Mutations in the cardiac troponin I gene associated with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM), the most common cause of sudden death in the young, is an autosomal dominant disease characterized by ventricular hypertrophy accompanied by myofibrillar disarrays. Linkage studies and candidate-gene approaches have demonstrated that about half of the patients have mutations in one of six disease genes: cardiac beta-myosin heavy chain (c beta MHC), cardiac troponin T (cTnT), alpha-tropomyosin (alpha TM), cardiac myosin binding protein C (cMBPC), ventricular myosin essential light chain (vMLC1) and ventricular myosin regulatory light chain (vMLC2) genes. Other disease genes remain unknown. Because all the known disease genes encode major contractile elements in cardiac muscle, we have systematically characterized the cardiac sarcomere genes, including cardiac troponin I (cTnI), cardiac actin (cACT) and cardiac troponin C (cTnC) in 184 unrelated patients with HCM and found mutations in the cTnI gene in several patients. Family studies showed that an Arg145Gly mutation was linked to HCM and a Lys206Gln mutation had occurred de novo, thus strongly suggesting that cTnI is the seventh HCM gene.

Early expression of a malignant phenotype of familial hypertrophic cardiomyopathy associated with a Gly716Arg myosin heavy chain mutation in a Korean family.

The clinical course and prognosis of familial hypertrophic cardiomyopathy (HCM) are different according to the type of mutation in the genes for sarcomere proteins. It has been disputed that a mutation, which occurs at a functionally important region in the sarcomere proteins, may increase the penetrance and expressivity of the disease. We searched for a causative mutation in an HCM family, which is characterized by early expression of clinical phenotype, high incidence of sudden death at young ages, and progressive heart failure in adults. Among the 32 family members in 4 generations, 13 were affected; 4 died suddenly before age 16, 2 children have already had full expression of the cardiac hypertrophy, and other adults have either progressive heart failure or poor left ventricular systolic functions. PCR-SSCP (polymerase chain reaction-single strand confirmation polymorphism) analysis of genomic DNAs isolated from peripheral blood leukocytes of the family members identified a Gly716Arg mutation in the cardiac beta-myosin heavy chain gene, which was cosegregated with the clinical phenotype. The mutation is localized near a functionally important site of the myosin heavy chain, the 2 active thiols, which contribute to the adenosine triphosphatase activity of myosin S1. This family provides further evidence that the mutation, which occurs at a functionally important site of the myosin heavy chain, is associated with the high penetrance and early expression of HCM.

AR intragenic deletions linked to androgen receptor splice variant expression and activity in models of prostate cancer progression.

Reactivation of the androgen receptor (AR) during androgen depletion therapy (ADT) underlies castration-resistant prostate cancer (CRPCa). Alternative splicing of the AR gene and synthesis of constitutively active COOH-terminally truncated AR variants lacking the AR ligand-binding domain has emerged as an important mechanism of ADT resistance in CRPCa. In a previous study, we demonstrated that altered AR splicing in CRPCa 22Rv1 cells was linked to a 35-kb intragenic tandem duplication of AR exon 3 and flanking sequences. In this study, we demonstrate that complex patterns of AR gene copy number imbalances occur in PCa cell lines, xenografts and clinical specimens. To investigate whether these copy number imbalances reflect AR gene rearrangements that could be linked to splicing disruptions, we carried out a detailed analysis of AR gene structure in the LuCaP 86.2 and CWR-R1 models of CRPCa. By deletion-spanning PCR, we discovered a 8579-bp deletion of AR exons 5, 6 and 7 in the LuCaP 86.2 xenograft, which provides a rational explanation for synthesis of the truncated AR v567es AR variant in this model. Similarly, targeted resequencing of the AR gene in CWR-R1 cells led to the discovery of a 48-kb deletion in AR intron 1. This intragenic deletion marked a specific CWR-R1 cell population with enhanced expression of the truncated AR-V7/AR3 variant, a high level of androgen-independent AR transcriptional activity and rapid androgen independent growth. Together, these data demonstrate that structural alterations in the AR gene are linked to stable gain-of-function splicing alterations in CRPCa.

