Grammatical representations of macromolecular structure.

Since the first application of context-free grammars to RNA secondary structures in 1988, many researchers have used both ad hoc and formal methods from computational linguistics to model RNA and protein structure. We show how nearly all of these methods are based on the same core principles and can be converted into equivalent approaches in the framework of tree-adjoining grammars and related formalisms. We also propose some new approaches that extend these core principles in novel ways.     

Molecular Characterization of Ethylene Response Sensor 1 (BoERS1) in <Emphasis Type="Italic">Bambusa oldhamii</Emphasis>

An ethylene receptor gene named BoERS1 was cloned from a bamboo (Bambusa oldhamii) cDNA library. The open reading frame of BoERS1 was 1,899 bp and encoded a 632-amino acid protein, which contains the five conserved motifs (H, N, G1, F, and G2 boxes) of the bacterial two-component system histidine kinases and shows high sequence similarity with other ethylene receptors in plants, such as rice and maize. Expression of BoERS1 in bamboo shoots increased with the growth of the emerging shoots. In an in vitro kinase assay, the expressed histidine kinase domain of BoERS1 (BHK) was phosphorylated in the presence of Mn²⁺, and LC-ESI-MS/MS analysis showed that four amino acids, namely T442, S444, S489, and S503, were phosphorylated. It is interesting to note that S489 and S503 are located in a loop region (L1) that is found only in plant histidine kinase-containing enzymes. The identification of multiple phosphorylation sites on BoERS1 provides a new avenue for future structure–function studies of the ethylene receptor protein family.     

A microtechnique for quantification of detergent-solubilized muscarinic and nicotinic acetylcholine receptors using a semi-automated cell harvestor

A specific method for the rapid assay of muscarinic acetylcholine receptors (mAChR), either detergent-solubilized or in neuroblastoma cells, is described. This method is also applicable to the assay of nicotinic acetylcholine receptors. The procedure employs a cell harvestor and microtiter plates, and has the advantage of requiring small quantities of radioligand, microgram quantities of detergent-solubilized cholinergic receptor or less cells. The binding parameters such as the equilibrium dissociation constants (Kd) of mAChR and nicotinic acetylcholine receptor (nAChR) and inhibition constants (Ki) for antagonists determined by the present method are in excellent agreement with values determined by other methods. This assay procedure for mAChR and nAChR should facilitate the rapid screening of cholinergic agonists/antagonists and also the further purification and characterization of mAChR.     

Comparative constitutive resistance in soybean lines to Mexican bean beetle

Constitutive (i.e., preformed) parameters of Mexican bean beetle (MBB) resistance were demonstrated in PI 227687 soybeans grown under highly controlled and reproducible biotron environmental conditions which favored optimal plant growth and development. Constitutive antifeedants in PI 227687 against the beetle were variously soluble in hexane, ethyl acetate and 100% methanol. Antifeedants also remained in the aqueous residue after the sequential organic extractions. PI 227687 and MBB-susceptible Davis soybean lines each showed characteristic temporal patterns of L-phenylalanine ammonia-lyase activity. Healthy PI 227687, but not healthy Davis, also showed a characteristic temporal pattern of L-tyrosine ammonia-lyase activity. Activities of the above two enzymes are positively correlated with plant phenylpropanoid metabolism. PI 227687 and Davis leaves did not differ in content of total phenols. Products from phenylpropanoid metabolism apparently contribute to PI 227687 resistance to MBB feeding.

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Résumé Les paramètres de la résistance spontanée (c.a.d. non induite) du soja à E. varivestis ont été examinés sur des PI 227687 cultivés dans des conditions contrôlées et reproductibles d'un phytotron assurant une croissance et un développement optimaux. Les antiappétants de PI 227687 contre E. varivestris présentaient différentes solubilités dans l'hexane, l'acétate, d'éthyle et le méthanol à 100%. Les produits du métabolisme phénylpropanoïde des plantes, y compris les phytoalexines, sont fortement solubles dans un ou plusieurs de ces solvants. Les antiappétants sont restés dans le résidu aqueux après les extractions organiques séquentielles. Les PI 227687 sains, ainsi que les Davis susceptibles ont présenté les caractéristiques temporelles de l'activité tyrosine-amonia lyase. Les niveaux d'activité de ces deux enzymes sont très significativement corrélés avec le métabolisme phenylpropanoïde de la plante. Les teneurs en phénols totax de PI 227687 et Davis ne présentaient pas de différences. Les résultats semblent compatibles avec la présence des produits aglycônes du métabolisme phénylpropanoïde comme paramètres spontanés principaux de la résistance de PI 227687 à E. varivestis.

