Biventricular hypertrophy in dogs with chronic AV block: effects of cyclosporin A on morphology and electrophysiology

Chronic atrioventricular (AV) block (CAVB) and biventricular hypertrophy in dogs increase susceptibility to drug-induced polymorphic ventricular tachycardia (PVT). In various rodent models, cyclosporin A (CsA) prevented hypertrophy. A similar effect in the CAVB model would allow us to determine whether hypertrophy represents an epiphenomenon, the cause of electrophysiological changes, and/or the anatomic substrate for PVTs. Upon AV node ablation, 6 dogs were studied acutely (AAVB), 25 dogs were kept for 6 (6W) and 12 wk (12W), receiving no treatment [CTL-CAVB-6W (n=6) and CTL-CAVB-12W (n=7)] or a daily oral dose of 10-20 mg/kg CsA directly (n=6, CsA-CAVB-6W) or 6 wk after radio-frequency ablation (n=6, CsA-CAVB-12W). For the final study, dogs were anesthetized, and 60 needles were inserted into both ventricles and connected to a multiplexer mapping system. Local effective refractory periods (ERPs) were determined at 56 +/- 22 randomly selected sites (extrastimulus technique, basic cycle length=800 ms). Arrhythmias within 30 min after application of almokalant (0.34 micromol/kg iv) were registered. The hearts were then excised to obtain the heart weight-body weight index (HBWI). Compared with AAVB, CTL-CAVB-6W and CTL-CAVB-12W showed increased HBWI and ERP associated with PVT inducibility in none of six AAVB dogs, four of six CTL-CAVB-6W dogs, and one of seven CTL-CAVB-12W dogs. Compared with CTL-CAVB-6W and CTL-CAVB-12W, CsA-CAVB-6W and CsA-CAVB-12W partially prevented hypertrophy or led to a regression of hypertrophy without reducing ERP prolongation. Despite ERP prolongation, PVTs were no longer inducible with CsA treatment. Thus prolongation of refractoriness seems to provide the trigger, but hypertrophy provides the essential substrate for the induction of PVTs in this model.     

Tuberculina-rusts: a unique basidiomycetous interfungal cellular interaction with horizontal nuclear transfer

Cellular interaction of the basidiomycete Tuberculina persicina with the haploid stages of two rusts Puccinia silvatica and Tranzschelia pruni-spinosae was analyzed by serial-section electron microscopy of chemically fixed samples and samples subjected to high-pressure freezing and freeze substitution. Tuberculina persicina is a contact parasite, forming neither haustoria nor other intracellular structures. However, at contact areas between T. persicina and its hosts, distinct interfungal interactions are present. At the beginning, a hyphal cell of T. persicina invades the host cell wall with a protuberance and the cell walls of both protuberance and host cell dissolve at the point of contact. Thus, the plasma membranes of the two organisms contact and fuse to form a pore that enlarges to a final diameter of approximately 1 μm. The membrane of the fusion pore is continuous with the plasma membranes of both cells, and Tuberculina nuclei and other organelles are transferred to the rust cells. Phylogenetic and functional aspects of this curious basidiomycetous interfungal interaction are discussed, and a hypothesis of the evolutionary derivation of the Tuberculina mycoparasitism from a sexual interaction is presented.     

Analysis of sequence, map position, and gene expression reveals conserved essential genes for iron uptake in Arabidopsis and tomato

Arabidopsis (Arabidopsis thaliana) and tomato (Lycopersicon esculentum) show similar physiological responses to iron deficiency, suggesting that homologous genes are involved. Essential gene functions are generally considered to be carried out by orthologs that have remained conserved in sequence and map position in evolutionarily related species. This assumption has not yet been proven for plant genomes that underwent large genome rearrangements. We addressed this question in an attempt to deduce functional gene pairs for iron reduction, iron transport, and iron regulation between Arabidopsis and tomato. Iron uptake processes are essential for plant growth. We investigated iron uptake gene pairs from tomato and Arabidopsis, namely sequence, conserved gene content of the regions containing iron uptake homologs based on conserved orthologous set marker analysis, gene expression patterns, and, in two cases, genetic data. Compared to tomato, the Arabidopsis genome revealed more and larger gene families coding for the iron uptake functions. The number of possible homologous pairs was reduced if functional expression data were taken into account in addition to sequence and map position. We predict novel homologous as well as partially redundant functions of ferric reductase-like and iron-regulated transporter-like genes in Arabidopsis and tomato. Arabidopsis nicotianamine synthase genes encode a partially redundant family. In this study, Arabidopsis gene redundancy generally reflected the presumed genome duplication structure. In some cases, statistical analysis of conserved gene regions between tomato and Arabidopsis suggested a common evolutionary origin. Although involvement of conserved genes in iron uptake was found, these essential genes seem to be of paralogous rather than orthologous origin in tomato and Arabidopsis.     