Oncolytic and immunostimulatory efficacy of a targeted oncolytic poxvirus expressing human GM-CSF following intravenous administration in a rabbit tumor model.

Targeted oncolytic poxviruses hold promise for the treatment of cancer. Arming these agents with immunostimulatory cytokines (for example, granulocyte-monocyte colony-stimulating factor; GM-CSF) can potentially increase their efficacy and/or alter their safety. However, due to species-specific differences in both human GM-CSF (hGM-CSF) activity and poxviruses immune avoidance proteins, the impact of hGM-CSF expression from an oncolytic poxvirus cannot be adequately assessed in murine or rat tumor models. We developed a rabbit tumor model to assess toxicology, pharmacodynamics, oncolytic efficacy and tumor-specific immunity of hGM-CSF expressed from a targeted oncolytic poxvirus JX-963. Recombinant purified hGM-CSF protein stimulated a leukocyte response in this model that paralleled effects of the protein in humans. JX-963 replication and targeting was highly tumor-selective after i.v. administration, and intratumoral replication led to recurrent, delayed systemic viremia. Likewise, hGM-CSF was expressed and released into the blood during JX-963 replication in tumors, but not in tumor-free animals. hGM-CSF expression from JX-963 was associated with significant increases in neutrophil, monocyte and basophil concentrations in the peripheral blood. Finally, tumor-specific cytotoxic T lymphocytes (CTL) were induced by the oncolytic poxvirus, and expression of hGM-CSF from the virus enhanced both tumor-specific CTL and antitumoral efficacy. JX-963 had significant efficacy against both the primary liver tumor as well as metastases; no significant organ toxicity was noted. This model holds promise for the evaluation of immunostimulatory transgene-armed oncolytic poxviruses, and potentially other viral species.

Systemic armed oncolytic and immunologic therapy for cancer with JX-594, a targeted poxvirus expressing GM-CSF.

Targeted oncolytic viruses and immunostimulatory therapeutics are being developed as novel cancer treatment platforms. These approaches can be combined through the expression of immunostimulatory cytokines from targeted viruses, including adenoviruses and herpesviruses. Although intratumoral injection of such viruses has been associated with tumor growth inhibition, eradication of distant metastases was not reported. The major limitations for this approach to date have been (1) inefficient intravenous virus delivery to tumors and (2) the lack of predictive, immunocompetent preclinical models. To overcome these hurdles, we developed JX-594, a targeted, thymidine kinase(-) vaccinia virus expressing human GM-CSF (hGM-CSF), for intravenous (i.v.) delivery. We evaluated two immunocompetent liver tumor models: a rabbit model with reproducible, time-dependent metastases to the lungs and a carcinogen-induced rat liver cancer model. Intravenous JX-594 was well tolerated and had highly significant efficacy, including complete responses, against intrahepatic primary tumors in both models. In addition, whereas lung metastases developed in all control rabbits, none of the i.v. JX-594-treated rabbits developed detectable metastases. Tumor-specific virus replication and gene expression, systemically detectable levels of hGM-CSF, and tumor-infiltrating CTLs were also demonstrated. JX-594 holds promise as an i.v.-delivered, targeted virotherapeutic. These two tumor models hold promise for the optimization of this approach.

Differential actions of AlF4- and vanadate on canine trachealis muscle.

Fluoride (F-) a known stimulator of G-protein, has been reported to inhibit "P"-type ATPase activity in smooth muscles. On the other hand, vanadate, a strong "P"-type ATPase inhibitor, has been reported to stimulate G-protein in some cells. This study was designed to compare the contractile actions of fluoroaluminate (AlF4-) and vanadate and to clarify their mechanisms of actions by measuring changes in the amount of cyclic adenosine monophosphate (cAMP) and inositol phosphates. F- and vanadate induced strong contractions in canine trachealis muscle. The F(-)-induced contraction was potentiated by the addition of aluminum (Al3+, 20 microM) and inhibited by deferoxamine (200 microM), a heavy metal chelator. Ca2+ removal and 10 microM verapamil inhibited the contraction induced by AlF4- and vanadate. AlF4- and vanadate increased 45Ca influx in the absence and presence of verapamil. AlF4(-)-induced contractions were partially relaxed by isoproterenol (38.2 +/- 7.4%) in contrast with those induced by vanadate (72.1 +/- 5.3%), which could be explained by a decrease of tissue cAMP content by AlF4- in forskolin-pretreated tissues. Vanadate increased inositol phosphate accumulation as did AlF4-, although the magnitude of the increase was smaller than that produced by AlF4-. The increases of inositol phosphate content by both drugs were not affected after the pretreatment by pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Apical and basolateral ATP stimulates tracheal epithelial chloride secretion via multiple purinergic receptors.

