Combined expression of S-VSPalpha in two different organelles enhances its accumulation and total lysine production in leaves of transgenic tobacco plants

Soybean vegetative storage proteins (S-VSPs) accumulate to high levels in vacuoles of both wild types and heterologous plants. Here it is shown that directing S-VSPalpha to two different organelles-chloroplasts and vacuoles-in a single transgenic plant significantly increased its accumulation. Accumulation of S-VSPalpha in heterologous plants correlated with total soluble lysine. Using this approach with essential amino-acid-rich transgene proteins may lead to a breakthrough in improving plant nutritional quality.     

Evaluation of turfgrass species and cultivars for potential resistance to twolined spittlebug (Hemiptera: Cercopidae)

Potential resistance to the twolined spittlebug, Prosapia bicincta (Say), was evaluated among 56 turfgrass genotypes. Greenhouse, laboratory, and field bioassays identified differences in spittlebug survival and development, host preference and damage levels, and turfgrass tolerance to and ability to recover from pest induced injury. All centipede grasses demonstrated high levels of susceptibility, followed by bermudagrasses, seashore paspalums, and zoysiagrasses. Average nymphal survival to the adult stage ranged from 1.5 to 78.1%. Development required 38.1-62.0 d under greenhouse conditions, depending on plant taxa. Among seashore paspalums, nymphal survival to the adult stage was lowest and duration of development was longest on HI-1, 'Sea Isle 2000', 561-79, and 'Mauna Kea'. Reduced spittlebug survival and increased developmental times were also observed on the bermudagrasses BERPC 91-15 and 'Tifway'. Although zoysiagrasses supported spittlebug development and survival to the adult stage, developmental times were extended on the zoysiagrass cultivars 'Emerald' and 'El Toro'. Spittlebug preference varied with generation evaluated. First-generation spittlebugs inflicted the greatest damage on TC201 (centipede grass), 'Primavera' (bermudagrass), and 'Emerald' (zoysiagrass) in choice tests. In the fall, second-generation spittlebugs damaged TC201 (centipedegrass) and 'Sea Isle 1' (paspalum) most severely, whereas 561-79 (paspalum) and 'Emerald'(zoysiagrass) were less severely affected. Among taxa included in field trials, HI-1, 'Mauna Kea', 'Sea Isle 2000',and AP-14 paspalums, 'Tifway' bermudagrass, and 'Emerald' zoysiagrass were most tolerant (demonstrated the best regrowth potential following twolined spittlebug feeding).     

Fertile transgenic pearl millet [<Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br.] plants recovered through microprojectile bombardment and phosphinothricin selection of apical meristem-, inflorescence-, and immature embryo-derived embryogenic tissues

Pearl millet [ Pennisetum glaucum (L.) R. Br.] is a drought-tolerant cereal crop used for grain and forage. Novel traits from outside of the gene pool could be introduced provided a reliable gene-transfer method were available. We have obtained herbicide-resistant transgenic pearl millet plants by microprojectile bombardment of embryogenic tissues with the bar gene. Embryogenic tissues derived from immature embryos, inflorescences and apical meristems from diploid and tetraploid pearl millet genotypes were used as target tissues. Transformed cells were selected in the dark on Murashige and Skoog medium supplemented with 2 mg/l 2,4-D and 15 mg/l phosphinothricin (PPT). After 3-10 weeks in the dark, herbicide-resistant somatic embryos were induced to germinate on MS medium containing 0.1 mg/l thidiazuron and 0.1 mg/l 6-benzylaminopurine. Plants were transferred to the greenhouse after they were rooted in the presence of PPT and had passed a chlorophenol red assay (the medium turned from red to yellow). Transgenic plants were recovered from bombardments using intact pAHC25 plasmid DNA, a gel-purified bar fragment, or a mixture of pAHC25 plasmid or bar fragment and a plasmid containing the enhanced green fluorescent protein ( gfp) gene (p524EGFP.1). Analyses by the polymerase chain reaction, Southern blot hybridization, GFP expression, resistance to herbicide application, and segregation of the bar and gfp genes confirmed the presence and stable integration of the foreign DNA. Transformed plants were recovered from all three explants, although transformation conditions were optimized using only the tetraploid inflorescence. Time from culture initiation to rooted transgenic plant using the tetraploid inflorescence ranged from 3-4 months. Seven independent DNA/gold precipitations were used to bombard 52 plates, 29 of which produced an average of 5.5 herbicide-resistant plants per plate. The number of herbicide-resistant plants recovered per successful bombardment ranged from one to 28 and the frequency of co-transformation with gfp ranged from 5% to 85%.     

