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.

Sialylation of beta 1 Integrins blocks cell adhesion to galectin-3 and protects cells against galectin-3-induced apoptosis

In previous studies, we determined that beta 1 integrins from human colon tumors have elevated levels of alpha 2-6 sialylation, a modification added by beta-galactosamide alpha-2,6-sialyltranferase I (ST6Gal-I). Intriguingly, the beta 1 integrin is thought to be a ligand for galectin-3 (gal-3), a tumor-associated lectin. The effects of gal-3 are complex; intracellular forms typically protect cells against apoptosis through carbohydrate-independent mechanisms, whereas secreted forms bind to cell surface oligosaccharides and induce apoptosis. In the current study, we tested whether alpha 2-6 sialylation of the beta 1 integrin modulates binding to extracellular gal-3. Herein we report that SW48 colonocytes lacking alpha 2-6 sialylation exhibit beta 1 integrin-dependent binding to gal-3-coated tissue culture plates; however, binding is attenuated upon forced expression of ST6Gal-I. Removal of alpha 2-6 sialic acids from ST6Gal-I expressors by neuraminidase treatment restores gal-3 binding. Additionally, using a blot overlay approach, we determined that gal-3 binds directly and preferentially to unsialylated, as compared with alpha 2-6-sialylated, beta 1 integrins. To understand the physiologic consequences of gal-3 binding, cells were treated with gal-3 and monitored for apoptosis. Galectin-3 was found to induce apoptosis in parental SW48 colonocytes ( unsialylated), whereas ST6Gal-I expressors were protected. Importantly, gal-3-induced apoptosis was inhibited by function blocking antibodies against the beta 1 subunit, suggesting that beta 1 integrins are critical transducers of gal-3-mediated effects on cell survival. Collectively, our results suggest that the coordinate up-regulation of gal-3 and ST6Gal-I, a feature that is characteristic of colon carcinoma, may confer tumor cells with a selective advantage by providing a mechanism for blockade of the pro-apoptotic effects of secreted gal-3.

Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pl 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

Evaluation of the insulin-like growth factors (IGF) IGF-I and IGF binding protein 3 in patients at high risk for breast cancer

Our data suggest that serum concentrations of insulin-like growth factor I and insulin-like growth factor binding protein 3 do not correlate with breast cancer development. (Fertil Steril (R) 2011;95:2753-5. (C)2011 by American Society for Reproductive Medicine.)

COL1A1 Sp1-binding site polymorphism as a risk factor for genital prolapse

The objective of this study was to verify the possible association between the Sp1-binding site polymorphism and genital prolapse. A case-control study was conducted in 107 patients with stages III and IV genital prolapse. The control group included 209 women with stages 0 and I. The polymorphism of type I collagen Sp1-binding site was identified by amplification of the first intron of the COL1A1 gene. We did not find differences in the prevalence of the GT and TT genotypes between the groups (p=0.34), even when we grouped patients with at least one polymorphic allele (GT and TT) and compared them with patients without the polymorphic allele (GG; p=0.17) The presence of at least one vaginal delivery, family history for prolapse, and macrosomatic fetus were independent risk factors for prolapse. In conclusion, the COL1A1 Sp1-binding site was not significantly associated with genital prolapse among our study subjects.

Mango starch degradation. II. The binding of alpha-amylase and beta-amylase to the starch granule

During mango ripening, soluble sugars that account for mango sweetening are accumulated through carbon supplied by both photosynthesis and starch degradation. The cultivar Keitt has a characteristic dependence on sugar accumulation during starch degradation, which takes place during ripening, only a few days after detachment from the tree. Most knowledge about starch degradation is based on seeds and leaves currently used as models. However, information about the mango fruit is scarce. This work presents the evaluation of alpha- and beta-amylases in the starch granule surface during fruit development and ripening. Extractable proteins were assayed for amylase activity and detected by immunofluorescence microscopy and correlated to gene expression. The results suggest that both amylases are involved in starch degradation during mango ripening, probably under the dependence of another signal triggered by the detachment from the mother-plant.

Angiotensin II Binding to Angiotensin I-Converting Enzyme Triggers Calcium Signaling

Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system. (Hypertension. 2011;57:965-972.) . Online Data Supplement

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination. (C) 2009 Elsevier B.V. All rights reserved.

