Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue.
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We recently generated a transgenic mouse line overexpressing spermidine/spermine N1-acetyltransferase (SSAT) gene under its own promoter. The tissue polyamine pools of these animals were profoundly affected and the mice were hairless from early age. We have now generated another transgenic-mouse line overexpressing the SSAT gene under the control of a heavy-metal-inducible mouse metallothionein I (MT) promoter. Even in the absence of heavy metals, changes in the tissue polyamine pools indicated that a marked activation of polyamine catabolism had occurred in the transgenic animals. As with the SSAT transgenic mice generated previously, the mice of the new line (MT-SSAT) suffered permanent hair loss, but this occurred considerably later than in the previous SSAT transgenic animals. Liver was the most affected tissue in the MT-SSAT transgenic animals, revealed by putrescine overaccumulation, significant decrease in spermidine concentration and >90% reduction in the spermine pool. Even though hepatic SSAT mRNA accumulated to massive levels in non-induced transgenic animals, SSAT activity was only moderately elevated. Administration of ZnSO4 further elevated the level of hepatic SSAT message and induced enzyme activity, but not more than 2- to 3-fold. Treatment of the transgenic animals with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) resulted in an immense induction, more than 40000-fold, of enzyme activity in the liver of transgenic animals, and minor changes in the SSAT mRNA level. Liver spermidine and spermine pools were virtually depleted within 1-2 days in response to the treatment with the analogue. The treatment also resulted in a marked mortality (up to 60%) among the transgenic animals which showed ultrastructural changes in the liver, most notably mitochondrial swelling, one of the earliest signs of cell injury. These results indicated that, even without its own promoter, SSAT is powerfully induced by the polyamine analogue through a mechanism that appears to involve a direct translational and/or heterogenous nuclear RNA processing control. It is likewise significant that overexpression of SSAT renders the animals extremely sensitive to polyamine analogues. (+info)
Metallothionein-null mice absorb less Zn from an egg-white diet, but a similar amount from solutions, although with altered intertissue Zn distribution.
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The influence of metallothionein (MT) on Zn transfer into non-gut tissues was investigated in MT-null (MT-/-) and normal (MT+/+) mice 4 h after oral gavage of aqueous 65ZnSO4solution at doses of 154, 385, 770 and 1540 nmol Zn per mouse. Zn transfer was not significantly different between MT+/+ and MT-/- mice and was directly proportional to the oral dose (slope = 0.127, r = 0.991; 0. 146, r = 0.994, respectively). Blood 65Zn and plasma Zn concentrations increased progressively in MT-/- mice at doses >154 nmol Zn, reaching levels of 2.4% of oral dose and 60 micromol/L, respectively, at the 1540 nmol Zn dose. The corresponding values for MT+/+ mice were approximately half, 1.0% and 29 micromol/L. Intergenotypic differences were found in tissue distribution of 65Zn within the body; MT-/- mice had higher 65Zn levels in muscle, skin, heart and brain, whereas MT+/+ mice retained progressively more Zn in the liver, in conjunction with a linear increase in hepatic MT up to the highest Zn dose. MT induction in the small intestine reached its maximum at an oral dose of 385 nmol Zn and did not differ at higher doses. Absorption of a 770 nmol 65Zn dose from a solid egg-white diet was only one fourth (MT+/+) and one eighth (MT-/-) of the Zn absorption from the same dose of 65Zn in aqueous solution. MT+/+ mice had greater (P < 0.05) Zn absorption from the egg-white diet than did MT-/- mice, indicating that gut MT confers an absorptive advantage, but only when Zn is incorporated into solid food. (+info)
Overexpression of the multidrug resistance-associated protein (MRP1) in human heavy metal-selected tumor cells.
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Cellular and molecular mechanisms involved in the resistance to cytotoxic heavy metals remain largely to be characterized in mammalian cells. To this end, we have analyzed a metal-resistant variant of the human lung cancer GLC4 cell line that we have selected by a step-wise procedure in potassium antimony tartrate. Antimony-selected cells, termed GLC4/Sb30 cells, poorly accumulated antimony through an enhanced cellular efflux of metal, thus suggesting up-regulation of a membrane export system in these cells. Indeed, GLC4/Sb30 cells were found to display a functional overexpression of the multidrug resistance-associated protein MRP1, a drug export pump, as demonstrated by Western blotting, reverse transcriptase-polymerase chain reaction and calcein accumulation assays. Moreover, MK571, a potent inhibitor of MRP1 activity, was found to markedly down-modulate resistance of GLC4/Sb30 cells to antimony and to decrease cellular export of the metal. Taken together, our data support the conclusion that overexpression of functional MRP1 likely represents one major mechanism by which human cells can escape the cytotoxic effects of heavy metals. (+info)
Polaprezinc protects gastric mucosal cells from noxious agents through antioxidant properties in vitro.
