Abstract
Familial hypercholesterolemia (FH) is characterized by a raised concentration of LDL in plasma that results in a significantly increased risk of premature atherosclerosis. In FH, impaired removal of LDL from the circulation results from inherited mutations in the LDL receptor gene or, more rarely, in the gene for apo B, the ligand for the LDL receptor. We have identified two unrelated clinically homozygous FH patients whose cells exhibit no measurable degradation of LDL in culture. Extensive analysis of DNA and mRNA revealed no defect in the LDL receptor, and alleles of the LDL receptor or apo B genes do not cosegregate with hypercholesterolemia in these families. FACS® analysis of binding and uptake of fluorescent LDL or anti–LDL receptor antibodies showed that LDL receptors are on the cell surface and bind LDL normally, but fail to be internalized, suggesting that some component of endocytosis through clathrin-coated pits is defective. Internalization of the transferrin receptor occurs normally, suggesting that the defective gene product may interact specifically with the LDL receptor internalization signal. Identification of the defective gene will aid genetic diagnosis of other hypercholesterolemic patients and elucidate the mechanism by which LDL receptors are internalized.
Authors
Dennis Norman, Xi-Ming Sun, Mafalda Bourbon, Brian L. Knight, Rossitza P. Naoumova, Anne K. Soutar
Abstract
The intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte plasma membrane. Clearly, the establishment and maintenance of the driving forces — transepithelial ion gradients — are vital for maximum nutrient absorption. The purpose of this study was to determine the nature of intracellular pH (pHi) regulation in response to H+-coupled transport at the apical membrane of human intestinal epithelial Caco-2 cells. Using isoform-specific primers, mRNA transcripts of the Na+/H+ exchangers NHE1, NHE2, and NHE3 were detected by RT-PCR, and identities were confirmed by sequencing. The functional profile of Na+/H+ exchange was determined by a combination of pHi, 22Na+ influx, and EIPA inhibition experiments. Functional NHE1 and NHE3 activities were identified at the basolateral and apical membranes, respectively. H+/solute-induced acidification (using glycylsarcosine or β-alanine) led to Na+-dependent, EIPA-inhibitable pHi recovery or EIPA-inhibitable 22Na+ influx at the apical membrane only. Selective activation of apical (but not basolateral) Na+/H+ exchange by H+/solute cotransport demonstrates that coordinated activity of H+/solute symport with apical Na+/H+ exchange optimizes the efficient absorption of nutrients and Na+, while maintaining pHi and the ion gradients involved in driving transport.
Authors
David T. Thwaites, Dianne Ford, Michael Glanville, Nicholas L. Simmons
Abstract
To understand the role of Na+/H+ exchanger 1 (NHE1) in intracellular pH (pHi) regulation and neuronal function, we took advantage of natural knockout mice lacking NHE1, the most ubiquitously and densely expressed NHE isoform in the central nervous system (CNS). CA1 neurons from both wild-type (WT) and NHE1 mutant mice were studied by continuous monitoring of pHi, using the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. In the nominal absence of CO2/HCO3–, steady-state pHi was higher in WT neurons than in mutant neurons. Using the NH4Cl prepulse technique, we also show that H+ flux in WT neurons was much greater than in mutant neurons. The recovery from acid load was blocked in WT neurons, but not in mutant neurons, by removal of Na+ from the extracellular solution or by using 100 μM 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate (HOE 694) in HEPES buffer. Surprisingly, in the presence of CO2/HCO3–, the difference in H+ flux between WT and mutant mice was even more exaggerated, with a difference of more than 250 μM/s between them at pH 6.6. H+ flux in CO2/HCO3– was responsive to diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) in the WT but not in the mutant. We conclude that (a) the absence of NHE1 in the mutant neurons tended to cause lower steady-state pHi and, perhaps more importantly, markedly reduced the rate of recovery from an acid load; and (b) this difference in the rate of recovery between mutant and WT neurons was surprisingly larger in the presence, rather than in the absence, of HCO3–, indicating that the presence of NHE1 is essential for the regulation and/or functional expression of both HCO3–-dependent and -independent transporters in neurons.
