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.

The carbohydrate-binding specificity and molecular modelling of Canavalia maritima and Dioclea grandiflora lectins

The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea grandiflora was studied by hapten-inhibition of haemagglutination using various sugars and sugar derivatives as inhibitors, including N-acetylneuraminic acid and N-acetylmuramic acid. Despite some discrepancies, both lectins exhibited a very similar carbohydrate-binding specificity as previously reported for other lectins from Diocleinae (tribe Phaseoleae, sub-tribe Diocleinae). Accordingly, both lectins exhibited almost identical hydropathic profiles and their three-dimensional models built up from the atomic coordinates of ConA looked very similar. However, docking experiments of glucose and mannose in their monosaccharide-binding sites, by comparison with the ConA-mannose complex used as a model, revealed conformational changes in side chains of the amino acid residues involved in the binding of monosaccharides. These results fully agree with crystallographic data showing that binding of specific ligands to ConA requires conformational chances of its monosaccharide-binding site.

Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases

Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2) in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.

Interaction of Diocleinae Lectins with Glycoproteins Based in Surface Plasmon Resonance

Interaction of glucose/mannose-binding lectins in solution with immobilized glycoproteins was followed in real time using surface plasmon resonance technology. The lectins which share many biochemical and structural features could be clearly differentiated in terms of their specificity for complex glycoconjugates. The most prominent interaction of the lectins with PHA-E comparing with soybean agglutinin, both glycoproteins exhibiting high mannose oligosaccharides, suggests that the whole structure of the glycoproteins themselves, may interfere in affinity. These findings also support the hypothesis that minor amino acid replacements in the primary sequence of the lectins might be responsible for their divergence in fine specificity and biological activities. This is the first report using surface plasmon resonance technology that evidences differences of Diocleinae lectins in respect their fine glycan-specificity.

Nonconventional amide bond formation catalysis: programming enzyme specificity with substrate mimetics

This article reports on the design and characteristics of substrate mimetics in protease-catalyzed reactions. Firstly, the basis of protease-catalyzed peptide synthesis and the general advantages of substrate mimetics over common acyl donor components are described. The binding behavior of these artificial substrates and the mechanism of catalysis are further discussed on the basis of hydrolysis, acyl transfer, protein-ligand docking, and molecular dynamics studies on the trypsin model. The general validity of the substrate mimetic concept is illustrated by the expansion of this strategy to trypsin-like, glutamic acid-specific, and hydrophobic amino acid-specific proteases. Finally, opportunities for the combination of the substrate mimetic strategy with the chemical solid-phase peptide synthesis and the use of substrate mimetics for non-peptide organic amide synthesis are presented.

Cycle modulation of insulin-like growth factor-binding protein 1 in human endometrium

Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1) interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP) have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5%) followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia.

HIV-1 binding and neutralizing antibodies of injecting drug users

Previous studies have demonstrated a stronger seroreactivity against some synthetic peptides responsible for inducing neutralizing antibodies in injecting drug users (IDU) compared to that of individuals sexually infected with HIV-1 (S), but the effectiveness in terms of the neutralizing ability of these antibodies has not been evaluated. Our objective was to study the humoral immune response of IDU by determining the specificity of their antibodies and the presence of neutralizing antibodies. The neutralization capacity against the HIV-1 isolate MN (genotype B), the primary HIV-1 isolate 95BRRJ021 (genotype F), and the seroreactivity with peptides known to induce neutralizing antibodies, from the V2 and V3 loops of different HIV-1 subtypes, were analyzed. Seroreactivity indicates that IDU plasma are more likely to recognize a broader range of peptides than S plasma, with significantly higher titers, especially of V3 peptides. Similar neutralization frequencies of the MN isolate were observed in plasma of the IDU (16/47) and S (20/60) groups in the 1:10 dilution. The neutralization of the 95BRRJ021 isolate was more frequently observed for plasma from the S group (15/23) than from the IDU group (15/47, P = 0.0108). No correlation between neutralization and seroreactivity with the peptides tested was observed. These results suggest that an important factor responsible for the extensive and broad humoral immune response observed in IDU is their infection route. There was very little difference in neutralizing antibody response between the IDU and S groups despite their differences in seroreactivity and health status.

Analysis of the specificity of human antibodies to antigens of Leishmania braziliensis braziliensis

The antigenicity of promastigotes of Leishmania braziliensis braziliensis (L. b.braziliensis) treated with 1% sodium desoxycholate in 10 mM Tris-Hcl pH 8.2 was analysed by immunoblot using as probes sera from American cutaneous leishmaniasis (ACL), American visceral leishmaniasis (AVL), schistosomiasis, malaria and Chagas' disease. The ACL sera reacted constantly with a 60 kD band. No reactivity to this protein was observed with sera from the other diseases above mentioned indicating that the 60 kD protein may be used in serodiagnosis for ACL.

The sensitivity, specificity and efficiency values of some serological tests used in the diagnosis of paracoccidioidomycosis

This work reports on the results of double immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF) and indirect immunofluorescence (IIF) techniques in the serodiagnosis of paracoccidioidomycosis. The study was undertaken on four groups of individuals: 46 patients with untreated paracoccidioidomycosis, 22 patients with other deep mycoses, 30 with other infectious diseases (tuberculosis and cutaneous leishmaniasis) and 47 blood donors as negative controls. Data were obtained using Paracoccidioides brasiliensis antigens, i.e.,a yeast culture filtrate for ID, CIE and CF, and a yeast cell suspension for IIF. The sensitivity, specificity and efficiency values were measured according to GALEN & GAMBINO8.The gel precipitation tests (ID and CIE) showed the greatest sensitivity (91.3 and 95.6%, respectively), maximum specificity (100%) and the highest efficiency values when compared to the CF and IIF tests.

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.

PCR-based identification of Burkholderia pseudomallei

DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.

Self-diagnosis of active head lice infestation by individuals from an impoverished community: high sensitivity and specificity

To compare sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of self-diagnosis for head lice infestation with visual inspection, we conducted a study in an urban slum in Brazil. Individuals were asked about active head lice infestation (self-diagnosis); we performed visual inspection and thereafter wet combing (gold standard). Of the 175 individuals included, 77 (44%) had an active head lice infestation. For self-diagnosis, sensitivity (80.5%), specificity (91.8%), PPV (88.6%) and NPV (85.7%) were high. Sensitivity of visual inspection was 35.1%. Public health professionals can use self-diagnosis as a diagnostic tool, to estimate accurately prevalence of pediculosis in a community, and to monitor ongoing intervention strategies.