Coordinated expression of galectin-3 and galectin-3-binding sites in malignant mammary tumors: implications for tumor metastasis

Galectin-3 is a glycan-binding protein that mediates cell-cell and/or cell-extracellular matrix (ECM) interactions. Although galectin-3 is implicated in the progression of various types of cancers, the mechanisms by which galectin-3 enhances metastasis remain unclear. In order to elucidate the role of galectin-3 in the complex multistage process of cancer metastasis, we examined galectin-3 and galectin-3-binding site expression in a series of 82 spontaneous canine mammary tumors (CMT) and two CMT cell lines. Benign CMT tumors exhibited strong nuclear/cytoplasmic galectin-3 immunostaining, whereas malignant CMT tumors and metastases exhibited dramatically decreased galectin-3 expression with the majority of the immunostaining confined to the cytoplasm. Interestingly, intravascular tumor cells overexpressed galectin-3 regardless of their location. CMT-U27 xenografts displayed the same pattern of galectin-3 expression found in spontaneous malignant CMT. In parallel with the downregulation of galectin-3, malignant CMT displayed an overall loss of galectin-3-binding sites in the ECM and focal expression of galectin-3-binding sites mainly detected in intravascular tumor cells and endothelium. Furthermore, loss of galectin-3-binding sites was correlated with the downregulation of GLT25D1, a beta (1-O) galactosyltransferase that modifies collagen, and upregulation of stromal galectin-1. Finally, GLT25D1 mRNA expression was strikingly downregulated in malignant CMT-U27 compared with the benign cell line, and its expression was further de-creased in a galectin-3 knockdown CMT-U27 cell line. We therefore hypothesized that the loss of galectin-3-binding sites in the ECM in conjunction with the overexpression of galectin-3 in specific tumor cell subpopulations are crucial events for the development of mammary tumor metastases.

In vitro osteogenesis induced by cells derived from sites submitted to sinus grafting with anorganic bovine bone

Objectives: This study evaluated key parameters of the in vitro osteogenesis induced by osteoblastic cells obtained from sites submitted to sinus grafting with anorganic bovine bone (ABB) in comparison with cells derived from bone sites of the same patients. Materials and methods: In three patients, the augmentation of maxillary sinus was carried out using ABB (Bio-Oss (R)). After at least 6 months, during the surgical intervention for titanium implants placement, biopsies were taken from these areas using trephine burs (grafted group). Bone fragments, of the same patients, from sites that had not received graft were also obtained with trephine burs and used as a control group. Osteoblastic cells were obtained from grafted and control groups by enzymatic digestion and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured in 24-well culture plates. Cell adhesion was evaluated at 24 h. For proliferation and viability assay, cells were cultured for 1, 3, 7, and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 3, 7, 10, 14, 17, and 21 days. Cultures were stained with Alizarin red S at 21 days, for detection of mineralized matrix. Data were compared by Student`s t-test. Results: Cell adhesion and viability were not affected by cell source (P>0.05). Total protein content was greater (P<0.05) for grafted group. Cell proliferation, ALP activity, and bone-like nodule formation were all greater (P<0.05) for the control group. Conclusions: Taken together, these results indicate that the in vivo long-term contact of cells with ABB downregulates the expression of osteoblast phenotype and consequently the in vitro osteogenesis.

Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases in hypertensive vascular remodeling

Structural vascular changes in two-kidney, one-clip (2K-1C) hypertension may result from increased matrix metalloproteinase (MMP)-2 activity. MMP-2 activation is regulated by other MMPs, including transmembrane-MMPs, and by tissue inhibitors of MMPs (TIMPs). We have investigated the localization of MMP-2, -9, -14, and TIMPs 1-4 in hypertensive aortas and measured their levels by zymography/Western blotting and immunohistochemistry. Gelatinolytic activity was assayed in tissues by in situ zymography. Sham-operated and 2K-1C hypertensive rats were treated with doxycycline (or vehicle) for 8 weeks, and the systolic blood pressure was monitored weekly. Doxycycline attenuated 2K-1C hypertension (165 +/- 11.7 mmHg versus 213 +/- 7.9 mm Hg in hypertensive controls, P<0.01), and completely prevented increase in the thicknesses of the media and the intima in 2K-1C animals (P<0.01). Increased amounts of MMP-2, -9, and -14 were found in hypertensive aortas, as well as enhanced gelatinolytic activity. A gradient in the localization of MMP-2, -9, and -14 was found, with increased amounts detected in the intima, at sites with higher gelatinolytic activity. Doxycycline attenuated hypertension induced increases in all the 3 investigated MMPs in both the media and the intima (all P<0.05). but it did not change the amounts of TIMPs 1-4 (P>0.05). Therefore, an imbalance between increased amounts of MMPs at the tissue level without a corresponding increase in the quantities of TIMPs, particularly in the intima and inner media layers, appears to account for the increased proteolytic activity found in 2K-1C hypertension-induced maladaptive vascular remodeling. (C) 2009 Elsevier B.V. All rights reserved.

