Small linear chloride channels are endogenous to nonepithelial cells

1992 ◽  
Vol 263 (3) ◽  
pp. C708-C713 ◽  
Author(s):  
S. E. Gabriel ◽  
E. M. Price ◽  
R. C. Boucher ◽  
M. J. Stutts
Keyword(s):  
Cell Lines ◽  
Chloride Channels ◽  
Single Channel ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Ovary Cells ◽  
3T3 Fibroblasts ◽  
Whole Cell Patch Clamp ◽  
Cl Channels

We used both single-channel and whole cell patch-clamp techniques to characterize chloride channels and currents endogenous to Sf9 cells, 3T3 fibroblasts, and Chinese hamster ovary cells. In cell-attached patches from these cell types, anion channels were observed with low ohmic conductance (4-11 ps), linear current-voltage relationships, and little time- or voltage-dependent behavior. These channels are very similar to the Cl- channels reported to appear concomitant with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in these cell lines. The presence of such endogenous channels suggests either that low levels of CFTR are present in all of these cell lines prior to transfection or that an endogenous non-CFTR channel is present in these cell types. Our results suggest that at least some of the channel behaviors attributed to expressed, recombinant CFTR in previous studies may have been due to these endogenous Cl- channels.

DNA methylation patterns associated with asparagine synthetase expression in asparagine-overproducing and -auxotrophic cells

1989 ◽  
Vol 9 (7) ◽  
pp. 2922-2927
Author(s):  
I L Andrulis ◽  
M T Barrett
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary Cells ◽  
Methylation Status ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Methylation Pattern ◽  
Asparagine Synthetase ◽  
Synthetase Activity ◽  
Dna Hypomethylation ◽  
Ovary Cells

In Chinese hamster ovary cells, the gene for asparagine synthetase, which spans 20 kilobase pairs, was found to contain a cluster of potential sites for CpG methylation in a 1-kilobase-pair region surrounding the first exon. Fourteen of the sites that could be assayed for methylation by MspI-HpaII digestions were found in this region, with an additional nine MspI sites spread throughout the remainder of the gene. The methylation status of the gene was analyzed in a series of cell lines that differed in the amount of asparagine synthetase activity. The level of expression showed a direct correlation with the extent of methylation of a subset of the MspI sites found in the 5' region of the gene. The rest of the gene was completely methylated in most cell lines. Wild-type cells, which expressed a basal level of asparagine synthetase activity, were partially demethylated in the 5' region. In contrast, asparagine-requiring N3 cells, which lacked detectable mRNA for asparagine synthetase, were methylated throughout the entire gene. Spontaneous revertants of strain N3, selected for growth in asparagine-free medium, exhibited extensive hypomethylation of the asparagine synthetase gene. The methylation pattern of the gene in cell lines that overproduced the enzyme was also examined. Albizziin-resistant cell lines, which had amplified copies of the gene, were extensively demethylated in the 5' region. Overexpression of asparagine synthetase in beta-aspartyl hydroxamate-resistant lines without amplified copies of the gene was also correlated with DNA hypomethylation.

Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles

1983 ◽  
Vol 3 (7) ◽  
pp. 1172-1181
Author(s):  
W E Bradley
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
High Frequency ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Low Frequency ◽  
Class Ii ◽  
Class I ◽  
Wild Type ◽  
Ovary Cells

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.

Cell-specific posttranslational processing of the surfactant-associated protein SP-B

1993 ◽  
Vol 264 (3) ◽  
pp. L290-L299 ◽  
Author(s):  
S. Hawgood ◽  
D. Latham ◽  
J. Borchelt ◽  
D. Damm ◽  
T. White ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Types ◽  
Specific Protein ◽  
Precursor Protein ◽  
Type Ii Cells ◽  
Posttranslational Processing ◽  
Type Ii ◽  
Clara Cells ◽  
Ovary Cells

Pulmonary surfactant-associated protein B (SP-B) is a 9-kDa lung-specific protein expressed in alveolar epithelial type II cells and Clara cells. The protein markedly increases the surface activity of phospholipids and is an active component in some surfactants in clinical use. SP-B is produced from a 43-kDa precursor protein by proteolytic cleavage of flanking regions from both the NH2- and COOH-terminal ends of the active protein. In this study we have compared the nature of the posttranslational processing of the SP-B precursor in type II cells and in a heterologous cell line transfected with the SP-B precursor. We found that isolated type II cells produce the 9-kDa form of SP-B from the precursor through a series of intermediates detectable in the cell lysates. In contrast Chinese hamster ovary cells stably transfected with the full-length human SP-B precursor produce the precursor and a 26-kDa intermediate but not the 9-kDa protein. The precursor protein in both cell types is glycosylated with NH2-linked sugars. Our results suggest there is cell specificity in the posttranslational processing of the SP-B precursor.

