Rapid Publication
Abstract
Aldosterone stimulates sodium transport in the renal collecting duct by activating the epithelial sodium channel (ENaC). To investigate the basis of this effect, we have developed a novel set of rabbit polyclonal antibodies to the 3 subunits of ENaC and have determined the abundance and distribution of ENaC subunits in the principal cells of the rat renal collecting duct. Elevated circulating aldosterone (due to either dietary NaCl restriction or aldosterone infusion) markedly increased the abundance of αENaC protein without increasing the abundance of the β and γ subunits. Thus, αENaC is selectively induced by aldosterone. In addition, immunofluorescence immunolocalization showed a striking redistribution in ENaC labeling to the apical region of the collecting duct principal cells. Finally, aldosterone induced a shift in molecular weight of γENaC from 85 kDa to 70 kDa, consistent with physiological proteolytic clipping of the extracellular loop as postulated previously. Thus, at the protein level, the response of ENaC to aldosterone stimulation is heterogenous, with both quantitative and qualitative changes that can explain observed increases in ENaC-mediated sodium transport.
Authors
Shyama Masilamani, Gheun-Ho Kim, Carter Mitchell, James B. Wade, Mark A. Knepper
Abstract
Therapeutic intervention with highly active antiretroviral therapy (HAART) can lead to suppression of HIV-1 plasma viremia to undetectable levels for 3 or more years. However, adherence to complex drug regimens can prove problematic, and subjects may temporarily discontinue HAART for variable periods. We studied 6 HIV-1–infected individuals who stopped therapy. Off HAART, levels of viremia were suppressed to fewer than 500 copies/mL in 2 subjects for more than 12 and more than 24 months, respectively, and in 1 subject for 4 months on 1 occasion. Three subjects failed to contain plasma viremia. Broad and strong HIV-1–specific immune responses were detected in subjects with prolonged suppression of viral replication. This longitudinal study suggests that containment of HIV-1 replication to low or undetectable levels after discontinuation of HAART is associated with strong virus-specific immune responses. Boosting of HIV-1–specific immune responses should be considered as an adjunctive treatment strategy for HIV-1–infected individuals on HAART.
Authors
Gabriel M. Ortiz, Douglas F. Nixon, Alexandra Trkola, James Binley, Xia Jin, Sebastian Bonhoeffer, Peter J. Kuebler, Sean M. Donahoe, Marie-Ange Demoitie, William M. Kakimoto, Tom Ketas, Brian Clas, Jonas J. Heymann, Linqi Zhang, Yunzhen Cao, Arlene Hurley, John P. Moore, David D. Ho, Martin Markowitz
Abstract
Many HIV-1 isolates at the late stage of disease are capable of using both CXCR4 and CCR5 in transfected cell lines, and are thus termed dual-tropic. Here we asked whether these dual-tropic variants also use both coreceptors for productive infection in a natural human lymphoid tissue microenvironment, and whether use of a particular coreceptor is associated with viral cytopathicity. We used 3 cloned dual-tropic HIV-1 variants, 89.6 and its chimeras 89-v345.SF and 89-v345.FL, which use both CCR5 and CXCR4 in transfected cell lines. In human lymphoid tissue ex vivo, one variant preferentially used CCR5, another preferentially used CXCR4, and a third appeared to be a true dual-tropic variant. The 2 latter variants severely depleted CD4+ T cells, whereas cytopathicity of the virus that used CCR5 only in lymphoid tissue was mild and confined to CCR5+/CD4+ T cells. Thus, (a) HIV-1 coreceptor usage in vitro cannot be unconditionally extrapolated to natural microenvironment of human lymphoid tissue; (b) dual-tropic viruses are not homogeneous in their coreceptor usage in lymphoid tissue, but probably comprise a continuum between the 2 polar variants that use CXCR4 or CCR5 exclusively; and (c) cytopathicity toward the general CD4+ T cell population in lymphoid tissue is associated with the use of CXCR4.
Authors
Svetlana Glushakova, Yanjie Yi, Jean-Charles Grivel, Anjali Singh, Dominique Schols, Erik De Clercq, Ronald G. Collman, Leonid Margolis
Abstract
LD78α and LD78β are 2 highly related nonallelic genes that code for different isoforms of the human CC chemokine macrophage inflammatory protein-1α (MIP-1α). Two molecular forms of natural LD78β (7.778 and 7.793 kDa) were identified from conditioned media of stimulated peripheral blood mononuclear cells. Although LD78α and LD78β only differ in 3 amino acids, both LD78β variants were 100-fold more potent chemoattractants for mouse lymphocytes than was LD78α. On the contrary, LD78β was only 2-fold more efficient than LD78α in chemoattracting human lymphocytes and monocytes. Using CC chemokine receptor–transfected cells, both molecular forms of LD78β proved to be much more potent than LD78α in inducing an intracellular calcium rise through CCR5. Compared with LD78α and RANTES, this preferential binding of LD78β to CCR5 resulted in a 10- to 50-fold higher potency in inhibiting infection of peripheral blood mononuclear cells by CCR5-using (R5) HIV-1 strains. To date, LD78β is the most potent chemokine for inhibiting HIV-1 infection, and can be considered as a potentially important drug candidate for the treatment of infection with R5 HIV-1 strains.