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BACKGROUND: Polaprezinc has been shown to exert an anti-oxidant property in a tube experiment, protect gastric mucosa from experimental ulcerations in vivo, and accelerate the healing of gastric ulcer in humans. AIM: To examine a possible protective effect of polaprezinc on oxidant-mediated injury in primary monolayer cultures of rat gastric fundic mucosa. METHODS: Cytotoxicity was quantified by measuring 51Cr release. Whether or not polaprezinc exerts an antioxidant property was investigated by determining the effect of this agent on hydrogen peroxide (H2O2)-induced injury. The effects of polaprezinc on superoxide (O2-. ) generation as well as on ethanol (EtOH)-induced injury were also examined. Generation of O2-. was assessed by the reduction in cytochrome c. RESULTS: H2O2 caused a time- and dose-dependent increase in 51Cr release. The dose-response curve of 51Cr release by H2O2 shifted to the right in the presence of polaprezinc. Polaprezinc, at submillimolar concentrations, prevented H2O2-induced 51Cr release. EtOH also caused a dose-dependent increase in 51Cr release, which was prevented by the addition of polaprezinc. The incubation of cells with EtOH caused an increase in cytochrome c reduction, as the concentrations of EtOH increased. Polaprezinc inhibited EtOH-induced cytochrome c reduction. Protection by polaprezinc was microscopically associated with the prevention of monolayer disruption. CONCLUSIONS: Polaprezinc is antioxidative and directly protects gastric mucosal cells from noxious agents through its antioxidant properties in vitro. This finding may provide the theoretical basis for the usage of an antiulcer drug with antioxidant properties for the treatment of gastric inflammation, such as that induced by ethanol. (+info)
Factors affecting dimensional instability of alginate impressions during immersion in the fixing and disinfectant solutions.
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To clarify the factors determining the dimensional stability of alginate impressions during immersion in disinfectant and fixing solution, the weight change of impressions in solutions of glutaraldehyde (GA), NaClO, Na2SO4, K2SO4, CaCl2, and ZnSO4 was measured. In the nonelectrolytic solution, GA, the weight decreased in proportion to concentration, possibly due to the gradient of osmotic pressure between the impression and solution. In monovalent metallic salt solutions the weight change decreased with increased concentration. Especially at lower concentrations the rate of weight loss was high. A chemical action of the solution might also be involved, in addition to the osmotic pressure difference. The weight loss in divalent metallic salt solutions was greater than in monovalent solutions, implicating crosslinking reactions between the impression and solution. (+info)
Bioavailability of zinc in several sources of zinc oxide, zinc sulfate, and zinc metal.
