Marisa M. Fernández

Modulation of endothelial cell migration and angiogenesis: a novel function for the “tandem-repeat” lectin galectin-8

Angiogenesis, the growth of new capillaries from preexisting blood vessels, is a complex process involving endothelial cell (EC) activation, disruption of vascular basement membranes, and migration and proliferation of ECs. Glycan‐mediated recognition has been proposed to play an instrumental role in mediating cell‐cell and cell‐matrix interactions. Ga‐lectins (Gal), a family of glycan‐binding proteins with affinity for β‐galactosides and a conserved sequence motif, can decipher glycan‐containing information and mediate cell‐cell communication. Galectin‐8 (Gal‐8), a member of this family, is a bivalent “tandem‐repeat”‐type galectin, which possesses 2 CRDs connected by a linker peptide. Here, we show that Gal‐8 is endowed with proangiogeneic properties. Functional assays revealed a critical role for this lectin in the regulation of capillary‐tube formation and EC migration. Moreover, Matrigel, either supplemented with Gal‐8 or vascular endothelial growth factor (VEGF), injected in mice resulted in induction of in vivo angiogenesis. Remarkably, Gal‐8 was expressed both in the cytoplasm and nucleus in ECs of normal and tumor vessels. Furthermore, CD166 [activated leukocyte cell adhesion molecule (ALCAM)] was identified as a specific Gal‐8‐binding partner in normal vascular ECs. Collectively, these data provide the first evidence demonstrating an essential role for Gal‐8 in the regulation of angiogenesis with critical implications in tumor biology.—CCárdenasrdenas Delgado, V. M., Nugnes, L. G., Colombo, L. L., Troncoso, M. F., Fernández, M. M., Malchiodi, E. L., Frahm, I., Croci, D. O., Compagno, D., Rabinovich, G. A., Wolfenstein‐Todel, C., Elola, M. T. Modulation of endothelial cell migration and angiogenesis: a novel function for the “tandem‐repeat” lectin galectin‐8. FASEB J. 25, 242–254 (2011). www.fasebj.org

Semen Clusterin Is a Novel DC-SIGN Ligand

The C-type lectin receptor dendritic cell-specific ICAM-3–grabbing nonintegrin (DC-SIGN) is an important player in the recognition of pathogens by dendritic cells. A plethora of pathogens including viruses, bacteria, parasites, and fungi are recognized by DC-SIGN through both mannose and fucose-containing glycans expressed on the pathogen surface. In this study, we identified semen clusterin as a novel DC-SIGN ligand. Semen clusterin, but not serum clusterin, expresses an extreme abundance of fucose-containing blood-type Ags such as Lex and Ley, which are both excellent DC-SIGN ligands. These motifs enable semen clusterin to bind DC-SIGN with very high affinity (Kd 76 nM) and abrogate the binding of HIV-1 to DC-SIGN. Depletion of clusterin from semen samples, however, did not completely prevent the ability of semen to inhibit the capture of HIV-1 by DC-SIGN, supporting that besides clusterin, semen contains other DC-SIGN ligands. Further studies are needed to characterize these ligands and define their contribution to the DC-SIGN–blocking activity mediated by semen. Clusterin is an enigmatic protein involved in a variety of physiologic and pathologic processes including inflammation, atherosclerosis, and cancer. Our results uncover an unexpected heterogeneity in the glycosylation pattern of clusterin and suggest that the expression of high concentrations of fucose-containing glycans enables semen clusterin to display a unique set of biological functions that might affect the early course of sexually transmitted infectious diseases.

