scholarly journals Adenine transport and binding in cultured mammalian cells deficient in adenine phosphoribosyltransferase.

1983 ◽  
Vol 3 (1) ◽  
pp. 82-90 ◽  
Author(s):  
M B Puziss ◽  
R M Wohlhueter ◽  
P G Plagemann
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster ◽  
High Capacity ◽  
Rate Equations ◽  
Cell Water ◽  
Adenine Phosphoribosyltransferase ◽  
Novikoff Hepatoma ◽  
L929 Cells ◽  
Equilibrium Exchange ◽  
Time Courses

Rapid kinetic techniques were employed to measure the transport of adenine in adenine phosphoribosyltransferase-deficient L929 and Chinese hamster ovary (CHO) cells in zero-trans entry and exit and equilibrium exchange procedures. The kinetic parameters of transport were computed by fitting appropriate integrated rate equations to time courses of transmembrane equilibration of radiolabeled adenine. Adenine transport conformed to the simple carrier model with directional symmetry and equal mobility of loaded and empty carrier. The Michaelis-Menten constants and maximum velocities for various strains of L929 cells fell between 2.3 and 3.5 mM and 90 and 150 pmol/microliters of cell water per s, respectively, values similar to those previously reported for CHO and Novikoff hepatoma cells. The corresponding values for hypoxanthine transport in L929 cells were 413 microM and 16 pmol/microliters of cell water per s. Adenine transport velocities were directly proportional to adenine concentrations between 0.03 and 50 microM in both CHO and Novikoff cells. The results indicate that adenine is transported in these cells by a single, low-affinity, high-capacity transporter. Adenine transport was inhibited by hypoxanthine in some cell strains, but not in others. Adenine also rapidly bound to L929 cells in a saturable manner (KD = 18 microM), presumably to the cell surface (about 3 X 10(7) sites per cell).

Adenine transport and binding in cultured mammalian cells deficient in adenine phosphoribosyltransferase

1983 ◽  
Vol 3 (1) ◽  
pp. 82-90
Author(s):  
M B Puziss ◽  
R M Wohlhueter ◽  
P G Plagemann
Keyword(s):  
Mammalian Cells ◽  
Chinese Hamster ◽  
High Capacity ◽  
Rate Equations ◽  
Cell Water ◽  
Adenine Phosphoribosyltransferase ◽  
Novikoff Hepatoma ◽  
L929 Cells ◽  
Equilibrium Exchange ◽  
Time Courses

Rapid kinetic techniques were employed to measure the transport of adenine in adenine phosphoribosyltransferase-deficient L929 and Chinese hamster ovary (CHO) cells in zero-trans entry and exit and equilibrium exchange procedures. The kinetic parameters of transport were computed by fitting appropriate integrated rate equations to time courses of transmembrane equilibration of radiolabeled adenine. Adenine transport conformed to the simple carrier model with directional symmetry and equal mobility of loaded and empty carrier. The Michaelis-Menten constants and maximum velocities for various strains of L929 cells fell between 2.3 and 3.5 mM and 90 and 150 pmol/microliters of cell water per s, respectively, values similar to those previously reported for CHO and Novikoff hepatoma cells. The corresponding values for hypoxanthine transport in L929 cells were 413 microM and 16 pmol/microliters of cell water per s. Adenine transport velocities were directly proportional to adenine concentrations between 0.03 and 50 microM in both CHO and Novikoff cells. The results indicate that adenine is transported in these cells by a single, low-affinity, high-capacity transporter. Adenine transport was inhibited by hypoxanthine in some cell strains, but not in others. Adenine also rapidly bound to L929 cells in a saturable manner (KD = 18 microM), presumably to the cell surface (about 3 X 10(7) sites per cell).

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Thermotropic lipid and protein transitions in Chinese hamster lung cell membranes: relationship to hyperthermic cell killing

1983 ◽  
Vol 61 (6) ◽  
pp. 421-427 ◽  
Author(s):  
James R. Lepock ◽  
Kwan-Hon Cheng ◽  
Hisham Al-Qysi ◽  
Jack Kruuv
Keyword(s):  
Mammalian Cells ◽  
Plasma Membranes ◽  
Chinese Hamster ◽  
Cell Killing ◽  
Water Soluble ◽  
Growth Data ◽  
Protein Fluorescence ◽  
Polar Groups ◽  
Spin Resonance ◽  
Intrinsic Protein Fluorescence

Exposure of mammalian cells to hyperthermic temperatures (ca. 41–45 °C) appears to act as a direct or triggering effect to produce some later response such as cell death, thermotolerance, or heat-shock protein synthesis. The high activation energy of cell killing indicates that the direct effect of hyperthermia might be a thermotropic transition in some cellular component, for this particular response. Both hyperthermic survival and growth data imply that the temperature for the onset of hyperthermic cell killing is 40–41.5 °C for Chinese hamster lung V79 cells. Studies using the electron spin resonance label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide and the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene show the existence of lipid transitions at approximately 7–8 and 23–36 °C (or a broad transition between these temperatures) in mitochondria and whole cell homogenates, that correlate well with changes in growth and hypothermic killing. No lipid transition was detected near 40–41.5 °C that could correlate with hyperthermic killing in either mitochondrial or plasma membranes, but measurements of intrinsic protein fluorescence and protein fluorophore to trans-paranaric acid energy transfer demonstrate the existence of an irreversible transition in protein structure or arrangement above ca. 40 °C in both mitochondrial and plasma membranes. This transition is due to protein rearrangement and (or) unfolding such that there is increased exposure of protein tryptophan and tyrosine residues to polar groups and to paranaric acid. The strength of the transition implies that a significant fraction of total membrane protein is involved in this transition, which may be analogous to the heat-induced denaturation of water-soluble proteins. This alteration in membrane structure above ca. 40 °C could cause many of the observed changes in plasma membrane and mitochondrial function, which may further be involved in cellular responses to hyperthermia.

