Correlation of neomycin, faecal neutral and acid sterols with colon carcinogenesis in rats.
(9/850)
High fat diets have been implicated in incidence of colon cancer both in epidemiological and animal studies. Present investigation deals with the incidence, location and numbers of large and small bowel tumours induced by 1,2-dimethyl hydrazine (DMH) in rats fed high fat diets and neomycin. Neomycin was used to modify the faecal sterol metabolism and the relationship of the high fat diet and faecal neutral and acid sterols to the large bowel tumorigenesis was evaluated. DMH administered rats were fed with (a) 20% safflower oil; (b) 20% safflower oil and neomycin; (c) 20% safflower oil, cholesterol and cholic acid; and (d) 20% safflower oil, cholesterol, cholic acid and neomycin. Neomycin was found to be associated with both increase and decrease of tumour numbers. The faecal sterols lithocholic and deoxycholic acids were found to have no participation, while cholesterol and cholic acid were found to decrease with increase in tumour numbers. However, faecal coprostanol has been found to have a significant positive correlation with tumorigenesis in all dietary groups. Therefore coprostanol might possibly be associated with colon carcinogenesis in DMH-fed rats and cholesterol metabolism in gut appears to be related to the development of tumours. (+info)
Enhancer-blocking activity within the DNase I hypersensitive site 2 to 6 region between the TCR alpha and Dad1 genes.
(10/850)
Although tightly linked, the TCR alpha and delta genes are expressed specifically in T lymphocytes, whereas the Dad1 gene is ubiquitously expressed. Between TCR alpha and Dad1 are eight DNase I hypersensitive sites (HS). HS1 colocalizes with the TCR alpha enhancer (Ealpha) and is T cell-specific; HS2, -3, -4, -5, and -6 map downstream of HS1 and are tissue-nonspecific. The region spanning HS2-6 was reported to display chromatin-opening activity and to confer copy number-dependent and integration site-independent transgene expression in transgenic mice. Here, we demonstrate that HS2-6 also displays enhancer-blocking activity, as it can block an enhancer from activating a promoter when located between the two in a chromatin-integrated context, and can do so without repressing either the enhancer or the promoter. Multiple enhancer-blocking elements are arrayed across HS2-6. We show that HS2-6 by itself does not activate transcription in chromatin context, but can synergize with an enhancer when located upstream of an enhancer and promoter. We propose that HS2-6 primarily functions as an insulator or boundary element that may be critical for the autonomous regulation of the TCR alpha and Dad1 genes. (+info)
Variation in the properties of a strain of Staphylococcus aureus isolated over three months from a single hospital.
(11/850)
A strain of Staphylococcus aureus has been isolated from a hospital environment over 3 months. Every isolate was lysed by phage 77, had high-level resistance to streptomycin, and was resistant to about 250 pg per ml of both tetracycline and sulphonamide; a combination of sulphamethoxazole and trimethoprim produced little bacteristatic synergy towards each isolate. All These organisms were thus considered to be "the same"; the variation in other properties was probably due to rapid evolutionary change in vivo. the variation in senxitivity to methicillin and neomycin, and the absence of penicillinase production in some isolates, probably indicated loss of the relevant genes. Several isolates had probably acquired resistance to lincomycin by a one-step mutatuon in vivo. The usefulness of lincomycin and analogues in treating staphylococcal infections seems limited. (+info)
Calcium release from InsP3-sensitive internal stores initiates action potential in Chara.
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Neomycin and U73122 are known to suppress inositol 1,4,5-trisphosphate (InsP3) production by inhibition of phospholipase C. We studied the effects of these inhibitors on the excitatory currents, Iex, in Chara corallina under voltage-clamp conditions. Computer simulations of the experimental effects by a minimum model for the excitatory reaction pathway allow the assignment of the inhibitory effects to one specific reaction step, i.e. the release of Ca2+ from InsP3-sensitive internal stores. In contrast, the inhibitory effect of La3+ on Iex suggests inactivation of Cl- channels. Furthermore, ryanodine-sensitive Ca2+ stores seem to be irrelevant for electrical excitation in Chara. (+info)
Transfer of activation-dependent gene expression into T cell lines by recombinant adeno-associated virus.