Clinical status and parasitic infection in a Wichí Aboriginal community in Salta, Argentina

In a study, carried out in 2000, of the clinical and parasitological status of a Wichi Aboriginal community living in the suburbs of Tartagal, northern Salta, Argentina, 154 individuals were screened for parasitic infections. Ninety-five faecal samples were also obtained from the same population. Ninety-three percent of the subjects were positive for 1 or more of the parasites investigated by direct test and 70.5% of them had parasitic superinfection. The most frequent helminths were Strongyloides stercoralis (50.5%) and hookworm (47.4%). We found low reinfection rates and a long reinfection period after treatment and provision of safe water and sanitation. Serum reactivity of these patients was analysed by enzyme-linked immunosorbent assay and indirect immunofluorescent assay and 22.1% of them had anti-Toxocara antibodies, 16.2% were positive for a complex antigen of Leishmania braziliensis, 29.9% were positive for a complex Trypanosoma cruzi antigen, and 17.5% were positive for a specific Trypanosoma cruzi antigen, Ag 163B6/cruzipain.

Partial characterization of enterocin MR99 from a corn silage isolate of Enterococcus faecalis

Aims:  To assess the inhibitory activity on Gram‐positive and Gram‐negative bacteria of several species of enterococci recovered from a natural corn silage.

Differential Effects of Paromomycin on Ribosomes of Leishmania mexicana and Mammalian Cells

ABSTRACT Paromomycin, an aminoglycoside antibiotic having low mammalian cell toxicity, is one of the drugs currently used in the chemotherapy of cutaneous and visceral leishmaniasis. In order to understand the mode of action of this antibiotic at the molecular level, we have investigated the effects of paromomycin on protein synthesis in Leishmania and its mammalian hosts. We were able to demonstrate that in vivo protein synthesis in the promastigote stage of the parasite and its proliferation rate are markedly inhibited by paromomycin while being only slightly affected by other aminoglycoside antibiotics, such as streptomycin and neomycin B. Furthermore, both in vitro polypeptide synthesis induced by poly(U) as mRNA and accuracy of translation are significantly decreased by paromomycin in cell-free systems containing ribosomal particles of Leishmania promastigotes. Conversely, when ribosomes from mammalian cells are used instead of the protozoan particles, polyphenylalanine synthesis is only barely reduced by the antibiotic and the translation misreading remains almost unaltered. Surface plasmon resonance analysis of the interaction between paromomycin and protozoan or mammalian cell ribosomal RNAs shows a strong binding of antibiotic to the parasite ribosomal decoding site and practically no interaction with the mammalian cell counterpart. Our results indicating differential effects of paromomycin on the translation processes of the Leishmania parasite and its mammalian hosts can explain the therapeutic efficiency of this antibiotic as an antileishmaniasis agent.

New Scheme of Intermittent Benznidazole Administration in Patients Chronically Infected with Trypanosoma cruzi: a Pilot Short-Term Follow-Up Study with Adult Patients.

ABSTRACT There is a clinical need to test new schemes of benznidazole administration that are expected to be at least as effective as the current therapeutic scheme but safer. This study assessed a new scheme of benznidazole administration in chronic Chagas disease patients. A pilot study with intermittent doses of benznidazole at 5 mg/kg/day in two daily doses every 5 days for a total of 60 days was designed. The main criterion of response was the comparison of quantitative PCR (qPCR) findings prior to and 1 week after the end of treatment. The safety profile was assessed by the rate of suspensions and severity of adverse effects. Twenty patients were analyzed for safety, while qPCR was tested for 17 of them. The average age was 43 ± 7.9 years; 55% were female. Sixty-five percent of treated subjects showed detectable qPCR results prior to treatment of 1.45 (0.63 to 2.81) and 2.1 (1.18 to 2.78) parasitic equivalents per milliliter of blood (par.eq/ml) for kinetoplastic DNA (kDNA) qPCR and nuclear repetitive sequence satellite DNA (SatDNA) qPCR, respectively. One patient showed detectable PCR at the end of treatment (1/17), corresponding to 6% treatment failure, compared with 11/17 (65%) patients pretreatment (P = 0.01). Adverse effects were present in 10/20 (50%) patients, but in only one case was treatment suspended. Eight patients showed mild adverse effects, whereas moderate reactions with increased liver enzymes were observed in two patients. The main accomplishment of this pilot study is the promising low rate of treatment suspension. Intermittent administration of benznidazole emerges a new potential therapeutic scheme, the efficacy of which should be confirmed by long-term assessment posttreatment.

