Characterization of monoclonal antibodies to O-antigen of lipopolysaccharide of Actinobacillus pleuropneumoniae serotype 2 and their use in the classification of field isolates

Abstract Monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 2 (reference strain Shope 4226 and field isolate F46) were produced. Twelve hybridoma clones were selected against both strains, and all the antibodies secreted were found to be reactive with whole-cell antigen of the homologous strain in ELISA, whereas only one mAb was reactive in slide agglutination test. The predominant antibody classes were IgG2b and IgG3, although IgG1 and IgM were also obtained. Immunoblot assay showed that mAbs could recognize a ladder band profile which is in accordance with the O-antigen of lipopolysaccharide. Most of the epitopes involved were resistant to proteinase K and also to boiling in the presence of sodium dodecyl sulfate and reducing conditions, but they were sensitive to periodic acid. The 12 mAbs recognized neither reference strains of the remaining A. pleuropneumoniae serotypes nor other taxonomically related Gram-negative organisms. The suitability of mAbs for serotyping of field isolates was also examined, and a high correlation (97.4%) was found between the results previously established by indirect hemagglutination with polyclonal rabbit sera and those obtained by ELISA with mAbs. The panel of mAbs described in this study was found to be extremely useful for identifying field isolates belonging to serotype 2 and could be used as a complementary serotyping method.     

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Sensitivity and specificity of parallel or serial serological testing for detection of canine Leishmania infection

In Brazil, human and canine visceral leishmaniasis (CVL) caused byLeishmania infantum has undergone urbanisation since 1980, constituting a public health problem, and serological tests are tools of choice for identifying infected dogs. Until recently, the Brazilian zoonoses control program recommended enzyme-linked immunosorbent assays (ELISA) and indirect immunofluorescence assays (IFA) as the screening and confirmatory methods, respectively, for the detection of canine infection. The purpose of this study was to estimate the accuracy of ELISA and IFA in parallel or serial combinations. The reference standard comprised the results of direct visualisation of parasites in histological sections, immunohistochemical test, or isolation of the parasite in culture. Samples from 98 cases and 1,327 noncases were included. Individually, both tests presented sensitivity of 91.8% and 90.8%, and specificity of 83.4 and 53.4%, for the ELISA and IFA, respectively. When tests were used in parallel combination, sensitivity attained 99.2%, while specificity dropped to 44.8%. When used in serial combination (ELISA followed by IFA), decreased sensitivity (83.3%) and increased specificity (92.5%) were observed. Serial testing approach improved specificity with moderate loss in sensitivity. This strategy could partially fulfill the needs of public health and dog owners for a more accurate diagnosis of CVL.     

Inactivation of (Na+ + K+)-stimulated ATPase by a cytotoxic protein from cobra venom in relation to its lytic effects on cells

The mechanism of action of the cytotoxic protein P₆ isolated from cobra venom (Naja naja) which shows preferential cytotoxicity particularly to Yoshida sarcoma cells has been studied by its effects on the membrane-bound enzyme (Na⁺ + K⁺)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of a variety of cell systems. Evidence obtained with Yoshida sarcoma cells, dog and human erythrocytes and three tissue culture cell lines KB (human oral carcinoma), Hela (human cervix carcinoma) and L-132 (human lung embryonic) shows that inhibition of (Na⁺ + K⁺)-ATPase by the P₆ protein can be correlated with its lytic activity. (Na⁺ + K⁺)-ATPase of Yoshida sarcoma membrane fragments inactivated by P₆ protein could be reconstituted by the addition of phosphatidylserine and phosphatidic acid. It is conceivable that lysis of cells by the P₆ protein may be due to an imbalance of K⁺ and Na⁺ in the cell which leads to swelling and disintegration of the membrane structure. Observations indicate that the P₆ protein combines with membrane constituents of susceptible cells. The overall evidence suggests that both the specificity of its protein structure and the highly basic nature of the P₆ protein are factors which enable it to compete with the lipid moiety maintaining the (Na⁺ + K⁺)-ATPase of the susceptible cells in proper conformation for activity.     

