scholarly journals Hydroxyeicosanoids Bind to and Activate the Low Affinity Leukotriene B4Receptor, BLT2

2001 ◽  
Vol 276 (15) ◽  
pp. 12454-12459 ◽  
Author(s):  
Takehiko Yokomizo ◽  
Kazuhiko Kato ◽  
Hiroshi Hagiya ◽  
Takashi Izumi ◽  
Takao Shimizu
Keyword(s):  
Inflammatory Diseases ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ligand Specificity ◽  
Hydroxyeicosatetraenoic Acid ◽  
Ovary Cells ◽  
High Affinity Receptor ◽  
Arachidonate Metabolite ◽  
Affinity Receptor ◽  
G Protein Coupled

Leukotriene B4, an arachidonate metabolite, is a potent chemoattractant of leukocytes involved in various inflammatory diseases. Two G-protein-coupled receptors for leukotriene B4have been cloned and characterized. BLT1 (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997)Nature387, 620–624) is a high affinity receptor exclusively expressed in leukocytes, and BLT2 (Yokomizo, T., Kato, K., Terawaki, K., Izumi, T., and Shimizu, T. (2000)J. Exp. Med.192, 421–432) is a low affinity receptor expressed more ubiquitously. Here we report the binding profiles of various BLT antagonists and eicosanoids to either BLT1 or BLT2 using the membrane fractions of Chinese hamster ovary cells stably expressing the receptor. BLT antagonists are grouped into three classes: BLT1-specific U-75302, BLT2-specific LY255283, and BLT1/BLT2 dual-specific ZK 158252 and CP 195543. We also show that 12(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroperxyeicosatetraenoic acid, and 15(S)-hydroxyeicosatetraenoic acid competed with [3H]LTB4binding to BLT2, but not BLT1, dose dependently. These eicosanoids also cause calcium mobilization and chemotaxis through BLT2, again in contrast to BLT1. These findings suggest that BLT2 functions as a low affinity receptor, with broader ligand specificity for various eicosanoids, and mediates distinct biological and pathophysiological roles from BLT1.

scholarly journals New Methods to Evaluate Colocalization of Fluorophores in Immunocytochemical Preparations as Exemplified by a Study on A2A and D2 Receptors in Chinese Hamster Ovary Cells

2005 ◽  
Vol 53 (8) ◽  
pp. 941-953 ◽  
Author(s):  
Luigi F. Agnati ◽  
Kjell Fuxe ◽  
Maria Torvinen ◽  
Susanna Genedani ◽  
Rafael Franco ◽  
...  
Keyword(s):  
Image Analysis ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Distribution Patterns ◽  
Receptor Interaction ◽  
Confocal Laser ◽  
High Concentration ◽  
Ovary Cells ◽  
G Protein Coupled

An important aspect of the image analysis of immunocytochemical preparations is the evaluation of colocalization of different molecules. The aim of the present study is to introduce image analysis methods to identify double-labeled locations exhibiting the highest association of two fluorophores and to characterize their pattern of distribution. These methods will be applied to the analysis of the cotrafficking of adenosine A2A and dopamine D2 receptors belonging to the G protein–coupled receptor family and visualized by means of fluorescence immunocytochemistry in Chinese hamster ovary cells after agonist treatment. The present procedures for colocalization have the great advantage that they are, to a large extent, insensitive to the need for a balanced staining with the two fluorophores. Thus, these procedures involve image processing, visualization, and analysis of colocalized events, using a covariance method and a multiply method and the evaluation of the identified colocalization patterns. Moreover, the covariance method offers the possibility of detecting and quantitatively characterizing anticorrelated patterns of intensities, whereas the immediate detection of colocalized clusters with a high concentration of labeling is a possibility offered by the multiply method. The present methods offer a new and sensitive approach to detecting and quantitatively characterizing strongly associated fluorescence events, such as those generated by receptor–receptor interaction, and their distribution patterns in dual-color confocal laser microscopy.

scholarly journals Development of a Ca2+-Activated Photoprotein, Photina®, and Its Application to High-Throughput Screening

2007 ◽  
Vol 12 (5) ◽  
pp. 694-704 ◽  
Author(s):  
Silvia Bovolenta ◽  
Maria Foti ◽  
Stefan Lohmer ◽  
Sabrina Corazza
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Signal To Noise Ratio ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Number ◽  
Ovary Cells ◽  
Mammalian Cell Lines ◽  
Calcium Dyes ◽  
G Protein Coupled

The present work describes the engineering and characterization of a new Ca2+-activated photoprotein (Photina®) and its use in mammalian cell lines for implementation of flash luminescence cell-based assays for high-throughput screening (HTS). When used to measure the activation of 2 G protein—coupled receptors (GPCRs), targeting Photina® to the mitochondria increased the signal strength as compared to the normal cytoplasmic expression of Photina.® The mitochondrial-targeted Photina® also produced a higher signal-to-noise ratio than conventional calcium dyes and a consistently stronger signal than aequorin when tested under equivalent conditions. MitoPhotina® provided strong and reliable results when used to measure the activity of purinergic receptors endogenously expressed in the Chinese Hamster Ovary cells and heterologously expressed GPCRs in response to their cognate ligands. Several different types of flash luminescence plate readers (FLIPR3, FLIPRTETRA, CyBi®-®Lumax flash HT, Lumilux®, Lumibox) in different plate formats (96, 384, 1536 wells) were used to validate the use of Photina in HTS. The cell number had to be adjusted to correspond to the qualities of the different readers, but once so adjusted, it provided equivalent results on each device. The results obtained show robust and reproducible light signals that offer new possibilities for application of photoproteins to the generation of cell-based assays for HTS. ( Journal of Biomolecular Screening 2007:694-704)

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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