Nilda E. Fink

Identification of an equilibrium intermediate in the unfolding process of galectin-1, which retains its carbohydrate-binding specificity

The unfolding process of galectin-1 (Gal-1) in the presence of a denaturing agent was examined using fluorescence and far-UV circular dichroism (CD) spectroscopy determinations, and was found to be completely reversible. The data showed that the transitions of guanidine hydrochloride (GdnHCl)-induced lectin unfolding, in the absence of ligand, were biphasic in nature, clearly showing the existence of at least one stable intermediate. On the other hand, the unfolding in the presence of disaccharide yielded data that could fit very well to a two-state model, indicating a stabilizing effect of the ligand. The folding intermediate was further characterized by size exclusion chromatography, near-UV CD and anilinonaphtalene sulfonate binding, and shown to belong to the molten globule type. Strikingly, this intermediate retained its carbohydrate-binding specificity, as evidenced by the tryptophan fluorescence changes detected upon its interaction with lactose.

Red blood cell osmotic fragility confidence intervals: a definition by application of a mathematical model.

Abstract The red blood cell osmotic fragility test is based on the measure of the resistance of red blood cells to lysis as a function of decreasing NaCl concentration. Up to now, several methods have been used for recording these data, but for the first time, the human red blood cell osmotic fragility confidence interval using the Orcutt mathematical model was determined. The absorbance of the hemoglobin measured at 540 nm, released by the red blood cells of 40 healthy adult individuals, was fitted to the equation Absorbance=p3 erfc ([NaCl] − p1/p2); p3 measures one half the absorbance produced by maximum red blood cell hemolysis, p1 is the [NaCl] producing 50% red blood cell hemolysis, and p2 is the dispersion in [NaCl] producing red blood cell hemolysis. Confidence intervals (mean±SD) for the three parameters were as follows: p1=4.2718±0.1848; p2= 0.1947±0.0391, and p3=0.5568+0.0426. The usefulness of this osmotic fragility data analysis method using two pathological samples (β-thalassemia minor and hereditary spherocytosis) was demonstrated. Parameters of the fitted data were compared with those obtained by the conventional recording method of Beutler.

Toxicological studies in adult amphibians: Effects of lead

This paper summarises the results of studies that examined the biochemical and physiological effects of lead (Pb) in an in vivo amphibian model (the South American toad Bufo arenarum) under laboratory conditions. Acute toxicity tests generated LD50s that indicated a high tolerance to Pb. In animals injected intra-lymphatically with a sublethal dose of Pb, the total numbers of red (RBC) and white blood cells (WBC) and differential leukocytes were altered in a dose-related fashion. RBC osmotic fragility was also affected; i.e. the concentration of NaCl necessary to provoke 50% haemolysis was significantly reduced in lead-injected toads, indicating an increase in the osmotic resistance of the cells. Other studies examined δ-Aminolevulinic acid dehydratase (δ-ALAD) activity, free erythrocyte protoporphyrin (FEP) levels and blood Pb. FEP and blood Pb were shown to be valuable biomarkers of chronic metal intoxication, the former being the marker with the highest sensitivity. In lead injected animals FEP increased almost 9-fold compared to controls. The highest concentration of Pb occurred in the liver at a level 382 times higher than the controls. Kidney, spleen, and femur also appeared as repository sites of the metal, although in lower proportions. Other studies examined immunocompetency parameters. Lead affected the function of the polymorphonuclear cells of B. arenarum. Phagocytic and lytic functions of the adherent blood cells incubated with suspensions of Candida pseudotropicalis were negatively affected after the lead administration. The decrease of the phagocytic activity was statistically correlated with blood Pb levels. Also, following administration of Pb the production of natural antibodies was significantly increased by 39%. In non-exposed control animals there was a non-significant increase. It was concluded that in B. arenarum Pb may act as an immunostimulating factor on the humoral immune system. Serum protein electrophoresis was performed. Administration of Pb provoked a significant decrease in both total proteins and the albumin fraction. Among the globulin fractions, the G3 fraction was augmented. These findings were interpreted as a consequence of Pb toxicity on toad hepatic cells, the kidney and some components of the immune system.

