Biochemical characterization of legume seeds as ingredients in animal feed

The current European protein deficit is estimated as high as 70% of present needs. Because of the high protein content of their seeds, grain legumes are attractive candidates for lowering the deficiency in plant protein production. The objective of this work was to identify new sources of vegetable protein that would reduce our high dependence of soy, the main source of protein in the manufacture of feedstuffs. To achieve this goal, we determined the proximate composition, the bioactive components, as well as the antinutritional factors present in the studied seeds. In general, the protein, fat and carbohydrates content of legume seeds studied were within the limits found in the literature. The bioactive compounds detected in all the seeds were α-galactosides, myoinositol phosphates, protease inhibitors and phenols. IP₆(phytic acid) was the main inositol phosphate form in all the samples. The highest protease inhibitors content was detected in both <em>Lathyrus cicera</em> cultivars. <em>Vicia ervilia</em> and <em>L. cicera</em> cultivars showed low haemagglutinating activity (20.4 HU/g). The γ-glutamyl-S-ethenyl-cysteine content in <em>Vicia narbonensis </em>was around 16.0 mg/g. Both <em>L. cicera </em>varieties presented similar β-N-oxalyl-L-α, β-diaminopropionic acid content (0.80 mg/g). The two <em>V. ervilia </em>varieties showed high canavanine concentration (1.93-5.28 mg/g). Vicine was only detected in <em>V. narbonensis </em>cultivars (0.3 mg/g). The biochemical characterization carried out in this study allows us to know the limits of inclusion of these minor crop seeds in feed formulations in order to replace the soybean.

Serological cross-reactions between four polyomaviruses of birds using virus-like particles expressed in yeast

Polyomaviruses are aetiological agents of fatal acute diseases in various bird species. Genomic analysis revealed that avian polyomavirus (APyV), crow polyomavirus (CPyV), finch polyomavirus (FPyV) and goose hemorrhagic polyomavirus (GHPyV) are closely related to each other, but nevertheless form separate viral species; however, their serological relationship was previously unknown. As only APyV can be grown efficiently in tissue culture, virus-like particles (VLPs) were generated by expression of the genomic regions encoding the major structural protein VP1 of these viruses in yeast; these were used to elicit type-specific antibodies in rabbits and as antigens in serological reactions. For increased VLP assembly, a nuclear-localization signal was introduced into APyV-VP1. VLPs derived from the VP1 of the monkey polyomavirus simian virus 40 served as control. APyV-, GHPyV- and CPyV-VLPs showed haemagglutinating activity with chicken and human erythrocytes. CPyV- and GHPyV-specific sera showed slight cross-reactions in immunoblotting, haemagglutination-inhibition assay and indirect ELISA. The FPyV-specific serum inhibited the haemagglutination activity of APyV-VLPs slightly and showed a weak cross-neutralizing activity against APyV in cell-culture tests. Generally, these data indicate that the four polyomaviruses of birds are serologically distinct. However, in accordance with genetic data, a relationship between CPyV and GHPyV as well as between APyV and FPyV is evident, and grouping into two different serogroups may be suggested. The haemagglutinating activity of APyV, CPyV and GHPyV may indicate similar receptor-binding mechanisms for these viruses. Our data could be useful for the development of vaccines against the polyomavirus-induced diseases in birds and for interpretation of diagnostic test results.

Interaction of Moringa oleifera seed lectin with humic acid

The aim of this work was to characterise the affinity of protein preparations from Moringa oleifera seeds, specifically extract (seeds homogenised with 0.15 M NaCl), fraction (extract precipitated with 390 mg mL−1 of ammonium sulphate) and cMoL (coagulant M. oleifera lectin) to bind humic acids using a haemagglutinating activity assay with rabbit erythrocytes and a radial diffusion assay in agarose gel. Specific haemagglutinating activity (SHA) decreased by 94 % for the extract and cMoL and by 50 % for the fraction in the presence of humic acid. Precipitation bands were observed in the diffusion gel. Both results suggested humic acid-cMoL binding. Carbohydrates, potassium, and calcium ions and pH affected the SHA of cMoL. As an example of application, cMoL was immobilised on a column packed with sepharose receiving 20 mg mL−1 of carbon humic acid solution, 30 mg of humic acid per gram of support was removed. This result suggested that protein preparations might be used in water treatment to remove humic acids.

Rapid affinity-purification and physicochemical characterization of pumpkin (Cucurbita maxima) phloem exudate lectin

The chito-oligosaccharide-specific lectin from pumpkin (Cucurbita maxima) phloem exudate has been purified to homogeneity by affinity chromatography on chitin. After SDS/PAGE in the presence of 2-mercaptoethanol, the pumpkin phloem lectin yielded a single band corresponding to a molecular mass of 23.7 kDa, whereas ESI-MS (electrospray ionization MS) gave the molecular masses of the subunit as 24645 Da. Analysis of the CD spectrum of the protein indicated that the secondary structure of the lectin consists of 9.7% α-helix, 35.8% β-sheet, 22.5% β-turn and 32.3% unordered structure. Saccharide binding did not significantly affect the secondary and tertiary structures of the protein. The haemagglutinating activity of pumpkin phloem lectin was mostly unaffected in the temperature range 4–70 °C, but a sharp decrease was seen between 75 and 85 °C. Differential scanning calorimetric and CD spectroscopic studies suggest that the lectin undergoes a co-operative thermal unfolding process centred at approx. 81.5 °C, indicating that it is a relatively stable protein.

