Abundance of phytoseiid mites on Vitis species: effects of leaf hairs,domatia, prey abundance and plant phylogeny

We observed the number of predatory mites (Phytoseiidae:Typhlodromus caudiglans) on the foliage of 20 North American species of grapes (Vitis spp) plus the domesticated EuropeanVitis vinifera, all grown in a common garden. We found relatively few phytophagous mites. The numbers of phytophagous mites were not correlated with the plant characteristics that we measured. We found approximately five times as many predatory mites as phytophagous mites and the numbers of these phytoseiid predators were not affected by the availability of prey. Similarly, numbers of phytoseiids were unaffected by plant gender and, hence, the availability of pollen, another source of food. The numbers of phytoseiids were not clustered according to the taxonomic grouping of the tested plant species. Leaf surface characteristics explained over 25% of the variance in the numbers of phytoseiids. Numbers of phytoseiids were positively associated with the density of vein hairs, the density of bristles in leaf axils, and the presence of leaf domatia. These results suggest that sheltered habitats rather than food availability may limit the numbers of phytoseiid mites on grapevines.     

The properties of transketolase from photosynthetic tissue

Transketolase (E.C. 2.2.1.1.) has been partially purified from wheat (Triticum aestivum, cv. Sappo) and spinach (Spinacia oleracea) leaves. The fully-active enzyme is a tetramer of relative molecular mass (M_r) of 150 kM_r requiring thiamin pyrophosphate for maximal activity, and dissociating into a 74 kM_r dimer in its absence or in dilute solution. The chloroplastic transketolase (over 75% of the cellular total) is magnesium-stimulated but the cytosolic form is magnesium-insensitive. Both chloroplastic and cytosolic transketolase showed similar broad specificities towards several ketose phosphate substrates including fructose 6-phosphate and sedoheptulose 7-phosphate. Wheat and spinach leaf transketolases are not light-activated and closely resemble the yeast enzyme in many of their properties.Abbreviations Mr relative molecular mass - TPP thiamin pyrophosphate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Pressurized gas transport in two Japanese alder species in relation to their natural habitats

Oxygen uptake measurements have shown that pressurized gas transport, resulting from the physical effect of thermo-osmosis of gases, improves oxygen supply to the roots of the seedlings in two alder speciesAlnus japonica (Thunb.) Steud. andAlnus hirsuta (Spach) Rupr., which are both native in Japan. When gas transport conditions were established by irradiation of the tree stems the internal aeration was increased to a level nearly equal to the oxygen demand of the root system in leafless seedlings ofA. hirsuta, but was higher inA. japonica so that excess oxygen was excreted into the environment. An increase of superoxide dismutase (SOD) activity, which protects plants from toxic oxygen radicals and post-anoxic injury, has been observed in root tissues ofA. japonica when the seedlings were flooded for 3 days. The increase of SOD activity, in concert with high gas transport rates, may enable this tree species to grow in wet sites characterized by low oxygen partial pressure in the soil and by varying water tables. A less effective gas transport, flood-induced reduction of SOD activity in root tissues, and reduced height growth in waterlogged soil may be responsible for the fact thatA. hirsuta is unable to inhabit wettland sites.     

Identification of a Cholinergic-Specific Antigen Chol-1 as a Ganglioside

Abstract: An antiserum specific for cholinergic terminals was used to identify an antigen conserved between Elasmobranchs and mammals. Immunohistochemistry and a cytotoxicity test were used to assay the binding of antibody to mammalian terminals. Torpedo electric organ gangliosides totally abolished antibody binding. The highest inhibitory activity was associated with a single polysialoganglioside band on TLC plates. Neuraminidase altered the migration of the inhibitory activity on TLC plates. Antibody binding was inhibited by ganglioside fractions derived from chicken and mammalian brains. A summary of those tissues in which the antigen has been detected is presented. The possible function of the antigen is discussed.     

