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PubMed1978

A diploid rat liver cell culture. IV. Malignant transformation by aflatoxin B1.

Chronic exposure of a cloned rat hepatocyte culture (RL-PR-C) to a subtoxic, sublethal dose of aflatoxin B1 resulted in malignant transformation. Continuous exposure to aflatoxin B1 caused increasing tumorigenic potential as tested by back injection into isogenic animals. Control cultures exhibited spontaneous transformation, although approximately 20 passages beyond the chemically induced event. Neither aflatoxin-treated nor control cultures exhibited cytopathological morphology, formation of cell foci, growth in soft agar, or irregular fibroblast-like growth patterns that could be specifically related to the onset of tumorigenic potential. In general, those parameters commonly used to monitor fibroblast cultures for transformation in vitro were not applicable for assessing the tumorigenic potential of these epithelial cells. Karyotypic analyses revealed no specific chromosomal aberrations associated with aflatoxin treatment; however, chromosomal instability was a property of the tumorigenic cell populations. Injection of both aflatoxin-treated and control cultures at passage 56 resulted in tumors indicative of both carcinoma and sarcoma indicating to us the multipotency of these epithelial cells transformed in vitro.

Aflatoxins↗
PubMed1995

Detection of oxidative mutagens in strains of Escherichia coli deficient in the OxyR or MutY functions: dependence on SOS mutagenesis.

The Escherichia coli strain IC3821, a delta oxyR derivative of WP2 uvrA trpE65, was more sensitive to mutagenicity promoted by t-butyl hydroperoxide and cumene hydroperoxide than the isogenic oxyR+ control. Mutagenicity of menadione, a redox cycling quinone, was clearly detected in the delta oxyR strain, whereas only a slight mutagenic response was observed in the oxyR+ strain. Plumbagin, another quinone structurally similar to menadione, was not mutagenic to any of the strains. These mutagenic responses appeared to involve the SOS processing of oxidative DNA lesions and were mediated by MucA/B proteins more efficiently than by UmuD/C. In cells lacking mutagenesis proteins, induction of SOS-independent mutations by the two alkyl hydroperoxides required a deficiency in the MutY DNA glycosylase and was increased by the presence of the delta oxyR mutation. In contrast, the two quinones assayed were unable to induce SOS-independent mutations in the MutY-deficient strains.

Benzene Derivatives↗
PubMed1993

Generation of atrial natriuretic peptide (ANP) in perfused lungs of spontaneously hypertensive rats (SHR). Comparison to Wistar-Kyoto (WKY) and Wistar (W) rat strains.

1. Lungs can take up from the vasculature, circulating forms of atrial natriuretic peptide (Turrin and Gillis, 1986, 1987) and also to synthesize ANP. 2. The lung peptide directly delivered by lungs into the lung vasculature could play a role in the local water/electrolytic balance. 3. Using Spontaneously Hypertensive Rats (SHR), isogenic normotensive controls, the Wistar-Kyoto strain (WKY), and the regular Wistar strain as second control (W), and using a highly sensitive RIA, we measured the immunoreactive IR-ANP content of extracted plasma, lung homogenate and lung perfusate, since there are references of altered ANP levels in this kind of hypertension. 4. The IR-ANP measured in the lung vasculature effluent collected throughout 32 min of Krebs perfusion, was significantly different in all of the three analyzed strains (SHR > WKY > W). 5. The results support the idea of a local function for the peptide hormone directly delivered into the lung vasculature of SHR, which could represent a local adaptation to haemodynamics SHR characteristics besides a genetic characteristic distinguishing WKY from W strains.

Animals↗
PubMed2001

Defective regulatory volume decrease in human cystic fibrosis tracheal cells because of altered regulation of intermediate conductance Ca2+-dependent potassium channels.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein has the ability to function as both a chloride channel and a channel regulator. The loss of these functions explains many of the manifestations of the cystic fibrosis disease (CF), including lung and pancreatic failure, meconium ileus, and male infertility. CFTR has previously been implicated in the cell regulatory volume decrease (RVD) response after hypotonic shocks in murine small intestine crypts, an effect associated to the dysfunction of an unknown swelling-activated potassium conductance. In the present study, we investigated the RVD response in human tracheal CF epithelium and the nature of the volume-sensitive potassium channel affected. Neither the human tracheal cell line CFT1, expressing the mutant CFTR-DeltaF508 gene, nor the isogenic vector control line CFT1-LC3, engineered to express the betagal gene, showed RVD. On the other hand, the cell line CFT1-LCFSN, engineered to express the wild-type CFTR gene, presented a full RVD. Patch-clamp studies of swelling-activated potassium currents in the three cell lines revealed that all of them possess a potassium current with the biophysical and pharmacological fingerprints of the intermediate conductance Ca(2+)-dependent potassium channel (IK, also known as KCNN4). However, only CFT1-LCFSN cells showed an increase in IK currents in response to hypotonic challenges. Although the identification of the molecular mechanism relating CFTR to the hIK channel remains to be solved, these data offer new evidence on the complex integration of CFTR in the cells where it is expressed.