Impaired cell volume regulation in Na(+)-H+ exchange-deficient mutants

1989 ◽  
Vol 257 (6) ◽  
pp. C1158-C1165 ◽  
Author(s):  
D. Rotin ◽  
S. Grinstein
Keyword(s):  
Chinese Hamster Ovary ◽  
Volume Regulation ◽  
Chinese Hamster Ovary Cells ◽  
Cell Volume Regulation ◽  
Chinese Hamster ◽  
Cell Types ◽  
Influx Rate ◽  
Volume Increase ◽  
Ovary Cells ◽  
Na Uptake

To elucidate the mechanism of regulatory volume increase (RVI) in Chinese hamster ovary cells, Na(+)-H+ exchange-deficient mutants, called AP-1, were derived from WT-5 cells, a wildtype subclone. The absence of functional antiports in AP-1 cells was established through measurements of intracellular pH (pHi) and Na+ uptake. Cells exposed to hypotonic medium initially swelled but regained near-normal volume within minutes. When isotonicity was then restored, WT-5 cells shrank immediately and then carried out RVI, which was inhibited by 0.1 mM amiloride. This amiloride-sensitive RVI was absent in the AP-1 mutants, suggesting involvement of Na(+)-H+ exchange. In some cell types, RVI is mediated by Na(+)-K(+)-2Cl- cotransport. Bumetanide-sensitive 86Rb+ (K+) influx was detectable in both WT-5 and AP-1 cells, suggesting the presence of Na(+)-K(+)-2Cl- cotransport. Bumetanide-sensitive influx was stimulated by osmotic shrinking in WT-5 cells, and only slightly in AP-1 cells. However, Na(+)-K(+)-2Cl- cotransport did not contribute to volume regulation, since bumetanide (50 microM) failed to inhibit RVI in osmotically shrunken WT-5 cells. The inability of cotransport to induce a volume gain in WT-5 cells was attributable to the simultaneous stimulation of Na(+)-K(+)-2Cl- efflux. The rate of efflux was similar in magnitude to the corresponding influx rate so that net Na(+)-K(+)-2Cl- cotransport was negligible. These results show that RVI in osmotically shrunken Chinese hamster ovary cells is mediated by the Na(+)-H+ antiport and that, although stimulated, Na(+)-K(+)-2Cl- cotransport does not contribute to anisosmotic volume regulation.

scholarly journals Comparison of the mRNA sequences for Pi class glutathione transferases in different hamster species and the corresponding enzyme activities with anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide

1996 ◽  
Vol 318 (2) ◽  
pp. 533-538 ◽  
Author(s):  
Stellan SWEDMARK ◽  
Bengt JERNSTRÖM ◽  
Dag JENSSEN
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Sequences ◽  
Cho Cell ◽  
Glutathione S Transferase ◽  
V79 Cells ◽  
Ovary Cells ◽  
Cdna Sequences ◽  
Chinese Hamster V79 Cells

Glutathione S-transferase (GST) of class Pi (GST Pi) is known to detoxify the mutagenic and carcinogenic (+)-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide [(+)-anti-BPDE] by conjugation with glutathione. Previously, we have shown that Chinese hamster V79 cells contain GST Pi, but seem to lack the capacity to conjugate (+)-anti-BPDE, although these cells do conjugate other substrates with GSH [Romert, Dock, Jenssen and Jernström (1989) Carcinogenesis 10, 1701–1707; Swedmark, Romert, Morgenstern and Jenssen (1992) Carcinogenesis 13, 1719–1723; Swedmark and Jenssen (1994) Gene 139, 251–256]. In the present study we have compared four cell lines derived from different hamster species with respect to GST cDNA sequences and capacity to conjugate (+)- or (-)-anti-BPDE. The cell lines were V79 and Chinese hamster ovary cells (CHO), Armenian hamster lung (AHL) cells and baby hamster kidney (BHK) cells. The sequencing revealed a complete homology between the V79 and CHO cDNA for GST Pi, whereas the corresponding amino acid sequences predicted from the corresponding AHL and BHK cDNAs differed by six and nine amino acids, respectively, from the predicted V79 sequence. None of these changes alone was found to influence the xenobiotic substrate-binding site. The cytosolic fractions from BHK and AHL cells were found to catalyse conjugation of (+)-anti-BPDE with GSH, whereas the corresponding activity in CHO cells was non-detectable. As shown previously, V79 cells were devoid of activity towards (+)-anti-BPDE. All the cell lines studied demonstrated appreciable GST activity towards 1-chloro-2,4-dinitrobenzene, but no activity with (-)-anti-BPDE. The latter result suggests that GST Pi is the sole or predominant GST in these cell lines. This was confirmed by HPLC analysis of purified enzymes obtained by affinity chromatography. However, when the catalytic activities of the pure enzymes were determined, all four different GST Pi enzymes were found to be highly capable of conjugating (+)-anti-BPDE with GSH. This observation indicates the existence of an intracellular factor that selectively inhibits conjugation of (+)-anti-BPDE, but not of 1-chloro-2,4-dinitrobenzene in the V79 and CHO cell lines. This new phenomenon seems to be specific for Chinese hamster, since both these cell lines originate from this species.

