Brugia malayi: recombinant antigens expressed by genomic DNA clones

A Brugia malayi genomic DNA expression library was screened with rabbit antiserum generated against live infective larvae and 33 clones were identified. Five randomly selected clones were characterized in detail by Western blot, DNA and RNA analyses. The fusion proteins produced by each of these recombinant DNA clones are expressed by different genomic sequences. A profile of antigenic cross-reactivities of all 33 recombinant clones was compiled using a battery of antisera, including sera from humans infected with B. malayi. A high percentage of clones were cross-reactive with antisera against the filarial parasites B. pahangi, Dirofilaria immitis, and Onchocerca volvulus. We have made a preliminary identification of three categories of recombinant clones encoding (1) antigens that were cross-reactive with some or all antisera tested, (2) antigens that were specific to the Brugia genus, and (3) antigens that appeared to be specific to B. malayi. These recombinant antigens are candidates for further studies in filarial immunoprophylaxis and diagnosis.     

The mechanism of protein splicing and its modulation by mutation.

Protein splicing results in the expression of two mature proteins from a single gene. After synthesis of a precursor protein, an internal segment (the intein) is excised and the external domains are joined together. A self-catalyzed mechanism for this cleavage-ligation reaction is presented, based on mutagenesis data and analysis of splicing intermediates. Mutations were used to block various steps in the protein splicing pathway, allowing each isolated step to be studied independently. A linear ester intermediate was identified and functional roles for the four conserved splice junction residues were determined. Understanding the mechanism of protein splicing provides a basis for protein engineering studies. For example, inteins can be constructed which fail to splice, but instead cleave the peptide bond at a chosen splice junction.     

Capuchins do cooperate: the advantage of an intuitive task

We used a cooperative pulling task to examine proximate aspects of cooperation in captive brown capuchin monkeys, Cebus apella. Specifically, our goal was to determine whether capuchins can learn the contingency between their partner's participation in a task and its successful completion. We examined whether the monkeys visually monitored their partners and adjusted pulling behaviour according to their partner's presence. Results on five same-sex pairs of adults indicate that (1) elimination of visual contact between partners significantly decreased success, (2) subjects glanced at their partners significantly more in cooperative tests than in control tests in which no partner-assistance was needed, and (3) they pulled at significantly higher rates when their partner was present rather than absent. Therefore, in contrast to a previous report by Chalmeau et al. (1997, Animal Behaviour, 54, 1215-1225), cooperating capuchins do seem able to take the role of their partner into account. However, the type of task used may be an important factor affecting the level of coordination achieved. Copyright 2000 The Association for the Study of Animal Behaviour.     

The Arthrobacter species FB24 Arth_1007 (DnaB) intein is a pseudogene

An Arthrobacter species FB24 gene (locus tag Arth_1007) was previously annotated as a putative intein-containing DnaB helicase of phage origin (Arsp-FB24 DnaB intein). However, it is not a helicase gene because the sequence similarity is limited to inteins. In fact, the flanking exteins total only 66 amino acids. Therefore, the intein should be referred to as the Arsp-FB24 Arth_1007 intein. The Arsp-FB24 Arth_1007 intein failed to splice in its native precursor and in a model precursor. We previously noted that the Arsp-FB24 Arth_1007 intein is the only putative Class 3 intein that is missing the catalytically essential Cys at position 4 of intein Motif F, which is one of the three defining signature residues of this class. Additionally, a catalytically essential His in position 10 of intein Motif B is also absent; this His is the most conserved residue amongst all inteins. Splicing activity was not rescued when these two catalytically important positions were 'reverted' back to their consensus residues. This study restores the unity of the Class 3 intein signature sequence in active inteins by demonstrating that the Arsp-FB24 Arth_1007 intein is an inactive pseudogene.     