Authors
Hang Yao, Enbo Ma, Xiang-Qun Gu, Gabriel G. Haddad
Abstract
Nitric oxide (NO) derived from the inducible isoform of NO synthase (iNOS) is an inflammatory product implicated both in secondary damage and in recovery from brain injury. To address the role of iNOS in experimental traumatic brain injury (TBI), we used 2 paradigms in 2 species. In a model of controlled cortical impact (CCI) with secondary hypoxemia, rats were treated with vehicle or with 1 of 2 iNOS inhibitors (aminoguanidine and L-N-iminoethyl-lysine), administered by Alzet pump for 5 days and 1.5 days after injury, respectively. In a model of CCI, knockout mice lacking the iNOS gene (iNOS–/–) were compared with wild-type (iNOS+/+) mice. Functional outcome (motor and cognitive) during the first 20 days after injury, and histopathology at 21 days, were assessed in both studies. Treatment of rats with either of the iNOS inhibitors after TBI significantly exacerbated deficits in cognitive performance, as assessed by Morris water maze (MWM) and increased neuron loss in vulnerable regions (CA3 and CA1) of hippocampus. Uninjured iNOS+/+ and iNOS–/– mice performed equally well in both motor and cognitive tasks. However, after TBI, iNOS–/– mice showed markedly worse performance in the MWM task than iNOS+/+ mice. A beneficial role for iNOS in TBI is supported.
Authors
Elizabeth H. Sinz, Patrick M. Kochanek, C. Edward Dixon, Robert S.B. Clark, Joseph A. Carcillo, Joanne K. Schiding, Minzhi Chen, Stephen R. Wisniewski, Timothy M. Carlos, Debra Williams, Steven T. DeKosky, Simon C. Watkins, Donald W. Marion, Timothy R. Billiar
Abstract
T-helper type 2 (Th2) cytokines have been implicated in the pathogenesis of the pulmonary inflammatory response and altered bronchial responsiveness in allergic asthma. To elucidate the mechanism of Th2-dependent mediation of altered airway responsiveness in the atopic asthmatic state, the expression and actions of specific cytokines were examined in isolated rabbit and human airway smooth muscle (ASM) tissues and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic sensitized ASM exhibited significantly enhanced maximal isometric contractility to acetylcholine and attenuated relaxation responses to isoproterenol. These proasthmatic changes in agonist responsiveness were ablated by pretreating the atopic sensitized tissues with either an IL-5 receptor blocking antibody (IL-5ra) or the human recombinant IL-1 receptor antagonist (IL-1ra), whereas an IL-4 neutralizing antibody had no effect. Moreover, relative to controls, atopic asthmatic sensitized ASM cells demonstrated an initial, early (after 3 hours of incubation) increased mRNA expression and protein release of IL-5. This was followed (after 6 hours of incubation) by an enhanced mRNA expression and release of IL-1β protein, an effect that was inhibited in sensitized cells pretreated with IL-5ra. Extended studies demonstrated that naive ASM exposed to exogenously administered IL-5 exhibited an induced upregulated mRNA expression and protein release of IL-1β associated with proasthmatic-like changes in ASM constrictor and relaxant responsiveness, and that these effects were ablated in tissues pretreated with IL-1ra. Taken together, these observations provide new evidence that (a) the Th2 cytokine IL-5 and the pleiotropic proinflammatory cytokine IL-1β are endogenously released by atopic asthmatic sensitized ASM and mechanistically interact to mediate the proasthmatic perturbations in ASM responsiveness; and (b) the nature of this interaction is given by an initial endogenous release of IL-5, which then acts to induce the autologous release of IL-1β by the sensitized ASM itself, resulting in its autocrine manifestation of the proasthmatic phenotype.
Authors
Hakon Hakonarson, Neil Maskeri, Carrie Carter, Sing Chuang, Michael M. Grunstein
Abstract
Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to the absence of the dystrophin protein in striated muscle. A significant number of these mutations are premature stop codons. On the basis of the observation that aminoglycoside treatment can suppress stop codons in cultured cells, we tested the effect of gentamicin on cultured muscle cells from the mdx mouse — an animal model for DMD that possesses a premature stop codon in the dystrophin gene. Exposure of mdx myotubes to gentamicin led to the expression and localization of dystrophin to the cell membrane. We then evaluated the effects of differing dosages of gentamicin on expression and functional protection of the muscles of mdx mice. We identified a treatment regimen that resulted in the presence of dystrophin in the cell membrane in all striated muscles examined and that provided functional protection against muscular injury. To our knowledge, our results are the first to demonstrate that aminoglycosides can suppress stop codons not only in vitro but also in vivo. Furthermore, these results raise the possibility of a novel treatment regimen for muscular dystrophy and other diseases caused by premature stop codon mutations. This treatment could prove effective in up to 15% of patients with DMD.