Force of infection and evolution of lesions of canine tegumentary leishmaniasis in Northwestern Argentina

A clinical-serological follow-up was carried out in a canine population in endemic foci of Leishmania braziliensis spread in northwestern Argentina. Each dog was studied in at least two visits, 309+/-15 days (X+/-SE) apart. Some initially healthy dogs (n=52) developed seroconversion or lesions. The clinical evolution of the disease in dogs resembles in many aspects the human disease. Similarities include the long duration of most ulcers with occasional healing or appearance of new ones and the late appearance of erosive snout lesions in some animals. Yearly incidence rates of 22.7% for seroconversion and of 13.5% for disease were calculated as indicators of the force of infection by this parasite upon the canine population.

Galectin-3 is essential for early wound healing and ventricular remodeling after myocardial infarction in mice☆☆☆

Fil: Gonzalez, German Esteban. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiopatologia Cardiovascular; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Bioquimica y Medicina Molecular; Argentina

Superantigen natural affinity maturation revealed by the crystal structure of staphylococcal enterotoxin G and its binding to T‐cell receptor Vβ8.2

The illnesses associated with bacterial superantigens (SAgs) such as food poisoning and toxic shock syndrome, as well as the emerging threat of purpura fulminans and community‐associated methicillin‐resistant S. aureus producer of SAgs, emphasize the importance of a better characterization of SAg binding to their natural ligands, which would allow the development of drugs or biological reagents able to neutralize their action. SAgs are toxins that bind major histocompatibility complex class II molecules (MHC‐II) and T‐cell receptors (TCR), in a nonconventional manner, inducing T‐cell activation that leads to production of cytokines such as tumor necrosis factor and interleukin‐2, which may result in acute toxic shock. Previously, we cloned and expressed a new natural variant of staphylococcal enterotoxin G (SEG) and evaluated its ability to stimulate in vivo murine T‐cell subpopulations. We found an early, strong, and widespread stimulation of mouse Vβ8.2 T‐cells when compared with other SAgs member of the SEB subfamily. In search for the reason of the strong mitogenic potency, we determined the SEG crystal structure by X‐ray crystallography to 2.2 Å resolution and analyzed SEG binding to mVβ8.2 and MHC‐II. Calorimetry and SPR analysis showed that SEG has an affinity for mVβ8.2 40 to 100‐fold higher than that reported for other members of SEB subfamily, and the highest reported for a wild type SAg‐TCR couple. We also found that mutations introduced in mVβ8.2 to produce a high affinity mutant for other members of the SEB subfamily do not greatly affect binding to SEG. Crystallographic analysis and docking into mVβ8.2 in complex with SEB, SEC3, and SPEA showed that the deletions and substitution of key amino acids remodeled the putative surface of the mVβ8.2 binding site without affecting the binding to MHC‐II. This results in a SAg with improved binding to its natural ligands, which may confer a possible evolutionary advantage for bacterial strains expressing SEG. Proteins 2007.

Plasma membrane calcium ATPase activity is regulated by actin oligomers through direct interaction

Background: Plasma membrane calcium ATPases interact dynamically with the submembrane actin cytoskeleton. Results: Biophysical and functional assays show that purified plasma membrane calcium ATPase binds to G-actin and is activated by short actin oligomers. Conclusion: Plasma membrane calcium ATPases are regulated by polymerizing actin independently of regulation by calmodulin. Significance: Dynamic actin participates in cytosolic Ca2+ homeostasis by regulating plasma membrane calcium ATPase activity. As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca2+ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca2+-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca2+-ATPase activity was related to an increase in the apparent affinity for Ca2+ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca2+ homeostasis.