(13/850)
We examined the ability of recombinant adeno-associated virus (rAAV) to transfer regulated gene expression into T cell lines. An AAV-based vector containing the neomycin resistance gene and expressing the firefly luciferase (luc) gene under the regulatory control of the interleukin 2 promoter (pAAV-luc) was generated and adenovirus-free rAAV (rAAV-luc) was produced from this vector. Transfection of pAAV-luc into the human T cell line Jurkat resulted in luciferase expression while infection of Jurkat T cells with rAAV-luc resulted in significant luciferase expression only after selection for neomycin-resistant cells. Long-term growth of transduced Jurkat T cells showed that there was no detectable constitutive expression of luciferase and that luciferase gene expression remained inducible for at least 180 days. Luciferase expression was activated by PMA and ionomycin and by anti-CD3 antibodies and was inhibited by cyclosporin A. Examination of G418-resistant clones showed that rAAV-luc had integrated into the host chromosomes but that some of the clones lost some of the transferred DNA or lost expression from the transferred DNA. These results indicate that rAAV can transfer and integrate regulated gene expression into T cell lines but that the transferred genetic material may be lost or its expression may be silenced over time. (+info)
Factors affecting de novo methylation of foreign DNA in mouse embryonic stem cells.
(14/850)
Integration of foreign DNA into an established host genome can lead to changes in methylation in both the inserted DNA and in host sequences and potentially alters transgene and cellular transcription patterns. This work addresses the questions of what factors influence de novo methylation, and whether the integration site or inserted DNA can affect de novo methylation. Homologous recombination was used to integrate foreign DNA into a specific gene, B lymphocyte kinase (BLK), in mouse embryonic stem (ES) cells. Two plasmids were chosen for integration; one contained the adenovirus type 2 E2AL promoter upstream of the luciferase reporter gene, and the second carried the early SV40 promoter. The methylation patterns were analyzed using HpaII and MspI restriction endonucleases for both homologously recombined and randomly integrated foreign DNA in the ES cell clones. Upon homologous reinsertion of the BLK gene into the genome of mouse ES cells, methylation patterns in this gene were reestablished. In DNA segments adjoined to the BLK gene, the de novo patterns of DNA methylation depended on the viral sequences in these clones and on the locations of the inserts, i.e. on whether the insertions resulted from homologously recombined or randomly integrated foreign DNA. In homologously recombined DNA, sequences carrying the adenovirus type 2 promoter were heavily methylated, and those with an SV40 promoter and an SV40 enhancer element remained unmethylated or hypomethylated. Upon removal of the enhancer element, these inserted constructs also became heavily methylated. In addition, all randomly integrated constructs were heavily methylated independently of the promoter and enhancer element present in the construct. These results indicate that modes and sites of integration as well as the inserted nucleotide sequence, possibly promoter strength, are factors affecting de novo methylation. (+info)
Simultaneous Cre catalyzed recombination of two alleles to restore neomycin sensitivity and facilitate homozygous mutations.
(15/850)
Cells homozygous for neo-expressing mutations can be derived by culturing heterozygotes with elevated G418. We demonstrate that this strategy is significantly less efficient if hyg is substituted for neo. Therefore, to introduce additional mutations Cre recombinase was used to remove floxed neo from both alleles of homozygotes at two different loci. The rate-determining step in Cre excision appeared independent of substrate copy number. Incorporating cytosine deaminase and Herpes simplex virus thymidine kinase allowed negative selection for both targeting and Cre excision. The resulting G418-sensitive homozygous mutants should allow mutagenesis at additional loci and avoid untoward effects of retained selection markers. (+info)
Mechanism of radiation-induced diacylglycerol production in primary cultured rat hepatocytes.
(16/850)
Protein kinase C (PKC) is known to be a key enzyme in radiation-induced signal transduction pathways. We have previously demonstrated that gamma-irradiation induces PKC activation and translocation from cytosol to membranes as a consequence of membrane lipid peroxidation in cultured rat hepatocytes (Int. J. Radiat. Biol. 70, 473-480, 1996). The present study was undertaken to investigate production of diacylglycerol, an endogenous activator of PKC, following gamma-irradiation of hepatocytes. Diacylglycerol content increased 3 min after irradiation, then decreased at 15 min and increased again at 30 min, indicating a biphasic pattern. This result implies participation of diacylglycerol in the radiation-induced activation of PKC in hepatocytes. In order to clarify the mechanism of the initial process of radiation-induced diacylglycerol production, the effects of reactive oxygens were investigated. Treatment of cells with hydroxyl radical, a major oxygen radical produced by radiation, induced diacylglycerol production without any change in the content of phosphatidylcholine, showing a peak at 1 min after treatment. No change in the diacylglycerol content was observed at that time by hydrogen peroxide treatment. Furthermore, the diacylglycerol production by hydroxyl radical was inhibited by pretreatment with neomycin sulfate, a phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor. These results suggest that radiation exerts PI-PLC activation through hydroxyl radical generation, followed by diacylglycerol production and PKC activation. (+info)