Stimulation of Cl- secretion across the airway epithelium by ATP or UTP as agonists has therapeutic implications for cystic fibrosis. Our results demonstrate that ATP stimulates Cl- secretion in rat tracheal epithelial cell monolayers in primary culture from the apical or basolateral side of the monolayer. Multiple types of ATP-sensitive Cl- conductances in intact monolayers were elucidated through inhibition by Cl- channel-blocking drugs. Multiple Cl- conductances stimulated by ATP and adenosine 3',5'-cyclic monophosphate (cAMP) (tested for comparison) were also deciphered more specifically by nystatin permeabilization of the basolateral membrane, subsequent imposition of symmetrical Cl-, I-, or Br- solutions to test halide permselectivity, inhibition by Cl- channel-blocking drugs, and construction of current-voltage plots to study time and voltage dependence of the currents. Apical ATP stimulates Cl- secretion through P2U (or P2Y2) purinergic receptors via both intracellular Ca2+ (Ca(2+)i)-dependent and Cai(2+)-independent signaling pathways by opening outwardly rectifying Cl- channels (ORCCs), cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, and Cai(2+)-dependent Cl- channels. Basolateral ATP stimulates Cl- secretion via a combination of receptor subtypes (P2T and P2U) or a novel type of receptor (P2Y3), independent of Cai2+ or cAMP signaling by opening only CFTR channels. cAMP also stimulated multiple types of Cl- conductances, consistent with simultaneous activation of CFTR and ORCCs. Together, these results suggest that ATP as an agonist stimulates Cl- secretion via multiple purinergic receptors and multiple signal transduction pathways activated in different membrane domains of tracheal epithelia.

Evidence for basolateral but not apical membrane localization of outwardly rectifying depolarization-induced Cl(-) channel in airway epithelia.

The rat primary cultured-airway monolayer had been an excellent model for deciphering the ion channel after nystatin permeabilization of its basolateral or apical membrane (Hwang et al., 1996). After apical membrane permeabilization of rat primary cultured-airway monolayer, 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive outwardly rectifying depolarization-induced Cl(-) (BORDIC) currents were observed across the basolateral membrane in symmetrical NMG-Cl solution in this study. No significant Cl(-) current induced by the application of voltage clamping was observed across the apical membrane in symmetrical NMG-Cl solution after basolateral membrane permeabilization. The halide permeability sequence for BORDIC current was Br(-) = I(-) > Cl(-). BORDIC current was not affected by basolaterally applied bumetanide (0.5 mm). Basolateral DIDS (0.2 mm) but not apical DIDS inhibited CFTR mediated short-circuit current (I(sc)) in an intact monolayer of rat airway epithelia, a T84 human colonal epithelial cell line, and a Calu-3 human airway epithelial cell line. This is the first report showing that depolarization induced Cl(-) current is present on the basolateral membrane of airway epithelia.

Stimulation of Cl- secretion by AlF4- and vanadate in T84 cells.