A haemagglutinin from the medicinal fungus Cordyceps militaris

There are only a few reports on agglutinins from ascomycete and medicinal fungi. An HA (haemagglutinin), with an N-terminal amino acid sequence different from those of known lectins, was isolated in the present study from dried fruiting bodies of the medicinal ascomycete fungus Cordyceps militaris. The purification protocol consisted of affinity chromatography, ion-exchange chromatography and gel filtration. The haemagglutinating activity of the HA could not be inhibited by simple sugars or heparin, and was stable over the pH range 2–13 and up to 60°C. Chemical modification of tryptophan and tyrosine residues had no effect. The HA exhibited some antiproliferative activity towards hepatoma (HepG2) cells and inhibited HIV-1 reverse transcriptase (IC50=10 μM). However, it did not exhibit antifungal activity, mitogenic activity towards splenocytes, nitric oxide-inducing activity towards macrophages or RNase activity. The results of the present study add to the meagre information pertaining to agglutinins from ascomycete and medicinal mushrooms. It is revealed in this study that C. militaris HA differs from other ascomycete mushroom HAs in a variety of biochemical characteristics.

Isolation and characterization of a lectin with potentially exploitable activities from caper (Capparis spinosa) seeds

A dimeric 62-kDa lectin exhibiting a novel N-terminal amino acid sequence was purified from caper (Capparis spinosa) seeds. The purification protocol involved anion-exchange chromatography, cation-exchange chromatography and, finally, gel filtration by FPLC on Superdex 75. Approx. 100-fold purification was achieved. The haemagglutinating activity of the lectin, which was stable in the pH range 1–12 and up to 40°C, could be inhibited by D(+) galactose, α-lactose, raffinose and rhamnose at 1 mM concentration, by 25 mM L(+)-arabinose and by 100 mM D(+)GlcN (glucosamine). The lectin potently inhibited HIV-1 reverse transcriptase with an IC50 of 0.28 μM and proliferation of both hepatoma HepG2 and breast cancer MCF-7 cells with an IC50 of approx. 2 μM. It induced apoptosis in HepG2 and MCF-7 cells. It manifested a weaker mitogenic activity on mouse splenocytes than ConA (concanavalin A). It inhibited mycelial growth in Valsa mali with an IC50 of 18 μM.

Identification of an RTX determinant of Burkholderia cenocepacia J2315 by subtractive hybridization

This study utilized suppressive subtractive hybridization between the clinical isolate Burkholderia cenocepacia J2315 and the closely related environmental isolate Burkholderia cepacia ATCC 25416T to isolate DNA fragments specific to B. cenocepacia J2315. Analysis of the resulting pools of B. cenocepacia-specific DNAs identified several fragments that may be part of putative virulence factors. Further in silico analysis of a single fragment indicated that it was internal to a gene of which the predicted product had characteristics of repeat in toxin (RTX)-like proteins and high similarity to proteins in other human or plant pathogens. In conjunction with this finding, phenotypic traits associated with known RTX proteins were assessed. A haemagglutinating activity of B. cenocepacia J2315 was identified that was absent in B. cepacia ATCC 25416T. The expression of this activity appeared to be growth phase-dependent. Analysis of the gene presence and haemagglutinating activity across the species of the B. cepacia complex showed that both were common to the ET12 lineage of B. cenocepacia, but were absent in the other species examined. Haemagglutinating activity was limited to isolates with the RTX-like gene. Expression studies utilizing quantitative PCR demonstrated an association between onset of haemagglutinating activity and increased expression of the gene, which suggests that the putative RTX determinant encodes a haemagglutinating activity.

Beak and feather disease virus haemagglutinating activity using erythrocytes from African Grey parrots and Brown-headed parrots : research communication

Psittacine beak and feather disease (PBFD) is a common viral disease of wild and captive psittacine birds characterized by symmetric feather loss and beak deformities. The causative agent, beak and feather disease virus (BFDV), is a small, circular single-stranded DNA virus that belongs to the genus Circovirus. BFDV can be detected by PCR or the use of haemagglutination (HA) and haemagglutination inhibition (HI) assays that detect antigen and antibodies respectively. Erythrocytes from a limited number of psittacine species of Australian origin can be used in these tests. In South Africa, the high cost of these birds makes them difficult to obtain for experimental purposes. Investigation into the use of erythrocytes from African Grey parrots and Brown-headed parrots yielded positive results showing the haemagglutinating activity of their erythrocytes with purified BFDV obtained from confirmed clinical cases of the disease. The HA activity was further confirmed by the demonstration of HI using BFDV antiserum from three different African Grey parrots previously exposed to the virus and not showing clinical signs of the disease.