An Anomaly in Potassium Accumulation by Barley Roots: II. Effect of Calcium Concentration and Rubidium-86 Labeling

Excised barley roots accumulated 40 to 50% more K⁺ from 0.04 mm than from 0.06 mm KCl when incubated for 24 hours in KCl solutions containing 0.2 mm CaSO₄. This phenomenon was not markedly influenced by the rate of absorption of the counteranion. The presence of Na⁺ in the treatment solutions decreased total K accumulation but did not alter the K⁺ concentration at which the accumulation peak occurred. Short interval studies indicated that this phenomenon is easily observable after 4 hours and begins to become apparent within 2 hours. In comparison with barley, accumulation of K⁺ by excised wheat roots decreased as KCl concentration was increased from 0.02 to 0.06 mm; but K⁺ accumulation curve for corn roots showed no peaks or depressions in the concentration range of 0.01 to 0.1 mm. A normal hyperbolic curve was noted for the accumulation of Na⁺ from 0.01 to 1 mm NaCl by barley roots.     

Identification of epsilon-N-mono-, epsilon-N-di-, and epsilon-N-trimethyllysine by high-performance liquid chromatography.

Dansyl derivatives of epsilon-N-mono-, epsilon-N-di-, and epsilon-N-trimethyllysine were resolved from other amino acids in proteins by the use of high-performance liquid chromatography. The system was tested with amino acid standard combinations as well as with acid-hydrolyzed proteins known to contain methylated residues. In all cases the methylated lysines were well resolved.     

Regulation of polyamine biosynthesis in tobacco. Effects of inhibitors and exogenous polyamines on arginine decarboxylase, ornithine decarboxylase, and S-adenosylmethionine decarboxylase

Treatment of tobacco liquid suspension cultures with methylglyoxal bis(guanylhydrazone) (MGBG) an inhibitor of S-adenosylmethionine decarboxylase, resulted in a dramatic overproduction of a 35-kDa peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Malmberg, R.L., and McIndoo, J. (1983) Nature 305, 623-625). MGBG treatment also resulted in a 20-fold increase in the activity of S-adenosylmethionine decarboxylase. Purification of S-adenosylmethionine decarboxylase from MGBG-treated cultures revealed that the overproduced 35-kDa peptide and S-adenosylmethionine decarboxylase are identical. Precursor incorporation experiments using [3H] methionine and [35S]methionine revealed that MGBG does not induce any increased synthesis of S-adenosylmethionine decarboxylase but rather stabilizes the protein to proteolytic degradation. The half-life of the enzyme activity was increased when MGBG was present in the growth medium. In addition to stabilizing S-adenosylmethionine decarboxylase, MGBG also resulted in the rapid and specific loss of arginine decarboxylase activity with little effect ornithine decarboxylase. The kinetics of this effect suggest that arginine decarboxylase synthesis was rapidly inhibited by MGBG. Exogenously added polyamines had little effect on ornithine decarboxylase, whereas S-adenosylmethionine and arginine decarboxylase activities rapidly diminished with added spermidine or spermine. Finally, inhibition of ornithine decarboxylase was lethal to the cultures, whereas inhibition of arginine decarboxylase was only lethal during initiation of growth in suspension culture.     

pH-dependent lysis of liposomes by adenovirus

Purified adenovirus induced a dose-dependent release of the water-soluble markers calcein and carboxyfluorescein from liposomes. Marker release was strongly dependent on pH, and at temperatures below 5 degrees C, the rate of release showed an optimum at a pH of about 6. This pH dependence parallels disruption of endocytic vesicles by adenovirus and the permeabilization that adenovirus induces on the cell surface. There did not seem to be a striking dependence on the lipid composition of the liposomes. Electron microscopy using a negative stain shows liposomes bound to adenovirus. In some cases, the liposomes were still intact, but many liposomes, which were attached to the vertices of the virus, appeared lysed. These data support the notion that adenovirus, which enters the host cell by receptor-mediated endocytosis, gains access to the cytoplasm by a subsequent pH-dependent disruption of the membrane of the endocytic vesicle.     

Electron microscopic localization of 5''-nucleotidase in the stratum intermedium and ameloblasts

Synopsis 5-nucleotidase was demonstrated at the fine structural level in the stratum intermedium and ameloblasts of the first mandibular molars of CD-1 mice. The enzyme was localized with the Wachstein & Meisel (1957) method along the plasma membranes of the cells of the stratum intermedium and ameloblasts. While 5-nucleotidase was present throughout the stratum intermedium, only the proximal region of the plasma membranes of ameloblasts was demonstrably active for this enzyme. 5-Nucleotidase has been implicated in transport of metabolites across cell membranes, and its localization in the present study supports this implication as well as the transport functions of the stratum intermedium and the stratum intermedium-ameloblastic interface.     

Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis

Toll-like receptors (TLRs) play a crucial role in host defense against microbial infection. The microbial ligands recognized by TLRs are not unique to pathogens, however, and are produced by both pathogenic and commensal microorganisms. It is thought that an inflammatory response to commensal bacteria is avoided due to sequestration of microflora by surface epithelia. Here, we show that commensal bacteria are recognized by TLRs under normal steady-state conditions, and this interaction plays a crucial role in the maintenance of intestinal epithelial homeostasis. Furthermore, we find that activation of TLRs by commensal microflora is critical for the protection against gut injury and associated mortality. These findings reveal a novel function of TLRs-control of intestinal epithelial homeostasis and protection from injury-and provide a new perspective on the evolution of host-microbial interactions.     

Interaction of Bacillus thuringiensis Vegetative Insecticidal Protein with Ribosomal S2 Protein Triggers Larvicidal Activity in Spodoptera frugiperda

Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.Insecticidal proteins produced by strains of Bacillus thuringiensis can broadly be classified into two major categories based on their site of accumulation. Category I consist of proteins that are deposited as crystals in sporangia and are referred to as insecticidal crystalline proteins (ICPs). The second category consists of recently described group of insecticidal proteins, called vegetative insecticidal proteins (8). These proteins are synthesized during the vegetative growth of Bacillus cells and are secreted into the culture medium. Irrespective of the site of accumulation of insecticidal proteins, their ingestion by susceptible insect larvae leads to disruption and lysis of epithelial tissue from the midgut, resulting in larval death (12). The mechanism of lysis of gut epithelial tissue by ICPs has been investigated in detail in several insects (16). Ingestion of ICPs triggers a sequence of biochemical cascade that involves its solubilization and subsequent activation by gut proteases. The activated toxin interacts with specific receptors located at the midgut epithelial tissue. In this sequence of events, the interaction with the receptor is the most significant event since subsequent to interaction, pore formation is initialized, and that leads to lysis of epithelial cells. The identification and characterization of receptors from various insect larvae has led to the identification of following molecules as receptor to ICPs, such as cadherinlike protein (21), glycosyl phosphatidylinositol (GPI)-anchored aminopeptidase N (APN) (1, 9, 11, 17, 19, 20), a GPI-anchored alkaline phosphatase (10, 14), and a 270-kDa glycoconjugate (see references 2, 7, 9, and 16 and references therein for an extensive list of receptors). In addition, certain glycopeptides have been identified as lysis-initiating receptor molecules. Although there is extensive information about the receptor-toxin interaction for ICPs, negligible work has been done toward the identification of receptors to vegetative insecticidal proteins. The ultrastructural changes induced at the midgut epithelial tissue, upon ingestion of ICPs or Vip3As, are common (12). Both ICPs and Vip3As interact at the epithelial layer of midgut, enlarging the affected cells due to osmotic imbalance and eventually causing lysis. In spite of inflicting nearly identical structural damage, the interacting receptor for the Vip3A is not identical (12). In fact, the receptor to Vip3As has not yet been characterized.Our group has been working on the identification, cloning, and evaluation of vegetative insecticidal proteins from strains of B. thuringiensis held in our collection. We have characterized the Vip3A (EMBL accession no. {"type":"entrez-nucleotide","attrs":{"text":"Y17158","term_id":"3135740","term_text":"Y17158"}}Y17158) class of protein and evaluated its toxicity profile (2, 8, 18). Vip3A is active against larvae of Spodoptera litura, among several other lepidopteran pests. In a parallel series of experiments, we identified APN as a receptor to the B. thuringiensis protein Cry1C in S. litura. The heterologously expressed APN did not interact with Vip3A, suggesting that Vip3A toxicity in this insect is not through interaction with APN (1). Our preliminary results on the toxicity of Vip3A revealed that purified insecticidal protein could lyse Sf21 cells, suggesting the presence of receptors in the insect cell line. In the present study, we identified the Vip3A interacting protein in Sf21 cells and the larvae of S. litura. The specificity of the interaction has been examined by a combination of ex vivo and in vitro assays. These assays identified ribosomal S2 protein as the interacting partner of Vip3A. The functional significance of S2-Vip3A protein interaction was examined by monitoring the reduction in Vip3A toxicity in Sf21 cells and larvae of S. litura by the RNA interference-induced knockdown of S2 protein. The results of these experiments are discussed in the context of colocalization of the S2-Vip3A protein interacting complex by confocal microscopy.     

Ethacrynic acid analogues lacking the α,β-unsaturated carbonyl unit—Potential anti-metastatic drugs

A series of ethacrynic acid analogues, lacking the α,β-unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, MCF-7/AZ. Several of the analogues were already active in the low micromolar range, whereas ethacrynic acid itself shows no potential to inhibit the migration of these cancer cells. Preliminary studies show that the presence of one or more methoxy groups at the phenyl ring of ethacrynic acid is important in order for the ethacrynic acid analogues to demonstrate an inhibitory effect on the migration.