Calcium↗
PubMed1987

Spontaneous mutagenesis and oxidative damage to DNA in Salmonella typhimurium.

Salmonella typhimurium strains containing deletions of oxyR, a positive regulator of defenses against oxidative stress, show 10- to 55-fold higher frequencies of spontaneous mutagenesis compared to otherwise isogenic oxyR+ control strains. The high spontaneous-mutation frequency in oxyR deletion strains is decreased by a factor of 3 when the strains are grown anaerobically. oxyR deletion strains show an increase in small deletion mutations and at least three of the six possible base-substitution mutations (T.A to A.T, C.G to T.A, and C.G to A.T). However, the largest increase in mutation frequency is observed for T.A to A.T transversions (40- to 146-fold), the base-substitution mutation most frequently caused by chemical oxidants. The introduction into oxyR deletion strains of multicopy plasmids carrying the oxyR-regulated genes for catalase (katG) or alkyl hydroperoxide reductase (ahp) results in overexpression of the respective enzyme activities and decreases the number of spontaneous mutants to wild-type levels. The introduction into oxyR deletions of a plasmid carrying the gene for superoxide dismutase (sodA) decreases the mutation frequency by a factor of 5 in some strain backgrounds. Strains that contain a dominant oxyR mutation and overexpress proteins regulated by oxyR show lower spontaneous-mutation frequencies by a factor of 2. These results indicate that oxyR and oxyR-regulated genes play a significant role in defense against spontaneous oxidative DNA damage. The role of oxidative damage to DNA in "spontaneous" mutagenesis is discussed.

Anaerobiosis↗
PubMed1988

Stable expression of the tax gene of type I human T-cell leukemia virus in human T cells activates specific cellular genes involved in growth.

Stable expression of the 40-kDa transactivator protein (Tax) from the type I human T-cell leukemia virus (HTLV-I) in Jurkat T cells leads to the activation and sustained expression of certain cellular genes that are transiently induced during normal T-cell growth. Cellular genes induced by Tax include those encoding the alpha subunit of the high-affinity interleukin 2 receptor (Tac), interleukin 2, and granulocyte/macrophage colony-stimulating factor. Tax induction of the interleukin 2 gene is synergistically amplified by mitogens that augment cytoplasmic levels of calcium. These changes in the pattern of cellular gene expression reflect a specific action of Tax, as they are undetectable in isogenically matched control cell lines expressing antisense tax cDNA. The spectrum of cellular genes regulated by Tax appears to be restricted: several other T-cell genes, either inducibly or constitutively expressed, are unaffected by this viral protein. These cell lines constitutively expressing Tax provide valuable reagents to explore the molecular basis for Tax action and to delineate the full spectrum of cellular genes regulated by this retroviral gene product.

Animals↗
PubMed1996

Role of the ceramide-signaling pathway in cytokine responses to P-fimbriated Escherichia coli.

Escherichia coli express fimbriae-associated adhesins through which they attach to mucosal cells and activate a cytokine response. The receptors for E. coli P fimbriae are the globoseries of glycosphingolipids; Gal alpha 1-->4Gal beta-containing oligosaccharides bound to ceramide in the outer leaflet of the lipid bilayer. The receptors for type 1 fimbriae are mannosylated glycoproteins rather than glycolipids. This study tested the hypothesis that P-fimbriated E. coli elicit a cytokine response through the release of ceramide in the receptor-bearing cell. We used the A498 human kidney cell line, which expressed functional receptors for P and type 1 fimbriae and secreted higher levels of interleukin (IL)-6 when exposed to the fimbriated strains than to isogenic nonfimbriated controls. P-fimbriated E. coli caused the release of ceramide and increased the phosphorylation of ceramide to ceramide 1-phosphate. The IL-6 response to P-fimbriated E. coli was reduced by inhibitors of serine/threonine kinases but not by other protein kinase inhibitors. In contrast, ceramide levels were not influenced by type 1-fimbriated E. coli, and the IL-6 response was insensitive to the serine/threonine kinase inhibitors. These results demonstrate that the ceramide-signaling pathway is activated by P-fimbriated E. coli, and that the receptor specificity of the P fimbriae influences this process. We propose that this activation pathway contributes to the cytokine induction by P-fimbriated E. coli in epithelial cells.