We investigated the mechanism of Cl- secretion by fluoroaluminate(AlF4-) and sodium orthovanadate(vanadate) using the human colonic T84 cell line. T84 cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure short circuit current(ISC). Serosal addition of AlF4- or vanadate to T84 monolayers produced a sustained increase in ISC. Removal of Ca2+ from the serosal bathing solution partially inhibited AlF4-(-)and vanadate-induced ISC, and readministration of Ca2+ restored AlF4-(-)and vanadate-induced ISC. Carbachol application in the presence of forskolin, AlF4- or vanadate induced a synergistic increase of ISC. Forskolin and vanadate significantly increased cellular cAMP level, while carbachol and AlF4- did not. Carbachol, AlF4- and vanadate significantly increased [Ca2+]i. After Na+ in mucosal bathing solution was replaced with K+, and the mucosal membrane of T84 cell was permeabilized with amphotericin B, AlF4-, vanadate, and carbachol increased K+ conductance, but forskolin did not. After sodium chloride in serosal bathing solution was replaced with sodium gluconate and the serosal membrane was permeabilized with nystatin, forskolin, AlF4-, and vanadate increased Cl- conductance, but carbachol did not. AlF4-(-)induced ISC was remarkably inhibited by the pretreatment of pertussis toxin(2 micrograms/ml) for 2 hours. These results indicate that AlF4- and vanadate can increase Cl- secretion via simultaneous stimulation of Cl- channel and K+ channel in T84 cells. However, the AlF4- action is mostly attributed to stimulation of pertussis toxin-sensitive G-proteins, whereas the vanadate action mostly results from G protein-independent mechanisms.

AlF4- and vanadate stimulate chloride secretion in rabbit colon by a Ca(2+)-dependent mechanism.

Stripped rabbit descending colon mucosae were studied in vitro in modified Ussing chambers to determine the effects of AlF4- and vanadate on Cl- transport. Serosal additions of AlF4- and vanadate increase short circuit current (Isc) and tissue conductance, while luminal addition of the agents is ineffective. Addition of aluminium potentiates the effect of NaF on Isc. AlF4- and vanadate increase serosal-to-mucosal flux of 36Cl without affecting mucosal-to-serosal flux. The effects of these agents on Isc are markedly inhibited by serosal addition of bumetanide and depend on the presence of Na+ in the serosal bathing solution. The effects of AlF4- and vanadate on Isc are dependent on the presence of Ca2+ in the bathing solution, and are completely inhibited by indomethacin, but the effect of forskolin is not affected by the removal of Ca2+ from the bathing solution and the addition of indomethacin. AlF4- and vanadate significantly increase the level of inositol phosphate metabolites. The results indicate that AlF4- and vanadate increase Cl- secretion in the rabbit colon via an increase in prostaglandin synthesis which is mediated by the increase of intracellular Ca2+ concentrations.

Genetic factors associated with endothelial dysfunction affect the early onset of coronary artery disease in Korean males.

The maintenance of balance between nitric oxide (NO) and the superoxide anion is required for proper functioning of the endothelium. To investigate the relationship between genetic factors associated with endothelial function and the development of coronary artery disease (CAD), endothelial nitric oxide synthase (ecNOS) gene a/b polymorphism and NADH/NADPH oxidase p22 phox gene C242T polymorphism were examined in 305 Korean male CAD patients and 215 healthy male control subjects. The beta-fibrinogen gene H1/H2 polymorphism was also analyzed. Both ecNOS a/b and p22 phox C242T polymorphisms were found to be associated with the development of CAD in the study population (p=0.020 and 0.011, respectively). When the association was analyzed by age, statistical significance was retained only in those <51 years (p=0.021 and 0.025 for the a/b and the C242T polymorphism, respectively) and not in those >51 years of age (p=0.155 and 0.278 respectively). However, the distribution of the beta-fibrinogen H1/H2 genotypes was not found to be associated with the development of CAD in either the < or =50 (p = 0.611) or >50 groups (p = 0.188). The ecNOS gene a/b polymorphism and the NADH/NADPH oxidase p22 phox gene C242T polymorphism were found to be significantly associated with the development of CAD in Korean male patients less than 51 years old.

Different expressivity of a ventricular essential myosin light chain gene Ala57Gly mutation in familial hypertrophic cardiomyopathy.

