cAMP-induced cytoskeleton rearrangement increases calcium transients through the enhancement of capacitative calcium entry

In this report we investigated the correlation between cell morphology and regulation of cytosolic calcium homeostasis. Type I astrocytes were differentiated to stellate process-bearing cells by a 100-min exposure to cAMP. Differentiation of cortical astrocytes increased the magnitude and duration of calcium transients elicited by phospholipase C-activating agents as measured by single cell Fura-2-based imaging. Calcium imaging showed differences in the spatial pattern of the response. In both differentiated and the control cells, the response originated in the periphery and gradually extended into the center of the cell. However, the elevation of cytosolic calcium concentration ([Ca(2+)](i)) was particularly evident within the processes and adjacent to the inner cell membrane of the differentiated astrocytes. In addition, differentiation significantly prolonged the duration of the [Ca(2+)](i) elevation. Potentiation of the calcium transients was mimicked by forskolin-induced differentiation and abolished by a specific protein kinase-A blocker. Conversely, the enhancement of the calcium transients was not mimicked by brief exposure to cAMP not causing morphological differentiation, and in PC12 cells that did not undergo morphological changes after 100 min of cAMP treatment. Impairing cAMP-induced cytoskeleton re-organization, by means of cytochalasin D and nocodazole, prevented the potentiation of the calcium transients in cAMP-treated astrocytes. Phospholipase C activity and sensitivity to inositol (1,4,5)-trisphosphate were not involved in the enhancement of the calcium responses. Also, potentiation of the calcium transients was dependent on extracellular calcium. Calcium storage and thapsigargin-depletable intracellular calcium reservoirs were analogously not increased in differentiated astrocytes. Rearrangement of the cell shape also caused a condensation of the endoplasmic reticulum and altered the spatial relationship between the endoplasmic reticulum and the cell membrane. In conclusion, morphological rearrangements of type I astrocytes increase the magnitude and the duration of agonist-induced calcium transients via enhancement of capacitative calcium entry and is associated with a spatial reorganization of the relationship between cell membrane and the endoplasmic reticulum structures.     

Heterologous expression of bovine and porcine beta-lactoglobulins in Pichia pastoris: towards a comparative functional characterisation

Bovine and porcine beta-lactoglobulins were cloned and expressed in host cells with the aim of developing the tools necessary for their structural, functional and conformational characterisation by NMR techniques. Both lipocalins were expressed in Pichia pastoris, where the use of a constitutive promoter turned out to allow the highest productivity. The yield of recombinant proteins was further improved through multiple integration of the encoding genes and by increasing aeration of the transformed cultures. Both proteins were obtained in the culture medium at the concentration of 200 microg/ml. Recombinant lipocalins were purified by ion-exchange chromatography from the culture medium. A preliminary NMR characterisation showed that both proteins were correctly folded.     

Low electromagnetic field (50 Hz) induces differentiation on primary human oral keratinocytes (HOK)

This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes.     

A phaseolin domain involved directly in trimer assembly is a determinant for binding by the chaperone BiP

The binding protein (BiP; a member of the heat-shock 70 family) is a major chaperone of the endoplasmic reticulum (ER). Interactions with BiP are believed to inhibit unproductive aggregation of newly synthesized secretory proteins during folding and assembly. In vitro, BiP has a preference for peptide sequences enriched in hydrophobic amino acids, which are expected to be exposed only in folding and assembly intermediates or in defective proteins. However, direct information regarding sequences recognized in vivo by BiP on real proteins is very limited. We have shown previously that newly synthesized monomers of the homotrimeric storage protein phaseolin associate with BiP and that phaseolin trimerization in the ER abolishes such interactions. Using different phaseolin constructs and green fluorescent protein (GFP) fusion proteins, we show here that one of the two alpha-helical regions of polypeptide contact in phaseolin trimers (35 amino acids located close to the C terminus and containing three potential BiP binding sites) effectively promotes BiP association with phaseolin and with secretory GFP fusions expressed in transgenic tobacco or in transfected protoplasts. We also show that overexpressed BiP transiently sequesters phaseolin polypeptides. We conclude that one of the regions of monomer contact is a BiP binding determinant and suggest that during the synthesis of phaseolin, the association with BiP and trimer formation are competing events. Finally, we show that the other, internal region of contact between monomers is necessary for phaseolin assembly in vivo and contains one potential BiP binding site.     

Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.     

Detection of integrin alpha IIbbeta 3 clustering in living cells

In platelets, bidirectional signaling across integrin alpha(IIb)beta(3) regulates fibrinogen binding, cytoskeletal reorganization, cell aggregation, and spreading. Because these responses may be influenced by the clustering of alpha(IIb)beta(3) heterodimers into larger oligomers, we established two independent methods to detect integrin clustering and evaluate factors that regulate this process. In the first, weakly complementing beta-galactosidase mutants were fused to the C terminus of individual alpha(IIb) subunits, and the chimeras were stably expressed with beta(3) in Chinese hamster ovary cells. Clustering of alpha(IIb)beta(3) should bring the mutants into proximity and reconstitute beta-galactosidase activity. In the second method, alpha(IIb) was fused to either a green fluorescent protein (GFP) or Renilla luciferase and transiently expressed with beta(3). Here, integrin clustering should stimulate bioluminescence resonance energy transfer between a cell-permeable luciferase substrate and GFP. These methods successfully detected integrin clustering induced by anti-alpha(IIb)beta(3) antibodies. Significantly, they also detected clustering upon soluble fibrinogen binding to alpha(IIb)beta(3). In contrast, no clustering was observed following direct activation of alpha(IIb)beta(3) by MnCl(2) or an anti-alpha(IIb)beta(3)-activating antibody Fab in the absence of fibrinogen. Intracellular events also influenced alpha(IIb)beta(3) clustering. For example, a cell-permeable, bivalent FK506-binding protein (FKBP) ligand stimulated clustering when added to cells expressing an alpha(IIb)(FKBP)(2) chimera complexed with beta(3). Furthermore, alpha(IIb)beta(3) clustering occurred in the presence of latrunculin A or cytochalasin D, inhibitors of actin polymerization. These effects were enhanced by fibrinogen, suggesting that actin-regulated clustering modulates alpha(IIb)beta(3) interaction with ligands. These studies in living cells establish that alpha(IIb)beta(3) clustering is modulated by fibrinogen and actin dynamics. More broadly, they should facilitate investigations of the mechanisms and consequences of integrin clustering.     

NMR solution structure of viscotoxin C1 from Viscum album species Coloratum ohwi: toward a structure-function analysis of viscotoxins

The high resolution three-dimensional structure of the newly discovered plant viscotoxin C1, from the Asiatic Viscum album ssp. Coloratum ohwi, has been determined in solution by (1)H NMR spectroscopy at pH 3.6 and 285 K. The viscotoxin C1-fold, consisting of a helix-turn-helix motif and a short stretch of an antiparralel beta-sheet is very similar to that found for the highly similar viscotoxins A2 and A3 and for other related thionins. Different functional properties of members of the thionin family are discussed here in light of the structural and electrostatic properties. Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from the other members because of its high toxicity against tumoral cells. Key residues for the modulation of viscotoxin cytotoxicity have been identified on the basis of sequence and structural alignment.     

Monitoring the effects of drug treatment in rat models of disease by serum protein analysis

In this review we list from literature investigations on rat serum proteins using electrophoretic techniques in connection with drug testing. From our own research work, we provide annotated two-dimensional maps of rat serum proteins under control and experimental conditions. Emphasis is on species-specific components and on the effects of acute and chronic inflammation. We discuss our project of structural proteomics on rat serum as a minimally invasive approach to pharmacological investigation, and we outline a typical experimental plan for drug testing according to the above guidelines. We then report in detail on the results of our trials of anti-inflammatory drugs on adjuvant arthritis, an animal model of disease resembling in many aspects human rheumatoid arthritis. We demonstrate a correlation between biochemical parameters and therapeutic findings and outline the advantages of the chosen methodological approach, which proved also sensitive in revealing "side effects" of the test drugs. In an appendix we describe our experimental protocol when performing two-dimensional electrophoresis of rat serum.