Diversity of abundant mRNA sequences and patterns of protein synthesis in etiolated and greened pea seedlings

The diversity of abundant mRNA sequences in various parts of 4-d etiolated pea seedlings (Pisum sativum L. var. Rondo CB) was compared by a cell-free translation of the mRNAs in the presence of [³⁵S]methionine and by an analysis of the products by two-dimensional electrofocussing/ electrophoresis (2D separation). The various parts of the seedlings were also examined for the pattern of protein synthesis in vivo. Proteins were labeled by injection of [³⁵S]methionine into the cotyledons, followed by 2D separation of the products. Over 95% of the abundant mRNA sequences and newly synthesized abundant polypeptides were shared by all parts of etiolated seedlings, including the cotyledons. However, a few distinct differences were observed when comparing mRNAs of roots and shoots; the most prominent among these were a group of six abundant mRNA sequences found exclusively in shoots. Only about 30% of the polypeptides synthesized on isolated RNA could be traced in equivalent positions on the gels as the polypeptides synthesized in vivo. Analysis of total RNA from light-grown pea seedlings showed the appearance of some twenty-five translation products not found with total RNA from etiolated seedlings, while about nine other translation products disappeared. At least ten of the light-induced RNA sequences were also present after growth in low-intensity red light (>600 nm) and are therefore thought to be controlled by the phytochrome system. Comparison of 11-d light-grown pea plants with 4-d light-grown seedlings did not reveal additional translatable RNA sequences, indicating that the major morphogenetic changes that occur after 4 d are not accompanied by significant changes in the pattern of abundant RNA sequences.     

High Leucine Diets Stimulate Cerebral Branched-Chain Amino Acid Degradation and Modify Serotonin and Ketone Body Concentrations in a Pig Model

In addition to its role as an essential protein component, leucine (Leu) displays several other metabolic functions such as activation of protein synthesis. This property makes it an interesting amino acid for the therapy of human muscle atrophy and for livestock production. However, Leu can stimulate its own degradation via the branched-chain keto acid dehydrogenase complex (BCKDH). To examine the response of several tissues to excessive Leu, pigs were fed diets containing two- (L2) and four-fold (L4) higher Leu contents than the recommended amount (control). We found that the L4 diet led to a pronounced increase in BCKDH activity in the brain (2.5-fold, P < 0.05), liver (1.8-fold, P < 0.05) and cardiac muscle (1.7-fold, P < 0.05), whereas we found no changes in enzyme activity in the pancreas, skeletal muscle, adipose tissue and intestinal mucosa. The L2 diet had only weak effects on BCKDH activity. Both high Leu diets reduced the concentrations of free valine and isoleucine in nearly all tissues. In the brain, high Leu diets modified the amount of tryptophan available: for serotonin synthesis. Compared to the controls, pigs treated with the high Leu diets consumed less food, showed increased plasma concentrations of 3-hydroxybutyrate and reduced levels of circulating serotonin. In conclusion, excessive Leu can stimulate BCKDH activity in several tissues, including the brain. Changes in cerebral tryptophan, along with the changes in amino acid-derived metabolites in the plasma may limit the use of high Leu diets to treat muscle atrophy or to increase muscle growth.     

Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq

Receptor-mediated modulation of KCNQ channels regulates neuronal excitability. This study concerns the kinetics and mechanism of M1 muscarinic receptor-mediated regulation of the cloned neuronal M channel, KCNQ2/KCNQ3 (Kv7.2/Kv7.3). Receptors, channels, various mutated G-protein subunits, and an optical probe for phosphatidylinositol 4,5-bisphosphate (PIP2) were coexpressed by transfection in tsA-201 cells, and the cells were studied by whole-cell patch clamp and by confocal microscopy. Constitutively active forms of Galphaq and Galpha11, but not Galpha13, caused a loss of the plasma membrane PIP2 and a total tonic inhibition of the KCNQ current. There were no further changes upon addition of the muscarinic agonist oxotremorine-M (oxo-M). Expression of the regulator of G-protein signaling, RGS2, blocked PIP2 hydrolysis and current suppression by muscarinic stimulation, confirming that the Gq family of G-proteins is necessary. Dialysis with the competitive inhibitor GDPbetaS (1 mM) lengthened the time constant of inhibition sixfold, decreased the suppression of current, and decreased agonist sensitivity. Removal of intracellular Mg2+ slowed both the development and the recovery from muscarinic suppression. When combined with GDPbetaS, low intracellular Mg2+ nearly eliminated muscarinic inhibition. With nonhydrolyzable GTP analogs, current suppression developed spontaneously and muscarinic inhibition was enhanced. Such spontaneous suppression was antagonized by GDPbetaS or GTP or by expression of RGS2. These observations were successfully described by a kinetic model representing biochemical steps of the signaling cascade using published rate constants where available. The model supports the following sequence of events for this Gq-coupled signaling: A classical G-protein cycle, including competition for nucleotide-free G-protein by all nucleotide forms and an activation step requiring Mg2+, followed by G-protein-stimulated phospholipase C and hydrolysis of PIP2, and finally PIP2 dissociation from binding sites for inositol lipid on the channels so that KCNQ current was suppressed. Further experiments will be needed to refine some untested assumptions.     

New magnetic nanoparticles for biotechnology

Paramagnetic carriers, which are linked to antibodies enable highly specific biological cell separations. With the colloidal synthesis of superparamagnetic Co and FeCo nanocrystals with superior magnetic moments the question about their potential to replace magnetite as the magnetically responsive component of magnetic beads is addressed. Starting from a magnetic analysis of the corresponding magnetophoretic mobility of Co and FeCo based alloys their synthesis and resulting microstructural and magnetic properties as function of the underlying particle size distribution are discussed in detail. The stability of the oleic acid ligand of Co nanocrystals has been investigated. The oxidation kinetics were quantified using magnetic measurements. As a result, this ligand system provides sufficient protection against oxidation. Furthermore, the kinetics of the synthesis of Fe(50)Co(50) nanoparticles has been monitored employing Fourier transform infra red (FT-IR) spectroscopy and is modeled using a consecutive decomposition and growth model. This model predicts the experimentally realized FeCo nanoparticle composition as a function of the particle size fairly well. High-resolution transmission electron microscopy (HRTEM) was performed to uncover the resulting microstructure and composition on a nanometer scale.     

SC3 and SC4 hydrophobins have distinct roles in formation of aerial structures in dikaryons of Schizophyllum commune

Two monokaryons of Schizophyllum commune can form a fertile dikaryon when the mating-type genes differ. Monokaryons form sterile aerial hyphae, while dikaryons also form fruiting bodies that function in sexual reproduction. The SC3 hydrophobin gene is expressed both in monokaryons and in dikaryons. The SC4 hydrophobin is dikaryon specific. In the monokaryon, SC3 lowers the water surface tension, coats aerial hyphae with a hydrophobic layer and mediates attachment of hyphae to hydrophobic surfaces. The SC4 protein lines gas channels within fruiting bodies with a hydrophobic membrane. Using gene disruptions, in this study, we show that in dikaryons SC3 fulfils the same roles as in monokaryons. SC4, on the other hand, has a role within fruiting bodies. In contrast to gas channels in fruiting bodies of the wild type, those of a DeltaSC4 strain easily filled with water. Thus, SC4 prevents gas channels filling with water under wet conditions, probably serving uninterrupted gas exchange. Other dikaryon-specific hydrophobin genes, SC1 and SC6, apparently do not substitute for the SC4 gene. In addition, by expressing the SC4 gene behind the SC3 promoter in a DeltaSC3 monokaryon, it was shown that SC4 cannot fully substitute for SC3, indicating that both hydrophobins evolved to fulfil specific functions.     