Animals↗
PubMed1989

A recombinational hotspot at the triplo-lethal locus of Drosophila melanogaster.

In the genome of Drosophila melanogaster there is only one locus, Tpl, that is triplo-lethal; it is also haplo-lethal. Previous work has identified 3 hypomorphic alleles of Tpl which rescue animals carrying a duplication of Tpl, but which are not dominant lethals as null mutations or deficiencies would be. We have found that all three hypomorphic alleles act as site-specific hotspots for recombination when heterozygous with a wild-type homolog. Recombination between the flanking markers ri and Ki is increased 6.5-10.5-fold in the presence of Tpl hypomorphic alleles. The increased recombination was found to occur between Tpl and Ki, while recombination in other adjacent regions is unchanged. The use of isogenic Tpl+ controls, and the use of flanking intervals in the mutant chromosomes allows us to rule out the interchromosomal effect as a cause. We have also observed premeiotic recombination occurring at the Tpl hypomorphic alleles in male heterozygotes. We hypothesize that transposons are responsible for both the hypomorphic phenotype and the high frequency of recombination.

Alleles↗
PubMed1999

Symposium overview: Characterization of xenobiotic metabolizing enzyme function using heterologous expression systems.

Genetically modified cell lines can be very useful models for assessing the toxicologic effects of modulation of expression of individual gene products in comparison to their isogenic parental control cell lines. This symposium begins with an overview of general issues related to development and utilization of model systems created by transfection of cell lines to induce elevated expression of metabolic enzymes of toxicologic relevance. Selected studies that illustrate the heterologous expression rationale and various approaches to transgenic-cell model construction are represented. Results to date with cells engineered to express specific transfected genes are discussed, with emphasis on the effects of expression of selected phase I or phase II enzymes on cellular sensitivity to several toxic end-points. The individual sections highlight the utility of these model cell lines for examining the role of enzyme catalysis and function in metabolism of biologically active xenobiotic or endobiotic compounds of interest in toxicology. Both activating and detoxifying enzymes are discussed, with principal emphasis on the latter. This symposium includes talks on transfected cells that express aldehyde dehydrogenases, superoxide dismutase, UDP-glycosyltransferases, glutathione transferases, and cytochrome P450 isozymes. In addition to the general toxicologic utility and advantages of these genetically engineered cell lines, this overview emphasizes their particular contributions to the insights obtained to date with the specific model cell lines.

Animals↗
PubMed1997

Mice overexpressing human lecithin: cholesterol acyltransferase are not protected against diet-induced atherosclerosis.

Lecithin: cholesterol acyltransferase (LCAT) (EC 2.3.1.43) is generally assumed to participate in reverse cholesterol transport, i.e., cholesterol transport from peripheral tissues to the liver. LCAT is secreted by the liver and transported in plasma mostly associated with high density lipoprotein. It catalyzes the esterification of cholesterol, mainly high density lipoprotein cholesterol, and produces cholesteryl ester and lysolecithin. Transgenic mice overexpression human LCAT on a C57BL/6 background have elevated high density lipoprotein cholesterol and markedly reduced low and very low density lipoprotein cholesterol and triglyceride levels in plasma, suggesting that such mice may be less susceptible to diet-induced atherosclerosis than isogenic nontransgenic controls. To determine if the apparent anti-atherogenic lipoprotein profile of the LCAT transgenics reduced their susceptibility to atherogenesis, the atherosclerotic lesions developing in transgenic LCAT mice and controls when fed an atherogenic diet were compared by histology and morphometry. Histological examination of the aortas from mice fed a high fat diet for 12, 17 and 22 weeks revealed that the aortic lesions were no smaller or less developed in the transgenic LCAT mice than in the C57BL/6 controls. After 17 weeks there were significantly more "fatty streaks" in the transgenic mice than in the controls. Thus, overexpression of human LCAT in transgenic mice, in spite of their very favourable blood lipoprotein and lipid profile, does not protect against development of atherosclerosis.