Percolation of colloids with distinct interaction sites

We generalize the Flory-Stockmayer theory of percolation to a model of associating (patchy) colloids, which consists of hard spherical particles, having on their surfaces f short-ranged-attractive sites of m different types. These sites can form bonds between particles and thus promote self-assembly. It is shown that the percolation threshold is given in terms of the eigenvalues of a m x m matrix, which describes the recursive relations for the number of bonded particles on the ith level of a cluster with no loops; percolation occurs when the largest of these eigenvalues equals unity. Expressions for the probability that a particle is not bonded to the giant cluster, for the average cluster size and the average size of a cluster to which a randomly chosen particle belongs, are also derived. Explicit results for these quantities are computed for the case f = 3 and m = 2. We show how these structural properties are related to the thermodynamics of the associating system by regarding bond formation as a (equilibrium) chemical reaction. This solution of the percolation problem, combined with Wertheim's thermodynamic first-order perturbation theory, allows the investigation of the interplay between phase behavior and cluster formation for general models of patchy colloids.

Shell variation in Patellid limpets: scales of spatial variability

World Congress of Malacology, Universidade dos Açores, Ponta Delgada, 21-28 de julho.

Novel Prostate Specific Antigen plastic antibody designed withcharged binding sites for an improved protein binding and itsapplication in a biosensor of potentiometric transduction

This work shows that the synthesis of protein plastic antibodies tailored with selected charged monomersaround the binding site enhances protein binding. These charged receptor sites are placed over a neutralpolymeric matrix, thus inducing a suitable orientation the protein reception to its site. This is confirmed bypreparing control materials with neutral monomers and also with non-imprinted template. This concepthas been applied here to Prostate Specific Antigen (PSA), the protein of choice for screening prostate can-cer throughout the population, with serum levels >10 ng/mL pointing out a high probability of associatedcancer.Protein Imprinted Materials with charged binding sites (C/PIM) have been produced by surfaceimprinting over graphene layers to which the protein was first covalently attached. Vinylben-zyl(trimethylammonium chloride) and vinyl benzoate were introduced as charged monomers labellingthe binding site and were allowed to self-organize around the protein. The subsequent polymerizationwas made by radical polymerization of vinylbenzene. Neutral PIM (N/PIM) prepared without orientedcharges and non imprinted materials (NIM) obtained without template were used as controls.These materials were used to develop simple and inexpensive potentiometric sensor for PSA. Theywere included as ionophores in plasticized PVC membranes, and tested over electrodes of solid or liq-uid conductive contacts, made of conductive carbon over a syringe or of inner reference solution overmicropipette tips. The electrodes with charged monomers showed a more stable and sensitive response,with an average slope of -44.2 mV/decade and a detection limit of 5.8 × 10−11mol/L (2 ng/mL). The cor-responding non-imprinted sensors showed lower sensitivity, with average slopes of -24.8 mV/decade.The best sensors were successfully applied to the analysis of serum, with recoveries ranging from 96.9to 106.1% and relative errors of 6.8%.

Haemoglobin smart plastic antibody material tailored with charged binding sites on silica nanoparticles: its application as an ionophore in potentiometric transduction

This work uses surface imprinting to design a novel smart plastic antibodymaterial (SPAM) for Haemoglobin (Hb). Charged binding sites are described here for the first time to tailor plastic antibody nanostructures for a large size protein such as Hb. Its application to design small, portable and low cost potentiometric devices is presented. The SPAM material was obtained by linking Hb to silica nanoparticles and allowing its ionic interaction with charged vinyl monomers. A neutral polymeric matrix was created around these and the imprinted protein removed. Additional materials were designed in parallel acting as a control: a neutral imprinted material (NSPAM), obtained by removing the charged monomers from the procedure, and the Non-Imprinted (NI) versions of SPAM and NSPAM by removing the template. SEM analysis confirmed the surface modification of the silica nanoparticles. All materials were mixed with PVC/plasticizer and applied as selective membranes in potentiometric transduction. Electromotive force (emf) variations were detected only for selective membranes having a lipophilic anionic additive in the membrane. The presence of Hb inside these membranes was evident and confirmed by FTIR, optical microscopy and Raman spectroscopy. The best performance was found for SPAM-based selective membranes with an anionic lipophilic additive, at pH 5. The limits of detection were 43.8 mg mL 1 and linear responses were obtained down to 83.8 mg mL 1, with an average cationic slope of +40 mV per decade. Good selectivity was also observed against other coexisting biomolecules. The analytical application was conducted successfully, showing accurate and precise results.

Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites.

BACKGROUND: Thy-1 is an abundant neuronal glycoprotein in mammals. Despite such prevalence, Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. In this paper, a ligand molecule for Thy-1 was identified, and the consequences of Thy-1 binding for astrocyte function were investigated. RESULTS: Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with beta3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4(-f), control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-beta3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However, neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn(2+)-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrin-derived peptide, but not fibronectin, also mediated Mn(2+)-dependent adhesion, suggesting the involvement of beta3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130(Cas) and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. CONCLUSIONS: Thy-1 binds to beta3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading.

Extra-cellular matrix changes in Schistosoma mansoni-infected Biomphalaria glabrata

Reactivity of snails against parasites exhibits a primitive focal reaction, with encapsulation, phagocytosis and destruction of parasite larvae by macrophage-like cells - the hemocytes. This reaction mimics granulomatous inflammation seen in higher animals. However, different from the latter, little is known about the participation of extra-cellular matrix in such snail defense reactions. Normal and Schistosoma mansoni-infected Biomphalaria glabrata of different strains were submitted to cytological, histological, ultrastructural and biochemical methods in order to investigate the behavior of extra-cellular tissues at the site of anti-parasite reactions. In spite of the presence of two cell-types in peripheral hemolymph, only one cell-type was present at the sites of tissue reactions. Although pre-existent collagen and elastic fibers and microfibrils sometimes appeared slightly compressed around focal reactions, no evidences of duplication, synthesis or deposition of connective-tissue extra-cellular components were observed within or around the zones of reactive cell accumulations. Thus, tissue reactions against S. mansoni in the snail B. glabrata appeared exclusively dependent on one specific population of hemocytes.

Nanopartícul·les core-shell estabilitzades en polímer amb propietats calítiques i bactericides medioambientalment segure

Aquest projecte de doctorat és un treball interdisciplinari adreçat a l’obtenció de nous nanocompòsits (NCs) funcionals sintetitzats a partir de materials polimèrics bescanviadors d’ions que són modificats amb nanopartícules metàl•liques (NPMs) de diferent composició. Els materials desenvolupats s’avaluen en funció de dues possibles aplicacions: 1) com a catalitzadors de reaccions orgàniques d’interès actual (NCs basats en pal•ladi) i, 2) la seva dedicació a aplicacions bactericides en el tractament d’aigües domèstiques o industrials (NCs basats en plata). El desenvolupament de nanomaterials és de gran interès a l’actualitat donades les seves especials propietats, l’aprofitament de les quals és la força impulsora per a la fabricació de nous NCs. Les nanopartícules metàl•liques estabilitzades en polímer (Polymer Stabilized Metal Nanoparticles, PSNPM) s’han preparat mitjançant la tècnica in-situ de síntesi intermatricial (Inter-matrix synthesis, IMS) que consisteix en la càrrega seqüencial dels grups funcionals de les matrius polimèriques amb ions metàl•lics, i la seva posterior reducció química dins de la matriu polimèrica de bescanvi iònic. L’estabilització en matrius polimèriques evita l’agregació entre elles (self-aggreagtion), un dels principals problemes coneguts de les NPs. Pel desenvolupament d’aquesta metodologia, s’han emprat diferents tipus de matrius polimèriques de bescanvi iònic: membrana Sulfonated PolyEtherEtherKetone, SPEEK, així com fibres sintètiques basades en polypropilè amb diferents tipus de grups funcionals, que ens permeten el seu ús com a filtres en la desinfecció de solucions aquoses o com a material catalitzador. Durant el projecte s’ha anat avançant en l’optimització del material nanocomposite final per a les aplicacions d’interès, en quant activitat i funcionalitat de les nanopartícules i estabilitat del nanocomposite. Així, s’ha optimitzat la síntesi de NPs estabilitzades en resines de bescanvi iònic, realitzant un screening de diferents tipus de resines i la seva avaluació en aplicacions industrials d’interès.

Mapping cell-matrix stresses during stretch reveals inelastic reorganization of the cytoskeleton.

The mechanical properties of the living cell are intimately related to cell signaling biology through cytoskeletal tension. The tension borne by the cytoskeleton (CSK) is in part generated internally by the actomyosin machinery and externally by stretch. Here we studied how cytoskeletal tension is modified during stretch and the tensional changes undergone by the sites of cell-matrix interaction. To this end we developed a novel technique to map cell-matrix stresses during application of stretch. We found that cell-matrix stresses increased with imposition of stretch but dropped below baseline levels on stretch release. Inhibition of the actomyosin machinery resulted in a larger relative increase in CSK tension with stretch and in a smaller drop in tension after stretch release. Cell-matrix stress maps showed that the loci of cell adhesion initially bearing greater stress also exhibited larger drops in traction forces after stretch removal. Our results suggest that stretch partially disrupts the actin-myosin apparatus and the cytoskeletal structures that support the largest CSK tension. These findings indicate that cells use the mechanical energy injected by stretch to rapidly reorganize their structure and redistribute tension.

