Functional fluorescent Ca2+ indicator proteins in transgenic mice under TET control

Genetically encoded fluorescent calcium indicator proteins (FCIPs) are promising tools to study calcium dynamics in many activity-dependent molecular and cellular processes. Great hopes—for the measurement of population activity, in particular—have therefore been placed on calcium indicators derived from the green fluorescent protein and their expression in (selected) neuronal populations. Calcium transients can rise within milliseconds, making them suitable as reporters of fast neuronal activity. We here report the production of stable transgenic mouse lines with two different functional calcium indicators, inverse pericam and camgaroo-2, under the control of the tetracycline-inducible promoter. Using a variety of in vitro and in vivo assays, we find that stimuli known to increase intracellular calcium concentration (somatically triggered action potentials (APs) and synaptic and sensory stimulation) can cause substantial and rapid changes in FCIP fluorescence of inverse pericam and camgaroo-2.     

Determination of NG,NG-dimethyl-L-arginine, an endogenous NO synthase inhibitor, by gas chromatography-mass spectrometry

A fully validated gas chromatographic-mass spectrometric (GC-MS) method for the accurate and precise quantification of NG,NG-dimethyl-L-arginine (asymmetric dimethylarginine, ADMA), an endogenous inhibitor of the NO synthase, in cell culture supernatants and in small volumes of plasma is described. ADMA was concentrated by solid phase extraction and converted to its methyl ester pentafluoropropionic amide derivative. The derivatives were analyzed without any further purification. Using gas chromatography-chemical ionization mass spectrometry, fragment ions at m/z 634 and m/z 640 were obtained for ADMA and for NG,NG-[2H6]-dimethyl-L-arginine ([2H6]-ADMA) as internal standard, respectively. [2H6]-ADMA was synthesized by reaction of L-ornithine fastened at bromcyan-agarose with dimethylamine. The limit of detection of the method was 2 fmol, while the limit of quantitation for cell culture supernatants was 0.05 microM. The method was validated in a concentration range of 0-1.2 microM in cell culture medium and 0-2 microM in 50 microl aliquots of human plasma. The precision was > or =97% and the accuracy was determined to be > or =94%. This method is fast, rugged and an alternative to high performance liquid chromatography (HPLC) analysis of ADMA in cell culture supernatants and small volumes of human plasma.     

Delineation of ligand binding and receptor signaling activities of purified P2Y receptors reconstituted with heterotrimeric G proteins

P2Y receptors are G protein coupled receptors that respond to extracellular nucleotides to promote a multitude of signaling events. Our laboratory has purified several P2Y receptors with the goal of providing molecular insight into their: (1) ligand binding properties, (2) G protein signaling selectivities, and (3) regulation by RGS proteins and other signaling cohorts. The human P2Y₁ receptor and the human P2Y₁₂ receptor, both of which are intimately involved in ADP-mediated platelet aggregation, were purified to near homogeneity and studied in detail. After high-level expression from recombinant baculovirus infection of Sf9 insect cells, approximately 50% of the receptors were successfully extracted with digitonin. Purification of nearly homogeneous epitope-tagged P2Y receptor was achieved using metal-affinity chromatography followed by other traditional chromatographic steps. Yields of purified P2Y receptors range from 10 to 100 g/l of infected cells. Once purified, the receptors were reconstituted in model lipid vesicles along with their cognate G proteins to assess receptor function. Agonist-promoted increases in steady-state GTPase assays demonstrated the functional activity of the reconstituted purified receptor. We have utilized this reconstitution system to assess the action of various nucleotide agonists and antagonists, the relative G protein selectivity, and the influence of other proteins, such as phospholipase C, on P2Y receptor-promoted signaling. Furthermore, we have identified the RGS expression profile of platelets and have begun to assess the action of these RGS proteins in a reconstituted P2Y receptor/G protein platelet model.     

The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential

The genome of Bacillus licheniformis DSM13 consists of a single chromosome that has a size of 4,222,748 base pairs. The average G+C ratio is 46.2%. 4,286 open reading frames, 72 tRNA genes, 7 rRNA operons and 20 transposase genes were identified. The genome shows a marked co-linearity with Bacillus subtilis but contains defined inserted regions that can be identified at the sequence as well as at the functional level. B. licheniformis DSM13 has a well-conserved secretory system, no polyketide biosynthesis, but is able to form the lipopeptide lichenysin. From the further analysis of the genome sequence, we identified conserved regulatory DNA motives, the occurrence of the glyoxylate bypass and the presence of anaerobic ribonucleotide reductase explaining that B. licheniformis is able to grow on acetate and 2,3-butanediol as well as anaerobically on glucose. Many new genes of potential interest for biotechnological applications were found in B. licheniformis; candidates include proteases, pectate lyases, lipases and various polysaccharide degrading enzymes.     

Recombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues

Plant-based expression systems are attractive for the large-scale production of pharmaceutical proteins. However, glycoproteins require particular attention as inherent differences in the N-glycosylation pathways of plants and mammals result in the production of glycoproteins bearing core-xylose and core-alpha(1,3)-fucose glyco-epitopes. For treatments requiring large quantities of repeatedly administered glycoproteins, the immunological properties of these non-mammalian glycans are a concern. Recombinant glycoproteins could be retained within the endoplasmic reticulum (ER) to prevent such glycan modifications occurring in the late Golgi compartment. Therefore, we analysed cPIPP, a mouse/human chimeric IgG1 antibody binding to the beta-subunit of human chorionic gonadotropin (hCG), fused to a C-terminal KDEL sequence, to investigate the efficiency of ER retrieval and the consequences in terms of N-glycosylation. The KDEL-tagged cPIPP antibody was expressed in transgenic tobacco plants or Agrobacterium-infiltrated tobacco and winter cherry leaves. N-Glycan analysis showed that the resulting plantibodies contained only high-mannose (Man)-type Man-6 to Man-9 oligosaccharides. In contrast, the cPIPP antibody lacking the KDEL sequence was found to carry complex N-glycans containing core-xylose and core-alpha(1,3)-fucose, thereby demonstrating the secretion competence of the antibody. Furthermore, fusion of KDEL to the diabody derivative of PIPP, which contains an N-glycosylation site within the heavy chain variable domain, also resulted in a molecule lacking complex glycans. The complete absence of xylose and fucose residues clearly shows that the KDEL-mediated ER retrieval of cPIPP or its diabody derivative is efficient in preventing the formation of non-mammalian complex oligosaccharides.     

Graph-based iterative Group Analysis enhances microarray interpretation

Background  

One of the most time-consuming tasks after performing a gene expression experiment is the biological interpretation of the results by identifying physiologically important associations between the differentially expressed genes. A large part of the relevant functional evidence can be represented in the form of graphs, e.g. metabolic and signaling pathways, protein interaction maps, shared GeneOntology annotations, or literature co-citation relations. Such graphs are easily constructed from available genome annotation data. The problem of biological interpretation can then be described as identifying the subgraphs showing the most significant patterns of gene expression. We applied a graph-based extension of our iterative Group Analysis (iGA) approach to obtain a statistically rigorous identification of the subgraphs of interest in any evidence graph.     

Dendritic polyglycerol sulfates as new heparin analogues and potent inhibitors of the complement system

Due to several limitations of heparin, a widely used antithrombotic drug, there is large interest to develop alternatives. The aim of the presented study was to produce fully synthetic highly branched heparin mimetics. For this purpose, a new type of 'treelike' polysulfated polymers based on dendritic polyglycerol was synthesized. An efficient synthetic approach has been chosen to prepare several polyglycerol sulfates with different molecular weights as well as a polyglycerol carboxylate analogue and to evaluate them for their anticoagulant and anticomplementary activities. In contrast to the nonderivatized and the carboxylated polyglycerols, the polyglycerol sulfates prolong the activated partial thromboplastin time (APTT) and thrombin time (TT) and inhibit both the classical (CCA) and alternative complement activation (ACA). Whereas their anticoagulant activity in the APTT and in the TT amounts to 5.7-8.1% and 15.7-33.6%, respectively, of that of unfractionated heparin (UFH), their CCA and ACA inhibitory activity is 13.4-23.9 and 2.7-3.7 times, respectively, higher. In contrast to sulfated polysaccharides, the activities are not clearly dependent on the molecular weight, which might be due to the globular 3D-structure of the dendritic molecules. Due to the coherence between coagulation, complement activation and inflammation in the pathophysiology of numerous diseases, polyglycerol sulfates with both anticoagulant and anticomplementary activities represent promising candidates for the development of potential drugs.     

