scholarly journals Human Acyl-Coenzyme A:Cholesterol Acyltransferase 1 (acat1) Sequences Located in Two Different Chromosomes (7 and 1) Are Required to Produce a Novel ACAT1 Isoenzyme with Additional Sequence at the N Terminus

2004 ◽  
Vol 279 (44) ◽  
pp. 46253-46262 ◽  
Author(s):  
Li Yang ◽  
Oneil Lee ◽  
Jia Chen ◽  
Jiang Chen ◽  
Catherine C. Y. Chang ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Monocyte ◽  
Functional Protein ◽  
Rare Form ◽  
Ovary Cells ◽  
Sds Page ◽  
Translation Initiation Codon ◽  
Additional Sequence ◽  
Additional Amino Acid

A rare form of human ACAT1 mRNA, containing the optional long 5′-untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomaltrans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7 (Li, B. L., Li, X. L., Duan, Z. J., Lee, O., Lin, S., Ma, Z. M., Chang, C. C., Yang, X. Y., Park, J. P., Mohandas, T. K., Noll, W., Chan, L., and Chang, T. Y. (1999)J. Biol. Chem.274, 11060–11071). To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5′-untranslated repeat. To produce the 56-kDa ACAT1,acat1sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1ORF. The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.

The extract of Ilex kudingcha inhibits atherosclerosis in apoE-deficient mice by suppressing cholesterol accumulation in macrophages

2021 ◽  
Author(s):  
Yukio Fujiwara ◽  
Shota Okada ◽  
Keisuke Uryu ◽  
Isafumi Maru ◽  
Yoshihiro Komohara
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Monocyte ◽  
Inhibitory Effects ◽  
Cholesterol Accumulation ◽  
Cholesterol Acyl Transferase ◽  
Ovary Cells ◽  
Acat Activity ◽  
Atherosclerotic Lesions ◽  
Deficient Mice

Abstract It was previously reported that oleanolic acid and ursolic acid, triterpenoid compounds occurring in Ilex kudingcha, ameliorate hyperlipidemia and atherosclerosis in apoE-deficient mice. In the present study, we investigated whether Ilex kudingcha extract exerts similar inhibitory effects on cholesterol accumulation in human monocyte-derived macrophages (HMDMs) and atherogenesis in apoE-deficient mice. Ilex kudingcha extract significantly inhibited cholesterol ester (CE) accumulation induced by acetyl-LDL (acetylated LDL) in HMDMs; however, it generated no effect on cell viability in HMDMs. Ilex kudingcha extract also suppressed CE accumulation in acyl-CoA: cholesterol acyl-transferase (ACAT)-overexpressing CHO (Chinese hamster ovary) cells, thereby indicating that it inhibits ACAT activity. Furthermore, the oral administration of Ilex kudingcha extract to apoE-deficient mice significantly decreased the levels of serum cholesterol, triglyceride, sLOX-1, as well as the regions of atherosclerotic lesions in the mice. Our study reveals crucial new-found evidence that Ilex kudingcha extract significantly inhibits ACAT activity and suppresses atherogenesis.

Purification of phosphatidylglycerophosphate synthase from Chinese hamster ovary cells

2001 ◽  
Vol 354 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Kiyoshi KAWASAKI ◽  
Osamu KUGE ◽  
Yoshio YAMAKAWA ◽  
Masahiro NISHIJIMA
Keyword(s):  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mitochondrial Fraction ◽  
Ovary Cells ◽  
Protein Levels ◽  
Defective Mutant ◽  
Sds Page