Defective suppressor cell function mediated by T8+ cell lines from patients with progressive multiple sclerosis

Activated suppressor cell function, induced with either concanavalin A or OKT3 and mediated by either unfractionated mononuclear cells or "panning" enriched T8+ cells, freshly isolated from peripheral blood, is reduced in patients with progressive multiple sclerosis (MS) as compared with control donors. In this study, we generated T8+ cell lines from the peripheral blood of these same patients and controls. Suppressor activity, mediated by T8+ cells exposed to OKT3 on days 1, 7, and 14 of culture and then treated with mitomycin C on day 16, was significantly reduced in the MS group (mean percent suppression 13% +/- 5) as compared with the control group (68% +/- 6, n = 8, p less than 0.001). No differences were noted in [3H]thymidine uptake by the OKT3-stimulated T8+ cell lines of MS and control groups. Mean percent suppression mediated by T4+ cell lines did not differ between MS and control groups (15% +/- 4, n = 3, vs 22% +/- 2, n = 4). These current data suggest that the previously observed defect in T8+ cell-mediated activated suppressor cell function in MS is a persistent one, favoring the postulate that the defect reflects intrinsic alterations in this cell population rather than a transient effect of serum factors on T8+ cell function.     

An indirect role of vitamin B12 in regulation of thymidylate synthase in Lactobacillus leichmannii.

Vitamin B12 augments thymidylate synthase function in L. leichmannii by facilitating indirectly the availability of suitable nonmethylpolyglutamylfolate cofactors. This is effected by the demethylation of trapped methyltetrahydrofolates, catalysed by a vitamin B12 requiring methionine synthase. Deoxyuridine supplemented cells, lacking in B12, have decreased levels of methionine synthase and thymidylate synthase. Addition of active and inactive conjugase preparation as a source of mono and polyglutamylfolates indicated that the latter are the preferred cofactors for thymidylate synthase.     

Screening and secretomic analysis of enthomopatogenic Beauveria bassiana isolates in response to cowpea weevil (Callosobruchus maculatus) exoskeleton

The production of cowpea (Vigna unguiculata), an important self-sustained crop in Latin America and Africa, is severely affected by damage by the cowpea weevil Callosobruchus maculatus. The presence of a single larva in stored seeds can lead to losses of almost 40%. Control of C. maculatus currently relies on the inefficient use of chemical insecticides and post-harvest treatments. The use of entomopathogenic fungus became a reliable alternative for coleopteran pest control and has been extensively investigated. Among them, Beauveria bassiana and Metarhizium anisopliae were widely evaluated in order to measure their virulence toward many insects. In this report, we evaluated the insecticidal activity of ten strains of B. bassiana and the most lethal fungi strains were analyzed for proteinaceous secretions by two dimensional electrophoresis and for enzyme activities, including chitinolytic, proteolytic and alpha-amylolytic activities. This study could, in the near future, help to establish novel biotechnological tools to use for cowpea weevil control.     

Influenza virus sequence feature variant type analysis: evidence of a role for NS1 in influenza virus host range restriction

Genetic drift of influenza virus genomic sequences occurs through the combined effects of sequence alterations introduced by a low-fidelity polymerase and the varying selective pressures experienced as the virus migrates through different host environments. While traditional phylogenetic analysis is useful in tracking the evolutionary heritage of these viruses, the specific genetic determinants that dictate important phenotypic characteristics are often difficult to discern within the complex genetic background arising through evolution. Here we describe a novel influenza virus sequence feature variant type (Flu-SFVT) approach, made available through the public Influenza Research Database resource (www.fludb.org), in which variant types (VTs) identified in defined influenza virus protein sequence features (SFs) are used for genotype-phenotype association studies. Since SFs have been defined for all influenza virus proteins based on known structural, functional, and immune epitope recognition properties, the Flu-SFVT approach allows the rapid identification of the molecular genetic determinants of important influenza virus characteristics and their connection to underlying biological functions. We demonstrate the use of the SFVT approach to obtain statistical evidence for effects of NS1 protein sequence variations in dictating influenza virus host range restriction.     

Production and Biochemical Characterization of Insecticidal Enzymes from Aspergillus fumigatus Toward Callosobruchus maculatus

In the present work, Aspergillus fumigatus is described as a higher producer of hydrolytic enzymes secreted in response to the presence of the Callosobruchus maculatus bruchid pest. This fungus was able to grow over cowpea weevil shells as a unique carbon source, secreting alkaline proteolytic and chitinolytic enzymes. Enzyme secretion in A. fumigatus was induced by both C. maculatus exoskeleton as well as commercial chitin, and alkaline proteolytic and chitinolytic activities were detected after 48 hours of growth. Furthermore, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed the production of specific proteins. Among them, two extracellular alkaline proteinases from culture enriched with C. maculatus exoskeleton were purified after chromatographic procedures using ion exchange and affinity columns. These proteins, named AP15 and AP30, had apparent molecular masses of 15,500 and 30,000 Da, respectively, as estimated by SDS-PAGE electrophoresis and mass spectrometry. AP30 was classified as a serine proteinase because it was inhibited by 5 mM phenylmethylsulfonyl fluoride (100%) and 50 μM leupeptin (67.94%).