Authors
Elisabeth R. Barton-Davis, Laurence Cordier, Daria I. Shoturma, Stuart E. Leland, H. Lee Sweeney
A novel therapy for colitis utilizing PPAR-γ ligands to inhibit the epithelial inflammatory response
Abstract
Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the nuclear hormone receptor superfamily originally shown to play a critical role in adipocyte differentiation and glucose homeostasis, has recently been implicated as a regulator of cellular proliferation and inflammatory responses. Colonic epithelial cells, which express high levels of PPAR-γ protein, have the ability to produce inflammatory cytokines that may play a role in inflammatory bowel disease (IBD). We report here that PPAR-γ ligands dramatically attenuate cytokine gene expression in colon cancer cell lines by inhibiting the activation of nuclear factor-κB via an IκB-α–dependent mechanism. Moreover, thiazolidinedione ligands for PPAR-γ markedly reduce colonic inflammation in a mouse model of IBD. These results suggest that colonic PPAR-γ may be a therapeutic target in humans suffering from IBD.
Authors
Chinyu G. Su, Xiaoming Wen, Shannon T. Bailey, Wen Jiang, Shamina M. Rangwala, Sue A. Keilbaugh, Anne Flanigan, Sreekant Murthy, Mitchell A. Lazar, Gary D. Wu
Abstract
Cardiac hypertrophy often presages the development of heart failure. Numerous cytosolic signaling pathways have been implicated in the hypertrophic response in cardiomyocytes in culture, but their roles in the hypertrophic response to physiologically relevant stimuli in vivo is unclear. We previously reported that adenovirus-mediated gene transfer of SEK-1(KR), a dominant inhibitory mutant of the immediate upstream activator of the stress-activated protein kinases (SAPKs), abrogates the hypertrophic response of neonatal rat cardiomyocytes to endothelin-1 in culture. We now report that gene transfer of SEK-1(KR) to the adult rat heart blocks SAPK activation by pressure overload, demonstrating that the activity of cytosolic signaling pathways can be inhibited by gene transfer of loss-of-function mutants in vivo. Furthermore, gene transfer of SEK-1(KR) inhibited pressure overload–induced cardiac hypertrophy, as determined by echocardiography and several postmortem measures including left ventricular (LV) wall thickness, the ratio of LV weight to body weight, cardiomyocyte diameter, and inhibition of atrial natriuretic factor expression. Our data suggest that the SAPKs are critical regulators of cardiac hypertrophy in vivo, and therefore may serve as novel drug targets in the treatment of hypertrophy and heart failure.
Authors
Gabriel Choukroun, Roger Hajjar, Stefanie Fry, Federica del Monte, Syed Haq, J. Luis Guerrero, Michael Picard, Anthony Rosenzweig, Thomas Force
Abstract
Parathyroid hormone (PTH) and parathyroid hormone–related peptide (PTHrP) bind to and activate the same PTH/PTHrP receptor. Deletion of either the PTHrP gene or the PTH/PTHrP receptor gene leads to acceleration of differentiation of growth plate chondrocytes. To explore further the functional relationships of PTHrP and the PTH/PTHrP receptor, bones of knockout mice were analyzed early in development, and the phenotypes of double-knockout mice were characterized.
Authors
Beate Lanske, Michael Amling, Lynn Neff, Jennifer Guiducci, Roland Baron, Henry M. Kronenberg
Abstract
Fcγ receptors on the surface of phagocytic cells bind the Fc region of IgG and mediate binding, phagocytosis, and destruction of particulate antigens opsonized by the antigen-specific IgG molecule. The present study evaluates the feasibility of converting lung epithelial cells into phagocytic cells using adenovirus (Ad) vector–mediated gene transfer of FcγRIIA cDNA to induce expression of the human FcγRIIA receptor. Binding and phagocytosis of opsonized sheep red blood cells (SRBCs) by the A549 human lung epithelial cell line after Ad-mediated FcγRIIA gene transfer was demonstrated using light and fluorescence microscopy and phagocytic assays with 51Cr-labeled SRBCs. When A549 cells were infected with an Ad vector expressing a FcγRIIA mutant in which 2 of 3 cytoplasmic tyrosines have been replaced with phenylalanine, only binding, but not phagocytosis, of opsonized SRBCs was observed. In vivo expression of FcγRIIA in the lung after intratracheal administration of the AdFcγRIIA enhanced clearance of opsonized Pseudomonas aeruginosa from the lung in normal rats and in mice deficient in Fcγ receptor expression. Similar results were observed with a chimeric FcγRIIA construct containing the extracellular domain of FcγRIIIA. Together, these data demonstrate that Ad-mediated FcγRIIA receptor cDNA expression can mediate the binding and phagocytosis of opsonized particulate antigens by normally nonphagocytic cells, suggesting that gene-transfer strategies might be used to utilize nonphagocytic cells to clear bacteria or other opsonized particulate antigens from the respiratory tract.
Authors
Stefan Worgall, Petr Bezdicek, Moo-Kyung Kim, Jong-Gu Park, Ravi Singh, Melpo Christofidou-Solomidou, Alice Prince, Imre Kovesdi, Alan D. Schreiber, Ronald G. Crystal
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