Phosphatidylglycerophosphate (PGP) synthase catalyses the committed step in the biosynthesis of phosphatidylglycerol and cardiolipin in mammalian cells. Recently we isolated a Chinese hamster ovary (CHO) PGS1 cDNA encoding PGP synthase. In the present study we purified this PGP synthase to near-homogeneity from the mitochondrial fraction of CHO-K1 cells; the final enzyme preparation gave a single 60kDa protein on SDS/PAGE. Polyclonal antibodies raised against a recombinant CHO PGS1 protein cross-reacted with the purified 60kDa protein and with CHO membrane proteins of 60kDa and 62kDa that increased after transfection with the PGS1 cDNA. The 60 and 62kDa protein levels in a PGP synthase-defective mutant of CHO-K1 cells were markedly lower than those in CHO-K1 cells. These results indicated that the purified 60kDa protein was PGP synthase encoded by the PGS1 gene. In addition we found that the purified PGP synthase had no PGP phosphatase activity, indicating that phosphatidylglycerol was produced from CDP-diacylglycerol through two steps catalysed by distinct enzymes, PGP synthase and PGP phosphatase.

scholarly journals The class I scavenger receptor CD163 promotes internalization of ADAMTS13 by macrophages

2017 ◽  
Vol 1 (5) ◽  
pp. 293-305 ◽  
Author(s):  
Fabian C. Verbij ◽  
Nicoletta Sorvillo ◽  
Paul H. P. Kaijen ◽  
Johana Hrdinova ◽  
Ivan Peyron ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Scavenger Receptor ◽  
Chinese Hamster ◽  
Human Monocyte ◽  
Mannose Receptor ◽  
Class I ◽  
Spectrometric Analysis ◽  
Ovary Cells ◽  
Macrophage Mannose Receptor ◽  
Partial Blocking

Abstract Internalization of ADAMTS13 by macrophages may contribute to its clearance from the circulation. Here we investigated endocytic mechanisms that contribute to the uptake of ADAMTS13 by macrophages. Human monocyte-derived macrophages were used to monitor the uptake of fluorescently labeled recombinant ADAMTS13 by flow cytometry. Internalization of ADAMTS13 was blocked upon addition of the cell-permeable dynamin inhibitor dynasore. Partial blocking of ADAMTS13 uptake was observed by using mannan; however, uptake was not affected by an antibody that blocked binding to the macrophage mannose receptor CD206, which suggests that other endocytic receptors contribute to the internalization of ADAMTS13 by macrophages. A pull-down with ADAMTS13 and subsequent mass spectrometric analysis identified the class I scavenger receptor CD163 as a candidate receptor for ADAMTS13. Blocking experiments with monoclonal anti-CD163 antibody EDHu-1 resulted in decreased ADAMTS13 internalization by macrophages. Pronounced inhibition of ADAMTS13 uptake by EDHu-1 was observed in CD163 high-expressing macrophages. In agreement with these findings, CD163-expressing Chinese hamster ovary cells were capable of rapidly internalizing ADAMTS13. Surface plasmon resonance revealed binding of ADAMTS13 to scavenger receptor cysteine-rich domains 1-9 and 1-5 of CD163. Taken together, our data identify CD163 as a major endocytic receptor for ADAMTS13 on macrophages.

Cloning and Expression of Canine Glycoprotein Ibα

1999 ◽  
Vol 82 (10) ◽  
pp. 1327-1333 ◽  
Author(s):  
Patricia Morateck ◽  
Scot Fahs ◽  
David Warltier ◽  
Robert Montgomery ◽  
Dermot Kenny
Keyword(s):  
Amino Acids ◽  
Physiological Function ◽  
Chinese Hamster Ovary Cells ◽  
Human Monoclonal Antibody ◽  
Chinese Hamster ◽  
Cloning And Expression ◽  
Ovary Cells ◽  
Translation Initiation Codon ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe interaction of the glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (vWF) is critical in initiation of haemostasis and thrombosis through platelet adhesion to damaged endothelium. The binding site for vWF resides within the GPIbα subunit of the complex. To further define the physiological function of platelet GPIbα we cloned and expressed the canine GPIbα cDNA. A canine platelet cDNA library was constructed and screened with a randomly primed 32P-labeled 1041-base-pair restriction fragment of the human GPIbα cDNA. Analysis of 23 clones demonstrated that the canine GPIbα cDNA is 2530 nucleotides in length and includes a short 5’ untranslated segment of 42 nucleotides followed by a signal peptide of 16 amino acids, a mature peptide of 645 amino acids and a 3’ noncoding region of 455 nucleotides. A single intron of 142 nucleotides, 6 nucleotides upstream from the ATG translation initiation codon was identified in the canine gene in a similar location to that present in the human gene.Chinese hamster ovary cells that stably express human GPIbα and GPIX were transfected with the canine GPIbα cDNA. Canine GPIbα was expressed on the surface of these cells and bound vWF in the presence of botrocetin. The binding of vWF was inhibited by an anti-vWF human monoclonal antibody known to inhibit vWF binding to GPIbα. The results of this investigation will allow the development of reagents to study the physiological function of GPIbα in an animal model.