Plasmodium vivax, P. cynomolgi, and P. knowlesi: identification of homologue proteins associated with the surface of merozoites

We have identified a Plasmodium vivax merozoite surface protein (MSP) that migrates on SDS-polyacrylamide gels at a Mr of about 185 kDa. This protein was recognized by a P. vivax monoclonal antibody (mAb) that localizes the protein by immunofluorescence to the surface of merozoites and also immunoprecipitates this protein from NP-40 detergent extracts of [35S]methionine metabolically radiolabeled P. vivax schizonts. The P. vivax MSP does not become biosynthetically radiolabeled with [3H]glucoamine, [3H]myristate, [3H]palmitate, or [3H]mannose, indicating that this P. vivax MSP is not posttranslationally modified and bound to the merozoite membrane by a glycosylphosphatidylinositol (GPI) lipid anchor. Thus, in this respect, this protein is different from members of the MSP-1 protein family and from MSP-2 and MSP-4 of P. falciparum. The mAb cross-reacts with and outlines the surface of P. cynomolgi merozoites and immunoprecipitates a 150-kDa P. cynomolgi homologue. The mAb was used as an affinity reagent to purify the native homologous MSP from NP-40 extracts of P. cynomolgi mature schizonts in order to develop a specific polyclonal antiserum. The resulting anti-PcyMSP rabbit antiserum cross-reacts strongly with the P. vivax 185-kDa MSP and also recognizes an analogous 110-kDa protein from P. knowlesi. We have determined via an immunodepletion experiment that the 110-kDa P. knowlesi MSP corresponds to the PK 110 protein partially characterized earlier (Perler et al. 1987). The potential of P. vivax MSP as a vaccine candidate was addressed by conducting in vitro inhibition of erythrocyte invasion assays, and the IgG fraction of both the P. vivax MSP mAb and the P. cynomolgi MSP rabbit antiserum significantly inhibited entry of P. vivax merozoites. We denote, on a preliminary basis, these antigenically related merozite surface proteins PvMSP-185, PcyMSP-150, and PkMSP-110.     

The identification of an Onchocerca-specific recombinant antigen containing a T cell epitope.

Recombinant Onchocerca volvulus Ag have been derived from expression libraries and examined for their ability to stimulate PBMC from patients infected with O. volvulus. Ten clones producing recombinant Ag were selected and plaque purified; lysogens were produced and found to express beta-galactosidase fusion proteins ranging in molecular mass from 115 to 138 kDa. When ammonium sulfate-precipitated lysates of these recombinant phage clones were examined for their ability to stimulate PBMC from a patient with onchocerciasis, all 10 recombinants produced stimulation above that to nonrecombinant phage. When individual fusion proteins, affinity purified on anti-beta-galactosidase linked to agarose, were used to stimulate PBMC from patients with onchocerciasis, only one of the recombinant Ag induced PBMC proliferation (stimulation index greater than 4) above that to Ag from nonrecombinant phage. Characterization of the DNA coding for this Ag showed it to be 1.2 kb in length with a small (90 bp) open reading frame; furthermore, it appears to be Onchocerca specific (on genomic dot blots) and single copy. Using overlapping peptides encompassing the entire open reading frame, one T cell epitope has been localized.     

InBase: the Intein Database

Inteins are self-catalytic protein splicing elements. InBase (http://www.neb.com/neb/inteins.html), the Intein Database and Registry, is a curated compilation of published and unpublished information about protein splicing. It presents general information as well as detailed data for each intein, including tabulated comparisons and a comprehensive bibliography. An intein-specific BLAST server is now available to assist in identifying new inteins.     

Feedback equilibrium control during human standing

Equilibrium maintenance during standing in humans was investigated with a 3-joint (ankle, knee and hip) sagittal model of body movement. The experimental paradigm consisted of sudden perturbations of humans in quiet stance by backward displacements of the support platform. Data analysis was performed using eigenvectors of motion equation. The results supported three conclusions. First, independent feedback control of movements along eigenvectors (eigenmovements) can adequately describe human postural responses to stance perturbations. This conclusion is consistent with previous observations (Alexandrov et al., 2001b) that these same eigenmovements are also independently controlled in a feed-forward manner during voluntary upper-trunk bending. Second, independent feedback control of each eigenmovement is sufficient to provide its stability. Third, the feedback loop in each eigenmovement can be modeled as a linear visco-elastic spring with delay. Visco-elastic parameters and time-delay values result from the combined contribution of passive visco-elastic mechanisms and sensory systems of different modalities