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insulin receptor kinase domain autophosphorylation regulates receptor enzymatic function.we have studied a series of insulin receptor molecules in which the 3 tyrosine residues which undergo autophosphorylation in the kinase domain of the beta-subunit (tyr1158, tyr1162, and tyr1163) were replaced individually, in pairs, or all together with phenylalanine or serine by in vitro mutagenesis. a single-phe replacement at each of these three positions reduced insulin-stimulated autophosphorylation of solubilized receptor by 45-60% of that observed with wild-type receptor. the double-phe r ...19921322912
effect of topoisomerase poisoning by antitumor drugs vm 26, fostriecin and camptothecin on dna repair replication by mammalian cell extracts.a recently developed in vitro excision-repair system was used to investigate the effect of the topoisomerase poisons vm 26, fostriecin and camptothecin on dna repair replication carried out by chinese hamster ovary cell extracts. vm 26 and camptothecin partially inhibit topoisomerases ii and i respectively, which are present in the repair-competent extracts, but have only slight effects on the repair efficiency. on the contrary, the antitumor drug fostriecin markedly affects repair replication b ...19921323426
effects of novel fluoroquinolones on the catalytic activities of eukaryotic topoisomerase ii: influence of the c-8 fluorine group.a previous study (m.j. robinson, b.a. martin, t.d. gootz, p.r. mcguirk, m. moynihan, j.a. sutcliffe, and n. osheroff, j. biol. chem. 266:14585-14592, 1991) demonstrated that novel 6,8-difluoroquinolones were potent effectors of eukaryotic topoisomerase ii. to determine the contribution of the c-8 fluorine to drug potency, we compared the effects of cp-115,955 [6-fluoro-7-(4-hydroxyphenyl)-1-cyclopropyl-4-quinolone-3-carboxylic acid] on the enzymatic activities of drosophila melanogaster topoisom ...19921323952
the gene for the dihydropyridine-sensitive calcium channel alpha 2 subunit (cchl2a) maps to the proximal region of mouse chromosome 5.a rat brain cdna probe for the gene encoding the alpha 2 subunit of the dihydropyridine-sensitive l-type calcium channel was used as a hybridization probe for the southern blot analysis of chinese hamster x mouse somatic cell hybrids and the progeny of an intersubspecies backcross. this gene, termed cchl2a, was mapped near the centromeric end of the chromosome 5 linkage group with gene order: centromere-pgy-1-cchl2a-il-6-pgm-1.19921324224
n-linked oligosaccharide chains of the insulin receptor beta subunit are essential for transmembrane signaling.insulin receptor (ir) is a glycoprotein possessing n-linked oligosaccharide side chains on both alpha and beta subunits. the present study focuses for the first time on the potential contribution of n-linked oligosaccharides of the beta subunit in the processing, structure, and function of the insulin receptor. to investigate this point, a receptor mutant (ir beta n1234) was obtained by stable transfection into chinese hamster ovary cells of an ir cdna modified by site-directed mutagenesis on th ...19921324936
is the mammalian cell plasma membrane a barrier to oxygen transport?oxygen transport in the chinese hamster ovary (cho) plasma membrane has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed in the lipid bilayer portion of the membrane at various distances from the membrane surface using the long-pulse saturation-recovery electron spin resonance (esr) technique. the collision rate was estimated for 5-, 12-, and 16-doxylstearic acids from spin-lattice relaxation times (t1) measured in the presence and absence of ...19921324973
comparison of recombinant tissue factor pathway inhibitors expressed in human sk hepatoma, mouse c127, baby hamster kidney, and chinese hamster ovary cells.recombinant tissue factor pathway inhibitor (rtfpi) has been expressed in four mammalian expression systems using human sk hepatoma, mouse c127, baby hamster kidney (bhk), and chinese hamster ovary (cho) cells as hosts. on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the immunoaffinity purified rtfpis all show broad bands and the mean molecular weight of sk hepatoma and c127 rtfpis (m(r) approximately 38,000) appear larger than those of bhk and cho rtfpis (m(r) approximately 35,000 ...19921325078
molecular cloning and expression of an adenosine a2b receptor from human brain.a novel receptor cdna was isolated from a human hippocampal cdna library. the encoded polypeptide contains structural features consistent with its classification as a g protein-coupled receptor and shares 45% homology with the human a1 and a2a adenosine receptors. chinese hamster ovary k1 cells expressing this receptor showed marked stimulation of adenylate cyclase when treated with 1mm adenosine. there was no response to ligands selective for a1 and a2a receptors but the general adenosine agoni ...19921325798
the insulin receptor-related receptor. tissue expression, ligand binding specificity, and signaling capabilities.in 1989, shier and watt identified a gene which was predicted to encode a new member of the insulin receptor (ir) family, and they called it the insulin receptor-related receptor (irr) (shier, p., and watt, v. m. (1989) j. biol. chem. 264, 14605-14608). however, the tissues expressing this receptor, its ligand binding specificity and its signaling capability have remained unknown. in the present studies we report northern blot analyses and polymerase chain reaction data, which indicate that the ...19921326521
interaction of the frog brain kainate receptor expressed in chinese hamster ovary cells with a gtp-binding protein.a protein that binds kainate with high affinity has been purified and cloned from frog brain (rana pipiens) and has approximately 35% sequence homology with mammalian non-n-methyl-d-aspartate glutamate receptors, some of which have been shown to be ligand-gated ion channels. frog brain membranes and membranes from chinese hamster ovary (cho) cells transfected with the cdna coding for the frog kainate-binding protein (cho-4 cells) bound kainate with essentially identical affinity (kd values of 1. ...19921326545
modulation of the cellular phenotype by integrated adeno-associated virus.the adeno-associated virus (aav) rep gene encodes a series of overlapping, multifunctional, nonstructural proteins (rep proteins) which regulate the viral life cycle and which are also capable of trans-regulating nonviral gene expressions (reviewed in berns, 1990, microbiol. rev. 54, 316-329). to investigate the expression of the aav rep gene in a cellular chromosomal context, sv40-transformed chinese hamster embryo (od4) cells were infected with an aav/neo hybrid virus and progeny resistant to ...19921326806
the rat neurotensin receptor expressed in chinese hamster ovary cells mediates the release of inositol phosphates.to study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. the cdna clone of this receptor was subcloned into the pcdna1neo expression vector. this construct was then used to transfect chinese hamster ovary (cho)-k1 cells. colony clones, selected for resistance to antibiotic g-418 sulfate, were isolated and grown separately. nineteen individual clones were screened for total [3h]neurotensin binding as an indication ...19921328536
cloning and pharmacological characterization of a novel human 5-hydroxytryptamine1d receptor subtype.the canine rdc4 gene was used to isolate two distinct human serotonin receptor genes. the receptor encoded by clone rh-6 was the species homolog of rdc4 and was identical to a human serotonin 5-hydroxytryptamine1d (5-ht1d) receptor that was recently reported [mol. pharmacol. 40:143-148 (1991)]. the receptor encoded by rh-2 was a novel 5-ht receptor that was 61% identical to rh-6 and showed the greatest homology with the rat 5-ht1b receptor sequence (94%). the rh-2 gene contained an intronless, 1 ...19921328844