Animals↗
PubMed2007

Native microbial colonization of Drosophila melanogaster and its use as a model of Enterococcus faecalis pathogenesis.

Enterococci are commensal organisms of the gastrointestinal (GI) tracts of a broad range of mammalian and insect hosts, but they are also leading causes of nosocomial infection. Little is known about the ecological role of enterococci in the GI tract consortia. To develop a tractable model for studying the roles of these organisms as commensals and pathogens, we characterized the Drosophila melanogaster microflora and examined the occurrence of enterococci in the gastrointestinal consortium of Drosophila. In a survey of laboratory-reared Drosophila and wild-captured flies, we found that Drosophila was naturally colonized by representatives of five bacterial phyla. Among these organisms were several species of enterococci, including Enterococcus faecalis, Enterococcus faecium, Enterococcus gallinaraum, and Enterococcus durans, as well as a previously detected but uncultured Enterococcus species. Drosophila could be cured of enterococcal carriage by antibiotic treatment and could be reassociated with laboratory strains. High-level colonization by a well-characterized strain expressing the enterococcal cytolysin was found to be detrimental to Drosophila compared to the effect of an isogenic, noncytolytic control. The anatomical distribution of enterococci in the Drosophila GI tract was determined by immunohistochemical staining of thin sections of naturally colonized and reassociated flies.

Animals↗
PubMed1980

Regulation of nitrogen utilization of hisT mutants of Salmonella typhimurium.

Mutations in the hisT gene of Salmonella typhimurium alter pseudouridine synthetase I, the enzyme that modifies two uridines in the anticodon loop of numerous transfer ribonucleic acid species. We have examined two strains carrying different hisT mutations for their ability to grow on a variety of nitrogen sources. The hisT mutants grew more rapidly than did hisT+ strains with either arginine or proline as the nitrogen source and glucose as the carbon source. The hisT mutations were transduced into new strains to show that these growth properties were due to the hisT mutations. The hisT mutations did not influence the growth of mutants having altered glutamine synthetase regulation. Assays of the three primary ammonia-assimilatory enzymes, glutamate dehydrogenase, glutamine synthetase, and glutamate synthase, showed that glutamate synthase activities were lower in hisT mutants than in isogenic hisT+ controls; however, the glutamate dehydrogenase activity was about threefold higher in the hisT strains grown in glucose-arginine medium. The results suggest that the controls for enzyme synthesis for nitrogen utilization respond either directly or indirectly to transfer ribonucleic acid species affected by the hisT mutation.

Glutamate Dehydrogenase↗
PubMed2026

A guide to selecting high-performing antibodies for TMEM175 (UniProt ID: Q9BSA9) for use in western blot, immunoprecipitation, and immunofluorescence.

TMEM175 is the pore-forming subunit of a lysosomal K+ channel complex that regulates lysosomal pH stability and membrane potential. To further investigate its cellular functions and implications in neurodegenerative diseases, antibody reagents are needed. Here we have characterized six TMEM175 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Humans↗
PubMed2026

A guide to selecting high-performing antibodies for DJ-1 ( PARK7) (Q99497) for use in western blot, immunoprecipitation, and immunofluorescence.

DJ-1 is a multifunctional protein that plays a pivotal role in cellular protection against oxidative stress and neurodegeneration. Mutations in the PARK7 gene are associated with early-onset familial Parkinson's disease. Here we have characterized sixteen DJ-1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Protein Deglycase DJ-1↗
PubMed2026

A guide to selecting high-performing antibodies for Syntenin-1 (O00560) for use in western blot, immunoprecipitation, and immunofluorescence.

Syntenin-1 is the Syndecan-binding protein 1 and a PDZ domain-containing adaptor protein that regulates diverse cellular processes through its interactions with transmembrane receptors, cytoskeletal components, and signaling molecules. Here we have characterized twelve Syntenin-1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Syntenins↗
PubMed2000

Effects of brown midrib 3 mutation in corn silage on productivity of dairy cows fed two concentrations of dietary neutral detergent fiber: 1. Feeding behavior and nutrient utilization.