scholarly journals An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

1994 ◽  
Vol 14 (1) ◽  
pp. 68-76 ◽  
Author(s):  
K W Caldecott ◽  
C K McKeown ◽  
J D Tucker ◽  
S Ljungquist ◽  
L H Thompson
Keyword(s):  
Dna Repair ◽  
Affinity Chromatography ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Affinity Purification ◽  
Alkylating Agents ◽  
Chinese Hamster ◽  
Dna Ligase ◽  
Base Excision ◽  
Dna Ligase Iii

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

scholarly journals Neurotrophin Receptor TrkC Is an Entry Receptor for Trypanosoma cruzi in Neural, Glial, and Epithelial Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 4081-4087 ◽  
Author(s):  
Craig Weinkauf ◽  
Ryan Salvador ◽  
Mercio PereiraPerrin
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Neuronal Cell ◽  
Host Cells ◽  
Neurotrophin Receptor ◽  
Content Type

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated byT. cruzisurfacetrans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used byT. cruzito enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant toT. cruzibecame highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore,trkCtransfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive toT. cruziafter transfection with thetrkCgene. Additionally, NT-3 specifically blockedT. cruziinfection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blockedT. cruziinfection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected byT. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broadT. cruziinfection bothin vitroandin vivo.

scholarly journals Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.

1988 ◽  
Vol 8 (7) ◽  
pp. 2698-2704 ◽  
Author(s):  
I W Caras ◽  
D W Martin
Keyword(s):  
Ribonucleotide Reductase ◽  
Mammalian Cells ◽  
Mutant Mouse ◽  
Chinese Hamster Ovary Cells ◽  
Spontaneous Mutation ◽  
Single Point ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Single Point Mutation ◽  
Mutator Phenotype

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.

scholarly journals Cell cycle-dependent effects on deoxyribonucleotide and DNA labeling by nucleoside precursors in mammalian cells.

1987 ◽  
Vol 7 (1) ◽  
pp. 532-534 ◽  
Author(s):  
J M Leeds ◽  
C K Mathews
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Ovary Cells ◽  
Dna Labeling ◽  
Metabolic Pool ◽  
Specific Activities ◽  
Cycle Dependent

dCTP pools equilibrated to equivalent specific activities in Chinese hamster ovary cells or in nuclei after incubation of cells with radiolabeled nucleosides, indicating that dCTP in nuclei does not constitute a distinct metabolic pool. In the G1 phase, [5-3H]deoxycytidine labeled dCTP to unexpectedly high specific activities. This may explain reports of replication-excluded DNA precursor pools.

scholarly journals Repair of abasic sites by mammalian cell extracts

1994 ◽  
Vol 304 (3) ◽  
pp. 699-705 ◽  
Author(s):  
G Frosina ◽  
P Fortini ◽  
O Rossi ◽  
F Carrozzino ◽  
A Abbondandolo ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Excision Repair ◽  
Chinese Hamster ◽  
Pyrimidine Dimer ◽  
Repair Synthesis ◽  
Repair Patch ◽  
Cell Extracts ◽  
Pyrimidine Dimers ◽  
Ap Sites ◽  
Ap Site

Hamster cell extracts that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers, were used to study the repair of apurinic/apyrimidinic (AP) sites and methoxyamine (MX)-modified AP sites. Plasmid molecules were heat-treated at pH 5 and incubated with MX when required. The amount of damage introduced ranged from 0.2 to 0.9 AP sites/kb. Extracts were prepared from the Chinese hamster ovary CHO-9 cell line and from its derivative, 43-3B clone which is mutated in the nucleotide excision repair (NER) ERCC1 gene. AP and MX-AP sites stimulated repair synthesis by CHO-9 cell extracts. The level of synthesis correlated with the number of lesions and was of similar magnitude to the repair stimulated by 4.3 u.v. photoproducts/kb. Repair of AP and MX-AP sites was faster than the repair of u.v. damage and was independent of ERCC1 gene product. The high level of repair replication was due to a very efficient and rapid incision of plasmids carrying AP or MX-AP sites, performed by abundant AP endonucleases present in the extract. The calculated average repair patch sizes were: 7 nucleotides per AP site; 10 nucleotides per MX-AP site; 28 nucleotides per (6-4) u.v. photoproduct or cyclobutane pyrimidine dimer. The data indicate that AP and MX-AP sites are very efficiently repaired by base-excision repair in mammalian cells and suggest that MX-AP sites may also be processed via alternative repair mechanisms.

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