Thermodynamic evaluation and modeling of proton and water exchange associated with benzamidine and berenil binding to ß-trypsin

Serine-proteases are involved in vital processes in virtually all species. They are important targets for researchers studying the relationships between protein structure and activity, for the rational design of new pharmaceuticals. Trypsin was used as a model to assess a possible differential contribution of hydration water to the binding of two synthetic inhibitors. Thermodynamic parameters for the association of bovine ß-trypsin (homogeneous material, observed 23,294.4 ± 0.2 Da, theoretical 23,292.5 Da) with the inhibitors benzamidine and berenil at pH 8.0, 25ºC and with 25 mM CaCl2, were determined using isothermal titration calorimetry and the osmotic stress method. The association constant for berenil was about 12 times higher compared to the one for benzamidine (binding constants are K = 596,599 ± 25,057 and 49,513 ± 2,732 M-1, respectively; the number of binding sites is the same for both ligands, N = 0.99 ± 0.05). Apparently the driving force responsible for this large difference of affinity is not due to hydrophobic interactions because the variation in heat capacity (DCp), a characteristic signature of these interactions, was similar in both systems tested (-464.7 ± 23.9 and -477.1 ± 86.8 J K-1 mol-1 for berenil and benzamidine, respectively). The results also indicated that the enzyme has a net gain of about 21 water molecules regardless of the inhibitor tested. It was shown that the difference in affinity could be due to a larger number of interactions between berenil and the enzyme based on computational modeling. The data support the view that pharmaceuticals derived from benzamidine that enable hydrogen bond formation outside the catalytic binding pocket of ß-trypsin may result in more effective inhibitors.

Solubilization of human erythrocyte membranes by ASB detergents

Understanding the membrane solubilization process and finding effective solubilizing agents are crucial challenges in biochemical research. Here we report results on the interaction of the novel linear alkylamido propyl dimethyl amino propanosulfonate detergents, ASB-14 and ASB-16, with human erythrocyte membranes. An estimation of the critical micelle concentration of these zwitterionic detergents (ASB-14 = 100 µM and ASB-16 = 10 µM) was obtained using electron paramagnetic resonance. The amount of proteins and cholesterol solubilized from erythrocytes by these detergents was then determined. The hemolytic activities of the ASB detergents were assayed and the detergent/lipid molar ratios for the onset of hemolysis (Re sat) and total lysis (Re sol) were calculated, allowing the determination of the membrane binding constants (Kb). ASB-14 presented lower membrane affinity (Kb = 7050 M-1) than ASB-16 (Kb = 15610 M-1). The amount of proteins and cholesterol solubilized by both ASB detergents was higher while Re sat values (0.22 and 0.08 detergent/lipid for ASB-14 and ASB-16, respectively) were smaller than those observed with the classic detergents CHAPS and Triton X-100. These results reveal that, besides their well-known use as membrane protein solubilizers to enhance the resolution of two dimensional electrophoresis/mass spectrometry, ASB-14 and ASB-16 are strong hemolytic agents. We propose that the physicochemical properties of ASB detergents determine their membrane disruption efficiency and can help to explain the improvement in the solubilization of membrane proteins, as reported in the literature.

Relationship between acute diarrhoea and low plasma levels of vitamin A and retinol binding protein

The objective of this study was to assess vitamin A status and association between acute diarrhoea and plasma levels of vitamin A through cross-sectional comparison in children. Plasma vitamin A was measured by colorimetric method of Neeld & Pearson and RBP by radial immunodiffusion technique. Seventy eight children (aged 18-119 months), 26 with current history of diarrhoea and 52 children as controls (outpatient from the Santa Casa de Misericórdia Hospital in metropolitan area of São Paulo City, Brazil) were studied. Children with history of diarrhoea showed significant low levels (mean ± s.e.) as compared to controls, vitamin A (15.87 ± 1.4 µg/dl vs. 21.14 ± 1.15 µg/dl, p < 0.007) and RBP (1.70 ± 0.2 mg/dl vs. 2.52 ±0.11 mg/dl). Multivariate logistic regression adjusted by sex, age, nutritional status and mother education revealed association between diarrhoea and inadequate levels of vitamin A and RBP.

