high pressure helium gas, have been studied in an epithelial-like cell line — Chinese hamster ovary (CHO) cells, using inhibition of cell growth as the cytotoxic endpoint. High pressure (175 bar) by itself reduced the net growth of CHO cells to 71.7 ± 5.5% (mean ± SD) of normobaric control growth. The combined effects of high pressure and metal salts were additive for the lowest concentrations of chromate, copper, manganese and barium, more than additive for the lowest concentration of nickel, and less than additive for vanadate and the highest concentrations of chromate, copper, manganese and barium. A ranking of the normobaric growth inhibition of the metal salts, estimated as the concentration giving 50% inhibition, was Cr>V>Mn>Ni>Cu>Ba. The growth inhibition of barium was less than 50% at a concentration of ImM BaCl2. The combined exposure to high pressure and the metal salt did not change the toxicity ranking.

Substrate specificity of the rat liver Na+-bile salt cotransporter inXenopus laevis oocytes and in CHO cells

1998 ◽  
Vol 274 (2) ◽  
pp. G370-G375 ◽  
Author(s):  
Alice Schroeder ◽  
Uta Eckhardt ◽  
Bruno Stieger ◽  
Ronald Tynes ◽  
Claudio D. Schteingart ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Cyclosporin A ◽  
Bile Salt ◽  
Rat Liver ◽  
Cho Cells ◽  
Bile Salts ◽  
Chinese Hamster ◽  
Xenopus Laevis Oocytes ◽  
Uptake System ◽  
Mammalian Cell Lines

It has been proposed that the hepatocellular Na+-dependent bile salt uptake system exhibits a broad substrate specificity in intact hepatocytes. In contrast, recent expression studies in mammalian cell lines have suggested that the cloned rat liver Na+-taurocholate cotransporting polypeptide (Ntcp) may transport only taurocholate. To characterize its substrate specificity Ntcp was stably transfected into Chinese hamster ovary (CHO) cells. These cells exhibited saturable Na+-dependent uptake of [3H]taurocholate [Michaelis constant ( K m) of ∼34 μM] that was strongly inhibited by all major bile salts, estrone 3-sulfate, bumetanide, and cyclosporin A. Ntcp cRNA-injected Xenopus laevis oocytes and the transfected CHO cells exhibited saturable Na+-dependent uptake of [3H]taurochenodeoxycholate ( K m of ∼5 μM), [3H]tauroursodeoxycholate ( K m of ∼14 μM), and [14C]glycocholate ( K m of ∼27 μM). After induction of gene expression by sodium butyrate, Na+-dependent transport of [3H]estrone 3-sulfate ( K m of ∼27 μM) could also be detected in the transfected CHO cells. However, there was no detectable Na+-dependent uptake of [3H]bumetanide or [3H]cyclosporin A. These results show that the cloned Ntcp can mediate Na+-dependent uptake of all physiological bile salts as well as of the steroid conjugate estrone 3-sulfate. Hence, Ntcp is a multispecific transporter with preference for bile salts and other anionic steroidal compounds.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Fouling Is the Beginning: Upcycling Biopolymer-Fouled Substrates for Fabricating High-Permeance Thin-Film Composite Polyamide Membranes

2020 ◽  
Author(s):  
Ruobin Dai ◽  
Hongyi Han ◽  
Tianlin Wang ◽  
Jiayi Li ◽  
Chuyang Y. Tang ◽  
...  
Keyword(s):  
Thin Film ◽  
High Pressure ◽  
End Of Life ◽  
Water Purification ◽  
Low Pressure ◽  
Polymeric Membranes ◽  
Membrane Recycling ◽  
Film Composite ◽  
Thin Film Composite ◽  
First Time

Commercial polymeric membranes are generally recognized to have low sustainability as membranes need to be replaced and abandoned after reaching the end of their life. At present, only techniques for downcycling end-of-life high-pressure membranes are available. For the first time, this study paves the way for upcycling fouled/end-of-life low-pressure membranes to fabricate new high-pressure membranes for water purification, forming a closed eco-loop of membrane recycling with significantly improved sustainability.

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

scholarly journals Induction of Promoter DNA Methylation Upon High-Pressure Spraying of Double-Stranded RNA in Plants

Agronomy ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 789
Author(s):  
Athanasios Dalakouras ◽  
Ioannis Ganopoulos
Keyword(s):  
High Pressure ◽  
De Novo ◽  
Epigenetic Changes ◽  
Double Stranded Rna ◽  
Rna Molecules ◽  
Promoter Sequences ◽  
Promoter Dna Methylation ◽  
Promoter Dna ◽  
First Time ◽  
De Novo Methylation

Exogenous application of RNA molecules is a potent method to trigger RNA interference (RNAi) in plants in a transgene-free manner. So far, all exogenous RNAi (exo-RNAi) applications have aimed to trigger mRNA degradation of a given target. However, the issue of concomitant epigenetic changes was never addressed. Here, we report for the first time that high-pressure spraying of dsRNAs can trigger de novo methylation of promoter sequences in plants.

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