Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO₃⁻/CO₂ stimulation to increase ciliary beat frequency (CBF). Because apical HCO₃⁻ exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO₃⁻/CO₂ exposure in part because of greater intracellular acidification from unbalanced CO₂ influx (estimated by 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO₃⁻/CO₂ perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTR_inh172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO₃⁻/CO₂ rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.     

The Interplay of Proton,Electron, and Metabolite Supply for Photosynthetic H2 Production in Chlamydomonas reinhardtii

To obtain a detailed picture of sulfur deprivation-induced H₂ production in microalgae, metabolome analyses were performed during key time points of the anaerobic H₂ production process of Chlamydomonas reinhardtii. Analyses were performed using gas chromatography coupled to mass spectrometry (GC/MS), two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC-TOFMS), lipid and starch analysis, and enzymatic determination of fermentative products. The studies were designed to provide a detailed metabolite profile of the solar Bio-H₂ production process. This work reports on the differential analysis of metabolic profiles of the high H₂-producing strain Stm6Glc4 and the wild-type cc406 (WT) before and during the H₂ production phase. Using GCxGC-TOFMS analysis the number of detected peaks increased from 128 peaks, previously detected by GC/MS techniques, to ∼1168. More detailed analysis of the anaerobic H₂ production phase revealed remarkable differences between wild-type and mutant cells in a number of metabolic pathways. Under these physiological conditions the WT produced up to 2.6 times more fatty acids, 2.2 times more neutral lipids, and up to 4 times more fermentation products compared with Stm6Glc4. Based on these results, specific metabolic pathways involving the synthesis of fatty acids, neutral lipids, and fermentation products during anaerobiosis in C. reinhardtii have been identified as potential targets for metabolic engineering to further enhance substrate supply for the hydrogenase(s) in the chloroplast.     

Linalool Dehydratase-Isomerase,a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ∼1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mm DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V_max) were 140 nanokatals mg⁻¹ for the linalool dehydratase and 410 nanokatals mg⁻¹ for the geraniol isomerase, with apparent K_m values of 750 μm and 500 μm, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2.1.X).     

Cardiac mitochondrial preconditioning by Big Ca2+-sensitive K+ channel opening requires superoxide radical generation

ATP-sensitive K+ channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance Ca2+-sensitive K+ channel (BK(Ca)) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BK(Ca) channel on inducing mitochondrial preconditioning during I/R and the role of O2-derived free radicals in modulating protection by putative mitochondrial (m)BK(Ca) channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide (O2(-*)), and m[Ca2+] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BK(Ca) channel opener 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of O2(-*), or an antagonist of the BK(Ca) channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less O2(-*) generation, decreased m[Ca2+], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mBK(Ca) channels; 2) NS effects are blocked by BK(Ca) antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.     

Freeze-thaw increases adeno-associated virus transduction of cells

A combination of gene and cell-based therapies may provide significant advantages over existing treatments in terms of their effectiveness. However, long-term efficient gene delivery has been difficult to achieve in many cell types, including endothelial cells. We developed a freeze-thaw technique which significantly increases the transduction efficiency of recombinant adeno-associated virus vectors in human aortic endothelial cells (23-fold) and in human renal proximal tubular epithelial cells (128-fold) in comparison to current methods for transduction. Freeze-thaw resulted in a transient but significant increase in cell surface area by 1,174 ± 69.8 µM² per cell. Reduction of cryogenic medium volume and repeated freeze-thaw further increased transduction efficiency by 2.8- and 2.4-fold, respectively. Trypsinization, dimethylsulfoxide, and cold temperatures, which are also involved in cell preservation, had no significant impact on transduction efficiency. Increased transduction was also observed in mesenchymal stem cells (42-fold) by the freeze-thaw method. The potential mechanism of this novel technique likely involves an increase in the net permeable area of biological membranes caused by water crystallization. These findings provide a new approach for gene delivery in various cell types, particularly in those resistant to transduction by conventional methods. gene therapy; endothelial cells; stem cells; cell therapy     

Experience-dependent formation and recruitment of large vesicles from reserve pool

The sizes and contents of transmitter-filled vesicles have been shown to vary depending on experimental manipulations resulting in altered quantal sizes. However, whether such a presynaptic regulation of quantal size can be induced under physiological conditions as a potential alternative mechanism to alter the strength of synaptic transmission is unknown. Here we show that presynaptic vesicles of glutamatergic synapses of Drosophila neuromuscular junctions increase in size as a result of high natural crawling activities of larvae, leading to larger quantal sizes and enhanced evoked synaptic transmission. We further show that these larger vesicles are formed during a period of enhanced replenishment of the reserve pool of vesicles, from which they are recruited via a PKA- and actin-dependent mechanism. Our results demonstrate that natural behavior can induce the formation, recruitment, and release of larger vesicles in an experience-dependent manner and hence provide evidence for an additional mechanism of synaptic potentiation.     