Characterization of mannose-binding lectin plasma levels and genetic polymorphisms in HIV-1-infected individuals

INTRODUCTION: The present study investigated the association between mannose-binding lectin (MBL) gene polymorphism and serum levels with infection by HIV-1. METHODS: Blood samples (5mL) were collected from 97 HIV-1-infected individuals resident in Belém, State of Pará, Brazil, who attended the Special Outpatient Unit for Infections and Parasitic Diseases (URE-DIPE). CD4+ T-lymphocyte count and plasma viral load were quantified. A 349bp fragment of exon 1 of the MBL was amplified via PCR, using genomic DNA extracted from controls and HIV-1-infected individuals, following established protocols. MBL plasma levels of the patients were quantified using an enzyme immunoassay kit. RESULTS: Two alleles were observed: MBL*O, with a frequency of 26.3% in HIV-1-infected individuals; and the wild allele MBL*A (73.7%). Similar frequencies were observed in the control group (p > 0.05). Genotype frequencies were distributed according to the Hardy-Weinberg equilibrium in both groups. Mean MBL plasma levels varied by genotype, with statistically significant differences between the AA and AO (p < 0.0001), and AA and OO (p < 0.001) genotypes, but not AO and OO (p = 0.17). Additionally, CD4+ T-lymphocytes and plasma viral load levels did not differ significantly by genotype (p > 0.05). CONCLUSIONS: The results of this study do not support the hypothesis that MBL gene polymorphism or low plasma MBL concentrations might have a direct influence on HIV-1 infection, although a broader study involving a large number of patients is needed.

Evaluation of the cytokine mannose-binding lectin as a mediator of periportal fibrosis progression in patients with schistosomiasis

INTRODUCTION : We hypothesized higher mannose-binding lectin level and classic factors (i.e., age, sex, alcohol consumption, exposure, and specific treatment) are associated with the severity of periportal fibrosis in schistosomiasis. METHODS : This cross-sectional study involved 79 patients infected with Schistosoma mansoni with severe or mild/moderate periportal fibrosis. Serum concentrations of mannose-binding lectin were obtained by enzyme-linked immunosorbent assay (ELISA). RESULTS: Higher serum level of mannose-binding lectin was significantly associated with advanced periportal fibrosis. CONCLUSIONS: Mannose-binding lectin may contribute to liver pathology in schistosomiasis and may represent a risk factor for advanced periportal fibrosis in the Brazilian population studied.

Studies on the relationship between lectin binding carbohydrates and different strains of Leishmania from the New World

The culture forms of L. mexicana pifanoi (LRC L-90), L. mexicana mexicana (LRC L-94, M-379); L. braziliensis braziliensis (LRC L-77, L-1, M-2903, H-LSS) and L. mexicana amazonensis (H-JMMO, M-JOF, H-21, H-PLL,M-1696) were tested with the following lectins: Canavalia ensiformis, Ricinus communis-120, Axinella polypoides, Phaseolus vulgaris, Evonymus europaeus, lotus tetragonolobus, Dolichos biflorus, Aaptos papillata II, Laburnum alpinum, Ulex europaeus, Arachis hypogaea and Soja hispida. All examined strains of Leishmania were agglutinated by C. ensiformis, R. communis-120 and A. popypoides. No agglutination reactions were observed with P. vulgaris, D.biflorus, A. papillata II, E. europaeus and L. tetragonolobus. Only L. m. pifanoi and the L. m. amazonensis strains H-JMMO and MJOF showed agglutination reactions with S. hispida, U. europaeus, L. alpinum and A. hypogaea, while L. m. mexicana (LRC L-94; M-379) strains, L. b. braziliensis H. LSS, LRC L-77; L-1; M-2903 and the L. m. amazonensis strains, H-PLL, H-21, M-1696 showed no agglutination reactions with these four lectins.

A simple method for assessing the binding of concanavalin A to mononuclear cell surfaces: no interference of visceral leishmaniasis serum on this binding

We report a simple method for evaluating the binding of concanavalin A (ConA) to human peripheral blood mononuclear cells (PBMC). The binding is evidenced by an immunoenzymic assay using peroxidase-conjugated immunoglobulins of a rabbit anti-ConA serum. Using the method we show that sera from patients with American leishmaniasis do not interfere with binding of ConA to PBMC.