Polyclonal anti-PrP auto-antibodies induced with dimeric PrP interfere efficiently with PrPSc propagation in prion-infected cells

Prion diseases are neurodegenerative infectious disorders for which no prophylactic regimens are known. In order to induce antibodies/auto-antibodies directed against surface-located PrP(c), we used a covalently linked dimer of mouse prion protein expressed recombinantly in Escherichia coli. Employing dimeric PrP as an immunogen we were able to effectively overcome autotolerance against murine PrP in PrP wild-type mice without inducing obvious side effects. Treatment of prion-infected mouse cells with polyclonal anti-PrP antibodies generated in rabbit or auto-antibodies produced in mice significantly inhibited endogenous PrP(Sc) synthesis. We show that polyclonal antibodies are binding to surface-located PrP(c), thereby interfering with prion biogenesis. This effect is much more pronounced in the presence of full IgG molecules, which, unlike Fab fragments, seem to induce a significant cross-linking of surface PrP. In addition, we found immune responses against different epitopes when comparing antibodies induced in rabbits and PrP wild-type mice. Only in the auto-antibody situation in mice an immune reaction against a region of PrP is found that was reported to be involved in the PrP(Sc) conversion process. Our data point to the possibility of developing means for an active immunoprophylaxis against prion diseases.     

The Ca-activated Cl channel and its control in rat olfactory receptor neurons

Odorants activate sensory transduction in olfactory receptor neurons (ORNs) via a cAMP-signaling cascade, which results in the opening of nonselective, cyclic nucleotide-gated (CNG) channels. The consequent Ca2+ influx through CNG channels activates Cl channels, which serve to amplify the transduction signal. We investigate here some general properties of this Ca-activated Cl channel in rat, as well as its functional interplay with the CNG channel, by using inside-out membrane patches excised from ORN dendritic knobs/cilia. At physiological concentrations of external divalent cations, the maximally activated Cl current was approximately 30 times as large as the CNG current. The Cl channels on an excised patch could be activated by Ca2+ flux through the CNG channels opened by cAMP. The magnitude of the Cl current depended on the strength of Ca buffering in the bath solution, suggesting that the CNG and Cl channels were probably not organized as constituents of a local transducisome complex. Likewise, Cl channels and the Na/Ca exchanger, which extrudes Ca2+, appear to be spatially segregated. Based on the theory of buffered Ca2+ diffusion, we determined the Ca2+ diffusion coefficient and calculated that the CNG and Cl channel densities on the membrane were approximately 8 and 62 micro m-2, respectively. These densities, together with the Ca2+ diffusion coefficient, demonstrate that a given Cl channel is activated by Ca2+ originating from multiple CNG channels, thus allowing low-noise amplification of the olfactory receptor current.     

The tumor suppressor cybL,a component of the respiratory chain,mediates apoptosis induction

A genetic screen was established to clone apoptosis-inducing genes in a high-throughput format. It led to the isolation of several proapoptotic genes whose proteins are localized to mitochondria. One of the isolated genes is cytochrome bL (cybL also known as SDHC, CII-3, or QPs-1), a component of the respiratory chain complex II. It was further investigated because both cybL and another component of complex II, cybS, have recently been identified as tumor suppressor proteins, some of which act by controlling apoptosis. Our studies reveal that cell death induction by cybL expression is concomitant with a transient inhibition of complex II and the generation of reactive oxygen species. Importantly, cells that are constitutively deficient in cybL are resistant to a variety of proapoptotic cytostatic drugs and to the effects of the Fas receptor. Our results therefore identify complex II as a sensor for apoptosis induction and could explain the unexpected observation that complex II is inactivated in tumors.     