An enzyme-linked immunosorbent assay for measuring anti-sheep red blood cells antibodies in lead-exposed toads

INTRODUCTION Immune function assays to screen immunotoxic effects of xenobiotics has recently become of major interest. In the framework of our studies, we standardized methods to quantify the humoral response of an amphibian species (Bufo arenarum, Amphibia, Anura) exposed to sublethal lead (as acetate). METHODS The levels of agglutinins to heterologous red blood cells (RBC) were measured in serum from adult B. arenarum. Since agglutinin titers were very low, a noncompetitive enzyme-linked immunosorbent assay (ELISA) method was carried out. As toad serum showed marked nonspecific binding, we developed a new ELISA on microtiter plates for the quantitative determination of the heterophile antibodies. The method was based on that described by Hirvonen et al. [Vox Sang. 69 (1995) 341], employing sheep red blood cells (SRBC) sensitized with amphibian antibodies that were transferred to microplates; later the measurement of bound immunoglobulins was performed. Different variables such as the amount of antigen, blocking agents, and other experimental conditions (fixing solution and commercial plates) were studied. Toads (n=22) received a weekly subcutaneous injection of 50 mg/kg lead (acetate) for 6 weeks, and the control ones (n=26) were injected with Na acetate at the same time. RESULTS The anti-sheep RBC antibodies titers of adult toads were obtained with the improved ELISA method, being the absorbance range 0.12 to 1.58 AU (1/200 diluted serum). Titers from lead-exposed toads were also determined, being the final titers (expressed as (-)x +/-S.E.M.) higher (0.79+/-0.06 AU), than those of Day 0 (0.57+/-0.06) (P<.01). DISCUSSION It was concluded that the ELISA technique we developed was useful for measuring the humoral immune response in this animal model and that in these preliminary studies, lead showed an immunostimulating action on the humoral immune system.

Purification and partial biochemical characterization of an S-type lectin from blastula embryos of Bufo arenarum

1. S-type lectin from Bufo arenarum embryos at blastula stage was purified by affinity chromatography. The molecule is a dimer with equal-sized monomers and the apparent subunit molecular weight was found to be 14.5 kDa. 2. Analytical isoelectric focusing of the pure lectin showed an acidic pI of 4.7. 3. Inhibition of the hemagglutination by mono- and oligosaccharides revealed a specificity for sugars bearing a beta-galactoside configuration. 4. Crossreactivity studies between the blastula lectin and the one purified earlier from adult ovary performed by immunodotting, ELISA and immunoblotting showed that these lectins share many epitopes.

Blood lead content in a peri-urban population of the South American toad Bufo arenarum

Blood Pb concentration in a South American toad Bufo arenarum population (n = 152) was determined over 10 samplings carried out between December 1996 and November 1999. The studied population lived in the surroundings of the La Plata City, the largest industrial-urban setting of the Buenos Aires Province, Argentina. The presence of the metal was detected in all the samples tested, the mean concentration range being 1.99-4.66 mg dl(-1). Some preliminary environmental data on soil content of Pb in the sampling area suggest the anthropogenic origin of the metal possibly due to high rate of Pb-containing gasoline utilisation until recently. The reported results may reflect a sequel of a sustained local air-soil-water pollution process.

Isolation of galectin-1 from human platelets: its interaction with actin.

Galectins are a family of animal lectins defined by their β-galactoside-binding specificity and a consensus sequence in their carbohydrate-recognition domain. Galectin-1 (Gal-1) is expressed as a non-covalently linked homodimer present in a variety of tissues. Here we describe its isolation from human platelets by a procedure involving ionic exchange chromatography and affinity chromatography on lactose-agarose. Platelet Gal-1 co-purifies with actin, forming an actin-Gal-1 complex which does no dissociate even after treatment with sodium dodecyl sulfate. The presence of both proteins was confirmed by Western blot and by trypsin digestion followed by mass spectrometry identification. By hemagglutination assays we studied the response of recombinant Gal-1/actin, mixed and pre-incubated in different proportions, and then tested against neuraminidase treated rabbit red blood cells. The complex formation was confirmed by confocal microscopy, showing that both proteins co-localised in resting platelets as well as in thrombin-activated ones. These results suggest that endogenous Gal-1 forms an intracellular complex with monomeric actin and that, after platelet activation, Gal-1 could play a role in the polymerization-depolymerization process of actin, which concludes in platelet aggregation.