expression cloning of a cdna encoding udp-glcnac:gal beta 1-3-galnac-r (glcnac to galnac) beta 1-6glcnac transferase by gene transfer into cho cells expressing polyoma large tumor antigen.a cdna encoding udp-glcnac:gal beta 1-3galnac-r (glcnac to galnac) beta 1-6glcnac transferase (ec 2.4.1.102), which forms critical branches in o-glycans, has been isolated by an expression cloning approach using chinese hamster ovary (cho) cells. increased activity of this enzyme and the concomitant occurrence of the o-glycan core 2 structure [gal beta 1-3(glcnac beta 1-6)galnac] has been observed in a variety of biological processes, such as t-cell activation and immunodeficiency due to the wis ...19921329093
human cytomegalovirus penetrates host cells by ph-independent fusion at the cell surface.biochemical, genetic, and morphological criteria were used to demonstrate that human cytomegalovirus penetrates permissive fibroblasts and nonpermissive chinese hamster ovary (cho) cells by ph-independent fusion between the virus envelope and the host cell plasma membrane and not by low ph-induced fusion within endosomes. viral immediate early (ie) gene expression and infectivity were unaffected by conditions which block various stages of endocytosis or agents that alter the acidic ph of the end ...19921329327
characterization of human tissue-type plasminogen activator variants with amino acid mutations in the kringle 1 domain.recombinant variants of tissue-type plasminogen activator (t-pa) were constructed by site-directed mutagenesis and expressed in chinese hamster ovary cells. five variants were designed to improve the function of t-pa by mutagenesis in the kringle 1 (k1) domain. the amino acids were replaced with the corresponding residues present in the kringle 2 (k2) domain of native t-pa. the t-pa mutants expressed were as follows: variant e94v.d95g with point mutations in glu94----val and asp95----gly; varian ...19921330023
substrate specificity of n-acetylglucosamine 1-phosphate transferase activity in chinese hamster ovary cells.the assembly pathway of the oligosaccharide chains of asparagine-linked glycoproteins in mammalian cells begins with the formation of glcnac-pp-dolichol in a reaction catalysed by the enzyme n-acetylglucosamine 1-phosphate transferase. we have investigated the efficiency of two lipid substrates for the transferase activity in an in vitro assay using chinese hamster ovary (cho) cell membranes as an enzyme source. experiments were carried out with varying concentrations of dolichyl phosphate or it ...19921330104
agonist analysis of 2-(carboxycyclopropyl)glycine isomers for cloned metabotropic glutamate receptor subtypes expressed in chinese hamster ovary cells.1. 2-(carboxycyclopropyl)glycines (ccgs) are conformationally restricted glutamate analogues and consist of eight isomers including l- and d-forms. the agonist potencies and selectivities of these compounds for metabotropic glutamate receptors (mglurs) were studied by examining their effects on the signal transduction of representative mglur1, mglur2 and mglur4 subtypes in chinese hamster ovary cells expressing the individual cloned receptors. 2. two extended isomers of l-ccg, l-ccg-i and l-ccg- ...19921330184
studies on glutathione transferases belonging to class pi in cell lines with different capacities for conjugating (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.the glutathione transferases (gst) belonging to class pi are primarily responsible for the intracellular detoxification of the highly mutagenic and carcinogenic compound (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (bpde). the aim of the present investigation was to study the nature and function of the gst pi gene in relation to the mutagenicity of bpde in different cell lines. the studies were performed on three cell lines commonly used in toxicological ...19921330340
investigation of the mutagenic specificity of x-rays using a retroviral shuttle vector in cho cells.in some biological systems ionizing radiation appears to induce large deletions and rearrangements, while in others point mutations predominate the mutational spectrum. moreover, while the point mutations are often randomly distributed, some systems exhibit "hot spots." retroviral shuttle vectors are particularly useful for investigating the basis of these differences since the genetic target can be conveniently analyzed in a variety of host backgrounds and genomic locations. we have studied the ...19921330546
certain tryptophan photoproducts are inhibitors of cytochrome p450-dependent mutagenicity.two photoproducts, derived from uv-irradiation of the amino acid l-tryptophan and with high ah (tcdd) receptor binding affinity, were tested for genotoxic and antimutagenic effects. the two indolo[3,2-b]carbazole derivatives, with the molecular weights of 284 and 312, respectively, were tested in saccharomyces cerevisiae strain d7 for mitotic gene conversion and reverse mutation and in strain rs112 for sister chromatid conversion and gene conversion. no significant (p > 0.05) genotoxic effects w ...19921330548
involvement of an asn/val cleavage site in the production of a soluble form of a human tumor necrosis factor (tnf) receptor. site-directed mutagenesis of a putative cleavage site in the p55 tnf receptor chain.soluble forms of receptors for tumor necrosis factor (tnf) might be important for regulating the actions of tnf. site-directed in vitro mutagenesis was employed to examine the processing of the p55 tumor necrosis factor receptor chain (tnf-r55) into soluble tnf-binding protein (tnf-r55-bp). an asn/val sequence close to the transmembrane region in tnf-r55 was indicated as a putative cleavage site for proteolytic processing. by mutagenesis, asn and val were replaced with gly and ala, respectively. ...19921330568
lack of intrinsic activity and significant subtype selectivity of sr 95639a at muscarinic receptors.we assessed the intrinsic activity of the purported selective muscarinic m1 receptor agonist sr 95639a (morpholinoethylamino-3-benzocyclohepta-(5,6-c)-pyridazine) in inducing several receptor-mediated signals. our results indicate that sr 95639a lacks the ability to activate phosphoinositide hydrolysis in rat cerebral cortex or in chinese hamster ovary cells transfected with the genes of the muscarinic m1 and m3 receptors. similarly, this compound did not exhibit intrinsic activity in stimulatin ...19921330650
stimulation of phosphatidylcholine breakdown by thrombin and carbachol but not by tyrosine kinase receptor ligands in cells transfected with m1 muscarinic receptors. rapid desensitization of phosphocholine-specific (pc) phospholipase d but sustained activity of pc-phospholipase c.in order to evaluate the possible contribution of phospholipase d (pld) stimulation to the mitogenic response, a screening of a variety of different compounds, some of which are known to be potent mitogens, was performed using the well characterized chinese hamster lung fibroblast (ccl39) cell line. in wild type ccl39 cells, or derivatives expressing high levels of either the human m1 muscarinic receptor (hm1) or the human epidermal growth factor (egf) receptor (39m1-81 and 39er22 clones, respec ...19921331066
activation of the na+/h+ antiporter during cell volume regulation. evidence for a phosphorylation-independent mechanism.a variety of cell types regulate their volume in anisotonic media by stimulating na+/h+ exchange. like growth factors, osmotic challenge activates the na+/h+ antiport by increasing its sensitivity to intracellular [h+]. to investigate the molecular mechanism underlying this shift in ph sensitivity, the antiporter of 32p-labeled human bladder carcinoma cells and of chinese hamster ovary cells was immunoprecipitated using antibodies raised against the cytosolic domain of the nhe-1 isoform of the n ...19921331102
