Swiss 3T3 mouse embryo fibroblasts transfected with a human prepro-GRP gene synthesize and secrete pro-GRP rather than GRP

A prepro-gastrin-releasing peptide (GRP) gene was introduced into Swiss 3T3 mouse embryo fibroblasts by DNA transfection in an attempt to establish autocrine growth stimulation. Clonal transfectants expressed varying amounts of GRP encoding mRNA. They synthesized and secreted a ～ 15-kd pro-GRP hormone but not fully processed 2.8-kd GRP. Accordingly, no changes in growth properties were associated with GRP gene expression. We postulate that Swiss 3T3 fibroblasts lack the enzymes necessary to process significantly pro-GRP into biologically active peptides and that this deficiency may be responsible for the failure to establish autocrine growth stimulation in the transfected cells.     

The Anatomy of the Developing Bud Union and its Relationship to Dwarfing in Apple

Light microscopy has been used to study the effect of dwarfingand semi-dwarfing apple rootstocks on the early developmentof bud-unions with 'Gala', and the anatomy of 2-year-old bud-unionsbetween 'Bramley' and the same rootstocks. The bridging of thecut edges of the cambia of bud and rootstock was achieved bydifferentiation of callus formed at an early stage in budding.New cambial cells were aligned at right angles to the pre-existingcambia, with their long axes horizontal. Subsequently-formedxylem adopted this arrangement, so that fibres and vessels werearranged obliquely to the axis of the stem. At the interfacebetween the bud and dwarfing rootstocks vessels with smallerthan normal diameter were formed, indicating the presence ofelevated levels of auxin in this region. In addition, littlexylem was produced in the adjacent rootstock tissue. In thecase of semi-dwarfing rootstocks, the rootstock produced normalxylem after a brief interruption. We suggest that failure ofauxin to cross the bud-union interface in the case of the dwarfingrootstocks leads to reduced rootstock xylem formation, and hencea poor supply of water and minerals to the scion, and this underliesthe dwarfing effect.Copyright 1994, 1999 Academic Press Apple, budding, dwarfing, anatomy, graft union     

Application for PCR technology to subtractive cDNA cloning: identification of genes expressed specifically in murine plasmacytoma cells.

We describe a simple method for preparing a renewable source of subtractive cDNA which can be used as a hybridization probe or as insert which can be cloned into a variety of convenient vectors. This has been done by ligating a double-stranded oligonucleotide to each end of double-stranded subtractive cDNA, and then using this oligonucleotide sequence to amplify the heterogeneous population of cDNA molecules using the polymerase chain reaction and thermostable Taq DNA polymerase. This method improves the chances for identifying cDNA clones representing low abundance mRNAs that are expressed differentially. Using this approach, we have identified cDNA clones which detect three different low abundance mRNAs that are expressed in mouse plasmacytoma cell lines but not in mouse pre-B or B lymphoma cell lines.     

A comparison of the metabolic fate of Fatty acids of different chain lengths in developing oilseeds

To determine if medium and long chain fatty acids can be appropriately metabolized by species that normally produce 16 and 18 carbon fatty acids, homogenates of developing Cuphea wrightii, Carthamus tinctorius, and Crambe abyssinica seeds were incubated with radiolabeled lauric, palmitic, oleic, and erucic acids. In all three species, acyl-CoA synthetase showed broad substrate specificity in synthesis of acyl-coenzyme A (CoA) from any of the fatty acids presented. In Carthamus, two- to fivefold less of the foreign FAs, lauric, and erucic acid was incorporated into acyl-CoAs than palmitic and oleic acid. Lauric and erucic acid also supported less glycerolipid synthesis in Carthamus than palmitic and oleic acid, but the rate of acyl-CoA synthesis did not control rate of glycerolipid synthesis. In all species examined, medium and long chain fatty acids were incorporated predominantly into triacylglycerols and were almost excluded from phospholipid synthesis, whereas palmitic and oleic acid were found predominantly in polar lipids. However, the rate of esterification of unusual fatty acids to triacylglycerol is slow in species that do not normally synthesize these acyl substrates.     

