Mobilization functions of the bacteriocinogenic plasmid pRJ6 of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Plasmid pRJ6 is the first known bacteriocinogenic mobilizable (Mob) plasmid of Staphylococcus aureus. Its Mob region is composed of four mob genes (mobCDAB) arranged as an operon, a genetic organization uncommon among S. aureus Mob plasmids. oriT _pRJ6 was detected in a region of 431 bp, positioned immediately upstream of mobC. This region, when cloned into pCN37, was able to confer mobilization to the re-combinant plasmid only in the presence of pRJ6. The entire Mob region, including oriT _pRJ6, is much more similar to Mob regions from several coagulase-negative staphylococci plasmids, although some remarkable similarities with S. aureus Mob plasmids can also be noted. These similarities include the presence within oriT _pRJ6 of the three mcb (MobC binding sites), firstly described in pC221 and pC223, an identical nick site also found in these same plasmids, and a nearly identical sra _pC223 site (sequence recognized by MobA). pRJ6 was successfully transferred to S. epidermidis by conjugation in the presence of the conjugative plasmid pGOl. Altogether these findings suggest that pRJ6 might have been originally a coagulase-negative staphylococci plasmid that had been transferred successfully to S. aureus.     

Synthesis and biological characterization of human monocyte chemoattractant protein 1 (MCP-1) and its analogs.

Novel analogs of human monocyte chemoattractant protein 1 (MCP-1) were designed, synthesized and characterized to be used as tools to generate monoclonal antibodies as potential human therapeutics. MCP-1 and three analogs were synthesized by step-wise Fmoc solid phase synthesis. After oxidation to form the two-disulfide bonds, affinity chromatography using an immobilized mouse anti-human MCP-1 monoclonal antibody (mAb) was utilized for a simple and highly effective purification procedure for the proteins. The final products were extensively characterized and compared with recombinant human MCP-1 (rhMCP-1). All proteins showed identical binding with mouse anti-human MCP-1 mAbs as measured by surface plasmon resonance. Synthetic MCP-1 and the analogs were comparable to recombinant MCP-1 in competition radio-ligand binding to CCR2 receptors on THP-1 cells, and MCP-1-induced, calcium mobilization and chemotaxis assays.     

Lignotuber reserves support regrowth following clipping of two Mediterranean shrubs

1. We investigated whether reserves stored in the lignotubers of two Mediterranean shrubs, Arbutus unedo and Erica arborea , were significantly mobilized to support the demands of regrowth and respiration after clipping the tops at different frequencies.
2. After a single clipping, Arbutus showed a 29% decrease of phosphorus concentration by the end of the first growing season. Two years after recovery from clipping, the starch levels remained lower than those of unclipped plants. Similarly, Erica showed depletion of starch, but no nutrient reserves were depleted significantly.
3. Regrowth after multiple clippings mobilized a large fraction of the starch and nutrients stored in the lignotuber. Mean starch concentrations were depleted by 87–93% after multiple clippings and concentrations of nitrogen, phosphorus, potassium and magnesium were depleted by 10–45%, 27–41%, 19–39% and 23–31%, respectively.
4. An average-sized lignotuber produced 288 resprouts for Arbutus and 1990 resprouts for Erica during a 27 month period of multiple clippings, at the end of which the first plants died.
5. Plant mortality after multiple clipping was 10% for Arbutus and 30% for Erica , and was primarily attributed to exhaustion of carbon reserves because starch concentrations decreased by 96% in dead plants.     

Role of Group III Metabotropic Glutamate Receptors in Excitotoxin-Induced Cerebellar Granule Cell Death

Abstract: The neuronal effects of the metabotropic glutamate receptor agonist (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid have been studied in cultured rat cerebellar granule cells, and compared with those of the endogenous excitotoxin glutamate, and the dietary excitotoxin β- N -methylamino- l -alanine. Glutamate, β- N -methylamino- l -alanine, and (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid all caused concentration-dependent cerebellar granule cell death over a 24-h exposure period. The metabotropic antagonist ( RS )-α-methyl-4-carboxyphenylglycine reduced glutamate-, β- N -methylamino- l -alanine-, and (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid-induced death by 50, 37, and 90%, respectively. (1 S ,3 R )-Aminocyclopentane-1,3-dicarboxylic acid-induced death was unaffected by the group I antagonist ( RS )-1-aminoindan-1,5-dicarboxylic acid, increased by the group II antagonist ethylglutamic acid, and markedly decreased by the group III antagonist ( RS )-α-methylserine- O -phosphate. Neither (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid nor the group I agonist ( RS )-3,5-dihydroxyphenylglycine caused an increase in intracellular free calcium levels. The group III agonist l -(+)-2-amino-4-phosphonobutyric acid also induced concentration-dependent cerebellar granule cell death, and so it was suggested that the group III metabotropic glutamate receptors were responsible for (1 S ,3 R )-aminocyclopentane-1,3-dicarboxylic acid-induced death. Blocking these receptors with ( RS )-α-methylserine- O -phosphate also prevented a proportion of glutamate- and β- N -methylamino- l -alanine-induced death.     

Role of P-Selectin Cytoplasmic Domain in Granular Targeting In Vivo and in Early Inflammatory Responses

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (ΔCT mice). The deletion did not affect the sorting of P-selectin into α-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The ΔCT–P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the ΔCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.     