binding and functional selectivity of himbacine for cloned and neuronal muscarinic receptors.the binding potencies for the putative m2-selective antagonist himbacine were determined in radioligand binding and in functional response assays in neuronal tissue and chinese hamster ovary cells containing transfected muscarinic receptors. himbacine was shown to bind to all five cloned muscarinic receptor subtypes in the order of potencies: hm2 = hm4 > hm3 > hm1 > hm5 (kd values were 4, 7, 59, 83 and 296 nm, respectively). himbacine was shown to bind to m2 receptors in rat heart and brain stem ...19921331410
phospholipase c activation by neurotensin and neuromedin n in chinese hamster ovary cells expressing the rat neurotensin receptor.the rat neurotensin receptor cdna sequence was transfected in chinese hamster ovary cells and cellular clones which stably express the corresponding protein were isolated and characterized. the scatchard analysis of the specific binding of [3h]neurotensin indicated a kd value of 0.45 +/- 0.08 nm and a bmax value of 3.27 +/- 0.29 pmol/mg of protein. displacement experiments using peptidic analogs of neurotensin and levocabastine confirmed that the transfected receptor exhibits the binding propert ...19921331689
functional properties of the rat and human beta 3-adrenergic receptors: differential agonist activation of recombinant receptors in chinese hamster ovary cells.beta 3-adrenergic receptors (beta 3ar) mediate lipolytic and thermogenic responses in rodent adipose tissues in vitro, and "atypical" beta ar agonists that active these receptors have potent therapeutic effects in in vivo rodent models of adult-onset diabetes and obesity. however, experiments with rodent cells that natively express the beta 3ar, as well as those with cells that express cloned rodent beta 3ar, have suggested that the pharmacological properties of the rodent and human beta 3ar dif ...19921331754
kinetic properties of alpha 1 beta 1 gamma-aminobutyric acida receptor channels expressed in chinese hamster ovary cells: regulation by pentobarbital and picrotoxin.single-channel recordings from excised outside-out patches were obtained from chinese hamster ovary cells stably transfected with plasmids containing bovine gamma-aminobutyric acid (gaba) type a (gabaa) receptor channel alpha 1 and beta 1 subunit cdnas. the predominant or main conductance level recorded had a 17-ps chord conductance. there were minor contributions made from 25-ps and 11-ps conductance levels. average open duration, burst duration, and openings/burst did not change as the gaba co ...19921331767
chromosomal assignments of mouse connexin genes, coding for gap junctional proteins, by somatic cell hybridization.the connexin genes cx31 and cx45 coding for proteins of gap junctional subunits have been assigned to mouse chromosomes 4 and 11 by southern blot hybridization of specific gene probes to dna from mouse x chinese hamster somatic cell hybrids. in addition, our results confirm the recent assignment of mouse connexin genes cx26, cx32, cx37, cx40, cx43, and cx46 to mouse chromosomes 14, x, 4, 3, 10, and 14, respectively, by analysis of interspecific backcrosses and by somatic cell hybridization. our ...19921332196
characterization of a minimal promoter element required for transcription of the mouse type ii beta regulatory subunit (rii beta) of camp-dependent protein kinase.the 5'-flanking dna of the mouse rii beta subunit of the camp-dependent protein kinase gene was characterized by transient transfection of rii beta-cat constructs into mouse neuroblastoma cells (nb2a) and chinese hamster ovary (cho) cells and by gel mobility shift and footprinting assays. the minimal promoter of the rii beta gene was composed of two adjacent functional elements. a 3'-element which supported enhanced cat activity was located between base pairs (bp) -267/-168 from the translation ...19921332964
establishment of a chinese hamster ovary cell line that expresses grp78 antisense transcripts and suppresses a23187 induction of both grp78 and grp94.grp78, a 78,000 dalton protein residing in the endoplasmic reticulum, is postulated to play important roles in protein folding and cell survival during calcium and other physiological stress. here we describe the construction of an eukaryotic expression vector for the constitutive expression of grp78 antisense rna and the creation of a cho cell line, 78wo, which expresses high levels of the grp78 antisense rna through amplification of the stably transfected antisense vector. we observed that whe ...19921332981
transcription enhances intrachromosomal homologous recombination in mammalian cells.the influence of transcription on homologous intrachromosomal recombination between direct and inverted repeats has been examined by using chinese hamster ovary cells. recombination was monitored between two integrated neomycin (neo) genes, including one silent allele and a second allele regulated by the inducible mouse mammary tumor virus promoter. transcription of mouse mammary tumor virus neo alleles was regulated with the glucocorticoid hormone dexamethasone. alleles transcribed at high leve ...19921333040
binding of recombinant variants of human tissue-type plasminogen activator (t-pa) to human umbilical vein endothelial cells.endothelial cells synthesize and secrete hemostatic components like tissue-type plasminogen activator (t-pa) which is thought to be the major determinant of fibrinolytic activity in the blood. most recently, a receptor protein for t-pa on human umbilical vein endothelial cells (huvec) in culture has been described (1); there are, however, in addition low affinity binding sites for t-pa on huvec. the sites of binding are of particular interest, because they are potential regulators of t-pa activi ...19921333104
dissociation of cytotoxicity and dna cleavage activity induced by topoisomerase ii-reactive intercalating agents in hamster-human somatic cell hybrids.previous studies using the mutant chinese hamster ovary cell line vpmr-5 indicate that its resistance to epipodophyllotoxins and intercalating agents is likely to be mediated through a qualitative change in type ii topoisomerase that confers resistance to drug-stimulated dna cleavage activity. in a further investigation of the genetic basis of drug resistance in vpmr-5 cells, we fused a hypoxanthine-guanine phosphoribosyl transferase-deficient subline of vpmr-5 (vtgm-6) with normal human lymphoc ...19921333369
endotoxin transduces ca2+ signaling via platelet-activating factor receptor.lipopolysaccharide (lps) is a pathogenic substance causing severe multiple organ failures and high mortality. although several lps binding proteins have been identified, the molecular mechanism underlying the lps signaling pathway still remains obscure. we have found that the lps-induced ca2+ increase in platelets and platelet aggregation is blocked by selective platelet-activating factor (paf) receptor antagonists, thus suggesting a cross-talk between lps and the paf receptor. next, we confirme ...19921333988
testosterone and 5 alpha-dihydrotestosterone interact differently with the androgen receptor to enhance transcription of the mmtv-cat reporter gene.testosterone and its 5 alpha-reduced derivative 5 alpha-dihydrotestosterone exert different actions in the male during embryogenesis and in postnatal life. nevertheless the two hormones bind to the same intracellular androgen receptor, and genetic and endocrinological studies in the tfm mouse suggest that the actions of both hormones are mediated by this single receptor. previous studies indicate that dihydrotestosterone binds more tightly to the androgen receptor but that the bmax of binding of ...19921334007
