Degradation of the immunogenic peptide gp100(280-288) by the monocyte-like U937 cell line

The possible degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) in the presence of the monocyte-like line U937, and the effect of degradation on the in vitro-measured immune recognition, were investigated by chromatographic techniques and immunological assays. Results indicate a rapid hydrolysis of the substrate in the presence of the model cells, which is consistent with the hypothesis that degradation of gp100(280-288) is caused by the activity of U937-expressed enzymes, specifically amino- and carboxypeptidases. On the other hand, these results do not support the involvement of other enzymes known to be expressed by U937 cells. From a functional point of view, these data indicate that the degradation of gp100(280-288) severely hampered recognition by specific CTL clones. The results obtained may provide a model for epitope degradation by the antigen-presenting cells found in defined anatomical compartments and may, at least in part, account for the low activity of peptide-based antineoplastic vaccines, as well as for the transience of the effects of subcutaneously administered peptides in general.     

Structural investigation of proapoptotic peptide by CD and NMR spectroscopy

We have performed a systematic investigation of the structural features of the peptides Int (a sequence able to cross cell membranes) and Int-H1(S6A,F8A) (which shows interesting antitumoral properties). After screening in aqueous solution at different ionic strength and pH values, we analyzed the structures of the peptides in different water/trifluoroethanol mixtures by Circular Dichroism and Nuclear Magnetic Resonance techniques.     

SPECs, small binding proteins for Cdc42

The Rho GTPase, Cdc42, regulates a wide variety of cellular activities including actin polymerization, focal complex assembly, and kinase signaling. We have identified a new family of very small Cdc42-binding proteins, designated SPECs (for Small Protein Effector of Cdc42), that modulates these regulatory activities. The two human members, SPEC1 and SPEC2, encode proteins of 79 and 84 amino acids, respectively. Both contain a conserved N-terminal region and a centrally located CRIB (Cdc42/Rac Interactive Binding) domain. Using a yeast two-hybrid system, we found that both SPECs interact strongly with Cdc42, weakly with Rac1, and not at all with RhoA. Transfection analysis revealed that SPEC1 inhibited Cdc42-induced c-Jun N-terminal kinase (JNK) activation in COS1 cells in a manner that required an intact CRIB domain. Immunofluorescence experiments in NIH-3T3 fibroblasts demonstrated that both SPEC1 and SPEC2 showed a cortical localization and induced the formation of cell surface membrane blebs, which was not dependent on Cdc42 activity. Cotransfection experiments demonstrated that SPEC1 altered Cdc42-induced cell shape changes both in COS1 cells and in NIH-3T3 fibroblasts and that this alteration required an intact CRIB domain. These results suggest that SPECs act as novel scaffold molecules to coordinate and/or mediate Cdc42 signaling activities.     

Signaling from E-cadherins to the MAPK pathway by the recruitment and activation of epidermal growth factor receptors upon cell-cell contact formation

E-cadherins are well characterized cell surface molecules expressed in epithelial cells, which play a major role in cell adhesion through the establishment of calcium-dependent homophilic interactions at sites of cell-cell contacts. They are also integral components of morphogenetic programs controlling the maintenance of the structural and functional integrity of epithelia. Accumulated evidence indicates that the E-cadherin-mediated cell adhesion system is highly regulated from inside the cells by a number of intracellular signaling pathways. Recently available information suggests that E-cadherins may also play a role in the transduction of signals from the outside of the cell to the cytoplasm. However, the nature of the biochemical routes regulated by E-cadherins is still largely unknown. In this study, we set out to explore the possibility that E-cadherins may regulate the activity of MAPK, a key signaling pathway involved in cell fate decisions, upon the formation of cell-cell contacts among neighboring cells. By using an immortalized non-tumorigenic keratinocyte cell line, HaCat, as a model system, we provide evidence that the assembly of calcium-dependent adherens junctions leads to a rapid and remarkable increase in the state of activation of MAPK and that this event is mediated by E-cadherins. Furthermore, we found that E-cadherins stimulate the MAPK pathway through the ligand-independent activation of epidermal growth factor receptors and the consequent activation of a biochemical route leading to the stimulation of MAPKs. These findings suggest that E-cadherins can initiate outside-in signal transducing pathways through the engagement of tyrosine kinase receptors for epidermal growth factor, thus providing a novel molecular mechanism whereby these cell adhesion molecules may ultimately control the fate of normal and transformed epithelial cells.     