Authors
Patricia Menten, Sofie Struyf, Evemie Schutyser, Anja Wuyts, Erik De Clercq, Dominique Schols, Paul Proost, Jo Van Damme
Abstract
We identified the α-cardiac actin gene (ACTC) as a novel disease gene in a pedigree suffering from familial hypertrophic cardiomyopathy (FHC). Linkage analyses excluded all the previously reported FHC loci as possible disease loci in the family studied, with lod scores varying between –2.5 and –6.0. Further linkage analyses of plausible candidate genes highly expressed in the adult human heart identified ACTC as the most likely disease gene, showing a maximal lod score of 3.6. Mutation analysis of ACTC revealed an Ala295Ser mutation in exon 5 close to 2 missense mutations recently described to cause the inherited form of idiopathic dilated cardiomyopathy (IDC). ACTC is the first sarcomeric gene described in which mutations are responsible for 2 different cardiomyopathies. We hypothesize that ACTC mutations affecting sarcomere contraction lead to FHC and that mutations affecting force transmission from the sarcomere to the surrounding syncytium lead to IDC.
Authors
Jens Mogensen, Ib C. Klausen, Anders K. Pedersen, Henrik Egeblad, Peter Bross, Torben A. Kruse, Niels Gregersen, Peter S. Hansen, Ulrik Baandrup, Anders D. Børglum
Abstract
The trefoil gene family of mucus cell–secreted proteins is a critical mediator of gastrointestinal mucosal restitution. Transcription of trefoil genes is induced during mucosal repair, but the regulatory mechanisms involved are unknown. Mice deficient in the intestine-specific peptide intestinal trefoil factor (ITF), in which colonic restitution is lethally impaired, showed reduced expression of the gastric trefoil genes SP and pS2, suggesting that trefoil peptides may individually regulate transcription of the entire family. In gastric cell lines, the trefoils were shown to act in a manner suggestive of immediate-early genes capable of auto- and cross-induction through cis-acting regulatory regions. Trefoil-mediated transcriptional regulation required activation of the Ras/MEK/MAP kinase signal transduction pathway. EGF receptor (EGF-R) activation was also necessary for trefoil auto- and cross-induction, and both spasmolytic polypeptide (SP) and ITF stimulation of gastric cell lines led to phosphorylation of EGF-R. Nevertheless, ITF and ITF-thioredoxin cell surface binding at 4°C colocalized not with EGF-R, but with CD71, which is found in clathrin-coated pits, suggesting that integration of trefoil peptide responses may occur after internalization. As EGF-R expression is itself strongly induced after mucosal damage, the trefoil/EGF-R relationship may be pivotal in the generation and maintenance of the mucosal repair phenotype.
Authors
Douglas Taupin, Deng-Chyang Wu, Woo-Kyu Jeon, Kathryn Devaney, Timothy C. Wang, Daniel K. Podolsky
Abstract
Heme oxygenase (HO) catalyzes the oxidation of heme to generate carbon monoxide (CO) and bilirubin. CO increases cellular levels of cGMP, which regulates vascular tone and smooth muscle development. Bilirubin is a potent antioxidant. Hypoxia increases expression of the inducible HO isoform (HO-1) but not the constitutive isoform (HO-2). To determine whether HO-1 affects cellular adaptation to chronic hypoxia in vivo, we generated HO-1 null (HO-1–/–) mice and subjected them to hypoxia (10% oxygen) for five to seven weeks. Hypoxia caused similar increases in right ventricular systolic pressure in wild-type and HO-1–/– mice. Although ventricular weight increased in wild-type mice, the increase was greater in HO-1–/– mice. Similarly, the right ventricles were more dilated in HO-1–/– mice. After seven weeks of hypoxia, only HO-1–/– mice developed right ventricular infarcts with organized mural thrombi. No left ventricular infarcts were observed. Lipid peroxidation and oxidative damage occurred in right ventricular cardiomyocytes in HO-1–/–, but not wild-type, mice. We also detected apoptotic cardiomyocytes surrounding areas of infarcted myocardium by terminal deoxynucleotide transferase–mediated dUTP nick end-labeling (TUNEL) assays. Our data suggest that in the absence of HO-1, cardiomyocytes have a maladaptive response to hypoxia and subsequent pulmonary hypertension.
