Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa

The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands. Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Galalpha1-->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps. Among the mammalian disaccharides tested by the inhibition assay, the human blood group Pkactive Galalpha1-->4Gal, was the best. It was 7.4-fold less active than melibiose (Galalpha1-->6Glc). PA-IL has a preference for the alpha-anomer in decreasing order as follows: Galalpha1-->6 >Galalpha1-->4 >Galalpha1-->3. Of the monosaccharides studied, the phenylbeta derivatives of Gal were much better inhibitors than the methylbeta derivative, while only an insignificant difference was found between the Galalpha anomer of methyl- and p -NO2-phenyl derivatives. From these results, it can be concluded that the combining size of the agglutinin is as large as a disaccharide of the alpha-anomer of Gal at nonreducing end and most complementary to Galalpha1-->6Glc. As for the combining site of PA-IL toward the beta-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding.      

Glycosome turnover in Leishmania major is mediated by autophagy

Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite''s autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ∼20 glycosomes per cell, whereas amastigotes contain ∼10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ∼15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes.     

Immunological similarities between specific chloroplast ribosomal proteins from Chlamydomonas reinhardtii and ribosomal proteins from Escherichia coli

Polyclonal antibodies were elicited against seven of the 33 different proteins of the large subunit of the chloroplast ribosome from Chlamydomonas reinhardtii. Three of these proteins are synthesized in the chloroplast and four are made in the cytoplasm and imported. In western blots, six of the seven antisera are monospecific for their respective large subunit ribosomal proteins, and none of these antisera cross-reacted with any chloroplast small subunit proteins from C. reinhardtii. Antisera to the three chloroplast-synthesized ribosomal proteins cross-reacted with specific Escherichia coli large subunit proteins of comparable charge and molecular weight. Only one of the four antisera to the chloroplast ribosomal proteins synthesized in the cytoplasm cross-reacted with an E. coli large subunit protein. None of the antisera cross-reacted with any E. coli small subunit proteins. On the assumption of a procaryotic, endosymbiotic origin for the chloroplast, those chloroplast ribosomal proteins still synthesized within the organelle appear to have retained more antigenic sites in common with E. coli ribosomal proteins than have those which are now the products of cytoplasmic protein synthesis. Antisera to this cytoplasmically synthesized group of chloroplast ribosomal proteins did not recognize any antigenic sites among C. reinhardtii cytoplasmic ribosomal proteins, suggesting that the genes for the cytoplasmically synthesized chloroplast ribosomal proteins either are not derived from the cytoplasmic ribosomal protein genes or have evolved to a point where no antigenic similarities remain.      

Evidence for effect of random genetic drift on G+C content after lateral transfer of fucose pathway genes to Escherichia coli K-12

The cps cluster of Escherichia coli K-12 comprises genes involved in synthesis of capsular polysaccharide colanic acid. Part of the E. coli K-12 cps region has been cloned and sequenced and compared to its Salmonella enterica LT2 counterpart. The cps genes from the two organisms are homologous; in the case of the LT2 genes, with G+C content of 0.61 and codons characteristic of high G+C species, it seems clear that they have been acquired relatively recently by lateral transfer from a high G+C species. The K-12 form of these cps genes is closely related to those of LT2 so must derive from the same high G+C species, but it appears to have transferred much earlier such that random genetic drift has brought P3 (the corrected G+C content of codon base 3) down from 0.77 to 0.64, more than halfway to the E. coli average of 0.57. We estimate, using an equation developed by Sueoka, that the lateral transfer to E. coli took place approximately 45 million years ago. This is the first report we are aware of demonstrating the expected adjustment of P3 after lateral transfer between species with different G+C content DNA.      

The Ndc80/HEC1 complex is a contact point for kinetochore-microtubule attachment

Kinetochores are multicomponent assemblies that connect chromosomal centromeres to mitotic-spindle microtubules. The Ndc80 complex is an essential core element of kinetochores, conserved from yeast to humans. It is a rod-like assembly of four proteins- Ndc80p (HEC1 in humans), Nuf2p, Spc24p and Spc25p. We describe here the crystal structure of the most conserved region of HEC1, which lies at one end of the rod and near the N terminus of the polypeptide chain. It folds into a calponin-homology domain, resembling the microtubule-binding domain of the plus-end-associated protein EB1. We show that an Ndc80p-Nuf2p heterodimer binds microtubules in vitro. The less conserved, N-terminal segment of Ndc80p contributes to the interaction and may be a crucial regulatory element. We propose that the Ndc80 complex forms a direct link between kinetochore core components and spindle microtubules.     