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Three zinc depletion-repletion assays were carried out with chicks to determine Zn bioavailability in five sources of ZnO, three sources of ZnSO4.H2O, and two sources of Zn metal. A standard 23% CP corn-soybean meal diet was fed during the first 3 d posthatching, after which it was replaced with a Zn-deficient soy concentrate diet (13.5 mg Zn/kg) until d 7. On d 8 after an overnight period of feed withdrawal, chicks were fed for 12 d the Zn-deficient basal diet containing 0, 4.76, and 9.90 (Assay 1); 0, 5.06, or 10.12 (Assay 2); or 0, 4.73, or 9.13 (Assay 3) mg/kg supplemental Zn from analytical grade (AG) ZnSO4.7H2O (22.7% Zn) to generate a standard response curve. The AG and feed-grade (FG) Zn sources being evaluated were then provided at a level that would fall within the standard curve. Weight gain (Assays 1, 2, and 3) and total tibia Zn (Assay 1) responded linearly (P<.01) to Zn supplementation from ZnSO4.7H2O. Weight gain regressed on supplemental Zn intake gave standard-curve equations with fits (r2) ranging from .94 to .97. In Assay 1, regression of total tibia Zn (Y, in micrograms) on supplemental Zn intake (X, in milligrams/12 d) gave the equation Y = 13.2+6.74X (r2 = .90). Standard-curve methodology was used to estimate relative Zn bioavailability (RBV), with RBV of Zn in the ZnSO4.7H2O standard set at 100%. Four sources of FG ZnO were evaluated: Source 1 (78.1% Zn, hydrosulfide process, U.S.), Source 2 (74.1% Zn, Waelz process, Mexico), Source 3 (69.4% Zn, China), and Source 4 (78.0% Zn, French process, Mexico). Analytical-grade ZnO (80.3% Zn) was also evaluated. Feed-grade ZnO Sources 1 and 4 as well as AG ZnO produced average RBV values that were not different (P>.10) from the standard, but average RBV values for FG Source 2 and FG Source 3 were only 34 (P<.05) and 46% (P<.05), respectively. All sources of ZnSO4.H2O, which included two FG sources (source 1, 36.5% Zn; source 2, 35.3% Zn) and one food-grade source (36.5% Zn), were not different (P>.10) in RBV from the ZnSO4.7H2O standard. Two Zn metal products, Zn metal dust (100% Zn) and Zn metal fume (91.5% Zn), were also evaluated, and they were found to have Zn RBV values of 67 (P<.05) and 36% (P<.05), respectively. Feed-grade sources of ZnO vary widely in color, texture, Zn content, and Zn bioavailability. (+info)
The caudal-related homeodomain protein Cdx1 inhibits proliferation of intestinal epithelial cells by down-regulation of D-type cyclins.
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Cdx1 is a homeodomain transcription factor that regulates intestine-specific gene expression. Experimental evidence suggests that Cdx1 may be involved in cell cycle regulation, but its role is ill defined and the mechanisms have not been explored. We used stable transfection of inducible constructs and transient expression with a replication-deficient adenovirus to induce Cdx1 expression in rat IEC6 cells, a non-transformed intestinal epithelial cell line that does not express Cdx1 protein. Expression of Cdx1 markedly reduced proliferation of IEC6 cells with accumulation of cells in the G(0)/G(1) phase of the cell cycle. Cell cycle arrest was accompanied by an increase in the hypophosphorylated forms of the retinoblastoma protein (pRb) and the pRb-related p130 protein. Protein levels of multiple cyclin-dependent kinase inhibitors were either unchanged (p16, p18, p21, p27, and p57) or were not detected (p15 and p19). Most significantly, levels of cyclins D1 and D2 were markedly diminished with Cdx1 expression, but not cyclins D3, E, or the G(1) kinases. Additionally, cyclin-dependent kinase-4 activity was decreased in association with decreased cyclin D protein. We conclude that Cdx1 regulates intestinal epithelial cell proliferation by inhibiting progression through G(0)/G(1), most likely via modulation of cyclin D1 and D2 protein levels. (+info)
Zn-depleted mice absorb more of an intragastric Zn solution by a metallothionein-enhanced process than do Zn-replete mice.
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The influence of metallothionein (MT)(2) on Zn absorption was investigated in MT-null (MT-/-) and normal (MT+/+) mice fed Zn-depleted (ZnD) diets for 7 d and compared with those fed Zn-replete (ZnR) diets in a previous study. Mice were starved for 20 h, then administered an oral gavage of aqueous (65)ZnSO(4) solution at doses of 154, 770 or 1540 nmol of Zn, and the amount transferred into nongut tissues was determined 4 h later. (65)Zn transfer did not differ between genotypes in ZnR mice. However ZnD MT+/+ mice had a 30-40% greater transfer from the 154 and 770 Zn doses compared to ZnR MT+/+ mice. This was not observed in MT-/- mice. In MT+/+ mice, Zn depletion enhanced the induction of MT by Zn in the intestine and pancreas. (65)Zn uptakes in the liver and pancreas were greater in MT+/+ than MT-/- mice, and this was greater (50%) at the 154 and 770 doses in mice fed ZnD diets. Plasma Zn concentrations were raised to a similar extent in ZnR and ZnD MT-/- mice. ZnR MT+/+ mice had significantly lower plasma Zn levels than MT-/-mice; this difference was less marked in the ZnD mice. We conclude that a MT-facilitated enhancement in Zn absorption occurs in response to dietary Zn deficiency. (+info)