Mitochondria-rich cells as experimental model in studies of epithelial chloride channels

The mitochondria-rich (mr) cell of amphibian skin epithelium is differentiated as a highly specialised pathway for passive transepithelial transport of chloride. The apical membrane of mr cells expresses several types of Cl(-) channels, of which the function of only two types has been studied in detail. (i) One type of channel is gated by voltage and external chloride concentration. This intriguing type of regulation leads to opening of channels only if [Cl(-)](o) is in the millimolar range and if the electrical potential is of a polarity that secures an inwardly directed net flux of this ion. Reversible voltage activations of the conductance proceed with long time constants, which depend on V in such a way that the rate of conductance activation increases when V is clamped at more negative values (serosal bath grounded). The gating seems to involve processes that are dependent on F-actin localised in the submembrane domain in the neck region of the flask-shaped mr cell. (ii) The other identified Cl(-) pathway of mr cells is mediated by small-conductance apical CFTR chloride channels as concluded from its activation via beta-adrenergic receptors, ion selectivity, genistein stimulation and inhibition by glibenclamide. bbCFTR has been cloned, and immunostaining has shown that the gene product is selectively expressed in mr cells. There is cross-talk between the two pathways in the sense that activation of the conductance of the mr cell by voltage clamping excludes activation via receptor occupation, and vice versa. The mechanism of this cross-talk is unknown.     

Functional and structural changes due to a serine to alanine mutation in the active-site flap of enolase

Crystallographic and kinetic methods have been used to characterize a site-specific variant of yeast enolase in which Ser 39 in the active-site flap has been changed to Ala. In the wild-type enzyme, the carbonyl and hydroxyl groups of Ser 39 chelate the second equivalent of divalent metal ion, effectively anchoring the flap over the fully liganded active site. With Mg(2+) as the activating cation, S39A enolase has <0.01% of wild-type activity as reported previously [J.M. Brewer, C.V. Glover, M.J. Holland, L. Lebioda, Biochim. Biophys. Acta 1383 (2) (1998) 351-355]. Measurements of (2)H kinetic isotope effects indicate that the proton abstraction from 2-phosphoglycerate (2-PGA) is significantly rate determining. Analysis of the isotope effects provides information on the relative rates of formation and breakdown of the enolate intermediate. Moreover, assays with different species of divalent metal ions reveal that with S39A enolase (unlike the case of wild-type enolase), more electrophilic metal ions promote higher activities. The kinetic results with the S39A variant support the notions that a rate-limiting product release lowers the activity of wild-type enolase with more electrophilic metal ions and that the metal ions are used to acidify the C2-proton of 2-PGA. The S39A enolase was co-crystallized with Mg(2+) and the inhibitor phosphonoacetohydroxamate (PhAH). The structure was solved and refined at a resolution of 2.1 A. The structure confirms the conjecture that the active-site flap is opened in the mutant protein. PhAH chelates to both Mg ions as in the corresponding structure of the wild-type complex. Positions of the side chains of catalytic groups, Lys 345 and Glu 211, and of "auxiliary" residues Glu 168 and Lys 396 are virtually unchanged relative to the complex with the wild-type protein. His 159, which hydrogen bonds to the phosphonate oxygens in the wild-type complex, is 5.7 A from the closest phosphonate oxygen, and the loop (154-166) containing His 159 is shifted away from the active center. A peripheral loop, Glu 251-Gly 275, also moves to open access to the active site.     