subtype-selective desensitization of alpha 2-adrenergic receptors. different mechanisms control short and long term agonist-promoted desensitization of alpha 2c10, alpha 2c4, and alpha 2c2.we have recently shown that the alpha 2c10 adrenergic receptor (ar) undergoes short term agonist-promoted desensitization, mediated by phosphorylation of sites in the third intracellular loop. there is significant divergence in the third loop amino acid sequences between alpha 2c10 and the other subtypes, alpha 2c4 and alpha 2c2. we therefore explored the mechanisms of alpha 2ar subtype desensitization by expressing each human subtype in chinese hamster ovary cells and subjecting them to short a ...19921334095
cells transfected with human interleukin 6 cdna acquire binding sites for the hepatitis b virus envelope protein.earlier studies revealed that human interleukin 6 (il-6) contains recognition sites for the hepatitis b virus (hbv) envelope (env) protein, and that il-6 and anti-il-6 antibodies, respectively, inhibited the interaction of cells expressing a receptor for hbv with the pres(21-47) segment of the hbv env protein, encompassing the complementary attachment site for il-6. this suggested that il-6 mediates hbv-cell interactions. we report that: (a) chinese hamster ovary cells transfected with human il- ...19921334115
insulin-dependent formation of a complex containing an 85-kda subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1.monoclonal antibodies raised against the 85-kda subunit (p85) of bovine phosphatidylinositol (pi) 3-kinase were found to recognize uncomplexed p85 or p85 in the active pi 3-kinase. immunoprecipitation studies of chinese hamster ovary cells, which overexpress the human insulin receptor when treated with insulin, showed increased amounts of p85 and pi 3-kinase activity immunoprecipitable with monoclonal anti-p85 antibody and no increase in the tyrosine phosphorylation of p85. insulin also induced ...19921334490
the gene for the alpha 1 subunit of the skeletal muscle dihydropyridine-sensitive calcium channel (cchl1a3) maps to mouse chromosome 1.cchl1a3 encodes the dihydropyridine-sensitive calcium channel alpha 1 subunit isoform predominantly expressed in skeletal muscle. mdg (muscular dysgenesis) has previously been implicated as a mutant allele of this gene. hybridization of a rat brain cdna probe for cchl1a3 to southern blots of dnas from a panel of chinese hamster x mouse somatic cell hybrids suggested that this gene maps to mouse chromosome 1. analysis of the progeny of an inbred strain cross-positioned cchl1a3 1.3 cm proximal to ...19921335956
analysis of the acp1 gene product: classification as an fmn phosphatase.the relationship between the acp1 gene product, an 18kda acid phosphatase (e.c. 3.1.3.2) postulated to function as a protein tyrosyl phosphatase, and the cellular flavin mononucleotide (fmn) phosphatase has been examined in vitro and by using cultured chinese hamster ovary (cho) cells. kinetic analysis indicated that at ph 6 the acid phosphatase utilized a variety of phosphate monoesters as substrates. while small molecules such as fmn were effectively utilized as substrates (kcat/km = 7.3 x 10( ...19921336375
expression of ca(2+)-induced ca2+ release channel activity from cardiac ryanodine receptor cdna in chinese hamster ovary cells.we constructed an expression plasmid (pmamcrr51) that carried the entire protein-coding sequence of the rabbit cardiac ryanodine receptor cdna, linked to the dexamethasone-inducible mouse mammary tumor virus promoter and escherichia coli xanthine-guanine phosphoribosyltransferase (gpt). chinese hamster ovary (cho) cells were transfected with pmamcrr51 and mycophenolic acid-resistant cells showing caffeine-induced intracellular ca2+ transients were selected. immunoprecipitation with a monoclonal ...19921337083
a complementary dna for human choline acetyltransferase induces two forms of enzyme with different molecular weights in cultured cells.complementary dna (cdna) clones containing the entire coding region of human choline acetyltransferase (chat) were isolated from cdna libraries prepared from the autopsied spinal cord. in the human cdna, the atg codon assigned to the putative initiation codon for pig, rat and mouse chat cdnas was replaced by acg. the human cdna contained an in-frame atg codon 324 nucleotides upstream of the acg codon. therefore, human chat cdna should code for a 748 amino acid polypeptide of 82.6 kda. this deduc ...19921337937
humanized monoclonal antibody campath-1h: myeloma cell expression of genomic constructs, nucleotide sequence of cdna constructs and comparison of effector mechanisms of myeloma and chinese hamster ovary cell-derived material.expression of campath-1h, a humanized moab directed against an abundant surface antigen on human lymphocytes, has been studied using transfected myeloma cells. as the site of chromosome integration of dna transfected into a cell is random we investigated the feasibility and frequency of hitting a 'jackpot' site for expression after co-transfection with campath-1h heavy and light chain constructs in genomic context. a cell line generating 40 micrograms/ml of campath-1h in spent culture supernatan ...19921339322
recombinant human secretory phospholipase a2: purification and characterization of the enzyme for active site studies.a secreted form of phospholipase a2 (pla2) is thought to play an important role in inflammatory diseases. to characterize this enzyme the cdna encoding a low molecular weight pla2 was cloned from a human placental cdna library. the cdna encoding the human pla2 was subcloned into an expression vector and subsequently transfected into chinese hamster ovary (cho) cells. a stable cho cell clone, secreting ca 1 mg/l of recombinant pla2 into the medium, was scaled up in culture to 180 l. the recombina ...19921339483
immediate cell membrane damage produced by entamoeba histolytica subcellular extracts.the alpha[14c]amino isobutyric acid is a synthetic amino acid with low molecular weight, not incorporated to protein. chinese hamster ovary (cho) cells were labeled with this radioactive compound and treated with the amebal subcellular fraction p30 at short times. we detected membrane damage in cho cells by specific radiolabel release from the first 30 sec of interaction between cell cultures and amebal fraction.19921340292
bile salts promote adherence-decreasing effect of colonic luminal hydrolases on entamoeba histolytica.e. histolytica trophozoites cultivated for > 7 h in the presence of glycosidases, produced by a subset of the colonic anaerobic bacteria of healthy humans, and pancreatic proteases show decreased adherence to chinese hamster ovary epithelial cells. since activities of the glycosidases are enhanced by bile salts we investigated whether bile salts would enhance the e. histolytica-cho cell adherence decreasing effects of the luminal hydrolases (glycosidases plus proteases). cho cell adherence of co ...19921340299
repair of dna strand breaks at hyperthermic temperatures in chinese hamster ovary cells.the repair of dna double-strand breaks was measured by ph 7.2 filter elution in cells incubated at 25-45 degrees c either before or after x-irradiation. exposure to 45 degrees c for 15 minutes immediately prior to x-irradiation significantly increased both the half-time for dna double-strand break closure and the number of dna double-strand breaks remaining in nuclear dna 180 minutes after irradiation. exposure to temperatures between 41 and 45 degrees c immediately after x-irradiation accelerat ...19921345930
cloning and functional characterization of a family of human and mouse somatostatin receptors expressed in brain, gastrointestinal tract, and kidney.somatostatin is a tetradecapeptide that is widely distributed in the body. it acts on multiple organs including brain, pituitary, gut, exocrine and endocrine pancreas, adrenals, thyroid, and kidneys to inhibit release of many hormones and other secretory proteins. in addition, it functions as a neuropeptide affecting the electrical activity of neurons. somatostatin exerts its biological effects by binding to specific high-affinity receptors, which appear in many cases to be coupled to gtp-bindin ...19921346068
