Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N‐terminal peptide (NT‐S100A8)

After isolating NT‐S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca²⁺]_i oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT‐S100A8. In NT‐S100A8 stimulated β‐TC6 (insulinoma cell line) culture medium, insulin and [Ca²⁺] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca²⁺]_i oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT‐S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT‐S100A8 induced a rapid insulin release and enhanced β‐TC6 [Ca²⁺]_i oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT‐S100A8, [Ca²⁺] in β‐TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT‐S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB‐α, but it independently activated Akt and NF‐κB signaling in PC cells. In conclusion, NT‐S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF‐κB signaling, NT‐S100A8 enhances [Ca²⁺]_i oscillations and insulin release, probably by inducing Ca²⁺ influx from the extracellular space, but it does not interfere with insulin signaling. J. Cell. Physiol. 226: 456–468, 2011. © 2010 Wiley‐Liss, Inc.     

Nodulation of Crotalaria podocarpa DC. by Methylobacterium nodulans displays very unusual features

Crotalaria are plants of the Fabaceae family whose nodulation characteristics have been little explored despite the recent discovery of their unexpected ability to be efficiently nodulated in symbiosis with bacteria of the genus Methylobacterium. It has been shown that methylotrophy plays a key role in this unusual symbiotic system, as it is expressed within the nodule and as non-methylotroph mutants had a depleting effect on plant growth response. Within the nodule, Methylobacterium is thus able to obtain carbon both from host plant photosynthesis and from methylotrophy. In this context, the aim of the present study was to show the histological and cytological impacts of both symbiotic and methylotrophic metabolism within Crotalaria podocarpa nodules. It was established that if Crotalaria nodules are multilobed, each lobe has the morphology of indeterminate nodules but with a different anatomy; that is, without root hair infection or infection threads. In the fixation zone, bacteroids display a spherical shape and there is no uninfected cell. Crotalaria nodulation by Methylobacterium displayed some very unusual characteristics such as starch storage within bacteroid-filled cells of the fixation zone and also the complete lysis of apical nodular tissues (where bacteria have a free-living shape and express methylotrophy). This lysis could possibly reflect the bacterial degradation of plant wall pectins through bacterial pectin methyl esterases, thus producing methanol as a substrate, allowing bacterial multiplication before release from the nodule.     

The Presence in tRNA Molecule Sequences of the Double Hairpin,an Evolutionary Stage Through Which the Origin of this Molecule is Thought to have Passed

We analyse 6,810 tRNAs, calculating the free energy of the corresponding double hairpin and ‘cigar’ secondary structures, for which we find a high thermodynamic and statistical significance. We also analyse these tRNAs for similarity and complementarity of their 5′ and 3′ halves or segments of them in intra-and inter-molecular comparisons. We find very clear signs that the two halves of tRNAs had an evident evolutionary relationship, although it is not totally clear whether this was a relationship of homology or complementarity between the 5′ and 3′ halves of tRNAs, even if there is strong evidence in favour of the homology hypothesis. Overall, these data favour models for the origin of the tRNA molecule postulating that a duplication event involving a hairpin structure as a precursor was involved in the origin of this molecule. Moreover, we interpret these results and favour the hypothesis that sees the assembly of two hairpin structures sharing a homology relationship as the intermediate evolutionary stage preceding the appearance of the cloverleaf structure of tRNA.     

Characterization of a novel biosurfactant producing Pseudomonas koreensis lineage that is endemic to Cuatro Ciénegas Basin

The aim of this work is the taxonomic characterization of three biosurfactant-producing bacterial isolates from the Churince system at Cuatro Ciénegas Basin (CCB) in the Mexican State of Coahuila, and the study of the possible role of biosurfactant production in their ecology and evolution. We determined that these isolates belong to a Pseudomonas koreensis lineage endemic to CCB, using standard taxonomical techniques, phylogenetic analysis of three chromosomal loci and phenotypic characterization. This new lineage has the distinct capacity to produce a biosurfactant when compared with previously reported P. koreensis isolates recovered from agricultural soils in Korea. We present evidence suggesting that the biosurfactant secreted by CCB P. koreensis strains is involved in their ability to compete with a CCB Exiguobacterium aurantiacum strain (m5-66) used as a model organism in competition experiments. Furthermore, the ethyl acetate extract of culture supernatant of CCB P. koreensis strains results in growth inhibition not only of E. aurantiacum m5-66, but also of a Bacillus subtilis type strain (ATCC6633). Based on these results we propose that the production of biosurfactant could be of ecological importance and could play a role in the separation of the P. koreensis CCB lineage.     

Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants

Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.     

Ganglioside GM1 mimics: lipophilic substituents improve affinity for cholera toxin

Ganglioside GM1 mimics including (R)-2-hydroxy-3-cyclohexylpropionic acid or (R)-2-hydroxy-3-phenylpropionic acid as replacements for NeuAc are stronger cholera toxin binders than the parent ligand 2, which includes (R)-2-hydroxy-propionic acid.     

The recycling of apolipoprotein E and its amino-terminal 22 kDa fragment: evidence for multiple redundant pathways

A portion of apolipoprotein E (apoE) internalized by hepatocytes is spared degradation and is recycled. To investigate the intracellular routing of recycling apoE, primary hepatocyte cultures from LDL receptor-deficient mice and mice deficient in receptor-associated protein [a model of depressed expression of LDL receptor-related protein (LRP)] were incubated with human VLDL containing 125I-labeled human recombinant apoE3. Approximately 30% of the internalized intact apoE was recycled after 4 h. The N-terminal 22 kDa fragment of apoE was also resecreted, demonstrating that this apoE domain contains sufficient sequence to recycle. The 22 kDa fragment has reduced affinity for lipoproteins, suggesting that apoE recycling is linked to the ability of apoE to bind directly to a recycling receptor. Finally, apoE was found to recycle equally well in the presence of brefeldin A, a drug that blocks transport from the endoplasmic reticulum and leads to collapse of the Golgi stacks. Our studies demonstrate that apoE recycling occurs 1) in the absence of the LDL receptor or under conditions of markedly reduced LRP expression; 2) when apoE lacks the carboxyl-terminal domain, which allows binding to the lipoprotein; and 3) in the absence of an intact Golgi apparatus. We conclude that apoE recycling occurs through multiple redundant pathways.