Dynamics of human DNA topoisomerases IIalpha and IIbeta in living cells

DNA topoisomerase (topo) II catalyses topological genomic changes essential for many DNA metabolic processes. It is also regarded as a structural component of the nuclear matrix in interphase and the mitotic chromosome scaffold. Mammals have two isoforms (alpha and beta) with similar properties in vitro. Here, we investigated their properties in living and proliferating cells, stably expressing biofluorescent chimera of the human isozymes. Topo IIalpha and IIbeta behaved similarly in interphase but differently in mitosis, where only topo IIalpha was chromosome associated to a major part. During interphase, both isozymes joined in nucleolar reassembly and accumulated in nucleoli, which seemed not to involve catalytic DNA turnover because treatment with teniposide (stabilizing covalent catalytic DNA intermediates of topo II) relocated the bulk of the enzymes from the nucleoli to nucleoplasmic granules. Photobleaching revealed that the entire complement of both isozymes was completely mobile and free to exchange between nuclear subcompartments in interphase. In chromosomes, topo IIalpha was also completely mobile and had a uniform distribution. However, hypotonic cell lysis triggered an axial pattern. These observations suggest that topo II is not an immobile, structural component of the chromosomal scaffold or the interphase karyoskeleton, but rather a dynamic interaction partner of such structures.     

Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout

Onehundred and forty-eight out of onehundred and fifty strains of Vibrio anguillarum isolated from vibriosis in Danish marine aquaculture produced bacterial communication signals, acylated homoserine lactones, eliciting a response in the Agrobacterium tumefaciens (pZLR4) monitoring system. One strain, a serotype O4, induced a strong response in the Chromobacterium violaceum (CV026) monitoring system. Profiles of AHLs determined by TLC separation revealed the presence of at least four AHLs and a compound similar to N-3-oxo-decanoyl homoserine lactone (3-oxo-C10-HSL) was present in all strains. The production rate of the presumed 3-oxo-C10-HSL followed the growth rate of V. anguillarum whereas the production rate of a small AHL (Rf value of 0.74) increased faster than the growth rate of V. anguillarum indicating autoinduction. AHLs were produced by all serotypes (O1 to O10) and by non-typable strains. During infection with V. anguillarum, AHLs could be extracted from liver, kidney and muscle of rainbow trout and AHLs were detected both in vitro and in vivo when cell numbers reached 10(7) per ml or gram. Preliminary investigations of interactions between AHLs and the fish immune system were carried out determining oxidative burst of fish macrophages exposed to 3-oxo-C10-HSL. No activation or suppression of the superoxide anion production in the head kidney macrophages was seen when treated with the AHL compound in concentrations of 1 nM-10 microM. Our data show that AHLs are produced by almost all V. anguillarum strains and that no clear pattern relating AHL production to disease or virulence appear.     

Cdk5 is essential for synaptic vesicle endocytosis

Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.     

PPARalpha /gamma ragaglitazar eliminates fatty liver and enhances insulin action in fat-fed rats in the absence of hepatomegaly

Peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma agonists lower lipid accumulation in muscle and liver by different mechanisms. We investigated whether benefits could be achieved on insulin sensitivity and lipid metabolism by the dual PPARalpha/gamma agonist ragaglitazar in high fat-fed rats. Ragaglitazar completely eliminated high-fat feeding-induced liver triglyceride accumulation and visceral adiposity, like the PPARalpha agonist Wy-14643 but without causing hepatomegaly. In contrast, the PPARgamma agonist rosiglitazone only slightly lessened liver triglyceride without affecting visceral adiposity. Compared with rosiglitazone or Wy-14643, ragaglitazar showed a much greater effect (79%, P < 0.05) to enhance insulin's suppression of hepatic glucose output. Whereas all three PPAR agonists lowered plasma triglyceride levels and lessened muscle long-chain acyl-CoAs, ragaglitazar and rosiglitazone had greater insulin-sensitizing action in muscle than Wy-14643, associated with a threefold increase in plasma adiponectin levels. There was a significant correlation of lipid content and insulin action in liver and particularly muscle with adiponectin levels (P < 0.01). We conclude that the PPARalpha/gamma agonist ragaglitazar has a therapeutic potential for insulin-resistant states as a PPARgamma ligand, with possible involvement of adiponectin. Additionally, it can counteract fatty liver, hepatic insulin resistance, and visceral adiposity generally associated with PPARalpha activation, but without hepatomegaly.     