The effects of digestibility of corn silage neutral detergent fiber (NDF) and dietary NDF content on feeding behavior, dry matter intake (DMI), and energy utilization were evaluated with eight multiparous high producing dairy cows in a duplicated 4 x 4 Latin square design with 21-d periods. Experimental diets contained corn silage from a brown midrib (bm3) hybrid or its isogenic normal control at two concentrations of dietary NDF (29 and 38%). Both low NDF diets and bm3 corn silage treatments increased DMI and milk yield. However, an interaction between corn silage treatment and dietary NDF content was observed for meal size and for interval between meals, implying that different mechanisms regulating DMI dominate depending on the fermentation characteristics of diets. The bm3 treatment depressed milk fat concentration when fed in a low NDF diet. The bm3 corn silage increased solids-corrected milk yield, but did not affect daily body weight gain compared with control, whereas low NDF diets increased daily body weight gain, but did not affect solids-corrected milk yield compared to high NDF diets. Both bm3 treatments and low NDF diets reduced ruminal pH, but low NDF treatments increased fluctuation of ruminal pH and bm3 treatments did not. Feeding bm3 corn silage increased the energy utilized for milk production, possibly because of a consistent supply of metabolic fuels from the rumen. The beneficial effects of bm3 corn silage on productivity of lactating cows were greater for the cows fed a high NDF diet.

Animals↗
PubMed2000

Effects of brown midrib 3 mutation in corn silage on productivity of dairy cows fed two concentrations of dietary neutral detergent fiber: 2. Chewing activities.

The effects of digestibility of corn silage neutral detergent fiber (NDF) and dietary NDF content on chewing activities were evaluated with eight multiparous high producing dairy cows in a duplicated 4 x 4 Latin square design with 21-d periods. Experimental diets contained corn silage from a brown midrib (bm3) hybrid or its isogenic normal control at two concentrations of dietary NDF (29 and 38%). The NDF digestibility estimated by 30-h in vitro fermentation was higher for bm3 corn silage by 9.4 units (55.9 vs. 46.5%). Feeding behavior of animals and ruminal pH were monitored continuously for 4 d in each period by a computerized data acquisition system. Ruminal digesta were evacuated twice per period to determine the ruminal NDF pool and its turnover rate. There was no effect of NDF digestibility on total chewing time either per day or per kilogram of NDF intake, or on ruminating time either per day or per kilogram of NDF intake. Although bm3 treatments increased the turnover rate of NDF in the rumen, they increased dry matter intake and had no effect on ruminal NDF pool size. Total chewing time and ruminating time per day were related positively to NDF intake and ruminal NDF pool size, but were not related to ruminal turnover rate of NDF. These results provide no evidence that enhanced NDF digestibility decreases the physical effectiveness of NDF of corn silage.

Animals↗
PubMed2000

Effects of brown midrib 3 mutation in corn silage on productivity of dairy cows fed two concentrations of dietary neutral detergent fiber: 3. Digestibility and microbial efficiency.

The effects of digestibility of corn silage neutral detergent fiber (NDF) and dietary NDF content on ruminal digestion kinetics, site of nutrient digestion, and microbial N production efficiency were evaluated with eight multiparous high producing dairy cows in a duplicated 4 x 4 Latin square design with 21-d periods. Experimental diets contained corn silage from a brown midrib (bm3) hybrid or its isogenic normal control at two concentrations of dietary NDF (29 and 38%). The NDF digestibility estimated by a 30-h in vitro fermentation was higher for bm3 corn silage by 9.4 units (55.9 vs. 46.5%). Neither ruminal nor total tract NDF digestibility was affected by corn silage treatment. The bm3 corn silage diet decreased starch digestibility in the rumen and in the total tract, but increased postruminal starch digestibility compared with control diet. The bm3 corn silage diets increased microbial N flow to the duodenum and tended to decrease ruminal ammonia concentration. Microbial efficiency was greater for cows fed bm3 corn silage in spite of lower ruminal pH. Higher efficiency of microbial nitrogen production might be attributed to faster passage rate of NDF for cows fed bm3 corn silage compared with those fed control corn silage. Higher in vitro NDF digestibility might predict enhanced NDF fragility and ease of NDF hydrolysis in vivo. Enhanced in vitro NDF digestibility does not necessarily result in increased NDF digestibility either in the rumen or in the total tract, but possibly increases rate of passage and DMI, improving efficiency of microbial N production.

Animal Feed↗