Essential role of TM V and VI for binding the C-terminal sequences of Des-Arg-kinins

In the kallikrein-kinin and renin-angiotensin systems the main receptors, B-1 and B-2 (kinin receptors) and AT(1) and AT(2) (angiotensin receptors) respectively, are seven-transmembrane domain G-protein-coupled receptors. Considering that the B, agonists Des-Arg(9)-BK (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe), Lys-desArg(9)-BK or Des-Arg(10)-KD (Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe) and the AT, agonist (Asp-Arg-Val-Tyr-lle-His-Pro-Phe) have the same two residues at the C-terminal region (i.e. Pro-Phe), we hypothesized that TM V and TM VI of the B-1 receptor could play an essential role in agonist binding and activity, being these regions receptor sites for binding the C-terminal sequences of Des-Arg-kinins similarly to that observed to AT, receptor. To investigate this hypothesis, we replaced Arg(212) for Ala at the top of the TM V and the sequence 274-282 (CPYHFFAFL) in TM VI of the rat kinin B, receptor by the 32 receptor homologous sequence, 289-297 (FPFQISTFL) and subsequently analyzed the consequences of these mutations by competition binding and functional assays. Despite correct expression, observed at the mRNA and protein level by RT-PCR and confocal microscopy, respectively, no agonist binding and function was verified for the mutated receptors. Therefore, our results suggest an important role for Arg(212) in the TM V and a region of TM VI of rat B, receptor in the interaction with the C-terminal residues of Des-Arg-kinins, similar to that observed with AngII. (c) 2007 Elsevier B.V. All rights reserved.

NOVEL 65 kDa RNA-BINDING PROTEIN IN SQUID PRESYNAPTIC TERMINALS

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Lectins and/or xyloglucans/alginate layers as supports for immobilization of dengue virus particles

Formation of stable thin films of mixed xyloglucan (XG) and alginate (ALG) onto Si/SiO2 wafers was achieved under pH 11.6, 50 mM CaCl2, and at 70 degrees C. XG-ALG films presented mean thickness of (16 +/- 2) nun and globules rich surface, as evidenced by means of ellipsometry and atomic force microscopy (AFM), respectively. The adsorption of two glucose/mannose-binding seed (Canavalia ensiformis and Dioclea altissima) lectins, coded here as ConA and DAlt, onto XG-ALG surfaces took place under pH 5. Under this condition both lectins present positive net charge. ConA and DAIt adsorbed irreversibly onto XG-ALG forming homogenous monolayers similar to(4 +/- 1)nm thick. Lectins adsorption was mainly driven by electrostatic interaction between lectins positively charged residues and carboxylated (negatively charged) ALG groups. Adhesion of four serotypes of dengue virus, DENV (1-4), particles to XG-ALG surfaces were observed by ellipsometry and AFM. The attachment of dengue particles onto XG-ALG films might be mediated by (i) H bonding between E protein (located at virus particle surface) polar residues and hydroxyl groups present on XG-ALG surfaces and (ii) electrostatic interaction between E protein positively charged residues and ALG carboxylic groups. DENV-4 serotype presented the weakest adsorption onto XG-ALG surfaces, indicating that E protein on DENV-4 surface presents net charge (amino acid sequence) different from E proteins of other serotypes. All four DENV particles serotypes adsorbed similarly onto lectin films adsorbed. Nevertheless, the addition of 0.005 mol/L of mannose prevented dengue particles from adsorbing onto lectin films. XG-ALG and lectin layers serve as potential materials for the development of diagnostic methods for dengue. (c) 2008 Elsevier B.V. All rights reserved.

Agglutinin isolated from the red marine alga Hypnea cervicornis J. Agardh reduces inflammatory hypernociception: Involvement of nitric oxide

Hypnea cervicornis agglutinin (HCA), a lectin isolated from the red marine alga has been previously shown to have an antinociceptive effect. In the present study in rats, mechanisms of action of HCA were addressed regarding mechanical hypernociception induced by carrageenan, ovalbumin (as antigen), and also by prostaglandin E(2) in rats. The lectin administered intravenously inhibited carrageenan- and antigen-induced hypernociception at 1,3, 5 and 7 h. This inhibitory effect was completely prevented when lectin was combined with mucin, demonstrating the role of carbohydrate-binding sites. The inhibition of inflammatory hypernociception by HCA was associated with the prevention of neutrophil recruitment to the plantar tissue of rats but was not associated with the inhibition of the release of pro-hypernociceptive cytokines (TNF-alpha, IL-1 beta and CINC-1). HCA also blocked mechanical hypernociception induced by PGE(2), which was prevented by the administration of nitric oxide synthase inhibitors. These results were corroborated by the increased circulating levels of NO metabolites following HCA treatment. These findings suggest that the anti-hypernociceptive effects of HCA are not associated with the inhibition of pro-inflammatory cytokine production. However, these effects seem to involve the inhibition of neutrophil migration and also the increase in NO production. (C) 2010 Elsevier Inc. All rights reserved.