Detecting recombination from gene trees

In this article, a method is proposed for detecting recombination in the sequences of a gene from a set of closely related organisms. The method, the Homoplasy Test, is appropriate when the sequences are rather similar, differing by 1%-5% of nucleotides. It is effective in detecting relatively frequent recombination between a set of rather similar strains, in contrast to previous methods which detect rare or unique transfers between more distant strains. It is based on the fact that, if there is no recombination and if no repeated mutations have occurred (homoplasy), then the number of polymorphic sites, v, is equal to the number of steps, t, in a most-parsimonious tree. If the number of "apparent homoplasies" in the most-parsimonious tree, h = t-v, is greater than zero, then either homoplasies have occurred by mutation or there has been recombination. An estimate of the distribution of h expected on the null hypothesis of no recombination depends on Se, the "effective site number," defined as follows: if ps is the probability that two independent substitutions in the gene occur at the same site, then Se = 1/ps. Se can be estimated if a suitable outgroup is available. The Homoplasy Test is applied to three bacterial genes and to simulated gene trees with varying amounts of recombination. Methods of estimating the rate, as opposed to the occurrence, of recombination are discussed.      

The bombesin receptor subtypes have distinct G protein specificities

We used an in situ reconstitution assay to examine the receptor coupling to purified G protein alpha subunits by the bombesin receptor family, including gastrin-releasing peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin receptor subtype 3 (BRS-3). Cells expressing GRP-R or NMB-R catalyzed the activation of squid retinal Galphaq and mouse Galphaq but not bovine retinal Galphat or bovine brain Galphai/o. The GRP-R- and NMB-R-catalyzed activations of Galphaq were dependent upon and enhanced by different betagamma dimers in the same rank order as follows: bovine brain betagamma > beta1gamma2 > beta1gamma1. Despite these qualitative similarities, GRP-R and NMB-R had distinct kinetic properties in receptor-G protein coupling. GRP-R had higher affinities for bovine brain betagamma, beta1gamma1, and beta1gamma2 and squid retinal Galphaq. In addition, GRP-R showed higher catalytic activity on squid Galphaq. Like GRP-R and NMB-R, BRS-3 did not catalyze GTPgammaS binding to Galphai/o or Galphat. However, BRS-3 showed little, if any, coupling with squid Galphaq but clearly activated mouse Galphaq. GRP-R and NMB-R catalyzed GTPgammaS binding to both squid and mouse Galphaq, with GRP-R activating squid Galphaq more effectively, and NMB-R also showed slight preference for squid Galphaq. These studies reveal that the structurally similar bombesin receptor subtypes, in particular BRS-3, possess distinct coupling preferences among members of the Galphaq family.     

Phosphorylation uncouples the gastrin-releasing peptide receptor from G(q)

Previous work on the desensitization of G protein-coupled receptors has focused on the role of arrestin binding following receptor phosphorylation. We have examined the hypothesis that phosphorylation alone contributes to desensitization. In this study we demonstrate that for the G(q)-coupled gastrin-releasing peptide receptor (GRP-R), phosphorylation by GRK2 to a stoichiometry of approximately 1 mol PO(4)/mol GRP-R is sufficient in the absence of arrestin to reduce the rate of receptor catalyzed G protein activation by approximately 80%. Furthermore, GRP-Rs exposed in vivo to agonist are rapidly phosphorylated to a similar stoichiometry and are desensitized to a similar degree. Finally, the molecular mechanism for both in vitro GRK2-induced and in vivo agonist-induced desensitization is primarily a decrease in the maximum velocity (V(max)) for the catalysis of guanine nucleotide exchange by the GRP-R rather than a change in the affinity of the receptor for the alpha(q) or betagamma subunits. Based on these results, we suggest that, for some G protein-coupled receptors, phosphorylation has a role in desensitization that is independent of arrestin.     

Annexins: multifunctional components of growth and adaptation

Plant annexins are ubiquitous, soluble proteins capable of Ca(2+)-dependent and Ca(2+)-independent binding to endomembranes and the plasma membrane. Some members of this multigene family are capable of binding to F-actin, hydrolysing ATP and GTP, acting as peroxidases or cation channels. These multifunctional proteins are distributed throughout the plant and throughout the life cycle. Their expression and intracellular localization are under developmental and environmental control. The in vitro properties of annexins and their known, dynamic distribution patterns suggest that they could be central regulators or effectors of plant growth and stress signalling. Potentially, they could operate in signalling pathways involving cytosolic free calcium and reactive oxygen species.