scholarly journals In vitro expression demonstrates impaired secretion of the γAsn319, Asp320 deletion variant fibrinogen

2005 ◽  
Vol 94 (07) ◽  
pp. 53-59 ◽  
Author(s):  
Satomi Kani ◽  
Fumiko Terasawa ◽  
Susan T. Lord ◽  
Minoru Tozuka ◽  
Hiroyoshi Ota ◽  
...  
Keyword(s):  
Culture Media ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fibrinogen Concentration ◽  
Ovary Cells ◽  
Cell Lysates ◽  
Sds Page ◽  
The Media ◽  
Kinetics Of

SummaryThe hypodysfibrinogenemia Otsu is caused by the two-residue deletion,γAsn319 and γAsp320. Analysis of plasma or purified fibrinogen from the heterozygous propositus revealed that the amount of variant γ-chain was lower than that of normal γ-chain. In order to examine the basis for this difference, we transfected Chinese hamster ovary cells and established stable cell lines that expressed both chains, γΔ/γN, only the normal chain, γN, and only the variant chain, γΔ. We measured fibrinogen concentration of confluent cultures by ELISA. We found the ratios of the concentrations in the media to the concentrations in the cell lysates of γΔ, γΔ/γN, and γN-cells were 0.42, 0.60, and 1.00, respectively. We measured the concentrations of the γΔ and γN chains by densitometric analysis of samples following separation by SDS-PAGE and found the fraction of γΔ-chains in cell lysates was always greater than the fraction in the respective culture media. We examined the kinetics of fibrinogen synthesis, assembly and secretion in pulse-chase experiments, and found that the γΔ-chain was assembled into intact fibrinogen at a rate similar to assembly of the γN-chain into normal fibrinogen, but was secreted into the medium at a slightly slower rate than normal fibrinogen. Considered together, these experiments indicate secretion of the variant fibrinogen was slightly impaired. These results suggest that the reduced level of γΔ319,320 fibrinogen in the plasma of the Otsu patient arises from modestly impaired secretion of this variant fibrinogen.

Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C

1993 ◽  
Vol 70 (03) ◽  
pp. 418-422 ◽  
Author(s):  
Masaharu Aritomi ◽  
Naoko Watanabe ◽  
Rika Ohishi ◽  
Komakazu Gomi ◽  
Takao Kiyota ◽  
...  
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Transmembrane Domain ◽  
Chinese Hamster Ovary Cells ◽  
Time Lag ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Soluble Thrombomodulin ◽  
Simulation Based ◽  
Recombinant Human Soluble Thrombomodulin

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K d = 26 nM) or PC activation (K d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.

Malignancy-related characteristics of wild type and drug-resistant chinese hamster ovary cells

Pathology ◽  
1993 ◽  
Vol 25 (3) ◽  
pp. 268-276 ◽  
Author(s):  
Wanda B. Mackinnon ◽  
Marlen Dyne ◽  
Rebecca Hancock ◽  
Carolyn E. Mountford ◽  
Adrienne J. Grant ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Drug Resistant ◽  
Wild Type ◽  
Ovary Cells

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.

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