scholarly journals A Sperm-Associated WD Repeat Protein Orthologous to Chlamydomonas PF20 Associates with Spag6, the Mammalian Orthologue of Chlamydomonas PF16

2002 ◽  
Vol 22 (22) ◽  
pp. 7993-8004 ◽  
Author(s):  
Zhibing Zhang ◽  
Rossana Sapiro ◽  
David Kapfhamer ◽  
Maja Bucan ◽  
Jeff Bray ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chromosome 1 ◽  
Chromosome 2 ◽  
Flagellar Motility ◽  
Protein Fusion ◽  
Ovary Cells ◽  
Central Apparatus ◽  
Green Fluorescent

ABSTRACT cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.

scholarly journals New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein

2012 ◽  
Vol 12 (1) ◽  
pp. 24 ◽  
Author(s):  
Yeon-Gu Kim ◽  
Byoungwoo Park ◽  
Jung Ahn ◽  
Joon-Ki Jung ◽  
Hong Lee ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Line ◽  
Chinese Hamster Ovary ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Line Development ◽  
Ovary Cells ◽  
New Cell Line ◽  
Green Fluorescent

scholarly journals Expression of TRPC3 in Chinese Hamster Ovary Cells Results in Calcium-activated Cation Currents Not Related to Store Depletion

1997 ◽  
Vol 138 (6) ◽  
pp. 1333-1341 ◽  
Author(s):  
Christof Zitt ◽  
Alexander G. Obukhov ◽  
Carsten Strübing ◽  
Andrea Zobel ◽  
Frank Kalkbrenner ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Membrane Receptors ◽  
Calcium Stores ◽  
Channel Activity ◽  
Ovary Cells ◽  
Cytosolic Side ◽  
Green Fluorescent ◽  
Inside Out

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.

A glycosylated antitumor ether lipid kills cells via paraptosis-like cell death

2009 ◽  
Vol 87 (2) ◽  
pp. 401-414 ◽  
Author(s):  
Pranati Samadder ◽  
Robert Bittman ◽  
Hoe-Sup Byun ◽  
Gilbert Arthur
Keyword(s):  
Cell Death ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ether Lipid ◽  
Protein Fusion ◽  
Protein Marker ◽  
Anticancer Properties ◽  
Lc3 Puncta ◽  
Green Fluorescent

Glycosylated antitumor ether lipids (GAELs) have superior anticancer properties relative to the alkyllysophospholipid class, but there have been no studies of the mechanisms of these compounds. The prototype GAEL, 1-O-hexadecyl-2-O-methyl-3-O-(2′-amino-2′-deoxy-β-d-glucopyranosyl)-sn-glycerol (Gln), effectively killed mouse embryonic fibroblasts (MEFs) lacking key molecules involved in caspase-dependent apoptosis, and cell death was not prevented by caspase inhibitors. Gln did not cause a loss of mitochondrial membrane potential, even in rounded-up dying cells. Gln stimulated the appearance and accumulation of LC3-II, a protein marker for autophagy, in a variety of cells, including wild-type MEFs, but not in MEFs lacking ATG5, a key protein required for autophagy. Gln induced LC3 puncta formation in Chinese hamster ovary cells stably expressing a LC3–green fluorescent protein fusion protein. Thus, Gln appears to induce autophagy. Autophagy was mTOR-independent and was not inhibited by 3-methyladenine or wortmannin. Although Gln is toxic, cellular ability to undergo autophagy was not essential for its toxicity. Furthermore, the GAEL analog 2-deoxy-C-Glc induced LC3 puncta formation but did not kill the cells. Gln, but not 2-deoxy-C-Glc, caused the accumulation of cytoplasmic acidic vacuoles in the cells. Our data suggest that GAELs may activate autophagy; however, GAELs do not kill cells by apoptosis or autophagy but rather by a paraptosis-like cell death mechanism.

Hands-on experiments on glycemia regulation and type 1 diabetes

2015 ◽  
Vol 39 (3) ◽  
pp. 232-239 ◽  
Author(s):  
M. Mingueneau ◽  
A. Chaix ◽  
N. Scotti ◽  
J. Chaix ◽  
A. Reynders ◽  
...  
Keyword(s):  
Type 1 Diabetes ◽  
Green Fluorescent Protein ◽  
Chinese Hamster Ovary ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Pancreatic Cells ◽  
Green Fluorescent