BACKGROUND: Familial hypertrophic cardiomyopathy (HCM) is a clinically and genetically heterogeneous disease of the sarcomere. Molecular genetic studies have shown that familial HCM involves mutations in 8 different genes that encode proteins of the myofibrillar apparatus. METHODS: We thoroughly searched these genes to find the mutations in 38 probands of unrelated families with familial HCM. RESULTS: We found a novel missense mutation that resulted in Ala57Gly amino acid substitution of the ventricular essential myosin light chain (vMLC1) gene in two unrelated Korean families with familial HCM and one Japanese patient. The mutated site is located in the putative helix-loop-helix region (named EF-hand domain) of the calcium-binding site that is highly conserved in vMLC1 isoforms across the various species. The phenotype of this mutation in the affected families is a classic asymmetric septal hypertrophy, and the disease penetrance in genotyped members older than 18 years is 78%. In one Korean family a 42-year-old woman and two brothers (34 and 38 years old) with the mutation had fully expressed the disease, but two sisters (39 and 29 years old) with the mutation had no phenotypic expression of HCM. CONCLUSIONS: Ala57Gly mutation in the vMLC1 gene may exhibit the classic form of familial HCM and widely different penetration of the disease phenotype in the family members with mutation, especially in women.

CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions to regulate both Cl- and Na+ conductive pathways; however, the cellular mechanisms whereby CFTR acts as a conductance regulator are unknown. CFTR and outwardly rectifying Cl- channels (ORCCs) are distinct channels but are linked functionally via an unknown regulatory mechanism. We present results from whole-cell and single-channel patch-clamp recordings, short-circuit current recordings, and [gamma-32P]ATP release assays of normal, CF, and wild-type or mutant CFTR-transfected CF airway cultured epithelial cells wherein CFTR regulates ORCCs by triggering the transport of the potent agonist, ATP, out of the cell. Once released, ATP stimulates ORCCs through a P2U purinergic receptor-dependent signaling mechanism. Our results suggest that CFTR functions to regulate other Cl- secretory pathways in addition to itself conducting Cl-.

cGMP stimulates sodium and chloride currents in rat tracheal airway epithelia.

To test the hypothesis that guanosine 3',5'-cyclic monophosphate (cGMP) regulates ion transport in airway epithelial cells, we measured short-circuit current (I(sc)) and (22)Na+ fluxes in primary cultured rat tracheal epithelial cells. In Cl- -containing Ringer solution, I(sc) was increased by approximately 17 microA/cm2 after application of 1 mM 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), whereas, in Cl- -free solutions, the Na+ -mediated component was approximately 5 microA/cm2, suggesting a cGMP stimulation of Cl-secretory current and a smaller Na+ absorptive current. Inward and net mucosal-to-serosal (22)Na+ flux was doubled in the presence of 2 mM 8-BrcGMP. To determine whether nucleotide-gated channels play a role in this transepithelial Na+ absorption, blockers of nucleotide-gated cation channels were used to inhibit I(sc). The cGMP-stimulated Na+-mediated I(sc) was blocked by as little as 500 nM dichlorobenzamil or 50 microM L-cis-diltiazem, which are known blockers for cyclic nucleotide-gated cation channels. These agents also blocked the basal (non-cGMP-stimulated) current when measured in the presence of 10 microM amiloride, which blocks current through 5-pS amiloride-sensitive Na+ channels. To document whether the distribution of nucleotide-gated nonselective cation channels was consistent with a role in airway epithelial transport, in situ hybridization was performed. In situ hybridization of mRNA encoding for nucleotide-gated cation channels was found in epithelial cell layers of rat trachea, bronchi, bronchioles, and alveolar cells but not in smooth muscle layers or tracheal cartilage. Reverse transcriptase-polymerase chain reaction, restriction enzyme analysis, and sequencing of the cDNA transcribed from mRNA of whole lung and tracheal epithelial cells indicate that a channel highly homologous to the retinal nucleotide-gated nonselective cation channel (CNG1) is present. Thus these data, along with evidence supporting the existence of signal transduction pathways elevating intracellular levels of cGMP, indicate that cGMP regulates transepithelial ion transport in lung epithelial tissues.

Aging affects the association between endothelial nitric oxide synthase gene polymorphism and acute myocardial infarction in the Korean male population.