Amine ion porter inChara australis: Effects of alkyl substitution and external pH

Summary The rate of transport of amine ions intoChara australis internodes is studied by measuring changes in membrane current when amine solutions are presented to voltage-clamped cells. The dependence of this rate on ion concentration is investigated for a series of alkyl-amine ions: methyl-, ethyl-, isopropyl-, dimethyl-, trimethyl- and tetramethylammonium. A Michaelis-Menten relationship is displayed by all except tri- and tetramethylammonium, where currents are irregular and difficult to reproduce. Evidence suggests that the different ions cross the plasmalemma via a common uniport.K _M values for this porter increase as the amine ion becomes more highly substituted. TheV _m values are similar for all amines and lie within the range 10 to 100 mA m^–2 (for cell potential at –200 mV). The changes inK _M indicate that hydrogen bonding may be involved in the binding interaction.V _m varies with external pH in a way which suggests that an ionizable group on the transport protein with pK_a5.8 directly affects the transport rate.K _M is independent of external pH over the range 4.5 to 10.5     

‘Invertebrate Learning and Memory’ Edited by Randolf Menzel and Paul R. Benjamin: A review

In a new volume in the Handbooks of behavioural neuroscience series (series editor Joseph P. Huston), Randolf Menzel and Paul Benjamin have assembled an excellent and authoritative account of the importance, value and contribution of invertebrate models to the analysis of the molecular basis of learning and memory consolidation.     

Evolutionary links as revealed by the structure of Thermotoga maritima S-adenosylmethionine decarboxylase

S-adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine biosynthetic pathway and belongs to a small class of pyruvoyl-dependent amino acid decarboxylases. Structural elucidation of the prokaryotic AdoMetDC is of substantial interest in order to determine the relationship between the eukaryotic and prokaryotic forms of the enzyme. Although both forms utilize pyruvoyl groups, there is no detectable sequence similarity except at the site of pyruvoyl group formation. The x-ray structure of the Thermatoga maritima AdoMetDC proenzyme reveals a dimeric protein fold that is remarkably similar to the eukaryotic AdoMetDC protomer, suggesting an evolutionary link between the two forms of the enzyme. Three key active site residues (Ser55, His68, and Cys83) involved in substrate binding, catalysis or proenzyme processing that were identified in the human and potato AdoMet-DCs are structurally conserved in the T. maritima AdoMetDC despite very limited primary sequence identity. The role of Ser55, His68, and Cys83 in the self-processing reaction was investigated through site-directed mutagenesis. A homology model for the Escherichia coli AdoMetDC was generated based on the structures of the T. maritima and human AdoMetDCs.     

Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae α-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN^{K252 + 255A}, SEN^K304A, SEN^K334A, SEN^K344E, SEN^K435L, and SEN^Δ434–435) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN^K344E, as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN^K344E had lost α-enolase activity, which was also reduced in SEN^K334A and SEN^Δ434–435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN^{K252 + 255A}, SEN^K435L, and SEN^Δ434–435. The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.Streptococcus pyogenes (group A Streptococcus, GAS)⁸ is a common bacterial pathogen, causing over 700 million human disease episodes each year (1). These range from serious life-threatening invasive diseases including necrotizing fasciitis and streptococcal toxic shock-like syndrome to non-invasive infections like pharyngitis and pyoderma. Invasive disease, in combination with postinfection immune sequelae including rheumatic heart disease and acute poststreptococcal glomerulonephritis, account for over half a million deaths each year (1). Although a resurgence of GAS invasive infections has occurred in western countries since the mid-1980s, disease burden is much greater in developing countries and indigenous populations of developed nations, where GAS infections are endemic (2–4).GAS is able to bind human plasminogen and activate the captured zymogen to the serine protease plasmin (5–17). The capacity of GAS to do this plays a critical role in virulence and invasive disease initiation (3, 17–19). The plasminogen activation system in humans is an important and highly regulated process that is responsible for breakdown of extracellular matrix components, dissolution of blood clots, and cell migration (20, 21). Plasminogen is a 92-kDa zymogen that circulates in human plasma at a concentration of 2 μm (22). It consists of a binding region of five homologous triple loop kringle domains and an N-terminal serine protease domain that flank the Arg⁵⁶¹–Val⁵⁶² site (23), where it is cleaved by tissue plasminogen activator and urokinase plasminogen activator to yield the active protease plasmin (20, 23). GAS also has the ability to activate human plasminogen by secreting the virulence determinant streptokinase. Streptokinase forms stable complexes with plasminogen or plasmin, both of which exhibit plasmin activity (20, 24). Activation of plasminogen by the plasmin(ogen)-streptokinase complex circumvents regulation by the host plasminogen activation inhibitors, α₂-antiplasmin and α₂-macroglobulin (11, 20). GAS can bind the plasmin(ogen)-streptokinase complex and/or plasmin(ogen) directly via plasmin(ogen) receptors at the bacterial cell surface (6). These receptors include the plasminogen-binding group A streptococcal M-like protein (PAM) (25), the PAM-related protein (19), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also known as streptococcal plasmin receptor, Plr, or streptococcal surface dehydrogenase) (9, 26), and streptococcal surface enolase (SEN or α-enolase) (27). Interactions with these GAS receptors occurs via lysine-binding sites within the kringle domains of plasminogen (6).In addition to its ability to bind human plasminogen, SEN is primarily the glycolytic enzyme that converts 2-phosphoglycerate to phosphoenolpyruvate (27–29). SEN is abundantly expressed in the cytosol of most bacterial species but has also been identified as a surface-located protein in GAS and other bacteria including pneumococci, despite lacking classical cell surface protein motifs such as a signal sequence, membrane-spanning domain, or cell-wall anchor motif (27, 28, 30, 31). The interaction between SEN and plasminogen is reported to be facilitated by the two C-terminal lysine residues at positions 434 and 435 (27, 32). In contrast, an internal binding motif containing lysines at positions 252 and 255 in the closely related α-enolase of Streptococcus pneumoniae has been shown to play a pivotal role in the acquisition of plasminogen in this bacterial species (33). The octameric pneumococcal α-enolase structure consists of a tetramer of dimers. Hence, potential binding sites could be buried in the interface between subunits. In fact, the crystal structure of S. pneumoniae α-enolase revealed that the two C-terminal lysine residues are significantly less exposed than the internal plasminogen-binding motif (34).In this study, we constructed an in silico model of GAS SEN, based on the pneumococcal octameric α-enolase crystal structure, and validated this model using ion mobility (IM) mass spectrometry (MS). Site-directed mutagenesis followed by structural and functional analyses revealed that Lys³⁴⁴ plays a crucial role in structural integrity and enzymatic function. Furthermore, we demonstrate that the plasminogen-binding motif residues Lys²⁵² and Lys²⁵⁵ and the C-terminal Lys⁴³⁴ and Lys⁴³⁵ residues are located adjacently in the GAS SEN structure and play a concerted role in the binding of human plasminogen.     

Staurosporines Disrupt Phosphatidylserine Trafficking and Mislocalize Ras Proteins

Oncogenic mutant Ras is frequently expressed in human cancers, but no anti-Ras drugs have been developed. Since membrane association is essential for Ras biological activity, we developed a high content assay for inhibitors of Ras plasma membrane localization. We discovered that staurosporine and analogs potently inhibit Ras plasma membrane binding by blocking endosomal recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine from plasma membrane to endomembrane. Staurosporines are more active against K-Ras than H-Ras. K-Ras is displaced to endosomes and undergoes proteasomal-independent degradation, whereas H-Ras redistributes to the Golgi and is not degraded. K-Ras nanoclustering on the plasma membrane is also inhibited. Ras mislocalization does not correlate with protein kinase C inhibition or induction of apoptosis. Staurosporines selectively abrogate K-Ras signaling and proliferation of K-Ras-transformed cells. These results identify staurosporines as novel inhibitors of phosphatidylserine trafficking, yield new insights into the role of phosphatidylserine and electrostatics in Ras plasma membrane targeting, and validate a new target for anti-Ras therapeutics.