Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates

Grape berries contain compounds that aggregate with and precipitate RNA in the presence of chaotropic agents or phenol. The procedure described here extracts RNA from finely ground tissues using mild denaturants, and selectively precipitates the aggregate-forming material with 30% ethanol. The resulting RNA is suitable for northern blot analysis and translationin vitro.     

High-level expression of murine terminal deoxynucleotidyl transferase inEscherichia coli grown at low temperature and overexpressingargU tRNA

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5′-triphosphates onto the 3′-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production inEscherichia coli of murine TdT with minimal proteolysis and high specific activity.     

NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol

The discovery of autosomal dominant hypercholesterolemic patients with mutations in the PCSK9 gene, encoding the proprotein convertase NARC-1, resulting in the missense mutations suggested a role in low density lipoprotein (LDL) metabolism. We show that the endoplasmic reticulum-localized proNARC-1 to NARC-1 zymogen conversion is Ca2+-independent and that within the zymogen autocatalytic processing site SSVFAQ [downward arrow]SIP Val at P4 and Pro at P3' are critical. The S127R and D374Y mutations result in approximately 50-60% and > or =98% decrease in zymogen processing, respectively. In contrast, the double [D374Y + N157K], F216L, and R218S natural mutants resulted in normal zymogen processing. The cell surface LDL receptor (LDLR) levels are reduced by 35% in lymphoblasts of S127R patients. The LDLR levels are also reduced in stable HepG2 cells overexpressing NARC-1 or its natural mutant S127R, and this reduction is abrogated in the presence of 5 mm ammonium chloride, suggesting that overexpression of NARC-1 increases the turnover rate of the LDLR. Adenoviral expression of wild type human NARC-1 in mice resulted in a maximal approximately 9-fold increase in circulating LDL cholesterol, while in LDLR-/- mice a delayed approximately 2-fold increase in LDL cholesterol was observed. In conclusion, NARC-1 seems to affect both the level of LDLR and that of circulating apoB-containing lipoproteins in an LDLR-dependent and -independent fashion.     

Identification of the protein kinase C phosphorylation site in neuromodulin

Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin [Alexander, K. A., Cimler, B. M., Meier, K. E., & Storm, D. R. (1987) J. Biol. Chem. 262, 6108-6113], and we have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar Km values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [gamma-32P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32P-labeled tryptic peptide was generated from phosphorylated neuromodulin. The sequence of this peptide was IQASFR. The serine in this peptide corresponds to position 41 of the entire protein, which is adjacent to or contained within the calmodulin binding domain of neuromodulin. A synthetic peptide, QASFRGHITRKKLKGEK, corresponding to the calmodulin binding domain with a few flanking residues, including serine-41, was also phosphorylated by protein kinase C. We conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmodulin to neuromodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

GABAB receptor expression and function in olfactory receptor neuron axon growth

Neurotransmitters have been implicated in regulating growth cone motility and guidance in the developing nervous system. Anatomical and electrophysiological studies show the presence of functional GABAB receptors on adult olfactory receptor neuron (ORN) nerve terminals. Using antisera against the GABAB R1a/b receptor isoforms we show that developing mouse olfactory receptor neurons express GABAB receptors from embryonic day 14 through to adulthood. GABAB receptors are present on axon growth cones from both dissociated ORNs and olfactory epithelial explants. Neurons in the olfactory bulb begin to express glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, from E16 through to adulthood. When dissociated ORNs were cultured in the presence of the GABAB receptor agonists, baclofen or SKF97541, neurite outgrowth was significantly reduced. Concurrent treatment of the neurons with baclofen and the GABAB receptor antagonist CGP54626 prevented the inhibitory effects of baclofen on ORN neurite outgrowth. These results show that growing ORN axons express GABAB receptors and are sensitive to the effects of GABAB receptor activation. Thus, ORNs in vivo may detect GABA release from juxtaglomerular cells as they enter the glomerular layer and use this as a signal to limit their outgrowth and find synaptic targets in regeneration and development.     

Structure-based tailoring of compound libraries for high-throughput screening: discovery of novel EphB4 kinase inhibitors

High-throughput docking is a computational tool frequently used to discover small-molecule inhibitors of enzymes or receptors of known three-dimensional structure. Because of the large number of molecules in chemical libraries, automatic procedures to prune multimillion compound collections are useful for high-throughput docking and necessary for in vitro screening. Here, we propose an anchor-based library tailoring approach (termed ALTA) to focus a chemical library by docking and prioritizing molecular fragments according to their binding energy which includes continuum electrostatics solvation. In principle, ALTA does not require prior knowledge of known inhibitors, but receptor-based pharmacophore information (hydrogen bonds with the hinge region) is additionally used here to identify molecules with optimal anchor fragments for the ATP-binding site of the EphB4 receptor tyrosine kinase. The 21,418 molecules of the focused library (from an initial collection of about 730,000) are docked into EphB4 and ranked by force-field-based energy including electrostatic solvation. Among the 43 compounds tested in vitro, eight molecules originating from two different anchors show low-micromolar activity in a fluorescence-based enzymatic assay. Four of them are active in a cell-based assay and are potential anti-angiogenic compounds.     

The interaction between nesprins and sun proteins at the nuclear envelope is critical for force transmission between the nucleus and cytoskeleton

Maintaining physical connections between the nucleus and the cytoskeleton is important for many cellular processes that require coordinated movement and positioning of the nucleus. Nucleo-cytoskeletal coupling is also necessary to transmit extracellular mechanical stimuli across the cytoskeleton to the nucleus, where they may initiate mechanotransduction events. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, formed by the interaction of nesprins and SUN proteins at the nuclear envelope, can bind to nuclear and cytoskeletal elements; however, its functional importance in transmitting intracellular forces has never been directly tested. This question is particularly relevant since recent findings have linked nesprin mutations to muscular dystrophy and dilated cardiomyopathy. Using biophysical assays to assess intracellular force transmission and associated cellular functions, we identified the LINC complex as a critical component for nucleo-cytoskeletal force transmission. Disruption of the LINC complex caused impaired propagation of intracellular forces and disturbed organization of the perinuclear actin and intermediate filament networks. Although mechanically induced activation of mechanosensitive genes was normal (suggesting that nuclear deformation is not required for mechanotransduction signaling) cells exhibited other severe functional defects after LINC complex disruption; nuclear positioning and cell polarization were impaired in migrating cells and in cells plated on micropatterned substrates, and cell migration speed and persistence time were significantly reduced. Taken together, our findings suggest that the LINC complex is critical for nucleo-cytoskeletal force transmission and that LINC complex disruption can result in defects in cellular structure and function that may contribute to the development of muscular dystrophies and cardiomyopathies.     

Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.