Authors
Shaw-Fang Yet, Mark A. Perrella, Matthew D. Layne, Chung-Ming Hsieh, Koji Maemura, Lester Kobzik, Philippe Wiesel, Helen Christou, Stella Kourembanas, Mu-En Lee
Abstract
The ε4 allele of apolipoprotein E (apo E) is associated with an increased risk for developing Alzheimer's disease (AD). This may be due to interactions between apo E and the amyloid-β protein (Aβ). To assess the effects of human apo E isoforms on Aβ deposition in vivo, we bred apo E3 and apo E4 hemizygous (+/–) transgenic mice expressing apo E by astrocytes to mice homozygous (+/+) for a mutant amyloid precursor protein (APPV717F) transgene that develop age-dependent AD neuropathology. All mice were on a mouse apo E null (–/–) background. By nine months of age, APPV717F+/–, apo E–/– mice had developed Aβ deposition, and, as reported previously, the quantity of Aβ deposits was significantly less than that seen in APPV717F+/– mice expressing mouse apo E. In contrast to effects of mouse apo E, similar levels of human apo E3 and apo E4 markedly suppressed early Aβ deposition at nine months of age in APPV717F+/– transgenic mice, even when compared with mice lacking apo E. These findings suggest that human apo E isoforms decrease Aβ aggregation or increase Aβ clearance relative to an environment in which mouse apo E or no apo E is present. The results may have important implications for understanding mechanisms underlying the link between apo E and AD.
Authors
David M. Holtzman, Kelly R. Bales, Shan Wu, Priyanka Bhat, Maia Parsadanian, Anne M. Fagan, Louis K. Chang, Yuling Sun, Steven M. Paul
Abstract
We have studied the influence of verapamil hydrochloride on the in vitro and in vivo effects of daunorubicin in Ehrlich ascites carcinoma. Daunorubicin-sensitive tumor was rendered resistant to daunorubicin by the continuous treatment of sequential generations of tumor-bearing BALB/c mice. The ability of daunorubicin to inhibit [3H]uridine and [3H]thymidine incorporation and the effect of daunorubicin on the mean survival time of host animals bearing daunorubicin-sensitive and daunorubicin-resistant Ehrlich ascites carcinoma were compared. The addition of verapamil to daunorubicin in vitro reduced the concentration of daunorubicin required to inhibit 50% of DNA and RNA synthesis in the daunorubicin-resistant tumor to that required in the daunorubicin-sensitive tumor, from 6 and 4.4 μg/ml to 1.5 and 1.3 μg/ml, respectively. Verapamil also restored drug sensitivity to daunorubicin-resistant Ehrlich ascites carcinoma in vivo. The 21.7±0.7 d mean survival time (MST) of BALB/c mice bearing daunorubicin-resistant tumor treated with daunorubicin alone rose to 44.0±0.7 d when the same tumor was treated with verapamil and daunorubicin, P < 0.001. This in vivo effect is specific for daunorubicin-resistant Ehrlich ascites carcinoma, since there is no alteration in MST of BALB/c mice bearing daunorubicin-sensitive or daunorubicin-resistant tumor when they are treated with verapamil alone or when BALB/c mice bearing daunorubicin-sensitive tumor are treated with daunorubicin and verapamil.
Authors
Lewis M. Slater, Sandra L. Murray, Martha W. Wetzel, Ronald M. Wisdom, Emily M. Duvall
Abstract
[3H]Nitrendipine, a potent calcium channel antagonist [3-ethyl-5-methyl-1-1,4-dihydro-2,6 - dimethyl - 4 - (3 - nitrophenyl) - 3,5 - pyridine carboxylate], was used to label high affinity binding sites on membranes prepared from bovine aortic smooth muscle. The binding of [3H]nitrendipine is rapid (t1/2 < 5 min) and reversible at 37°C. The binding sites have a high affinity for [3H]nitrendipine with an equilibrium dissociation constant of 2.1 nM. The density of sites is 40-60 fmol/mg of membrane protein. Analogues of nitrendipine compete for the binding sites with affinities consistent with their known biological effects as calcium antagonists. Nisoldipine, [isobutyl methyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine carboxylate], a calcium antagonist more potent than nifedipine [2,6-dimethyl-3,5-dicarbomethoxy-4-(2-nitrophenyl)-1,4-dihydropyridine] in relaxing vascular smooth muscle, has an affinity three-fold higher than that of nifedipine in competing for the binding sites. A biologically inactive derivative of nifedipine does not compete for [3H]nitrendipine binding. Verapamil (α-isopropyl-α[(N-methyl - N-homoveratryl) -α-aminopropyl]-3,4-dimethyoxyphenyl acetonitrile), a structurally different calcium antagonist, only partially (25%) inhibits binding at high concentrations (1 μM). Prazosin, an alpha adrenergic antagonist does not compete for [3H]nitrendipine binding sites. The binding of [3H]nitrendipine is not affected by 1.5 mM calcium. Canine cardiac membranes also have high affinity [3H]nitrendipine binding sites, (KD = 6 nM) but bovine erythrocytes do not. The relative affinities of nisoldipine and nifedipine for the cardiac membrane binding sites reflect the relative activities of these compounds as calcium channel antagonists. These results suggest that the [3H]nitrendipine binding sites are the sites through which dihydropyridines act as calcium channel antagonists.
Authors
Lewis T. Williams, Patrice Tremble
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