Complex modular cis-acting elements regulate expression of the cardiac specifying homeobox gene Csx/Nkx2.5.
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The murine homeobox gene Csx/Nkx2.5 is an evolutionarily highly conserved gene related to the Drosophila tinman gene, which specifies cardiac and visceral mesoderm. Since Csx/Nkx2.5 plays an essential role in heart development, studying its regulation is essential for the better understanding of molecular mechanisms of cardiogenesis and the pathogenesis of congenital heart disease in humans. In this study, we characterized the murine Csx/Nkx2.5 gene and identified two novel untranslated exons, 1a, and 1b, resulting in three different Csx/Nkx2.5 transcripts. To examine the tissue-specific transcriptional regulation in vivo, we analyzed a total of 23 kb of Csx/Nkx2.5 upstream and downstream sequences by generating transgenic embryos carrying lacZ reporter constructs containing various lengths of flanking sequence. With 14 kb of 5' flanking sequence, lacZ expression was observed in the cardiac crescent at E7.5, and in the outflow tract, the interatrial groove, the atrioventricular canal and right and left ventricles, as well as in pharyngeal floor, thyroid primordia, and stomach at E10.5. In adult animals, lacZ expression of the transgene was limited to the atrioventricular junction and the subendocardium of the ventricular septum. Reducing the size of flanking sequence to 3.3 kb of intron 2 restricted lacZ expression to the outflow tract and the basal part of the right ventricle in E10.5 embryos. In contrast, the addition of 6 kb of 3' flanking sequence caused strong expression of the reporter gene in the entire right ventricle. Interestingly, Csx/Nkx2. 5 seems to be negatively regulated by its own gene product, because when lacZ was "knocked-in" to replace the entire coding exons, lacZ expression was much higher in the heart of homozygous embryos than that in the heterozygote. These results indicate that the transcriptional regulatory elements of Csx/Nkx 2.5 seems unexpectedly highly modular, and is temporally regulated in a dynamic manner by different enhancer regions. Since Csx/Nkx2.5-like genes are expressed in all species having a heart, their complex modular organization with multiple enhancers probably reflects progressive addition of regulatory elements during the evolution from a simple heart tube to a complex four-chambered organ. (+info)
Caspase-1 is not involved in experimental hepatitis in mouse.
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Experimental hepatitis induced by tumor necrosis factor in D-(+)-galactosamine-sensitized mice or by an agonistic anti-Fas antibody in normal mice is accompanied by dramatic apoptosis of hepatocytes. Apoptosis is the final result of activation of a cascade of caspases. We used caspase-1-/- mice, generated by gene targeting, to study the role of this protease in TNF- and anti-Fas-induced lethal hepatitis. We found that mutant mice exhibited the typical caspase-1-/- phenotype, since they resisted to a lethal injection of LPS and released no interleukin-1beta in the circulation, in contrast to wild-type littermates. When caspase-1-/- mice were challenged with different doses of tumor necrosis factor/D-(+)-galactosamine or with anti-Fas, no increased survival was observed compared with control mice. Furthermore, apoptosis in the livers of these mice and serum levels of alanine aminotransferase were not reduced. These data indicate that caspase-1 deficiency does not lead to reduced apoptosis in these models, either because caspase-1 is irrelevant in this model or because of functional redundancy. (+info)
Fc receptor beta subunit is required for full activation of mast cells through Fc receptor engagement.
(75/61166)
The high-affinity IgE receptor (Fc epsilonRI) and the low-affinity IgG receptor (Fc gammaRIII) on mast cells are the key molecules involved in triggering the allergic reaction. These receptors share the common beta subunit (FcRbeta) which contains an immunoreceptor tyrosine-based activation motif and transduces the signals of these receptors' aggregation. In rodents, FcRbeta is essential for the cell surface expression of the Fc epsilonRI. In humans, the FcRbeta gene was reported to be one of the candidate genes causing atopic diseases. However, the role of FcRbeta in vivo still remains ambiguous. To elucidate the functions of FcRbeta, we developed the mice lacking FcRbeta [FcRbeta(-/-)]. The FcRbeta(-/-) mice lacked the expression of the Fc epsilonRI on mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) was not induced in FcRbeta(-/-) mice as was expected. In these mice, the expression of IgG receptors on mast cells was augmented but the IgG-mediated PCA reaction was attenuated. Although with bone marrow-derived cultured mast cells from FcRbeta(-/-), adhesion to fibronectin and Ca2+ flux upon aggregation of IgG receptors were enhanced, mast cells co-cultured with 3T3 fibroblasts exhibited impaired degranulation on receptor aggregation. These observations indicate that FcRbeta accelerates the degranulation of mature mast cells via the IgG receptor in connective tissues. (+info)
Marking IL-4-producing cells by knock-in of the IL-4 gene.
(76/61166)
IL-4 is a cytokine which can be expressed by a number of cell types including Th2 cells, mast cells and a population of CD4+ NK1.1+ NK T cells. Although phenotypic markers exist for identifying each of these cell types, there is at present no known cell surface marker common to all IL-4-producing cells. Using gene targeting in embryonic stem cells, we have modified the IL-4 locus by knock-in of a transmembrane domain to generate mice that express a membrane-bound form of IL-4 (mIL-4). Flow cytometry using an IL-4-specific mAb allowed the detection of IL-secreting Th2 cells, mast cells and NK T cells from mIL-4 mice. Furthermore, the analysis of immune responses in mIL-4 mice following immunization with anti-CD3 and anti-IgD has allowed us to identify distinct subpopulations of IL-4-producing NK T cells. Thus, the expression of IL-4 in a membrane-bound form provides a novel method for the identification and characterization of IL-4-producing cells. (+info)
Iron-deficient diet reduces atherosclerotic lesions in apoE-deficient mice.