Modeling of the phase equilibria of polystyrene in methylcyclohexane with semi-empirical quantum mechanical methods I

A method for calculating interaction parameters traditionally used in phase-equilibrium computations in low-molecular systems has been extended for the prediction of solvent activities of aromatic polymer solutions (polystyrene+methylcyclohexane). Using ethylbenzene as a model compound for the repeating unit of the polymer, the intermolecular interaction energies between the solvent molecule and the polymer were simulated. The semiempirical quantum chemical method AM1, and a method for sampling relevant internal orientations for a pair of molecules developed previously were used. Interaction energies are determined for three molecular pairs, the solvent and the model molecule, two solvent molecules and two model molecules, and used to calculated UNIQUAC interaction parameters, a _ij and a _ji . Using these parameters, the solvent activities of the polystyrene 90,000 amu+methylcyclohexane system, and the total vapor pressures of the methylcyclohexane+ethylbenzene system were calculated. The latter system was compared to experimental data, giving qualitative agreement. Figure Solvent activities for the methylcylcohexane(1)+polystyrene(2) system at 316 K. Parameters a _ij (blue line) obtained with the AM1 method; parameters a _ij (pink line) from VLE data for the ethylbenzene+methylcyclohexane system. The abscissa is the polymer weight fraction defined as ₂(x ₁)=(1–x ₁)M ₂/[x ₁ M ₁+(1–x ₁)M ₂], where x ₁ is the solvent mole fraction and M _i are the molecular weights of the components.An erratum to this article can be found at     

Validation of the Hsp150 polypeptide carrier and HSP150 promoter in expression of rat alpha2,3-sialyltransferase in yeasts

Heterologous glycoproteins usually do not fold properly in yeast cells and fail to leave the endoplasmic reticulum. Here we show that the Hsp150Delta polypeptide carrier promoted proper folding and secretion of the catalytic ectodomain of rat alpha2,3-sialyltransferase (ST3Ne) in Pichia pastoris. The efficiency of the Hsp150Delta carrier in P. pastoris and Saccharomyces cerevisiae was at least as high as that of the MFalpha carrier. Most of Hsp150Delta-ST3Ne and MFalpha-ST3Ne remained noncovalently attached to the cell wall via the ST3Ne portion. The strength of the HSP150 promoter was found to be comparable to that of the GAL1 promoter.     

Modulation of adhesion,spreading and cytoskeleton organization of 3T3 fibroblasts by sulfonic groups present on polymer surfaces

The early phase of 3T3 fibroblast interaction with sulfonated styrene copolymer surfaces, of two sulfonic group densities and thus of differing wettability, was studied. The sulfonic groups present on copolymer surfaces affected the behaviour of cells, i.e. they stimulated cell adhesion, activated cell spreading and influenced cytoskeleton reorganization. The relative number of adhering cells correlated, while the number of spreading cells inversely correlated, with the surface density of sulfonic groups. Cell shape and the pattern of distribution of F-actin, alpha-actinin and vinculin in the interacting cells also depend on the surface density of sulfonic groups. On surfaces of high sulfonic group density, highly polarized cells were observed with F-actin bundles. On surfaces of low sulfonic group density, the cells spread with a square-like morphology with F-actin organized in stress fibres. In contrast, the cells spread poorly on nonsulfonated surfaces and cell adhesion was unaffected by surface wettability. The contribution of alpha(5)beta(1), alpha(4), and beta(5)integrins to the cell interaction with fibronectin (FN) and vitronectin (VN) adsorbed from serum-containing medium on polymer surfaces was examined. Our results suggest that surface sulfonic groups influence the conformation of FN and VN adsorbed on polymer surfaces and, in turn, determine the integrins that are involved in cell adhesion.     

CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112-2117, 2002). Investigation of the effects of phorbol ester (12-o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 microM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.     

Ventricular cardiotrophin-1 activation precedes BNP in experimental heart failure

Both cardiotrophin-1 (CT-1) and B-type or brain natriuretic peptide (BNP) are activated by cardiomyocyte stretch, and gene expression of CT-1 and BNP are augmented in the heart in experimental and human congestive heart failure (CHF). The goal of this study was to define cardiac gene expression of CT-1 and BNP by Northern blot analysis in normal (n=5), early left ventricular dysfunction (ELVD, n=5) and overt CHF dogs (n=5), in which ventricular function is progressively decreased. CT-1 mRNA was detected in both atria and ventricles in normal dogs. Ventricular CT-1 mRNA production increased in ELVD, and it further increased in overt CHF. Ventricular BNP mRNA remained below or at the limit of detection in normal and ELVD models, and it markedly increased in overt CHF. This study reports differential regulation of gene expression of CT-1 and BNP in the heart during the progression of CHF, and demonstrates that ventricular CT-1 gene activation precedes ventricular BNP gene activation.