B-type natriuretic peptide limits infarct size in rat isolated hearts via KATP channel opening

B-type natriuretic peptide (BNP) has been reported to be released from the myocardium during ischemia. We hypothesized that BNP mediates cardioprotection during ischemia-reperfusion and examined whether exogenous BNP limits myocardial infarction and the potential role of ATP-sensitive potassium (K(ATP)) channel opening. Langendorff-perfused rat hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. The control infarct-to-risk ratio was 44.8 +/- 4.4% (means +/- SE). BNP perfused 10 min before ischemia limited infarct size in a concentration-dependent manner, with maximal protection observed at 10(-8) M (infarct-to-risk ratio: 20.1 +/- 5.2%, P < 0.01 vs. control), associated with a 2.5-fold elevation of myocardial cGMP above the control value. To examine the role of K(ATP) channel opening, glibenclamide (10(-6) M), 5-hydroxydecanoate (5-HD; 10(-4) M), or HMR-1098 (10(-5) M) was coperfused with BNP (10(-8) M). Protection afforded by BNP was abolished by glibenclamide or 5-HD but not by HMR-1098, suggesting the involvement of putative mitochondrial but not sarcolemmal K(ATP) channel opening. We conclude that natriuretic peptide/cGMP/K(ATP) channel signaling may constitute an important injury-limiting mechanism in myocardium.     

Effect of NO synthase inhibition on myocardial metabolism during moderate ischemia

Nitric oxide (NO) is involved in the control of myocardial metabolism. In normoperfused myocardium, NO synthase inhibition shifts myocardial metabolism from free fatty acid (FFA) toward carbohydrate utilization. Ischemic myocardium is characterized by a similar shift toward preferential carbohydrate utilization, although NO synthesis is increased. The importance of NO for myocardial metabolism during ischemia has not been analyzed in detail. We therefore assessed the influence of NO synthase inhibition with N(G)-nitro-l-arginine (l-NNA) on myocardial metabolism during moderate ischemia in anesthetized pigs. In control animals, the increase in left ventricular pressure with l-NNA was mimicked by aortic constriction. Before ischemia, l-NNA decreased myocardial FFA consumption (MV(FFA); P < 0.05), while consumption of carbohydrate and O(2) (MVo(2)) remained constant. ATP equivalents [calculated with the assumption of complete oxidative substrate decomposition (ATP(eq))] decreased with l-NNA (P < 0.05), associated with a decrease of regional myocardial function (P < 0.05). In contrast, aortic constriction had no effect on MV(FFA), while MVo(2) increased (P < 0.05) and ATP(eq) and regional myocardial function remained constant. During ischemia, alterations in myocardial metabolism were similar in control and l-NNA-treated animals: MV(FFA) decreased (P < 0.05) and net lactate consumption was reversed to net lactate production (P < 0.05). Regional myocardial function was decreased (P < 0.05), although more markedly in animals receiving l-NNA (P < 0.05). We conclude that the efficiency of oxidative metabolism was impaired by l-NNA per se, paralleled by impaired regional myocardial function. During ischemia, l-NNA had no effect on myocardial substrate consumption, indicating that NO synthases were no longer effectively involved in the control of myocardial metabolism.     

Stress kinase phosphorylation is increased in pacing-induced heart failure in rabbits

In hearts with chronic left ventricular (LV) systolic dysfunction secondary to hypertension or myocardial infarction, MAPK phosphorylation and/or activity are increased. Whether other settings of LV dysfunction not associated with ischemia-reperfusion are also characterized by increased MAPK phosphorylation or activity is unknown. After 3 wk of rapid LV pacing (400 beats/min), eight rabbits displayed clinical signs of heart failure (HF), and echocardiography revealed an increase in LV end-diastolic diameter from 15.6 +/- 0.7 (means +/- SE) to 18.8 +/- 0.7 mm and a reduced shortening fraction from 31 +/- 1to10 +/- 2% (both P < 0.05). Morphological alterations in HF included increased numbers of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cardiomyocytes, extent of fibrosis, and cross-sectional cardiomyocyte area. Total p38 MAPK did not differ between failing and normal hearts (n = 8). However, p38 MAPK phosphorylation [164,488 +/- 29,323 vs. 43,565 +/- 14,817 arbitrary units (AU), P < 0.05, densitometry] and the activities of p38 MAPK-alpha and -beta were increased in failing compared with normal hearts (149,441 +/- 38,381 and 170,430 +/- 32,952 vs. 68,815 +/- 28,984 and 81,788 +/- 22,774 AU, respectively, both P < 0.05). In failing compared with normal hearts, total and phosphorylated JNK46 and JNK54 MAPK were increased, whereas total and phosphorylated ERK MAPK remained unchanged. In pacing-induced HF, p38 and JNK MAPK phosphorylation as well as p38 MAPK activity was increased. Further studies will have to define whether or not chronic specific blockade of MAPK activity can interfere with apoptosis/fibrosis and thereby attenuate the progression of HF.