mapping of 262 dna markers into 24 intervals on human chromosome 11.we have extended our mapping effort on human chromosome 11 to encompass a total of 262 dna markers, which have been mapped into 24 intervals on chromosome 11; 123 of the markers reveal rflps. these clones are scattered throughout the chromosome, although some clustering occurs in r-positive bands (p15.1, p11.2, q13, and q23.3). fifty-two of the markers were found to contain dna sequences conserved in chinese hamster, and some of these 52 also cross-hybridized with dna from other mammals and/or c ...19921346079
differential effect of interleukin 1 on naive and memory cd4+ t cells.freshly derived murine cd4+ t cells are divided into naive and memory cells based on the expression of cd45 isoforms. cross-linking the t cell receptor cd3 complex either by plastic-bound anti-cd3 antibodies or the antibody presented on non-lymphoid fc gamma receptor type ii-positive chinese hamster ovary cells in absence of competent antigen-presenting cells fails to activate naive cells to either secrete cytokines or to proliferate. in contrast, memory cells secrete their characteristic cytoki ...19921346115
sdz 280-446, a novel semi-synthetic cyclopeptolide: in vitro and in vivo circumvention of the p-glycoprotein-mediated tumour cell multidrug resistance.sdz 280-446 is a semi-synthetic derivative of a natural cyclic peptolide. its ability to sensitise in vitro tumour cells whose resistance is due to p-glycoprotein-mediated anticancer-drug efflux was shown using four different pairs of parental drug-sensitive (par-) and multidrug-resistant (mdr-) cell lines, from three different species (mouse, human, chinese hamster) representing four different cell lineages (monocytic leukaemia, nasopharyngeal epithelial carcinoma, colon epithelial carcinoma, o ...19921346365
antagonism by antimuscarinic and neuroleptic compounds at the five cloned human muscarinic cholinergic receptors expressed in chinese hamster ovary cells.we determined the affinity and selectivity of binding for 24 compounds: nine antimuscarinics (including some antiparkinson drugs) and 15 neuroleptics (including the atypical compounds clozapine, fluperlapine, melperone, rilapine, risperidone, tenilapine, tiosperone and zotepine) at the five human muscarinic receptor subtypes expressed in chinese hamster ovary cells. equilibrium dissociation constants (kd) were obtained from competitive radioligand binding studies with [3h]quinuclidinyl benzilate ...19921346637
level of dna double-strand break rejoining in chinese hamster xrs-5 cells is dose-dependent: implications for the mechanism of radiosensitivity.rejoining of dna double-strand breaks (dsb) was measured in a dsb repair-deficient mutant of cho cells, xrs-5, after exposure to various doses of x-rays in the range between 15 and 50 gy. for the experiments plateau-phase cultures were employed and dsb assayed by a pulsed field gel electrophoresis assay, the asymmetric field inversion gel electrophoresis (afige). the half-times of dsb rejoining were larger in xrs-5 than in parental cho cells and increased in both cell lines with increasing dose ...19921347063
chromosome damage induced by restriction endonucleases recognizing thymine-rich dna sequences in electroporated cho cells.the influence of brdu substitution of dna in chinese hamster cells on the frequencies of chromosomal aberrations induced by three restriction endonucleases which recognize thymine-rich sequences in dna has been studied. the restriction enzymes chosen were eco ri (recognition site g/aattc), sca i (agt/act), and dra i (ttt/aaa). a restriction enzyme that does not have thymine in the recognition sequence, hae iii (gg/cc), was also tried. these enzymes were introduced into cells by electroporation a ...19921347064
a potential pitfall in the use of electroporation: cellular radiosensitization by pulsed high-voltage electric fields.chinese hamster ovary cells have been exposed to high-voltage electric fields causing electroporation (ep) and the interaction between ep and radiation-induced cell lethality investigated. there was a voltage-dependent decrease in plating efficiency, assessed immediately following ep, and cell viability, assessed at 24 h. a linear decrease was seen for both. these decreases were accompanied by a voltage-dependent increase in cell volume, assessed immediately following ep. a good correlation betw ...19921347065
distinct regulation of beta 1- and beta 2-adrenergic receptors in chinese hamster fibroblasts.the agonist-induced reduction of beta-adrenergic receptor (beta ar) cell surface density is a well documented phenomenon. the mechanisms responsible for this regulation have been well characterized for the beta 2ar. they include a rapid sequestration of the receptor away from the cell surface in a vesicular compartment and a longer term down-regulation of the total beta 2ar number. in contrast, very little is known about the cell surface regulation of the beta 1ar. in the present study, we have ...19921347641
a single base change at a splice acceptor site leads to a truncated cad protein in urd-a mutant chinese hamster ovary cells.we have previously reported the isolation and characterization of mutant chinese hamster ovary (cho-k1) cells of the urd-a complementation group, which require uridine for growth, are deficient in the activities of the first three enzymes of de novo ump biosynthesis, and produce markedly reduced amounts of a truncated form of the multifunctional protein cad, which contains these three enzyme activities. we report here that a single base change of g to a at a highly conserved rna splice acceptor ...19921347664
effect of lengthening lymphocyte function-associated antigen 3 on adhesion to cd2.the effect of lengthening the distance in an adhesion molecule between the receptor binding site and the membrane anchor was studied by inserting four ig-like domains into the two ig domain lymphocyte function-associated antigen 3 (lfa-3) molecule. the extended molecule expressed in chinese hamster ovary (cho) cells bound to cd2 on t lymphocytes 4- to 20-fold more efficiently than the wild-type molecule at 4 degrees c. treatment of the cho clones with neuraminidase to remove sialic acid, or with ...19921348006
peptide transport by the multidrug resistance pump.the membrane p-glycoprotein (p170) is an atp-hydrolyzing transmembrane pump, and elevated levels of p170, due to higher expression with or without amplification of the multidrug resistance gene (mdr1), result in resistance to a variety of chemotherapeutic agents in mammalian cells. the function of the p170 pump has been proposed as a protection against toxic substances present in animal diets. here we describe a chinese hamster ovary cell line that was selected for resistance to a synthetic trip ...19921348245
effect of p-glycoprotein expression on the accumulation and cytotoxicity of topotecan (sk&f 104864), a new camptothecin analogue.topotecan (tpt, 9-dimethylaminomethyl-10-hydroxycamptothecin) is the first topoisomerase i-directed cytotoxic agent to enter clinical trials in the united states in two decades. the effect of p-glycoprotein (pgp) overexpression on tpt cytotoxicity was examined in chrc5 (colchicine-resistant) and auxb1 (parental) chinese hamster ovary cells. examination of the ic50 values observed in colony-forming assays revealed that the chrc5 cells were 15-fold (sd, +/- 3; n = 3) resistant to tpt after a 1-h e ...19921348448