Immunohistochemical localisation of a galectin from Bufo arenarum ovary

Galectins are a group of soluble animal lectins that exhibit specificity for beta-galactosides and conserve sequence homology in the carbohydrate-recognition domain. The galectin from Bufo arenarum ovary showed a strong cross-reaction with the lectin of 14.5 kDa purified from embryos at early blastula stage. In this paper, we studied the immunohistochemical localisation of the galectin of 14.5 kDa from ovary of the toad B. arenarum in adult ovary sections. We also analysed the immunohistochemical localisation of the embryonic lectin during early development using the antiserum anti-ovary galectin. In the ovary, oocytes in the previtellogenic stage showed strong reactivity in the nucleus and the cortex but not in the cytoplasm. Oocytes in the stage of primary vitellogenesis exhibited a similar pattern in the nuclear and cortical areas but showed immunostaining in the cytoplasm. Intense nuclear staining was detected in oocytes in the stage of late vitellogenesis and in mature oocytes, which also presented strong reactions in the yolk platelets that completely covered the cytoplasm. In blastula embryos the staining was found in the blastomeres, the yolk platelets and the blastocoele. Each lectin localisation is discussed in relation to potential biological roles in the corresponding tissues.

IFCC handbook on Master Program in clinical laboratory sciences.

This document summarizes the steps in development of a new IFCC Master Program and outlines some of the ways the IFCC would assist in setting up such a regional Program. This is intended to be of assistance to those who wish to embark on such an endeavour.

Reference Intervals for Erythropoietin Measurement by the JCL EIA Assay Need to Be Determined Locally

The measurement of erythropoietin (EPO) is useful for diagnosis and follow up of several pathologies which produce anemia. It can be also used in the differentiation of primary and secondary polycythemias. Simple cost-effective and sensitive methodologies for the EPO determination in clinical laboratories have been developed in the last two decades (1, 2). In our laboratory we have evaluated some previously described (3) performance characteristics of an EPO enzyme-linked immunoassay (EPO-EIA) alkaline phosphatase conjugate, marketed by JCL Clinical Research (Knowxville, USA; Cat. N° JCL 2–500MCT). We have also verified the manufacturer’s reference interval. Three kits (Lot. N° 700138) were used. Tests were done by duplicates as recommended by the manufacturer (4), and the absorbance (A) was measured at 410 nm in an E-max ELISA reader (Molecular Device Corporation, Sunnyvale, USA). To verify that the reader functioned correctly, the photometric linearity at 405 nm was assessed with reference solutions of p-nitrophenol at four different concentrations. These data (A: 0.210, 0.420, 0.833, and 1.633) correlated with the A values (0.193, 0.390, 0.771 and 1.520) obtained in a diode array Hewlett Packard spectrophotometer (correlation coefficient r = 0.99995). Each EIA duplicate was within ± 0.022 AU. Variation was lower than that claimed by the manufacturer (± 0.05 AU). Calibration curves with the five standards included in the kit were determined. Results are shown in Table 1. Our CV which illustrate plate-to-plate and day-to-day variability (intralaboratory assay data) were higher than those described previously (12.8, 6.6, 9.5, 12.0, and 10.9% for 1, 10, 50, 100, and 500 mIU/ml, respectively) (3). We also assayed an EPO control kit with low (L), mid (M) and elevated (E) concentrations (Cat. N° JCL 1–101; Lot. N° 000443), in the same conditions as standards and specimens (Table 1). In both the calibration curve standards as well as the L and E controls, values were lower than those described in JCL brochures, which indicate a defect in the calibration or in the standards employed. The latter seems less probable as it is claimed that the preparations have been checked against laboratory preparations of EPO previously calibrated against the International Reference Preparation IRP # 1 (Standard B) for erythropoietin. We also introduced an internal control (normal serum); a CV of 7.2% was obtained which, as expected, is lower than those from commercial controls. Subsequently, we verified the manufacturer’s reference interval (range: 22–54 mIU/ml, 150 individuals). For this purpose we obtained serum samples from 41