     

Opposite and selective effects of epidermal growth factor and human platelet transforming growth factor-beta on the production of secreted proteins by murine 3T3 cells and human fibroblasts

Growth regulators such as epidermal growth factor (EGF) and type beta transforming growth factor (TGF-beta) regulate the synthesis and secretion of certain proteins by cells in culture. The secretion pattern of each cell line and the effect of growth regulators on the secretion pattern are unique. EGF increased the secreted and intracellular levels of mitogen-regulated protein (MRP) and major excreted protein (MEP) by Swiss 3T3 cells. MRP is related by sequence to prolactin. MEP is a thiol protease located intracellularly in the lysosomes. EGF also selectively induced a 52,000-dalton mitogen-induced protein (MIP 52) secreted by human fibroblasts. Two types of TGF-betas were tested for their effects on the expression of secreted proteins in mouse and human fibroblasts: TGF-beta from human platelets and a growth inhibitor (GI/TGF-beta) secreted by BSC-1 cells. Each selectively decreased the levels of the two secreted proteins induced by growth factors in mouse embryo 3T3 cells and one secreted protein induced by growth factors in human fibroblasts. Platelet TGF-beta and GI/TGF-beta also induced one 48,000-dalton protein secreted by human fibroblasts. Synthesis of DNA and the incorporation of [35S]methionine into total protein in Swiss 3T3 cells were not affected by platelet TGF-beta or GI/TGF-beta. Thus, the inhibitory effect of platelet TGF-beta on the synthesis and secretion of these three proteins is due to a specific effect of platelet TGF-beta on the regulation of MRP and MEP that does not interfere with the ability of EGF to stimulate DNA or protein synthesis.     

Effects of bradykinin, GTP gamma S, R59022 and N-ethylmaleimide on inositol phosphate production in NG108-15 cells

Accumulation of inositol phosphates in NG108-15 neuroblastoma x glioma hybrid cells, pre-labeled for 24h to equilibrium, was stimulated by bradykinin, guanosine 5'-O-(3-thiotriphosphate) and the diacylglycerol kinase inhibitor R59022. Only the stimulation by bradykinin was inhibited by the bradykinin receptor antagonist [D-Arg0, Hyp3, Phe7, Thi5,8] bradykinin. Neither bradykinin nor R059022 increased the labeling of the inositol phospholipids. The sulfhydryl-alkylating reagent N-ethylmaleimide at 100 microM essentially abolished the stimulation caused by all three agents, possibly by preventing the binding of GTP to a guanine nucleotide-binding regulatory protein of as yet unknown size.     

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660, 631)

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. When tested in cultured human liver Hep G2 cells the compound inhibited the incorporation of 14C-acetate and 14C-octanoate into sterols 56% and 48% respectively at 3 x 10(-6) M with no effect on fatty acid synthesis. No noticeable effect was seen on fatty acid biosynthesis. This strongly suggests that the locus of inhibition of acetate incorporation into sterols found with this compound is the acetoacetyl-CoA thiolase step in the cholesterol biosynthesis pathway.     

Stability of pBR322-derived plasmids

The stability of pBR322-derived plasmids was studied during growth of their Escherichia coli host in the absence of antibiotics. Plasmid pBR322, as well as its delta rom and delta bla derivatives, were lost from their host within 60 generations, but a number of delta tet derivatives were quite stable under the same conditions. An evaluation of the data indicated that primary plasmid loss due to random partitioning corresponds to the generation of a plasmid-free cell about every 10(4) divisions (probability P0; = "intrinsic" instability). Secondary loss of plasmid-carrying cells resulted from a growth advantage of the plasmid-free cells when bacteria die, perhaps due to unrepaired lethal damage in the DNA, under conditions of stationary incubation (= "apparent" instability). This cell death also occurred in the absence of plasmids but was accelerated by the presence of extra plasmid DNA in the cell and further accelerated by a functional tet gene. This was the reason for the differential apparent stabilities of delta bla and delta tet plasmids. There was no indication that an accumulation of plasmid multimers contributed to the plasmid instability, as has been suggested in the literature. The value of P0 = 10(-4) is 14 orders of magnitude greater than expected under the assumption of a random (Poisson) distribution of plasmid copy numbers in a population of cells.