OBJECTIVES: The aging process affects responsiveness and other functions of endothelium and vascular smooth muscle cells, predisposing the old vessels to the development of atherosclerotic lesions. Endothelial nitric oxide synthase (ecNOS) gene polymorphisms were shown to affect the occurrence of acute myocardial infarction (AMI). We hypothesized that aging may affect the association between the ecNOS gene polymorphism and AMI. METHODS: We investigated the age-related distribution of the ecNOS gene a/b polymorphism in 121 male AMI patients and 206 age-matched healthy male controls. RESULTS: The aa, ab and bb genotypes were found in 1, 49 and 156 cases among the control subjects and 5, 23 and 93 cases among the AMI patients, respectively. There was a significant correlation between the ecNOS polymorphism and AMI (p = 0.045). When the correlation was analyzed by age, the significance remained only in the group below the age of 51 (p = 0.009). The proportion of smokers was increased in the young patients when compared to the old patients (p = 0.033), indicating that smoking also has greater effect on the younger population. The incidences of hypertension and diabetes mellitus, however, were similar in both populations. CONCLUSION: Our work provides the first evidence that links ecNOS polymorphism to the risk of AMI in relation to age. Young persons who smoke or have ecNOSaa genotype may have an increased risk of developing AMI. The functional as well as structural changes associated with aging in the vascular endothelium may mask the effect of the ecNOS polymorphism in the development of AMI in old persons.

Fenofibrate lowers abdominal and skeletal adiposity and improves insulin sensitivity in OLETF rats.

The effect of peroxisome proliferator-activated receptor (PPAR)-alpha activators on the liver is well established, but the other effects on muscle and adipose tissue about lipid metabolism and insulin sensitivity are not clear. We investigated whether PPAR-alpha activation affects adiposity of skeletal muscle as well as adipose tissue and improves insulin sensitivity in spontaneous type 2 diabetes model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Thirty-three weeks of aged, 20 male OLETF rats were divided into two groups. Control group (n=10) was fed with chow and treatment group (n=10) with chow contained fenofibrate for 7 weeks. At the age of 40 weeks, all rats were examined with MRI, intravenous glucose tolerance test, and then sacrificed for measurement of fat mass and RNA analyses. The total fat (the sum of subcutaneous, mesenteric, epididymal, and retroperitoneal fat pads) measured by dissection was significantly reduced in treatment group. The signal intensity of muscular adiposity was significantly decreased in treatment group. The mRNA levels of FAT/CD36 and mitochondrial carnitine palmitoyltransferase I (M-CPT I) in liver were remarkably increased. Fasting plasma insulin and leptin levels, insulin response after intravenous glucose loading and homeostasis model assessment insulin resistance (HOMA(IR)) index were lowered in treatment group. Fenofibrate increase mitochondrial fatty acid beta-oxidation in liver but not in skeletal muscle and lower the plasma levels of triglyceride and free fatty acid. It might result in reduction of adiposity of truncal adipose tissue and skeletal muscle. We suggest that reduction of adiposity in trunk and skeletal muscle might improve insulin sensitivity.

Characterization of inwardly rectifying K(+) conductance across the basolateral membrane of rat tracheal epithelia.

The rat primary cultured-airway monolayer has been an excellent model for deciphering the ion channel after nystatin permeabilization of its basolateral or apical membrane. Inwardly rectifying K(+) currents were characterized across the basolateral membrane in symmetrical HCO(-)(3)-free high K(+) Ringer's solution (125 mM) in this study. The potency of K(+) channel inhibitors against K(+) conductance was Ba(2+) (IC(50) = 5 microM) > Cs(+) (IC(50) = 2 mM) >> glybenclamide (IC(50) > 5 mM) >> TEA (IC(50) >> 100 mM). The application of basolateral Cs(+) changed K(+) conductance into an oscillating current, and its frequency (holding voltage = -100 mV) increased with increase in concentration of basolateral Cs(+) (0.05-5 mM) and in degree of hyperpolarization. Addition of basolateral Cs(+) blocked inward current strongly at -100 mV and hardly at all at -60 mV, giving a sharp curvature to the I-V relation of the IRK current. RT-PCR, Western blotting, and immunohistochemical analyses showed that Kir2.1 might be present in basolateral membrane of tracheal epithelia and plasma membrane of pulmonary alveolar cells.