Ventricular cardiotrophin-1 activation precedes BNP in experimental heart failure

Both cardiotrophin-1 (CT-1) and B-type or brain natriuretic peptide (BNP) are activated by cardiomyocyte stretch, and gene expression of CT-1 and BNP are augmented in the heart in experimental and human congestive heart failure (CHF). The goal of this study was to define cardiac gene expression of CT-1 and BNP by Northern blot analysis in normal (n=5), early left ventricular dysfunction (ELVD, n=5) and overt CHF dogs (n=5), in which ventricular function is progressively decreased. CT-1 mRNA was detected in both atria and ventricles in normal dogs. Ventricular CT-1 mRNA production increased in ELVD, and it further increased in overt CHF. Ventricular BNP mRNA remained below or at the limit of detection in normal and ELVD models, and it markedly increased in overt CHF. This study reports differential regulation of gene expression of CT-1 and BNP in the heart during the progression of CHF, and demonstrates that ventricular CT-1 gene activation precedes ventricular BNP gene activation.     

Change in substrate metabolism in isolated mouse hearts following ischemia-reperfusion

Several genetic and transgenic mouse models are currently being used for studying the regulation of myocardial contractility under normal conditions and in disease states. Little information has been provided, however, about myocardial energy metabolism in mouse hearts. We measured glycolysis, glucose oxidation and palmitate oxidation (using ³H-glucose, ¹⁴C-glucose and ³H-palmitate) in isolated working mouse hearts during normoxic conditions (control group) and following a 15 min global no-flow ischemic period (reperfusion group). Fifty min following reperfusion (10 min Langendorff perfusion + 40 min working heart perfusion) aortic flow, coronary flow, cardiac output, peak systolic pressure and heart rate were 44 ± 4, 88 ± 4, 57 ± 4, 94 ± 2 and 81 ± 4% of pre-ischemic values. Rates of glycolysis and glucose oxidation in the reperfusion group (13.6 ± 0.8 and 2.8 ± 0.2 mol/min/g dry wt) were not different from the control group (12.3 ± 0.6 and 2.5 ± 0.2 mol/min/g dry wt). Palmitate oxidation, however, was markedly elevated in the reperfusion group as compared to the control group (576 ± 37 vs. 357 ± 21 nmol/min/g dry wt, p < 0.05). This change in myocardial substrate utilization was accompanied by a marked fall in cardiac efficiency measured as cardiac output/oxidative ATP production (136 ± 10 vs. 54 ± 5 ml/mol ATP, p < 0.05, control and reperfusion group, respectively). We conclude that ischemia-reperfusion in isolated working mouse hearts is associated with a shift in myocardial substrate utilization in favour of fatty acids, in line with previous observations in rat.     

Enhanced ethylene responsiveness in the Arabidopsis eer1 mutant results from a loss-of-function mutation in the protein phosphatase 2A A regulatory subunit,RCN1

Ethylene signaling in Arabidopsis begins with a family of five ethylene receptors that regulate the activity of the Raf-like kinase, CTR1. Recent work to identify novel factors required for modulating ethylene signaling resulted in the isolation of enhanced ethylene response 1 (eer1), a mutant that displays both increased sensitivity and increased amplitude of response to ethylene. Molecular cloning of eer1 reveals that its mutant phenotype results from a loss-of-function mutation in the previously characterized RCN1, one of three PP2A A regulatory subunits in Arabidopsis. Our analysis shows that neither RCN1 expression nor PP2A activity is regulated by ethylene. Instead, we found that Arabidopsis PP2A-1C, a PP2A catalytic subunit previously characterized as interacting with RCN1, associates strongly with the kinase domain of CTR1 in vitro. This likely represents a role for PP2A in modulation of CTR1 activity because an in vitro kinase assay did not reveal phosphorylation of either RCN1 or PP2A-1C by CTR1, indicating that neither of them is a substrate for CTR1. PP2A activity is required for Ras-dependent activation of mammalian Raf, with reductions in PP2A activity significantly compromising the effectiveness of this mechanism. Our genetic and biochemical results suggest that a similar requirement for PP2A activity exists for ethylene signaling, with loss-of-function mutations affecting PP2A activity possibly reducing the effectiveness of CTR1 activation, thus lowering the threshold required for manifestation of ethylene response.