A short peptide domain of platelet factor 4 blocks angiogenic key events induced by FGF-2.

Platelet factor 4 (PF-4) is a CXC-chemokine with strong anti-angiogenic properties. We have shown previously that PF-4 inhibits angiogenesis by associating directly with fibroblast growth factor 2 (FGF-2), inhibiting its dimerization, and blocking FGF-2 binding to endothelial cells. We now have characterized a small peptide domain (PF-447-70) derived from the C-terminus of PF-4, which conserves anti-angiogenic effects of the parent protein. PF-447-70 inhibited internalization of 125I-FGF-2 by endothelial cells in a time-dependent manner. The peptide reduced FGF-2-stimulated cell migration to control levels in wounded monolayers of bovine capillary endothelial cells. PF-447-70 also reduced FGF-2 induced phosphorylation of MAP kinases ERK-1 and ERK-2, which are essential for migration and survival of endothelial cells. In a serum-free ex vivo angiogenesis assay, the peptide blocked microvessel outgrowth by 89%. A single amino acid substitution within PF-447-70 abolished all inhibitory activities. To simulate a real anti-angiogenic treatment situation, we administered PF-447-70 systemically to mice implanted subcutaneously with FGF-2 containing gelatin sponges with the result of sparse, scattered, and immature vessel growth. The small peptide fragment derived from the angio-inhibitory CXC-chemokine PF-4 might be used as a starting point to develop anti-angiogenic designer drugs for angiogenesis-dependent pathologies such as cancer, diabetic retinopathy, and rheumatoid arthritis.     

The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

Protecting capacity against malaria of chemically defined tetramer forms based on the Plasmodium falciparum apical sushi protein as potential vaccine components

Developing novel generations of subunit-based antimalarial vaccines in the form of chemically-defined macromolecule systems for multiple antigen presentation represents a classical problem in the field of vaccine development. Many efforts involving synthesis strategies leading to macromolecule constructs have been based on dendrimer-like systems, the condensation of large building blocks and conventional asymmetric double dimer constructs, all based on lysine cores.     

Enzymatic synthesis of 3'-hydroxyacetaminophen catalyzed by tyrosinase

3'-Hydroxyacetaminophen, a catechol metabolite of N-acetyl-p-aminophenol (acetaminophen) and N-acetyl-m-aminophenol (a structural analogue of acetaminophen and considered as a possible alternative because it is not hepatotoxic), is enzymatically synthesized for the first time using mushroom tyrosinase. Although reported to be weakly hepatotoxic in vivo, this catechol derivative of acetaminophen is not commercially available. This compound was obtained from its monophenolic precursor, acetaminophen, using the enzyme tyrosinase in the presence of an excess of ascorbic acid, thus reducing back the o-quinone product of catalytic activity to the catechol acetaminophen derivative. A mathematical model of the system is proposed, which is also applicable to the tyrosinase-mediated synthesis of any o-diphenolic compound from its corresponding monophenol. This synthesis procedure is continuous, easy to perform and control, and adaptable to a bioreactor with the immobilized enzyme for industrial purposes in a nonpolluting way.     

A kinetic study of the melanization pathway between L-tyrosine and dopachrome

In the pathway of melanin biosynthesis originating from L-tyrosine, the dopachrome accumulation at physiological pH is produced with a pronounced lag period, during which the level of L-dopa increases, following a sigmoidal kinetics to reach a steady-state. A kinetic model has been proposed for the overall pathway of melanization from L-tyrosine to dopachrome; it explains the lag period present during the dopachrome accumulation as well as the influence of L-tyrosine and tyrosinase over this lag period. Use of this model is also valid to explain the kinetics of L-dopa accumulation in the reaction medium, as has been tested by simulation.     

Kinetic analysis of deactivation of immobilized alpha-chymotrypsin by water-miscible organic solvent in kyotorphin synthesis.

Two different immobilized chymotrypsin derivatives were used to synthesize kyotorphin, using N-benzoyl-L-tyrosine ethyl ester and L-arginine ethyl ester as substrates, in water-DMF media. The first was adsorbed onto Celite particles and the second was multipoint covalently attached into polyacrylamide gel. In all cases, the conversion of the carboxyl substrate was carried out in first-order reaction conditions. For the adsorbed enzyme, the reaction kinetics deviated from first-order likely due to a fast irreversible inactivation of enzyme during the reaction time even at low DMF concentration (15-20% v/v). The covalent attachment of enzyme resulted in elimination of irreversible activity loss by organic solvent up to 60% (v/v) of DMF. The catalytic activity of the covalent derivative was conserved as appropriate for performing a synthetic reaction up to 60% v/v of DMF (in comparison to 30% v/v for the adsorbed derivative), showing a clear improvement in its stability against reversible denaturation by this solvent. The selectivity of the synthetic reaction was slightly enhanced (from 40-50%) with the increase in DMF concentration to 80% v/v, but it was significantly improved (to 80%) when L-argininamide was used as nucleophile.     

The Atapuerca sites and their contribution to the knowledge of human evolution in Europe

Over the last two decades, the Pleistocene sites of the Sierra de Atapuerca (Spain) have provided two extraordinary assemblages of hominin fossils that have helped refine the evolutionary story of the genus Homo in Europe. The TD6 level of the Gran Dolina site has yielded about one hundred remains belonging to a minimum of six individuals of the species Homo antecessor. These fossils, dated to the end of the Lower Pleistocene (800 kyr), provide the earliest evidence of hominin presence in Western Europe. The origin of these hominins is unknown, but they may represent a speciation event from Homo ergaster/Homo erectus. The TD6 fossils are characterized by a significant increase in cranial capacity as well as the appearance of a “sapiens” pattern of craniofacial architecture. At the Sima de los Huesos site, more than 4,000 human fossils belonging to a minimum of 28 individuals of a Middle Pleistocene population (ca. 500–400 kyr) have been recovered. These hominins document some of the oldest evidence of the European roots of Neanderthals deep in the Middle Pleistocene. Their origin would be the dispersal out of Africa of a hominin group carrying Mode 2 technologies to Europe. Comparative study of the TD6 and Sima de la Huesos hominins suggests a replacement model for the European Lower Pleistocene population of Europe or interbreeding between this population and the new African emigrants.