Exploration of receptor binding of Bacillus thuringiensis toxins

Wild type and mutant toxins of Bacillus thuringiensis delta-endotoxins were examined for their binding to midgut brush border membrane vesicles (BBMV). CryIAa, CryIAb, and CryIAc were examined for their binding to Gypsy moth (Lymantria dispar) BBMV. The binding of CryIAa and CryIAc was directly correlated with their toxicity, while CryIAb was observed to have lower binding than expected from its toxicity. The latter observation confirms the observation of Wolfersberger (1990). The "rule" of reciprocity of binding and toxicity is apparently obeyed by CryIAa and CryIAc, but broken by CryIAb on L. dispar. Alanine substitutions were made in several positions of the putative loops of CryIAa to test the hypothesis that the loops are intimately involved in binding to the receptor. The mutant toxins showed minor shifts in heterologous binding to Bombyx mori BBMV, but not enough to conclude that the residues chosen play critical roles in receptor binding.

Comparison of purified 12 kDa and recombinant 15 kDa Fasciola hepatica antigens related to a Schistosoma mansoni fatty acid binding protein

Vaccines in schistosomiasis using homologous antigens have been studied extensively in experimentally infected mammalian hosts. Vaccines using heterologous antigens have received comparatively less attention. This review summarizes recent work on a heterologous 12 kDa Fasciola hepatica antigenic polypeptide which cross reacts with Schistosoma mansoni. A cDNA has been cloned and sequenced, and the predicted amino acid sequence of the recombinant protein has been shown to have significant (44) identity with a 14 kDa S. mansoni fatty acid binding protein. Thus in the parasitic trematodes fatty acid binding proteins may be potential vaccine candidates. The F. hepatica recombinant protein has been overexpressed and purified and denoted rFh15. Preliminary rFh15 migrates more slowly (i.e. may be slightly larger) than nFh12 on SDS-PAGE and has a predicted pI of 6.01 vs. observed pI of 5.45. Mice infected with F. hepatica develop antibodies to nFh12 by 2 weeks of infection vs. 6 weeks of infection to rFh15; on the other hand, mice with schistosomiasis mansoni develop antibodies to both nFh12 and rFh15 by 6 weeks of infection. Both the F. hepatica and S. mansoni cross-reactive antigens may be cross-protective antigens with the protection inducing capability against both species.

Hemagglutinating and fusogenic activities of Newcastle disease virus: studies on receptor binding specificity and pH-induced conformational changes

Vaccinal and wild strains of Newcastle Disease virus (NDV) were analyzed for cell receptor binding and fusogenic biological properties associated with their HN (hemagglutinin-neuraminidase) and F (fusion protein) surface structures respectively. The evaluation of the biological activities of HN and F was carried out respectively by determination of hemagglutinating titers and hemolysis percentages, using erythrocytes from various animal origins at different pH values. Significant differences in hemagglutination titers for some strains of NDV were detected, when interacting with goose, sheep, guinea-pip and human "O" group erythrocytes at neutral pH. Diversity of hemolysis percentagens was observed between different NDV strains at acid pH. These analysis were developed to evaluate particular aspects of the actual influence of the receptor specifity and pH on the receptor binding and fusogenic processes of Newcastle Disease viruses.

V3 peptide binding pattern and HIV-1 transmission route in Rio de Janeiro

To characterize antibody binding to a panel of V3 loop peptides representing diverse HIV-1 neutralization epitopes, 149 HIV-1 infected individuals from Rio de Janeiro (RJ) were investigated. Results were analyzed with respect to risk factors for infection and other epidemiological and clinical data. Peptide reactivity was not associated with sex, clinical status, CD4 counts, antigenemia or ß2-microglobulin serum level. A segregation of peptide reactivity according to route of infection was encountered. This finding suggests that more then one viral strain may be circulating in RJ, in subjects with different risk factors for HIV-1 infection. An investigation of prevalent HIV-1 genotypes, serotypes and immunotypes may be of importance for the design and selection of potential vaccines to be used in Brazil as well as for the selection of populations to be included in future vaccine efficacy trials.