MOBILIZATION OF β-1,3-GLUCAN AND BIOSYNTHESIS OF AMINO ACIDS INDUCED BY NH4+ ADDITION TO N-LIMITED CELLS OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

Mobilization of the reserve β-1,3-glucan (chrysolaminaran) in N-limited cells of the marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) was investigated. The diatom was grown in pH-regulated batch cultures with a 14:10-h light:dark cycle until N depletion. In a pulse-chase experiment, the cells were first incubated in high light (200 μmol photons·m ^{− 2}·s ^{− 1}) with ¹⁴C-bicarbonate until dissolved inorganic carbon was exhausted. Unlabeled bicarbonate (1 mM) was then added, and the cells were incubated in the dark and subsequently in low light (20 μmol photons·m ^{− 2}·s ^{− 1}) with additions of 40 μM NH₄ ⁺ . In the ¹⁴C pulse phase with high light and N depletion, β-1,3-glucan accumulated and accounted for 85% of incorporated ¹⁴C. In the subsequent ¹⁴C chase phases, added NH₄ ⁺ was assimilated at an N-specific rate of 0.11 h ^{− 1} in both the dark and low light, and in both cases it caused a significant mobilization of β-1,3-glucan (dark, 26%; low light, 19%). Biochemical fractionation of organic ¹⁴C showed that free amino acids were most rapidly labeled in the early stage of NH₄ ⁺ assimilation, whereas proteins and polysaccharides were labeled more rapidly after 1.2 h. Analysis of the cellular free amino acids strongly indicated that de novo biosynthesis was occurring, with a Gln:Glu ratio increasing from 0.4 to 10 within 1.2 h. After the NH₄ ⁺ was exhausted, the cellular pools of glucan and amino acids became constant or slowly decreased. In another experiment, N-limited cells were first incubated in high light until dissolved inorganic carbon was exhausted and were further incubated in high light with 150 μM NH₄ ⁺ under inorganic carbon limitation. Added NH₄ ⁺ was assimilated at an N-specific rate of 0.023 h ^{− 1}, and cellular β-1,3-glucan decreased by 15% within 6 h. Hence, β-1,3-glucan was mobilized during NH₄ ⁺ assimilation, even though inorganic carbon was modifying the metabolic rates. The results provide new evidence of β-1,3-glucan supplying essential precursors for biosynthesis of amino acids and other components in S. costatum in both the dark and subsaturating light and even saturating light under inorganic carbon limitation.     

Adipokinetic hormone-induced influx of extracellular calcium into insect fat body cells is mediated through depletion of intracellular calcium stores

Adipokinetic hormone I (AKH I) needs extracellular Ca²⁺ for its activating action on glycogen phosphorylase in locust fat body in vitro. TMB-8 reduces this AKH effect significantly, indicating that for a major part, hormone action also requires the mobilization of Ca²⁺ from intracellular stores. Using ⁴⁵Ca²⁺, AKH was shown to stimulate both the influx and the efflux of Ca²⁺. Thapsigargin also enhances the influx of extracellular Ca²⁺ into the fat body cells, indicating that the stimulating effect of AKH on Ca²⁺ influx may be mediated through depletion of intracellular Ca²⁺ stores as well. AKH is known to enhance cAMP levels in locust fat body. We show that elevation of cAMP with forskolin or theophylline leads to activation of glycogen phosphorylase, both in the presence and in the absence of extracellular Ca²⁺. The present data are discussed in an attempt to elucidate further the mechanism underlying transduction of the hormonal signal in locust fat body.     

Homologous Desensitization of Serotonin 5-HT2 Receptor-Stimulated Intracellular Calcium Mobilization in C6BU-1 Glioma Cells via a Mechanism Involving a Calmodulin Pathway

Abstract: Serotonin 5-HT₂ receptor-mediated intracellular Ca²⁺ mobilization was investigated in rat glioma C6BU-1 cells. The receptors became desensitized after previous exposure to 5-HT in a time-and concentration-dependent manner. The desensitization of 5-HT₂ receptor-mediated intracellular signaling appeared to be homologous because previous exposure to 5-HT did not alter the response to other transmitters such as thrombin or isoproterenol and because previous exposure to thrombin or isoproterenol did not diminish the response to 5-HT. The desensitization induced by pretreatment with 5-HT was potently prevented by the naphthalenesulfonamide derivative W-7, a calmodulin antagonist, when it was cosupplied with 5-HT. Furthermore, the preventive effect of W-7 was greater than that of W-5, a weak analogue of W-7, and than that of H-7, a nonselective inhibitor of protein kinases. These results suggest that 5-HT₂ receptor-mediated Ca²⁺ mobilization can be desensitized homologously after prolonged exposure to 5-HT in a calmodulin-dependent manner in rat glioma C6BU-1 cells.     

Cyclic ADP-ribose: A calcium mobilizing metabolite of NAD+

Mobilization of Ca⁺² from intracellular stores is a signalling mechanism that is of fundamental importance to many cellular processes. It is mediated by two major mechanisms, the inositol 1,4,5-trisphosphate pathway and the Ca⁺²-induced Ca⁺² release process. A naturally occurring metabolite of NAD⁺ called cyclic ADP-ribose has been discovered recently and shown to be as effective as inositol 1,4,5-trisphosphate in mobilizing Ca⁺² stores in sea urchin eggs, a marine invertebrate cell, as well as several mammalian cells. This article reviews the accumulating evidence that indicates cyclic ADP-ribose may function as a physiological regulator of the Ca⁺²-induced Ca⁺² release process and the current knowledge about its receptor as well as the enzymes involved in its metabolism.