Forced expression of a dominant-negative chimeric tropomyosin causes abnormal motile behavior during cell division

Forced expression of the chimeric human fibroblast tropomyosin 5/3 (hTM5/3) in CHO cell was previously shown to affect cytokinesis [Warren et al., 1995: J. Cell Biol. 129:697-708]. To further investigate the phenotypic consequences of misexpression, we have compared mitotic spindle organization and dynamic 2D and 3D shape changes during mitosis in normal cells and in a hTM5/3 misexpressing (mutant) cell line. Immunofluorescence microscopy of wild type and mutant cells stained with monoclonal anti-tubulin antibody revealed that the overall structures of mitotic spindles were not significantly different. However, the axis of the mitotic spindle in mutant cells was more frequently misaligned with the long axis of the cell than that of wild type cells. To assess behavioral differences during mitosis, wild type and mutant cells were reconstructed in 2D and 3D and motion analyzed with the computer-assisted 2D and 3D Dynamic Image Analysis Systems (2D-DIAS, 3D-DIAS). Mutant cells abnormally formed large numbers of blebs during the later stages of mitosis and took longer to proceed from the start of anaphase to the start of cytokinesis. Furthermore, each mutant cell undergoing mitosis exhibited greater shape complexity than wild type cells, and in every case lifted one of the two evolving daughter cells off the substratum and abnormally twisted. These results demonstrate that misexpression of hTM5/3 in CHO cells leads to morphological instability during mitosis. Misexpression of hTM5/3 interferes with normal tropomyosin function, suggesting in turn that tropomyosin plays a role through its interaction with actin microfilaments in the regulation of the contractile ring, in the localized suppression of blebbing, in the maintenance of polarity and spatial symmetry during cytokinesis, and in cell spreading after cytokinesis is complete.     

The internal phosphodiesterase RegA is essential for the suppression of lateral pseudopods during Dictyostelium chemotaxis

Dictyostelium strains in which the gene encoding the cytoplasmic cAMP phosphodiesterase RegA is inactivated form small aggregates. This defect was corrected by introducing copies of the wild-type regA gene, indicating that the defect was solely the consequence of the loss of the phosphodiesterase. Using a computer-assisted motion analysis system, regA(-) mutant cells were found to show little sense of direction during aggregation. When labeled wild-type cells were followed in a field of aggregating regA(-) cells, they also failed to move in an orderly direction, indicating that signaling was impaired in mutant cell cultures. However, when labeled regA(-) cells were followed in a field of aggregating wild-type cells, they again failed to move in an orderly manner, primarily in the deduced fronts of waves, indicating that the chemotactic response was also impaired. Since wild-type cells must assess both the increasing spatial gradient and the increasing temporal gradient of cAMP in the front of a natural wave, the behavior of regA(-) cells was motion analyzed first in simulated temporal waves in the absence of spatial gradients and then was analyzed in spatial gradients in the absence of temporal waves. Our results demonstrate that RegA is involved neither in assessing the direction of a spatial gradient of cAMP nor in distinguishing between increasing and decreasing temporal gradients of cAMP. However, RegA is essential for specifically suppressing lateral pseudopod formation during the response to an increasing temporal gradient of cAMP, a necessary component of natural chemotaxis. We discuss the possibility that RegA functions in a network that regulates myosin phosphorylation by controlling internal cAMP levels, and, in support of that hypothesis, we demonstrate that myosin II does not localize in a normal manner to the cortex of regA(-) cells in an increasing temporal gradient of cAMP.     

Interfacial self-assembly of fungal hydrophobins of the lichen-forming ascomycetes Xanthoria parietina and X. ectaneoides

In the symbiotic phenotype of the lichen-forming ascomycetes Xanthoria parietina and X. ectaneoides, a conglutinate, hydrophilic cortex surrounds a system of aerial hyphae with hydrophobic wall surfaces. In X. parietina freeze-fracture electron microscopy showed that a rodlet layer covers the fungal and algal wall surfaces. Extracts of hot SDS-insoluble wall residues isolated from both species contained a protein that revealed a rodlet layer upon interfacial self-assembly. The N-terminal sequence of the 10-kDa protein of X. ectaneoides served to clone cDNA fragments of XEH1 (H1 of X. ectaneoides) and XPH1 (H1 of X. parietina) by RT-PCR. Genomic DNA blot analysis with both lichenized species and the aposymbiotically cultured symbionts of X. parietina showed that XPH1 and XEH1 are fungal single copy genes. The deduced amino acid sequences of the two encoded proteins were 96% identical and showed the characteristics of class I hydrophobins.