Physical and chemical attributes of archaeological soils developed from shell middens in the Região dos Lagos, Rio de Janeiro, Brazil

In prehistoric times, innumerous shell middens, called "sambaquis", consisting mainly of remains of marine organisms, were built along the Brazilian coast. Although the scientific community took interest in these anthropic formations, especially since the nineteenth century, their pedological context is still poorly understood. The purpose of this study was to characterize and identify the physical and chemical changes induced by soil-forming processes, as well as to compare the morphology of shell midden soils with other, already described, anthropogenic soils of Brazil. Four soil profiles developed from shell middens in the Região dos Lagos - RJ were morphologically described and the physical and chemical properties determined. The chemical analysis showed that Ca, Mn, Mg, and particularly P and Zn are indicators of anthropic horizons of midden soils, as in the Amazon Dark Earths (Terras Pretas de Índio). After the deposition of P-rich material, P reaction and leaching can mask or disturb the evidence of in situ man-made strata, but mineralogical and chemical studies of phosphate forms can elucidate the apparent complexity. Lower phosphate-rich strata without direct anthropic inputs indicate P leaching and precipitation in secondary forms. The total and bioavailable contents of Ca, Mg, Zn, Mn, Cu, P, and organic C of midden soils were much higher than of regional soils without influence of ancient human settlements, demonstrating that the high fertility persisted for long periods, at some sites for more than 4000 years. The physical analysis showed that wind-blown sand contributed significantly to increase the sand fraction in the analyzed soils (texture classes sand, sandy loam and sandy clay loam) and that the aeolian sand accumulation occurred simultaneously with the midden formation.

Semiclassical evaluation of average nuclear one- and two-body matrix elements

Thomas-Fermi theory is developed to evaluate nuclear matrix elements averaged on the energy shell, on the basis of independent particle Hamiltonians. One- and two-body matrix elements are compared with the quantal results, and it is demonstrated that the semiclassical matrix elements, as function of energy, well pass through the average of the scattered quantum values. For the one-body matrix elements it is shown how the Thomas-Fermi approach can be projected on good parity and also on good angular momentum. For the two-body case, the pairing matrix elements are considered explicitly.

Distorted-wave calculation of cross sections for inner-shell ionization by electron and positron impact

The relativistic distorted-wave Born approximation is used to calculate differential and total cross sections for inner shell ionization of neutral atoms by electron and positron impact. The target atom is described within the independent-electron approximation using the self-consistent Dirac-Fock-Slater potential. The distorting potential for the projectile is also set equal to the Dirac-Fock-Slater potential. For electrons, this guarantees orthogonality of all the orbitals involved and simplifies the calculation of exchange T-matrix elements. The interaction between the projectile and the target electrons is assumed to reduce to the instantaneous Coulomb interaction. The adopted numerical algorithm allows the calculation of differential and total cross sections for projectiles with kinetic energies ranging from the ionization threshold up to about ten times this value. Algorithm accuracy and stability are demonstrated by comparing differential cross sections calculated by our code with the distorting potential set to zero with equivalent results generated by a more robust code that uses the conventional plane-wave Born approximation. Sample calculation results are presented for ionization of K- and L-shells of various elements and compared with the available experimental data.

Experimental analysis and computer prediction of CTF/NFI transcription factor DNA binding sites.

Accurate prediction of transcription factor binding sites is needed to unravel the function and regulation of genes discovered in genome sequencing projects. To evaluate current computer prediction tools, we have begun a systematic study of the sequence-specific DNA-binding of a transcription factor belonging to the CTF/NFI family. Using a systematic collection of rationally designed oligonucleotides combined with an in vitro DNA binding assay, we found that the sequence specificity of this protein cannot be represented by a simple consensus sequence or weight matrix. For instance, CTF/NFI uses a flexible DNA binding mode that allows for variations of the binding site length. From the experimental data, we derived a novel prediction method using a generalised profile as a binding site predictor. Experimental evaluation of the generalised profile indicated that it accurately predicts the binding affinity of the transcription factor to natural or synthetic DNA sequences. Furthermore, the in vitro measured binding affinities of a subset of oligonucleotides were found to correlate with their transcriptional activities in transfected cells. The combined computational-experimental approach exemplified in this work thus resulted in an accurate prediction method for CTF/NFI binding sites potentially functioning as regulatory regions in vivo.