(77/61166)
BACKGROUND: Iron deposition is evident in human atherosclerotic lesions, suggesting that iron may play a role in the development of atherosclerosis. To test this idea, the correlation between the extent of iron deposition and the severity of atherosclerosis in apolipoprotein E (apoE)-deficient mice was investigated. Furthermore, the effect of a low-iron diet on the progression of atherosclerotic lesions in these animals was evaluated. METHODS AND RESULTS: Iron deposition in tissues of apoE-deficient mice was examined by Perls' staining method. The results clearly demonstrated that iron deposits are present in atherosclerotic lesions and tissue sections of heart and liver in an age-dependent manner. When the young mice received a low-iron diet for 3 months, the hematocrit, serum iron, hemoglobin, and cholesterol concentrations were not significantly altered compared with those of littermates placed on a chow diet. However, the serum ferritin level of animals in the iron-restricted group was 27% to 30% lower than that of the control group in either sex. Furthermore, the lipoproteins isolated from the iron-restricted group exhibited greater resistance to copper-induced oxidation. Histological examination revealed that atherosclerotic lesions developed in mice fed a low-iron diet were significantly smaller than those found in control littermates. Likewise, the iron deposition as well as tissue iron content was much less in aortic tissues of the iron-restricted animals. Circulating autoantibodies to oxidized LDL and immunostains for epitopes of malondialdehyde-modified LDL detected on lesions were also significantly lower in mice fed a low-iron diet. CONCLUSIONS: Iron deposition is closely associated with the progression of atherosclerosis in apoE-deficient mice. Restriction in dietary iron intake leads to significant inhibition of lesion formation in these animals. These results suggest that the beneficial effect of a low-iron diet may be mediated, at least in part, by the reduction of iron deposition as well as LDL oxidation in vascular lesions. (+info)
Nitric oxide-mediated regulation of transepithelial sodium and chloride transport in murine nasal epithelium.
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Transepithelial ion transport is regulated by a variety of cellular factors. In light of recent evidence that nitric oxide (NO) production is decreased in cystic fibrosis airways, we examined the role of NO in regulating sodium and chloride transport in murine nasal epithelium. Acute intervention with the inducible NO synthase (iNOS)-selective inhibitor S-methylisothiourea resulted in an increase of amiloride-sensitive sodium absorption observed as a hyperpolarization of nasal transepithelial potential difference. Inhibition of iNOS expression with dexamethasone also hyperpolarized transepithelial potential difference, but only a portion of this increase proved to be amiloride sensitive. Chloride secretion was significantly inhibited in C57BL/6J mice by the addition of both S-methylisothiourea and dexamethasone. Mice lacking iNOS expression [NOS2(-/-)] also had a decreased chloride-secretory response compared with control mice. These data suggest that constitutive NO production likely plays some role in the downregulation of sodium absorption and leads to an increase in transepithelial chloride secretion. (+info)
Impaired invariant chain degradation and antigen presentation and diminished collagen-induced arthritis in cathepsin S null mice.
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Cathepsins have been implicated in the degradation of proteins destined for the MHC class II processing pathway and in the proteolytic removal of invariant chain (Ii), a critical regulator of MHC class II function. Mice lacking the lysosomal cysteine proteinase cathepsin S (catS) demonstrated a profound inhibition of Ii degradation in professional APC in vivo. A marked variation in the generation of MHC class II-bound Ii fragments and presentation of exogenous proteins was observed between B cells, dendritic cells, and macrophages lacking catS. CatS-deficient mice showed diminished susceptibility to collagen-induced arthritis, suggesting a potential therapeutic target for regulation of immune responsiveness. (+info)
Mucosal immunity to influenza without IgA: an IgA knockout mouse model.
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IgA knockout mice (IgA-/-) were generated by gene targeting and were used to determine the role of IgA in protection against mucosal infection by influenza and the value of immunization for preferential induction of secretory IgA. Aerosol challenge of naive IgA-/- mice and their wild-type IgA+/+ littermates with sublethal and lethal doses of influenza virus resulted in similar levels of pulmonary virus infection and mortality. Intranasal and i.p. immunization with influenza vaccine plus cholera toxin/cholera toxin B induced significant mucosal and serum influenza hemagglutinin-specific IgA Abs in IgA+/+ (but not IgA-/-) mice as well as IgG and IgM Abs in both IgA-/- and IgA+/+ mice; both exhibited similar levels of pulmonary and nasal virus replication and mortality following a lethal influenza virus challenge. Monoclonal anti-hemagglutinin IgG1, IgG2a, IgM, and polymeric IgA Abs were equally effective in preventing influenza virus infection in IgA-/- mice. These results indicate that IgA is not required for prevention of influenza virus infection and disease. Indeed, while mucosal immunization for selective induction of IgA against influenza may constitute a useful approach for control of influenza and other respiratory viral infections, strategies that stimulate other Igs in addition may be more desirable. (+info)