Cation channels trigger apoptotic death of erythrocytes

Erythrocytes are devoid of mitochondria and nuclei and were considered unable to undergo apoptosis. As shown recently, however, the Ca(2+)-ionophore ionomycin triggers breakdown of phosphatidylserine asymmetry (leading to annexin binding), membrane blebbing and shrinkage of erythrocytes, features typical for apoptosis in nucleated cells. In the present study, the effects of osmotic shrinkage and oxidative stress, well-known triggers of apoptosis in nucleated cells, were studied. Exposure to 850 mOsm for 24 h, to tert-butyl-hydroperoxide (1 mM) for 15 min, or to glucose-free medium for 48 h, all elicit erythrocyte shrinkage and annexin binding, both sequelae being blunted by removal of extracellular Ca(2+) and mimicked by ionomycin (1 microM). Osmotic shrinkage and oxidative stress activate Ca(2+)-permeable cation channels and increase cytosolic Ca(2+) concentration. The channels are inhibited by amiloride (1 mM), which further blunts annexin binding following osmotic shock, oxidative stress and glucose depletion. In conclusion, osmotic and oxidative stress open Ca(2+)-permeable cation channels in erythrocytes, thus increasing cytosolic Ca(2+) activity and triggering erythrocyte apoptosis.     

Phosphorylation of eukaryotic initiation factor 2 by heme-regulated inhibitor kinase-related protein kinases in Schizosaccharomyces pombe is important for fesistance to environmental stresses

Protein synthesis is regulated by the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) in response to different environmental stresses. One member of the eIF2alpha kinase family, heme-regulated inhibitor kinase (HRI), is activated under heme-deficient conditions and blocks protein synthesis, principally globin, in mammalian erythroid cells. We identified two HRI-related kinases from Schizosaccharomyces pombe which have full-length homology with mammalian HRI. The two HRI-related kinases, named Hri1p and Hri2p, exhibit autokinase and kinase activity specific for Ser-51 of eIF2alpha, and both activities were inhibited in vitro by hemin, as previously described for mammalian HRI. Overexpression of Hri1p, Hri2p, or the human eIF2alpha kinase, double-stranded-RNA-dependent protein kinase (PKR), impeded growth of S. pombe due to elevated phosphorylation of eIF2alpha. Cells from strains with deletions of the hri1(+) and hri2(+) genes, individually or in combination, exhibited a reduced growth rate when exposed to heat shock or to arsenic compounds. Measurements of in vivo phosphorylation of eIF2alpha suggest that Hri1p and Hri2p differentially phosphorylate eIF2alpha in response to these stress conditions. These results demonstrate that HRI-related enzymes are not unique to vertebrates and suggest that these eIF2alpha kinases are important participants in diverse stress response pathways in some lower eukaryotes.     

Peroxynitrite-induced inhibition and nitration of cardiac myofibrillar creatine kinase

Although cardiac peroxynitrite formation and attendant protein nitration is an established event in both acute and chronic settings of cardiac failure, the putative intracellular targets involved remain incompletely defined. We have recently shown that the myofibrillar isoform of creatine kinase (a critical energetic controller of cardiomyocyte contractility) may be a particularly sensitive target of peroxynitrite-induced nitration and inactivation in vivo. However, the kinetic and mechanistic aspects of this interaction remain undefined. Here we tested the hypothesis that myofibrillar creatine kinase is sensitive to inhibition by peroxynitrite, and investigated the mechanistic role for tyrosine nitration in this process. Peroxynitrite potently and irreversibly inhibited myofibrillar creatine kinase capacity (Vmax), at concentrations as low as 100 nM, while substrate affinity (Km) was unaffected. Concentration-dependent nitration of myofibrillar creatine kinase was observed. The extent of nitration was linearly related to peroxynitrite concentration and highly correlated to the extent of myofibrillar creatine kinase inhibition. This inhibition was not reversible by treatment with free cysteine (250 microM), but pre-incubation with substrate (phosphocreatine and/or ATP) provided significant protection of MM-CK from both nitration and inhibition. These results suggest that myofibrillar creatine kinase is a highly sensitive target of peroxynitrite-mediated inhibition, and that nitration may mediate this inhibition.