Redox-active metals in commercial preparations of lipopolysaccharide: implications for studies of cellular responses to bacterial products

The mechanisms by which lipopolysaccharide (LPS) activates cells have been the subject of intense investigation for many years. Whereas much information on this process has been collected for mammalian species, little is known about the signalling path-ways operative in other animals. One general mode of cellular activation that has been recently pro-posed for pathways independent of the primary mammalian LPS receptor, CD14, involves reactive oxygen species (ROS) as intermediates in LPS-induced signalling pathways. Therefore, we used 2',7'-dichlorodihydrofluorescein, a fluorogenic probe of redox activity, to examine LPS-induced oxidative responses of a macrophage-like cell line from the rainbow trout, RTS11. Lipopolysaccharide dose-dependently increased oxidation of this probe by RTS11 cells, and a variety of other cell lines. This process was inhibited by catalase, superoxide dismutase and NG-methylarginine citrate, an inhibitor of nitric oxide synthases, suggesting the involvement of a diverse assortment of cellular ROS. More careful dissection of this phenomenon led us to conclude that the increase in oxidation was, in fact, due almost entirely to metals, particularly copper, in some LPS preparations, which is something to consider when experimenting with LPS.     

Influence of the length of the phosphate chain in mRNA 5' cap analogues on their interaction with eukaryotic initiation factor 4E

The recognition of the 5'mRNA cap structure m7G(5')ppp(5')N by one of the components of the initiation translation machinery, the eIF4E factor, plays a pivotal role in regulation of the protein synthesis. In the present study we have shown two opposing roles of the cap phosphate chain in the specific eIF4E-cap interaction. The extension of the phosphate chain enhances the binding of the cap to the unphosphorylated eIF4E but destabilises the eIF4E-cap complex in case of the phosphorylated protein.     

Binding studies of eukaryotic initiation factor eIF4E with novel mRNA dinucleotide cap analogues

Studies on the interaction of the murine translation initiation factor 4E with two new-synthesized cap-analogues, modified at C2' of 7-methylguanosine, have been performed by means of the fluorescence titration method. No difference in the binding affinity for eIF4E was observed compared with the "anti reversed" cap analogues, possessing the analogous modifications at C3'. Potential significance of the novel caps as research tools for examination of the nuclear cap binding complex CBC80/20 has been discussed.     

Serological characterization of anti-endotoxin serum directed against the conjugate of oligosaccharide core of Escherichia coli type R4 with tetanus toxoid

The covalent conjugate of oligosaccharide core of Escherichia coli type R4 with tetanus toxoid was prepared using reaction of reductive amination. The neoglycoconjugate was a good immunogen in rabbits yielding a high level of anti-lipopolysaccharide (LPS) antibodies of the IgG class. It was found that antiserum was able to react with the smooth LPS molecules of identical (R4) or related (R1) core type. The reactions were shown in the enzyme-linked immunosorbent assay and the immunoblotting test. Flow cytometry showed that anti-core antibodies reacted with LPS present on intact, live, smooth bacteria labelling more than 90% of cells. The anti-OS R4-TT serum used for in vitro studies showed high endotoxin neutralization activity. The serum inhibited endotoxin-induced tumor necrosis factor alpha and nitric oxide synthesis by the J-774A.1 cell line and attenuated pulmonary retention of YAC-1 cells.     

Infection of clover by plant growth promoting Pseudomonas fluorescens strain 267 and Rhizobium leguminosarum bv. trifolii studied by mTn5-gusA

Plant growth promoting Pseudomonas fluorescens strain 267, isolated from soil, produced pseudobactin A, 7-sulfonic acid derivatives of pseudobactin A and several B group vitamins. In coinoculation with Rhizobium leguminosarum bv. trifolii strain 24.1, strain 267 promoted clover growth and enhanced symbiotic nitrogen fixation under controlled conditions. To better understand the beneficial effect of P. fluorescens 267 on clover inoculated with rhizobia, the colonization of clover roots by mTn5-gusA marked bacteria was studied in single and mixed infections under controlled conditions. Histochemical assays combined with light and electron microscopy showed that P. fluorescens 267.4 (i) efficiently colonized clover root surface; (ii) was heterogeneously distributed along the roots without the preference to defined root zone; (iii) formed microcolonies on the surface of clover root epidermis; (iv) penetrated the first layer of the primary root cortex parenchyma and (v) colonized endophytically the inner root tissues of clover.     

Association between GM3 and CD4-Ick complex in human peripheral blood lymphocytes

The aim of this study was to further elucidate our previous observation on molecular interaction of GM3, CD4 and p56^lck in microdomains of human peripheral blood lymphocytes (PBL). We analyzed GM3 distribution by immunoelectron microscopy and the association between GM3 and CD4-p56^lck complex by scanning confocal microscopy and co-immunoprecipitation experiments. Scanning confocal microscopy analysis showed an uneven signal distribution of GM3 molecules over the surface of human lymphocytes. Nearly complete colocalization areas indicated that CD4 molecules were distributed in GM3-enriched plasma membrane domains. Co-immunoprecipitation experiments revealed that CD4 and p56^lck were immunoprecipitated by IgG anti-GM3, demonstrating that GM3 tightly binds to the CD4-p56^lck complex in human PBL. In order to verify whether GM3 association with CD4 molecules may depend on the presence of p56^lck, we analyzed this association in U937, a CD4+and p56^lck negative cell line. The immunoprecipitation with anti-GM3 revealed the presence of a 58[emsp4 ]kDa band immunostained with anti-CD4 Ab, suggesting that the GM3-CD4 interaction does not require its association with p56^lck. These findings support the view that GM3 enriched-domains may represent a functional multimolecular complex involved in signal transduction and cell activation.     

Involvement of Pituitary Adenylate Cyclase-Activating Polypeptide II Vasoactive Intestinal Peptide 2 Receptor in Mouse Neocortical Astrocytogenesis

Abstract: At the end of neuronal migration, the neopallial germinative zone produces glial cells destined to colonize the upper layers of neocortex. High densities of binding sites for vasoactive intestinal peptide (VIP) have been found in the rodent germinative zone just after completion of neuronal migration, suggesting a possible role of VIP in neocortical astrocytogenesis. In the present study, administration of a VIP antagonist at embryonic days 17 and 18 to pregnant mice was followed by a dramatic depletion of astrocytes in the upper cortical layer of the offspring. The depletion of astrocytes was dose-dependent, with a 42% reduction in the density of astrocytes observed with 50 µg of antagonist. The antagonist effect was reversed by cotreatment with VIP or pituitary adenylate cyclase-activating polypeptide (PACAP), suggesting the involvement of a receptor common to these two neuropeptides. VIP antagonist-induced inhibition of astrocytogenesis was also blocked by Ro 25-1553, a long-acting cyclic VIP analogue selective for the PACAP II VIP2 receptor subclass. Our results demonstrate that VIP and/or PACAP play a crucial physiological role in neocortical astrocytogenesis, possibly through interaction with PACAP II VIP2 receptors.     

Computer Modeling of the Solution Conformation of Cyclic Enkephalins

The probable conformations of two cyclic enkephalin analogs, DNS-cyclo[D-Dab-Gly-Trp-Leu] (I) and DNS-cyclo[D-Dab-Gly-Trp-D-Leu] (II) (DNS = dansyl), were determined by combining the results of NOE, vicinal coupling constant and fluorescence energy transfer measurements with theoretical calculations. The common feature of the conformations for both peptides is the presence of a -turn at residues 2 and 3.