Interaction of the amyloid precursor like protein 1 with the alpha2A-adrenergic receptor increases agonist-mediated inhibition of adenylate cyclase

Alpha2-adrenergic receptor agonists exert potent analgesic and sedative/hypnotic effects. In addition, they have been shown to be neuroprotective, but the mechanisms of these actions are still poorly defined. To isolate proteins that may control alpha2-adrenergic receptor function or trafficking, we performed a two-hybrid screen using the carboxy-terminal fourth intracellular tail of the alpha2A-adrenergic receptor as bait. This screen identified the amyloid precursor like protein 1 (APLP1), a homologue of the beta-amyloid precursor protein involved in Alzheimer's disease, as alpha2A-adrenergic receptor-binding protein. GST affinity chromatography revealed that APLP1 specifically interacts with all three human alpha2-adrenergic receptor subtypes and deletion mutant analysis confined the APLP1 domain involved in binding to alpha2-adrenergic receptors to the 13 amino acid residues Ser599-Ala611. Coimmunoprecipitations of transiently transfected cells with epitope-tagged APLP1 and alpha2-adrenergic receptors confirmed the interaction. Agonist treatment tended to increase the amount of alpha2A-adrenergic receptor associated with APLP1 while coimmunoprecipitations were not affected by the state of receptor phosphorylation or cotransfection of arrestin-3. Confocal laser microscopy showed that APLP1 causes a considerable shift of the alpha2A-adrenergic receptor localization from plasma membrane to intracellular compartments. Furthermore, cotransfection of alpha2A-adrenergic receptor and APLP1 into HEK293 cells significantly increased norepinephrine mediated inhibition of adenylate cyclase activity. These results suggest a possible role of APLP1 in regulation of alpha2A-adrenergic receptor trafficking. Moreover, we speculate that this interaction may present one mechanism by which alpha2-adrenergic receptor agonists exert their neuroprotective effects.     

Transcription of human neuronal nitric oxide synthase mRNAs derived from different first exons is partly controlled by exon 1-specific promoter sequences

The human neuronal nitric oxide synthase (NOS1) gene is subject to extensive splicing. A total of 12 NOS1 mRNA species have been identified. They differ in their 5' ends and are derived from 12 different first exons (termed exons 1a to 1l). Various cell lines whose NOS1 first exon expression patterns were representative of human brain, skin, and skeletal muscle were identified. These included A673 neuroepithelioma cells, SK-N-MC neuroblastoma cells, HaCaT keratinocyte-like cells, and C2C12 myocyte-like cells. In these cell lines, correlations were found between the exon 1 variants preferentially expressed and the promoter activities of their cognate 5' flanking sequences. These data demonstrate that expression of the different exon 1-related splice variants of NOS1 mRNA is controlled directly (at least in part) by the associated 5' flanking sequences.     

Relative importance of cellular uptake and reactive oxygen species for the toxicity of alloxan and dialuric acid to insulin-producing cells

The diabetogenic agent alloxan is selectively accumulated in insulin-producing cells through uptake via the GLUT2 glucose transporter in the plasma membrane. In the presence of intracellular thiols, especially glutathione, alloxan generates "reactive oxygen species" (ROS) in a cyclic reaction between this substance and its reduction product, dialuric acid. The cytotoxic action of alloxan is initiated by free radicals formed in this redox reaction. Autoxidation of dialuric acid generates superoxide radicals (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), and finally hydroxyl radicals ((*)OH). Thus, while superoxide dismutase (SOD) only reduced the toxicity, catalase, in particular in the presence of SOD, provided complete protection of insulin-producing cells against the cytotoxic action of alloxan and dialuric acid due to H(2)O(2) destruction and the prevention of hydroxyl radical ((*)OH) formation, indicating that it is the hydroxyl radical ((*)OH) which is the ROS ultimately responsible for cell death. After selective accumulation in pancreatic beta cells, which are weakly protected against oxidative stress, the cytotoxic glucose analogue alloxan destroys these insulin-producing cells and causes a state of insulin-dependent diabetes mellitus through ROS-mediated toxicity in rodents and in other animal species, which express this glucose transporter isoform in their beta cells.