In the present article, we describe a 3-day experimental workshop on glycemia regulation and type 1 diabetes that engages students in open-ended investigations and guided experiments leading to results that are not already known to them. After an initial questioning phase during which students observe PowerPoint slides depicting the glycemia (blood glucose levels) of individuals in various situations, students design, execute, and interpret experiments to address one of the following questions: 1) Which criteria must an animal model of diabetes fulfill? 2) How do pancreatic cells maintain glycemia constant? and 3) Is there a way to produce an insulin protein similar to the one released by human pancreatic cells? Students then 1) measure glycemia and glycosuria in control mice and in a mouse model of type 1 diabetes (Alloxan-treated mice), 2) measure the release of insulin by pancreatic β-cells (INS-1 cell line) in response to different concentrations of glucose in the extracellular medium, and 3) transfect Chinese hamster ovary cells with a plasmid coding for green fluorescent protein, observe green fluorescent protein fluorescence of some of the transfected Chinese hamster ovary cells under the microscope, and observe the characteristics of human insulin protein and its three-dimensional conformation using RASMOL software. At the end of the experimental session, students make posters and present their work to researchers. Back at school, they may also present their work to their colleagues.

scholarly journals α4β1 Integrin Regulates Lamellipodia Protrusion via a Focal Complex/Focal Adhesion-independent Mechanism

2002 ◽  
Vol 13 (9) ◽  
pp. 3203-3217 ◽  
Author(s):  
Karen A. Pinco ◽  
Wei He ◽  
Joy T. Yang
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Time Lapse ◽  
Random Motility ◽  
Protein Fusion ◽  
Green Fluorescent

α4β1 integrin plays an important role in cell migration. We show that when ectopically expressed in Chinese hamster ovary cells, α4β1 is sufficient and required for promoting protrusion of broad lamellipodia in response to scratch-wounding, whereas α5β1 does not have this effect. By time-lapse microscopy of cells expressing an α4/green fluorescent protein fusion protein, we show that α4β1 forms transient puncta at the leading edge of cells that begin to protrude lamellipodia in response to scratch-wounding. The cells expressing a mutant α4/green fluorescent protein that binds paxillin at a reduced level had a faster response to scratch-wounding, forming α4-positive puncta and protruding lamellipodia much earlier. While enhancing lamellipodia protrusion, this mutation reduces random motility of the cells in Transwell assays, indicating that lamellipodia protrusion and random motility are distinct types of motile activities that are differentially regulated by interactions between α4β1 and paxillin. Finally, we show that, at the leading edge, α4-positive puncta and paxillin-positive focal complexes/adhesions do not colocalize, but α4β1 and paxillin colocalize partially in ruffles. These findings provide evidence for a specific role of α4β1 in lamellipodia protrusion that is distinct from the motility-promoting functions of α5β1 and other integrins that mediate cell adhesion and signaling events through focal complexes and focal adhesions.

scholarly journals DNA amplification in multidrug, cross-resistant Chinese hamster ovary cells: molecular characterization and cytogenetic localization of the amplified DNA.

1986 ◽  
Vol 103 (4) ◽  
pp. 1159-1166 ◽  
Author(s):  
L D Teeter ◽  
S Atsumi ◽  
S Sen ◽  
T Kuo
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dna Amplification ◽  
Chromosome 1 ◽  
Cosmid Library ◽  
Cross Resistance ◽  
Northern Hybridization ◽  
Animal Cells ◽  
Ovary Cells

Vincristine-resistant (VCR) Chinese hamster ovary (CHO) cells have been established by stepwise selection in increasing concentrations of vincristine. These cells exhibit multidrug cross-resistance to a number of drugs that have no structural or functional similarities. Cytogenetic analyses of resistant cells revealed the presence of double minutes and expanded chromosomal segments, thus implicating gene amplification as a possible mechanism of resistance. An amplified DNA segment isolated from other multidrug cross-resistant CHO cell lines (Roninson, I. B., H. T. Abelson, D. E. Housman, N. Howell, and A. Varshavsky, 1984, Nature (Lond.), 309:626-628) is also amplified in our VCR lines. This DNA segment was used as a probe to screen a cosmid library of VCR genomic DNA, and overlapping clones were retrieved. All of these segments, totaling approximately 45 kilobases (kb), were amplified in VCR cells. Using in situ hybridization, we localized the amplification domain to the long arm of CHO chromosome 1 or Z1. Northern hybridization analysis revealed that a 4.3-kb mRNA was encoded by this amplified DNA domain and was over-produced in the VCR cells. Suggestions for the involvement of these amplified DNA segments in the acquisition of multidrug cross-resistance in animal cells are also presented.

scholarly journals Linkage of the MBG locus to another functionally hemizygous gene locus (IDH2) on chromosome Z3 in Chinese hamster ovary cells.

