We tested the hypothesis that angiotensin-converting enzyme (ACE) inhibitor therapy prevents volume-overload hypertrophy in dogs with chronic mitral regurgitation (MR). Seven adult mongrel dogs receiving ramipril (R; 10 mg orally, twice/day) for 4 mo were compared with 11 dogs receiving no R (N) for 4 mo after induction of MR. Cine-magnetic resonance imaging demonstrated that left ventricular (LV) mass increased in the R-MR dogs [80 +/- 4 (SE) to 108 +/- 7 g, P < 0.01] and in the N-MR dogs (92 +/- 7 to 112 +/- 8 g, P < 0.001). LV myocyte cell length was greater in the R-MR and N-MR dogs (203 +/- 6 and 177 +/- 10 microns, respectively) than in normal (144 +/- 4 microns, P < 0.05) dogs. There was significant loss of the collagen weave pattern by scanning electron microscopy in both R-MR and N-MR dogs. LV ACE and chymase activities were significantly elevated in R-MR and N-MR compared with normal dogs. LV angiotensin II (ANG II) levels in the R-MR dogs (28 +/- 12 pg/g) were reduced to levels seen in normal dogs (28 +/- 4 pg/g) compared with N-MR dogs (72 +/- 11 pg/g, P < 0.05). Steady-state AT1-receptor mRNA levels decreased 66% in N-MR compared with normal dogs (P < 0.001) and increased 1.5-fold in R-MR compared with normal dogs (P < 0.01). Thus upregulation of the AT1 receptor in the R-MR hearts may provide a mechanism by which normal intracardiac ANG II levels could continue to mediate LV hypertrophy. However, the mechanism of dissolution collagen weave in both N-MR and R-MR hearts may be related to the stretch of volume overload.

Our meta-analysis suggests evidence for cigarette smoking as an independent risk factor for incident CKD. Future studies are required to investigate whether smoking cessation can decrease incident CKD in the general adult population.

SummaryThe Nur77/Nurrl family of DNA binding proteins has been reported to be required for the signal transduction of CD3/T cell receptor (TCR)-mediated apoptosis in T cell hybridomas. To determine the role of this family of DNA-binding proteins in thymic clonal deletion, transgenic (Tg) mice bearing a dominant negative mutation were produced. The transgene consisted of a truncated Nur77 (ANur77) gene encoding the DNA-binding domain of Nur77 ligated to a TCR-[3 enhancer resulting in early expression in thymocytes. Apoptosis of CD4+CD8 + thymocytes mediated by CD3/TCR signaling was greatly inhibited in the ANur77 Tg mice, compared with non-Tg littermates, after treatment with anti-CD3 or anti-TCR antibody in vivo and in vitro. Clonal deletion of self-reactive T cells was investigated in ANur77-Db/HY TCR-ot/~3 double Tg mice. There was a five-fold increase in the total number of thymocytes expressing self-reactive Db/HY TCR-et/~3 in the ANur77-TCR-ot/[3 double Tg male mice. Deficient clonal deletion of self-reactive thymocytes was demonstrated by a 10-fold increase in the CD4+CD8 + thymocytes that expressed Tg TCR-a/[~. There was an eight-fold increase in CD8 +, DB/Hy TCR-ot/[3 T cells in the lymph nodes (LN) of ANur77-Db/Hy TCR-o_/[3 double Tg compared with Db/Hy TCR-0~/I3 Tg male mice. In spite of defective clonal deletion, the T cells expressing the Tg TCR were functionally anergic. In vivo analysis revealed increased activation and apoptosis of T cells associated with increased expression of Fas and Fas ligand in LN of ANur77-Db/Hy TCR-oJI3 double Tg male mice. These results indicate that inhibition of Nur77/Nurrl DNA binding in T cells leads to inefficient thymic clonal deletion, but T cell tolerance is maintained by Fas-dependent clonal deletion in LN and spleen.

Native amiloride-sensitive Na ؉ channels exhibit a variety of biophysical properties, including variable sensitivities to amiloride, different ion selectivities, and diverse unitary conductances. The molecular basis of these differences has not been elucidated. We tested the hypothesis that co-expression of ␦-epithelial sodium channel (ENaC) underlies, at least in part, the multiplicity of amiloride-sensitive Na ؉ conductances in epithelial cells. For example, the ␦-subunit may form multimeric channels with ␣␤␥-ENaC. Reverse transcription-PCR revealed that ␦-ENaC is co-expressed with ␣␤␥-subunits in cultured human lung (H441 and A549), pancreatic (CFPAC), and colonic epithelial cells (Caco-2). Indirect immunofluorescence microscopy revealed that ␦-ENaC is co-expressed with ␣-, ␤-, and ␥-ENaC in H441 cells at the protein level. Measurement of current-voltage relationships revealed that the cation selectivity ratios for Na ؉ /Li ؉ /K ؉ /Cs ؉ /Ca 2؉ /Mg 2؉ , the apparent dissociation constant (K i ) for amiloride, and unitary conductances for ␦␣␤␥-ENaC differed from those of both ␣␤␥-and ␦␤␥-ENaC (n ‫؍‬ 6). The contribution of the ␦ subunit to P Li /P Na ratio and unitary Na ؉ conductance under bi-ionic conditions depended on the injected cRNA concentration. In addition, the EC 50 for proton activation, mean open and closed times, and the self-inhibition time of ␦␣␤␥-ENaC differed from those of ␣␤␥-and ␦␤␥-ENaC. Co-immunoprecipitation of ␦-ENaC with ␣-and ␥-subunits in H441 and transfected COS-7 cells suggests an interaction among these proteins. We, therefore, concluded that the interactions of ␦-ENaC with other subunits could account for heterogeneity of native epithelial channels.

Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the β cells of the islets of Langerhans. β cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in β cell destruction, including Fas, perforin, and TNF-α. Evidence for Fas-mediated lysis of β cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on β cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.

Among the multiple organ disorders caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), acute lung failure following atypical pneumonia is the most serious and often fatal event. We hypothesized that two of the hydrophilic structural coronoviral proteins (S and E) would regulate alveolar fluid clearance by decreasing the cell surface expression and activity of amiloride-sensitive epithelial sodium (Na(+)) channels (ENaC), the rate-limiting protein in transepithelial Na(+) vectorial transport across distal lung epithelial cells. Coexpression of either S or E protein with human alpha-, beta-, and gamma-ENaC in Xenopus oocytes led to significant decreases of both amiloride-sensitive Na(+) currents and gamma-ENaC protein levels at their plasma membranes. S and E proteins decreased the rate of ENaC exocytosis and either had no effect (S) or decreased (E) rates of endocytosis. No direct interactions among SARS-CoV E protein with either alpha- or gamma-ENaC were indentified. Instead, the downregulation of ENaC activity by SARS proteins was partially or completely restored by administration of inhibitors of PKCalpha/beta1 and PKCzeta. Consistent with the whole cell data, expression of S and E proteins decreased ENaC single-channel activity in oocytes, and these effects were partially abrogated by PKCalpha/beta1 inhibitors. Finally, transfection of human airway epithelial (H441) cells with SARS E protein decreased whole cell amiloride-sensitive currents. These findings indicate that lung edema in SARS infection may be due at least in part to activation of PKC by SARS proteins, leading to decreasing levels and activity of ENaC at the apical surfaces of lung epithelial cells.

Inositolsphingolipid phospholipase C (Isc1p) is theCeramide is a bioactive lipid that in eukaryotic cells functions as a mediator of a variety of extracellular signals through the regulation of several downstream effectors which in turn control basic cellular functions such as cell growth, cell cycle arrest, apoptosis, and senescence (1-6). Sphingomyelinases, which hydrolyze the phosphodiester linkage of sphingomyelin (SM) 1 to produce ceramide and phosphorylcholine, function as key regulators of the intracellular levels of ceramide in mammalian cells.Sphingolipids are also important regulatory molecules in yeast where they are known to be required for viability (7), optimal life span (8), cell cycle regulation (9), endocytosis (10), and regulation of responses of yeast cells to stress (1, 11). Although Saccharomyces cerevisiae do not contain SM, they do contain inositol phosphoceramides, and recently we identified a homologue of neutral sphingomyelinase in S. cerevisiae, Isc1p, which acts on phosphoceramides to generate ceramide. This enzyme displays ϳ30% identity to neutral sphingomyelinase 2 and shares with it several common features (12) including hydrolytic activity on SM, the requirement of Mg 2ϩ for optimal activity, optimal neutral pH, and the presence of a newly discovered domain that is conserved in the entire family of sphingomyelinases, the P-loop-like domain (13), which appears to be important for substrate binding and/or catalysis.In addition, both enzymes demonstrate an absolute requirement for anionic phospholipids for in vitro activation. Thus, Isc1p is activated selectively in vitro by cardiolipin (CL), phosphtidylglycerol (PG), or phosphatidylserine. In a recent study we demonstrated that the enzyme binds these anionic phospholipids, and we identified the second transmembrane domain and the C terminus of Isc1p as required for this binding, and it was proposed that this interaction plays a critical role in enzyme function through a novel tethering mechanism of enzyme activation by lipid cofactors (14).Given these specific requirements for anionic phospholipids in vitro, it became important to determine whether the enzyme requires phospholipids for activation in vivo. Our attention was particularly directed to the possible roles of PG and CL as the deletion of PGS1, the gene that encodes the enzyme responsible for the synthesis of PG phosphate, and subsequent synthesis of PG and CL was shown to cause a late defect during growth in glucose media similar to that of ISC1. Moreover, these lipids are almost exclusively present in mitochondria, and we have shown recently that Isc1p localizes preferentially to mitochondria in the post-diauxic phase of growth.Synthesis of CL in yeast requires three sequential reactions.

Protein tyrosine dephosphorylation after Fas cross-linking occurred in Fas apoptosis-sensitive CEM-6 cells but not in Fas apoptosis-resistant MOLT-4 cells, and apoptosis in the CEM-6 cells could be inhibited by the protein tyrosine phosphatase inhibitor, pervanadate. The time course and level of dephosphorylation were correlated with increased hematopoietic cell protein tyrosine phosphatase (HCP) activity, but not with the activity of two other tyrosine phosphatases. The level of expression of HCP was correlated with Fas apoptosis function in eleven human and murine Fas-positive lymphoid cell lines. Expression of recombinant HCP in the MOLT-4 cell line converted this Fas apoptosis-resistant cell line to Fas apoptosis sensitive. HCP-mutant mev/mev mice exhibited increased expression of Fas but decreased Fas-mediated apoptosis function in lymphoid organs after anti-mouse Fas antibody treatment in vivo. Thus, HCP-mediated protein dephosphorylation is involved in the delivery of the Fas apoptosis signal in lymphoid cells.