intrinsic resistance to anticancer agents in the murine pancreatic adenocarcinoma panc02.panc02 is a ductal adenocarcinoma of the pancreas that is resistant to every known class of clinically active antitumor agent. to study the mechanism(s) underlying the intrinsic drug resistance of this tumor, a mammary adenocarcinoma (ca-755) that also grows in c57/bl mice and is known to be drug sensitive was used for comparison. panc02 resistance and ca-755 sensitivity to several antitumor agents and to x-ray therapy was confirmed in mice, and panc02 also demonstrated relative resistance in ti ...19921348974
localization of the nucleotide excision repair gene ercc6 to human chromosome 10q11-q21.we have cloned the human dna excision repair gene ercc6 by virtue of its ability to correct the uv sensitivity of chinese hamster overy cell mutant uv61. this mutant is a member of complementation group 6 of the nucleotide excision repair-deficient rodent mutants. by means of in situ hybridization and southern blot analysis of mouse x human somatic cell hybrids, the gene was localized to human chromosome 10q11-q21. an rflp detected within the ercc6 locus can be helpful in linkage analysis.19921349298
relationships between dna damage and the survival of radiosensitive mutant chinese hamster cell lines exposed to gamma-radiation. part 1: intrinsic radiosensitivity.in this study we used the neutral elution technique (at ph 9.6) to compare the efficiency of dna double-strand break (dsb) induction and cell killing in two radiosensitive chinese hamster ovary cell lines, em9 and nm2, and their parent cell line, aa8, after exposure to low doses (2.5-12 gy) of 137cs gamma-radiation. the survival curves indicated that at all radiation doses examined em9 cells were the most radiosensitive and aa8 cells were the least radiosensitive. we found significant difference ...19921349328
ablation of stimulation of a camp-responsive promoter in cho cell lines defective in their camp-dependent protein kinase system.we have studied the requirement for an intact camp-dependent protein kinase (pka) system to regulate camp-mediated gene transcription in chinese hamster ovary (cho) cells. wild-type cho cells and mutant cho cell lines selected for their resistance to the growth inhibitory effect of 8-br-camp and defective in their pka system were transiently transfected with reporter plasmids containing 2.5 and 3.0 kb of the 5'-flanking sequence of the rat tyrosine aminotransferase (tat) gene promoter. this segm ...19921349445
resistance of cho cells expressing p-glycoprotein to cyclopropylpyrroloindole (cpi) alkylating agents.several new antitumor agents belonging to the class of minor groove binders that are able to form covalent bonds with dna via a cyclopropylpyrroloindole (cpi) group are susceptible to a multidrug resistance (mdr) phenotype in chinese hamster ovary (cho) cells. the multidrug resistant cchr-c5 cell line was 16-, 23- and 13-fold more resistant to the analogs u-73,975, u-77,779 and u-80,244, respectively, although its cytotoxic response to the parent compound cc-1065 was similar to the response of t ...19921349478
direct comparison of biological effectiveness of protons and alpha-particles of the same let. ii. mutation induction at the hprt locus in v79 cells.mutation induction at the hprt locus has been studied in v79-4 chinese hamster cells irradiated with mono-energetic protons and alpha-particles with let of 20.3 and 23 kev microns-1. the mutation frequency increased linearly with the dose for all the four radiation qualities investigated, so that effectiveness for mutation induction could be expressed by the slope of the relevant curve. this effectiveness did not significantly change with the small change in let of each kind of particle, while s ...19921349626
direct comparison of biological effectiveness of protons and alpha-particles of the same let. iii. initial yield of dna double-strand breaks in v79 cells.the results reported form part of a series of experiments to substantiate and extend the findings by belli et al. (1989) that protons are more biologically effective at cell killing than alpha-particles of the same let. the irradiations were carried out using the variable energy cyclotron (vec) at the harwell laboratories. v79-4 chinese hamster cells were exposed to alpha-particles and protons with lets of 20 and 23 kev microns-1 in the dose range 40-150 gy. x-rays were also used for comparison. ...19921349627
perinatal changes in the coupling of beta 1- and beta 3 adrenergic receptors to brown fat adenylyl cyclase.the stimulation of adenylyl cyclase by catecholamines in neonatal brown adipose tissue (bat) is markedly biphasic, suggesting the existence of receptors that have both high and low affinities for catecholamines. the identities of these receptors were examined by comparing responses in neonatal bat membranes to those of chinese hamster ovary cells which had been transfected to express the cloned rat beta 1 and beta 3 receptors. the results from these experiments indicate that high-affinity stimul ...19921349643
isolation and characterization of putative intrinsic multidrug resistant chinese hamster ovary cells by fluorescence activated cell sorting.most multidrug resistant cell lines reported in the literature were established by long-term continuous exposure of cells to stepwise increasing concentrations of antitumor drugs. however, these resistant cell lines may not be relevant to the majority of clinically resistant cells. in this study, we described the establishment of doxorubicin (dox)-resistant chinese hamster ovary cells by repeated flow cytometric cell sorting using the intrinsic fluorescence of dox. in each sorting, the 15% least ...20131349795
sequence and expression of rat icam-1.we have isolated cdna clones-coding for rat intercellular adhesion molecule-1 (ricam-1) from a cdna library constructed from rat ax cells stimulated with il-1 beta using the mouse icam-1 cdna as a hybridization probe. the ricam-1 sequence shows 79.1% homology with mouse icam-1 and 55.6% homology with human icam-1 at the nucleic acid level. in order to examine the expression of ricam-1 on chinese hamster ovary (cho) cells, we constructed the vector, psv-ricam1-neo, containing the sv40 promoter. f ...19921349828
mapping of ornithine aminotransferase gene sequences to mouse chromosomes 7, x, and 3.ornithine aminotransferase (oat), a mitochondrial matrix enzyme, is deficient in patients with gyrate atrophy of the choroid and retina. in human, the oat structural gene maps to chromosome (chr) 10q26 and several oat-related sequences, some of which are known to be processed pseudogenes, which map to xp11.3-11.21. here, we report chromosomal localization in the mouse of the oat gene and related sequences. genomic dna blot analysis of a well-characterized panel of chinese hamster x mouse somatic ...19921349842
engagement of class i major histocompatibility complex molecules by cell surface cd8 delivers an activation signal.recent evidence has demonstrated that cross-linking class i major histocompatibility complex (mhc) molecules on human t cells with monoclonal antibodies (mab) triggers t cell activation. the only known natural ligand for mhc class i molecules is cd8. therefore, the possibility that cd8+ t cells might provide activation signals to other t cells by engaging mhc class i molecules was examined by culturing cd4+ peripheral blood t cells with chinese hamster ovary cells (cho) cells that had been trans ...19921350981
postnatal development of organic cation transport and mdr gene expression in mouse kidney.the apical surface of the proximal tubular epithelium is the site of both p-glycoprotein localization and postulated active secretion of organic cations in the mammalian kidney. p-glycoprotein has been shown to act as a pleiotropic drug efflux pump across the cell membrane of tumor cells expressing the multidrug resistance phenotype, whereas the renal organic anion and organic cation secretory systems serve the function of pleiotropic drug transport across the proximal tubule epithelium. because ...19921351097