scholarly journals Evidence for intracellular partitioning of serine and glycine metabolism in Chinese hamster ovary cells

1996 ◽  
Vol 313 (3) ◽  
pp. 991-996 ◽  
Author(s):  
Michael R. NARKEWICZ ◽  
S. David SAULS ◽  
Susan S. TJOA ◽  
Cecilia TENG ◽  
Paul V. FENNESSEY
Keyword(s):  
Cell Lines ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Mitochondrial Protein ◽  
Chinese Hamster ◽  
Serine Hydroxymethyltransferase ◽  
Cho Cell ◽  
Ovary Cells ◽  
Glycine Metabolism

Serine hydroxymethyltransferase (SHMT) is the primary enzyme in the interconversion of serine and glycine. The roles of mitochondrial and cytosolic SHMT in the interconversion of serine and glycine were determined in two Chinese hamster ovary (CHO) cell lines that both contain cytosolic SHMT but either have (CHOm+) or lack (CHOm-) mitochondrial SHMT. Mitochondrial SHMT activity was significantly reduced in CHOm- (0.24±0.11 nmol/min per mg of mitochondrial protein) compared with CHOm+ (3.21±0.66 nmol/min per mg of mitochondrial protein; P = 0.02) cells, whereas cytosolic SHMT activity was similar in CHOm- and CHOm+ cells (1.09±0.31 and 1.53±0.12 nmol/min per mg of cytosolic protein respectively; P = 0.57). In CHOm+ and CHOm- cells, the relative flux of glycine to serine measured with either [1-13C]- or [2-13C]-glycine was similar (CHOm-: 538±82 nmol/24 per mg of DNA; CHOm+: 616±88 nmol/24 h per mg of DNA; P = 0.42). In contrast, the relative flux of serine to glycine measured with [1-13C]serine was low in CHOm- cells (80±28 nmol/24 h per mg of DNA) compared with CHOm+ cells (3080±320 nmol/24 h per mg of DNA; P = 0.0001). The rate of glycine production determined by UA-2[1-13C]glycine dilution was lower in CHOm- (1200±200 nmol/24 h per mg of DNA) than CHOm+ (10200±1800 nmol/24 h per mg of DNA; P = 0.03) cells, whereas glycine utilization was similar in the two cell lines. Serine production was similar in the two cell lines but serine utilization was lower in CHOm- (3800±1200 μmol/24 h per mg of DNA) than CHOm+ (6600±1000 nmol/24 h per mg of DNA; P = 0.0002) cells. Increasing the serine concentration in the medium resulted in an increase in glycine production in CHOm+ but not in CHOm- cells. Intracellular studies with [1-13C]serine confirm the findings of decreased glycine production from serine. In CHO cells there is partitioning of intracellular serine and glycine metabolism. Our data support the hypothesis that mitochondrial SHMT is the primary pathway for serine into glycine interconversion.

scholarly journals Zephgrabetaine: A New Betaine-type Amaryllidaceae Alkaloid from Zephyranthes grandiflora

2013 ◽  
Vol 8 (2) ◽  
pp. 1934578X1300800 ◽  
Author(s):  
Deepali Katoch ◽  
Dharmesh Kumar ◽  
Upendra Sharma ◽  
Neeraj Kumar ◽  
Yogendra S. Padwad ◽  
...  
Keyword(s):  
Cell Lines ◽  
Spectroscopic Data ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glioma Cells ◽  
Cytotoxic Activities ◽  
Ovary Cells ◽  
Rat Glioma ◽  
Dose Dependent

Zephgrabetaine (1), a new betaine type Amaryllidaceae alkaloid, along with seven known alkaloids, lycorine, galanthine, lycoramine, hamayne, haemanthamine, tortuosine, and ungeremine were isolated from the bulbs of Zephyranthes grandiflora and their structures elucidated by spectroscopic data analysis. The isolated alkaloids were tested for in vitro cytotoxic activities against two cell lines, C-6 (rat glioma cells) and CHO-K1 (Chinese hamster ovary cells). A dose dependent cytotoxic effect was exhibited by all the alkaloids on these two cancer cell lines with prominent activity of lycorine and haemanthamine.

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