1985 ◽  
Vol 5 (1) ◽  
pp. 109-113 ◽  
Author(s):  
G M Adair ◽  
M J Siciliano
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Gene Locus ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chromosome 8 ◽  
Chromosome 2 ◽  
Ovary Cells ◽  
Resistance Markers ◽  
Linkage Relationships

To gain insight into the nature of hemizygosity in Chinese hamster ovary (CHO) cells and the mechanisms by which it has arisen, we are attempting to map and determine linkage relationships for as many hemizygous loci as possible. In this study, we have shown by segregation analysis of intraspecific somatic cell hybrids that the hemizygous gene locus associated with resistance to methylglyoxalbisguanyl hydrazone (MBG) in CHO cells is linked to the hemizygous IDH2 locus on chromosome Z3. Nine of the ten autosomal hemizygous gene loci that have been mapped to date in CHO cells are clustered on three chromosomes, with five such markers on chromosome 2, two on chromosome 8, and now two on the Z3 chromosome. With the mapping of MBG to the Z3 chromosome, selectable drug resistance markers are now available on eight different CHO chromosomes.

Production of Chimeric Recombinant Single Domain Antibody—Green Fluorescent Fusion Protein in Chinese Hamster Ovary Cells

Hybridoma ◽  
2007 ◽  
Vol 26 (1) ◽  
pp. 1-9 ◽  
Author(s):  
M. Rajabi Bazl ◽  
M.J. Rasaee ◽  
M. Foruzandeh ◽  
A. Rahimpour ◽  
J. Kiani ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Ovary Cells ◽  
Domain Antibody ◽  
Green Fluorescent

scholarly journals A single-wavelength flow cytometric approach using redox-sensitive green fluorescent protein probes for measuring redox stress in live cells

BioTechniques ◽  
2021 ◽  
Author(s):  
Elizabeth R Denn ◽  
Joseph M Schober
Keyword(s):  
Oxidative Stress ◽  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Recovery Phase ◽  
Live Cells ◽  
Redox Stress ◽  
Green Fluorescent

Cellular redox changes are common in apoptosis, immune function, signaling pathways and cancer. The authors aimed to develop a single-wavelength method using the superior fluorescence sensitivity of a flow cytometer for measuring redox-sensitive green fluorescent protein signal during oxidative stress in cell lines. The single-wavelength method was able to discern small differences in oxidative stress between cell lines and between the cytoplasmic and mitochondrial compartments within the same cell line. In Chinese hamster ovary cells, the mitochondrial matrix compartment was more sensitive to oxidative stress compared with MDA-MB-231 cells, and the rapid changes in redox state were followed by a slow recovery phase. The authors conclude that this simplified method is useful and preferred for studies where alterations in overall redox-sensitive green fluorescent protein expression are controlled.

Molecular characterization of volume-sensitive SKCachannels in human liver cell lines

2002 ◽  
Vol 282 (1) ◽  
pp. G116-G122 ◽  
Author(s):  
Richard Roman ◽  
Andrew P. Feranchak ◽  
Marlyn Troetsch ◽  
Jeffrey C. Dunkelberg ◽  
Gordon Kilic ◽  
...  
Keyword(s):  
Cell Volume ◽  
Human Liver ◽  
Fluorescent Protein ◽  
Recombinant Adenovirus ◽  
Chinese Hamster Ovary Cells ◽  
Cell Volume Regulation ◽  
Chinese Hamster ◽  
Protein Fusion ◽  
Fusion Construct ◽  
Human Liver Cell

In human liver, Ca2+-dependent changes in membrane K+permeability play a central role in coordinating functional interactions between membrane transport, metabolism, and cell volume. On the basis of the observation that K+conductance is partially sensitive to the bee venom toxin apamin, we aimed to assess whether small-conductance Ca2+-sensitive K+(SKCa) channels are expressed endogenously and contribute to volume-sensitive K+efflux and cell volume regulation. We isolated a full-length 2,140-bp cDNA (hSK2) highly homologous to rat brain rSK2 cDNA, including the putative apamin-sensitive pore domain, from a human liver cDNA library. Identical cDNAs were isolated from primary human hepatocytes, human HuH-7 hepatoma cells, and human Mz-ChA-1 cholangiocarcinoma cells. Transduction of Chinese hamster ovary cells with a recombinant adenovirus encoding the hSK2-green fluorescent protein fusion construct resulted in expression of functional apamin-sensitive K+channels. In Mz-ChA-1 cells, hypotonic (15% less sodium glutamate) exposure increased K+current density (1.9 ± 0.2 to 37.5 ± 7.1 pA/pF; P < 0.001). Apamin (10–100 nM) inhibited K+current activation and cell volume recovery from swelling. Apamin-sensitive SKCachannels are functionally expressed in liver and biliary epithelia and likely contribute to volume-sensitive changes in membrane K+permeability. Accordingly, the hSK2 protein is a potential target for pharmacological modulation of liver transport and metabolism through effects on membrane K+permeability.

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