a comparison of the chemical repair rates of free radical precursors of dna damage and cell killing in chinese hamster v79 cells.one of the important temporal stages of radiation action in cellular systems is the chemical phase, where oxygen fixation reactions compete with chemical repair reactions involving reducing agents such as gsh. using the gas explosion technique it is possible to follow the kinetics of these fast (greater than 1 ms) reactions in intact cells. we have compared the chemical repair kinetics of the oxygen-dependent free radical precursors leading to dna single-strand and double-strand breaks, measured ...19921351520
the effect of fluoride on photodynamic-induced fluorescence changes of aluminium phthalocyanine in chinese hamster cells.fluence-dependent changes in the fluorescence of aluminium phthalocyanine (alpc) were measured in chinese hamster ovary (cho) cells using digital fluorescence microscopy of single cells and spectrofluorimetry of cell suspensions. during illumination the fluorescence initially increased and later progressively decreased. in the presence of fluoride, which protects against phototoxicity of alpc by forming a fluoroaluminium complex, there was no initial increase in fluorescence: it decreased about ...19921351525
identification of a processed protein related to the human chaperonins (hsp 60) protein in mammalian kidney.the chaperonin family of proteins, which includes groel protein of e. coli, yeast heat shock protein (hsp-60) and the ribulose-1-5-bisphosphate carboxylase (rubis co.) subunit binding protein of plant chloroplasts, shows strong sequence homology to the chinese hamster ovary (cho) mitochondrial p1 protein. we have identified a 60 kda protein from bovine kidney which by n-terminal sequencing gives the amino acid sequence akdvkfgadarallmlqgvdllada. bovine whole kidney membranes were delipidated, so ...19921351721
effect of heat on induction and repair of dna strand breaks in x-irradiated cho cells.chinese hamster ovary cells were exposed to various heat treatments followed by x-irradiation, and the induction and repair of dna strand breaks was studied using the alkaline unwinding technique. heat treatments alone were found to cause dna strand breakage only for temperatures greater than or equal to 43 degrees c, whereas the number of radiation-induced strand breaks was unaffected by additional heating. strand break repair was studied for irradiated cells preheated at temperatures ranging f ...19921351910
effect of thermotolerance and step-down heating on thermal radiosensitization in cho cells.chinese hamster ovary cells were exposed to single or fractionated heat treatments followed by irradiation on ice with graded doses of x-rays. the dose-response curves obtained were fitted by the linear-quadratic equation -ln(s/s0) = alpha d + beta d2 and analysed in terms of ter10%, alpha and beta. thermal enhancement ratio, ter10%, was reduced when heat sensitivity was lowered either by chronic (pretreatment 40 degrees c, 16 h) or acute (43 degrees c, 45 min-37 degrees c, 10 h) thermotolerance ...19921351911
participation of prostaglandin e2 in dopamine d2 receptor-dependent potentiation of arachidonic acid release.stimulation of dopamine d2 receptors potentiates ca2+ ionophore- or atp-induced arachidonic acid (aa) release in d2 receptor cdna-transfected chinese hamster ovary (cho) cells [cho(d2)]. by using a combination of chromatographic, biochemical, and radioimmunochemical techniques, we show here that prostaglandin (pg) e2 is a major product of aa metabolism in cho(d2) cells stimulated with the ca2+ ionophore a23187. formation of this pg was markedly increased by the concomitant application of quinpir ...20061351931
characterization of a new hybrid mink-mouse clone panel: chromosomal and regional assignments of the glo, acy, np, ckbb, adh2, and me1 loci in mink (mustela vison).to expand the mink map, we established a new panel consisting of 23 mink-mouse clones. on the basis of statistical criteria (wijnen et al. 1977; burgerhout 1978), we developed a computer program for choice of clones of the panel. assignments of the following mink genes were achieved with the use of the hybrid panel: glyoxalase (glo), chromosome (chr) 1; acetyl acylase (acy), chr 5; creatine phosphokinase b (ckbb), chr 10; alcohol dehydrogenase-2 (subunit b) (adh2), chr 8. using a series of clone ...19921352156
substitution of the insulin receptor transmembrane domain with the c-neu/erbb2 transmembrane domain constitutively activates the insulin receptor kinase in vitro.to examine the role of the transmembrane domain (tm) of the insulin receptor in insulin-induced receptor kinase activation, we prepared four mutated insulin receptors: 1) a val938----asp substitution (ir/tmv----d), 2) insertion of a 3-amino acid repeat (val938-phe939-leu940) (ir/tm+3), or the entire tm was replaced by the corresponding domain of either the 3) platelet-derived growth factor (pdgf) receptor (ir/tmpdgfr) or 4) c-neu/erbb2 proto-oncogene product (ir/tmc-neu). each mutant receptor wa ...20061352286
reduced mrna levels for the multidrug-resistance genes in camp-dependent protein kinase mutant cell lines.we have previously shown that in chinese hamster ovary (cho) cells, a mutant cell line with a defective regulatory subunit (ri) for the camp-dependent protein kinase (abraham et al: mol. cell. biol., 7:3098-3106, 1987), and a transfectant cell line expressing the same mutant kinase, showed increased sensitivity to a number of drugs that are known to be substrates for the multidrug transporter (p-glycoprotein). in the current study we have investigated the mechanism by which camp-dependent protei ...19921352302
reversal of typical multidrug resistance by cyclosporin and its non-immunosuppressive analogue sdz psc 833 in chinese hamster ovary cells expressing the mdr1 phenotype.the new non-immunosuppressive cyclosporin derivative sdz psc 833 (psc) is a potent agent used to overcome typical multidrug resistance (mdr) associated with overexpression of the mdr1 gene encoding for a p-170 glycoprotein. in the present study, the efficacy of psc as compared with cyclosporin was determined in chinese hamster ovary cell lines exhibiting different levels of resistance to colchicine (0, 0.1, 0.2 and 10 micrograms/ml, respectively). low concentrations of psc (8.2 nm) increased the ...19921352739
pharmacological properties of two cloned somatostatin receptors.previous studies have shown that at least two subtypes of somatostatin (srif) receptors (srif1 and srif2) are expressed in mammalian cells. srif1 receptors have high affinity for mk 678, whereas srif2 receptors have no affinity for mk 678 but selectively bind peptides with structures similar to that of cgp 23996. recently, two srif receptor genes have been cloned from human and mouse genomic libraries. in the present study, the pharmacological properties of these two cloned srif receptors, expre ...19921353250
alternative polyadenylation of the amyloid protein precursor mrna regulates translation.the sequence of several cdnas encoding the amyloid protein precursor showed that two polyadenylation sites of the mrna are utilized; rna blot analysis with different riboprobes indicated that this explains the difference between the two major 3.2 and 3.4 kb mrnas found in the human brain. these two mrnas, which contain the whole sequence of the natural molecules, were synthesized by in vitro transcription and translated in xenopus oocytes. the long mrna using the second polyadenylation site prod ...19921353447