Mechanical Predictors of Discomfort during Load Carriage

Discomfort during load carriage is a major issue for activities using backpacks (e.g. infantry maneuvers, children carrying school supplies, or outdoor sports). It is currently unclear which mechanical parameters are responsible for subjectively perceived discomfort. The aim of this study was to identify objectively measured mechanical predictors of discomfort during load carriage. We compared twelve different configurations of a typical load carriage system, a commercially available backpack with a hip belt. The pressure distribution under the hip belt and the shoulder strap, as well as the tensile force in the strap and the relative motion of the backpack were measured. Multiple linear regression analyses were conducted to investigate possible predictors of discomfort. The results demonstrate that static peak pressure, or alternatively, static strap force is a significant (p<0.001) predictor of discomfort during load carriage in the shoulder and hip region, accounting for 85% or more of the variation in discomfort. As an additional finding, we discovered that the regression coefficients of these predictors are significantly smaller for the hip than for the shoulder region. As static peak pressure is measured directly on the body, it is less dependent on the type of load carriage system than static strap force. Therefore, static peak pressure is well suited as a generally applicable, objective mechanical parameter for the optimization of load carriage system design. Alternatively, when limited to load carriage systems of the type backpack with hip belt, static strap force is the most valuable predictor of discomfort. The regionally differing regression coefficients of both predictors imply that the hip region is significantly more tolerant than the shoulder region. In order to minimize discomfort, users should be encouraged to shift load from the shoulders to the hip region wherever possible, at the same time likely decreasing the risk of low back pain or injury.     

LESS IS MORE: SELECTIVE ADVANTAGES CAN EXPLAIN THE PREVALENT LOSS OF BIOSYNTHETIC GENES IN BACTERIA

Bacteria that have adapted to nutrient‐rich, stable environments are typically characterized by reduced genomes. The loss of biosynthetic genes frequently renders these lineages auxotroph, hinging their survival on an environmental uptake of certain metabolites. The evolutionary forces that drive this genome degradation, however, remain elusive. Our analysis of 949 metabolic networks revealed auxotrophies are likely highly prevalent in both symbiotic and free‐living bacteria. To unravel whether selective advantages can account for the rampant loss of anabolic genes, we systematically determined the fitness consequences that result from deleting conditionally essential biosynthetic genes from the genomes of Escherichia coli and Acinetobacter baylyi in the presence of the focal nutrient. Pairwise competition experiments with each of 20 mutants auxotrophic for different amino acids, vitamins, and nucleobases against the prototrophic wild type unveiled a pronounced, concentration‐dependent growth advantage of around 13% for virtually all mutants tested. Individually deleting different genes from the same biosynthesis pathway entailed gene‐specific fitness consequences and loss of the same biosynthetic genes from the genomes of E. coli and A. baylyi differentially affected the fitness of the resulting auxotrophic mutants. Taken together, our findings suggest adaptive benefits could drive the loss of conditionally essential biosynthetic genes.     

Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer

While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here, we identify the PIP(3)-Gβγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gβγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector pathway that is crucial for breast cancer progression.     

The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation

Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins mediate organelle fusion in the secretory pathway. Different fusion steps are catalyzed by specific sets of SNARE proteins. Here we have used the SNAREs mediating the fusion of early endosomes and exocytosis, respectively, to investigate how pairing specificity is achieved. Although both sets of SNAREs promiscuously assemble in vitro, there is no functional crosstalk. We now show that they not only colocalize to overlapping microdomains in the membrane of early endosomes of neuroendocrine cells, but also form cis-complexes promiscuously, with the proportion of the different complexes being primarily dependent on mass action. Addition of soluble SNARE molecules onto native membranes revealed preference for cognate SNAREs. Furthermore, we found that SNAREs are laterally segregated at endosome contact sites, with the exocytotic synaptobrevin being depleted. We conclude that specificity in endosome fusion is mediated by the following two synergistically operating mechanisms: (i) preference for the cognate SNARE in 'trans' interactions and (ii) lateral segregation of SNAREs, leading to relative enrichment of the cognate ones at the prospective fusion sites.