Can the Insulin Sensitivity Index (ISI) in Association with Insulin-like Growth Factor Binding Protein-1 Identify Insulin Resistance Early in Overweight Children?

Association between insulin resistance (IR) and non-alcoholic fatty liver disease (NAFLD) has been reported. This prompted us to evaluate the power of the insulin sensitivity index (ISI) in association with IGFBP-1 to identify IR early in obese children/adolescents. OGTT was performed in 34 obese/overweight children/adolescents. Glucose, insulin and IGFBP-1 were measured in serum samples and ISI was calculated. Considering the presence of three or more risk factors for IR as a criterion for IR, ISI <4.6 showed 87.5% sensitivity and 94.5% specificity in diagnosing IR. IGFBP-1 was lower in the group with ISI <4.6 (p <0.01). In this group, three patients had higher than expected IGFBP-1, suggesting hepatic IR, while three patients with ISI >4.6 showed very low IGFBP-1 levels. Conclusion: ISI <4.6 is a good indicator of early peripheral IR and, associated with IGFBP-1, can identify increased risk of hepatic IR. Low IGFBP-1 levels among non-IR children may indicate increased portal insulin levels.

The-202 A Allele of Insulin-Like Growth Factor Binding Protein-3 (IGFBP3) Promoter Polymorphism Is Associated with Higher IGFBP-3 Serum Levels and Better Growth Response to Growth Hormone Treatment in Patients with Severe Growth Hormone Deficiency

Context: Genetic factors that influence the response to recombinant human GH (rhGH) therapy remain mostly unknown. To date, only the GH receptor gene has been investigated. Objective: The aim of the study was to assess the influence of a polymorphism in the IGF-binding protein-3 (IGFBP-3) promoter region (-202 A/C) on circulating IGFBP-3 levels and growth response to rhGH therapy in children with GH deficiency (GHD). Design and Patients: -202 A/C IGFBP3 genotyping (rs2854744) was correlated with data of 71 children with severe GHD who remained prepubertal during the first year of rhGH treatment. Main Outcome Measures: We measured IGFBP-3 levels and first year growth velocity (GV) during rhGH treatment. Results: Clinical and laboratory data at the start of treatment were indistinguishable among patients with different -202 A/C IGFBP3 genotypes. Despite similar rhGH doses, patients homozygous for the A allele presented higher IGFBP-3 SD score levels and higher mean GV in the first year of rhGH treatment than patients with AC or CC genotypes (first year GV, AA = 13.0 +/- 2.1 cm/yr, AC = 11.4 +/- 2.5 cm/yr, and CC = 10.8 +/- 1.9 cm/yr; P = 0.016). Multiple linear regression analyses demonstrated that the influence of -202 A/C IGFBP3 genotype on IGFBP-3 levels and GV during the first year of rhGH treatment was independent of other variables. Conclusion: The -202 A allele of IGFBP3 promoter region is associated with increased IGFBP-3 levels and GV during rhGH treatment in prepubertal GHD children. (J Clin Endocrinol Metab 94: 588-595, 2009)

Cutting Edge: Nucleotide-Binding Oligomerization Domain 1-Dependent Responses Account for Murine Resistance against Trypanosoma cruzi Infection

An effective innate immune recognition of the intracellular protozoan parasite Trypanosoma cruzi is critical for host resistance against Chagas disease, a severe and chronic illness that affects millions of people in Latin America. In this study, we evaluated the participation of nucleotide-binding oligomerization domain (Nod)like receptor proteins in host response to T cruzi infection and found that Nod1-dependent, but not Nod2-dependent, responses are required for host resistance against infection. Bone marrow-derived macrophages from Nod1(-/-) mice showed an impaired induction of NF-kappa B-dependent products in response to infection and failed to restrict T cruzi infection in presence of IFN-gamma. Despite normal cytokine production in the sera, Nod1(-/-) mice were highly susceptible to T cruzi infection, in a similar manner to MyD88(-/-) and NO synthase 2(-/-) mice. These studies indicate that Nod1-dependent responses account for host resistance against T cruzi infection by mechanisms independent of cytokine production. The Journal of Immunology, 2010, 184: 1148-1152.