multifactorial resistance to antineoplastic agents in drug-resistant p388 murine leukemia, chinese hamster ovary, and human hela cells, with emphasis on the role of dna topoisomerase ii.the role of dna topoisomerase ii in multifactorial resistance to antineoplastic agents is reviewed. we have previously observed that in adriamycin (adr) resistant p388 murine leukemia cells, dna topoisomerase ii enzyme content and cleavage and catalytic activities were all reduced and correlated with drug sensitivity. a subsequent study provided evidence for an allelic mutation of the gene for dna topoisomerase ii as a possible molecular mechanism underlying the enzyme alterations. to ascertain ...19921353968
amino acid transport in multidrug-resistant chinese hamster ovary cells.in the process of assessing the effect of anthracycline drugs on cellular membrane function in cultured multidrug resistant (mdr) and its parental cells, experiments were undertaken to investigate the kinetics of neutral amino acid membrane transport (the sodium dependent a and asc systems). p-glycoprotein, a high molecular weight energy requiring integral membrane protein responsible for actively pumping drugs out of cells, has been shown to be overexpressed in mdr cells. it was our hypothesis ...19921354438
n-(5,5-diacetoxypent-1-yl)doxorubicin: a new intensely potent doxorubicin analogue.n-(5,5-diacetoxypent-1-yl)doxorubicin (dapdox) (3), a new, water-soluble analogue of doxorubicin, has been synthesized by coupling doxorubicin with 5-oxopentane-1,1-diacetate in the presence of nabh3cn. this analogue was designed to be converted to the corresponding aldehyde, n-(5-oxopent-1-yl)doxorubicin, in the presence of carboxylate hydrolases, enzymes that are ubiquitous in tissue. dapdox had a half-life of several days in 0.05 m phosphate or 0.05 m acetate buffer solution at ph 4.0. howeve ...19921354750
diphenylhydantoin is not genotoxic in a battery of short-term cytogenetic assays.5,5-diphenylhydantoin (dph) is an antiepileptic drug associated with an increase in malformations in infants born to women taking dph during pregnancy. positive and negative results have been reported by various investigators for in vivo and in vitro chromosome aberration (cab) assays, in vivo and in vitro sister chromatid exchange (sce) assays, and in vivo micronucleus tests (mnt). in this laboratory, dph was tested in an in vitro cab assay using chinese hamster ovary cells with and without an ...19921354899
doxorubicin-loaded nanospheres bypass tumor cell multidrug resistance.we have demonstrated that in vitro resistance of tumor cells to doxorubicin (dox) can be fully circumvented by using doxorubicin-loaded nanospheres (dox-ns), consisting of biodegradable polyisohexylcyanoacrylate polymers of 300 nm diameter and containing 2.83 mg of dox per 31.5 mg of polymer. five different multidrug-resistant cell lines, characterized by mdr1 amplification, were used in this study: dox-r-mcf7, a human breast adenocarcinoma; skvbl1, a human ovarian adenocarcinoma; k562-r, a huma ...19921354963
a naturally occurring tyrosine to histidine replacement at residue 33 of human thymidylate synthase confers resistance to 5-fluoro-2'-deoxyuridine in mammalian and bacterial cells.structural changes in the macromolecular targets of pharmacological agents can result in alterations in the efficacy of these agents. in previous studies, we identified a variant structural form of thymidylate synthase (ts) that is associated with relative resistance to 5-fluoro-2'-deoxyuridine, in a human colonic tumor cell line. we now report on the use of dna transfer techniques to examine directly the effects of each ts form on drug response. ts cdna constructs, corresponding to the normal o ...19921355260
genetic aspects of multidrug resistance.mammalian cells exposed to a single cytotoxic natural product drug, such as vincristine or dactinomycin, can develop resistance to the selective agent and cross-resistance to a broad spectrum of structurally and functionally distinct antibiotics and alkaloids. this phenomenon, termed multidrug resistance (mdr), has been widely studied experimentally. the most consistent feature of cells with high-level mdr is amplification and overexpression of genes encoding an integral plasma membrane protein ...19921355404
atpase activity of partially purified p-glycoprotein from multidrug-resistant chinese hamster ovary cells.in vitro studies of multidrug-resistant cell lines have shown that a membrane protein, the p-glycoprotein, is responsible for resistance to a wide range of structurally and functionally dissimilar anti-cancer drugs. the amino-acid sequence of p-glycoprotein (pgp) indicates two consensus sequences for atp binding and the purified protein has been reported to possess a low level of atpase activity. as part of our goal to further characterize the atpase activity of p-glycoprotein, we have developed ...19921355666
transport properties of p-glycoprotein in plasma membrane vesicles from multidrug-resistant chinese hamster ovary cells.multidrug resistant (mdr) cells overexpress a 170-180 kda membrane glycoprotein, the p-glycoprotein, which is believed to export drugs in an atp-dependent manner. plasma membrane vesicles from the mdr chrc5 cell line, but not the auxb1 drug-sensitive parent, showed uptake of [3h]colchicine and [3h]vinblastine that was stimulated by the presence of atp and an atp-regenerating system. steady-state uptake of drugs was achieved by 10 min and was stable for greater than 30 min. non-hydrolysable atp a ...19921355667
molecular cloning and chromosomal localization of a human gene encoding d-amino-acid oxidase.genomic clones covering the entire sequence of the gene encoding human d-amino-acid oxidase (dao) (ec 1.4.3.3), one of the principal and characteristic flavoenzymes of peroxisomes, were isolated from human placental genomic libraries with the aid of a previously cloned cdna for human dao as a probe. nucleotide sequence analysis revealed that the gene, present as a single copy in the human genome, comprises 11 exons and spans 20 kilobase pairs. the protein sequences containing the catalytically i ...19921356107
the role of non-protein sulphydryls in determining the chemical repair rates of free radical precursors of dna damage and cell killing in chinese hamster v79 cells.chinese hamster v79 fibroblasts were irradiated in the gas explosion apparatus and the chemical repair rates of the oxygen-dependent free radical precursors of dna double-strand breaks (dsb) and lethal lesions measured using filter elution (ph 9.6) and a clonogenic assay. depletion of cellular gsh levels, from 4.16 fmol/cell to 0.05 fmol/cell, by treatment with buthionine sulphoximine (50 mumol dm-3; 18 h), led to sensitization as regards dna dsb induction and cell killing. this was evident at a ...19921356131
chinese hamster ovarian cell glycoproteins that mediate type 1 piliated gram-negative bacterial adherence.we used chinese hamster ovary (cho) cell lines to define the structures of glycoproteins responsible for type 1 piliated bacterial adherence. csh 50 escherichia coli, a type 1 piliated bacteria, adhered significantly better than an isogenic nonpiliated e. coli to all cho lines tested. csh 50 e. coli adhered least well to cho cells expressing intact complex type oligosaccharides on cell surface glycoproteins. csh 50 adherence increased when shorter oligosaccharides were present and was maximal wh ...19921356377
total body and hepatic clearance in rats of recombinant tissue-type plasminogen activator expressed in mouse c127 and chinese hamster ovary cells.the total body and hepatic clearance of two recombinant human tissue-type plasminogen activators (t-pa) produced in mouse c127 cells [t-pa(c127)] and in chinese hamster ovary cells [t-pa(cho)] were determined in rats. recombinant t-pa was administered as a constant iv or intraportal venous infusion for 60 min. the plasma t-pa level over 120 min was measured by an elisa. whole-body pharmacokinetics were characterized in terms of the total body clearance (clt), total volume of distribution, and me ...19921356732
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