Structure of a lectin from Canavalia gladiata seeds: new structural insights for old molecules

Background

Lectins are mainly described as simple carbohydrate-binding proteins. Previous studies have tried to identify other binding sites, which possible recognize plant hormones, secondary metabolites, and isolated amino acid residues. We report the crystal structure of a lectin isolated from Canavalia gladiata seeds (CGL), describing a new binding pocket, which may be related to pathogen resistance activity in ConA-like lectins; a site where a non-protein amino-acid, α-aminobutyric acid (Abu), is bound.

Results

The overall structure of native CGL and complexed with α-methyl-mannoside and Abu have been refined at 2.3 Å and 2.31 Å resolution, respectively. Analysis of the electron density maps of the CGL structure shows clearly the presence of Abu, which was confirmed by mass spectrometry.

Conclusion

The presence of Abu in a plant lectin structure strongly indicates the ability of lectins on carrying secondary metabolites. Comparison of the amino acids composing the site with other legume lectins revealed that this site is conserved, providing an evidence of the biological relevance of this site. This new action of lectins strengthens their role in defense mechanisms in plants.     

Ethnobiology of snappers (Lutjanidae): target species and suggestions for management

In this study, we sought to investigate the biology (diet and reproduction) and ethnobiology (fishers knowledge and fishing spots used to catch snappers) of five species of snappers (Lutjanidae), including Lutjanus analis, Lutjanus synagris, Lutjanus vivanus, Ocyurus chrysurus, and Romboplites saliens at five sites along the northeast (Riacho Doce, Maceió in Alagoas State, and Porto do Sauípe, Entre Rios at Bahia State) and the southeast (SE) Brazilian coast (Paraty and Rio de Janeiro cities at Rio de Janeiro State, and Bertioga, at São Paulo State.). We collected 288 snappers and interviewed 86 fishermen. The stomach contents of each fish were examined and macroscopic gonad analysis was performed. Snappers are very important for the fisheries of NE Brazil, and our results indicated that some populations, such as mutton snapper (L. analis) and lane snapper (L. synagris), are being caught when they are too young, at early juvenile stages. Local knowledge has been shown to be a powerful tool for determining appropriate policies regarding management of target species, and artisanal fishermen can be included in management processes. Other suggestions for managing the fisheries are discussed, including proposals that could provide motivation for artisanal fishermen to participate in programs to conserve resources, such as co-management approaches that utilize local knowledge, the establishment of fishing seasons, and compensation of fishermen, through 'payment for environmental services'. These suggestions may enhance the participation of local artisanal fishermen in moving to a more realistic and less top-down management approach of the fish population.     

Prediction of heterosis using genome-wide SNP-marker data: application to egg production traits in white Leghorn crosses

Prediction of heterosis has a long history with mixed success, partly due to low numbersof genetic markers and/or small data sets. We investigated the prediction of heterosisfor egg number, egg weight and survival days in domestic white Leghorns, using∼400 000 individuals from 47 crosses and allele frequencies on∼53 000 genome-wide single nucleotide polymorphisms (SNPs). When heterosis isdue to dominance, and dominance effects are independent of allele frequencies, heterosisis proportional to the squared difference in allele frequency (SDAF) between parental purelines (not necessarily homozygous). Under these assumptions, a linear model includingregression on SDAF partitions crossbred phenotypes into pure-line values and heterosis,even without pure-line phenotypes. We therefore used models where phenotypes of crossbredswere regressed on the SDAF between parental lines. Accuracy of prediction was determinedusing leave-one-out cross-validation. SDAF predicted heterosis for egg number and weightwith an accuracy of ∼0.5, but did not predict heterosis for survival days. Heterosispredictions allowed preselection of pure lines before field-testing, saving∼50% of field-testing cost with only 4% loss in heterosis. Accuraciesfrom cross-validation were lower than from the model-fit, suggesting that accuraciespreviously reported in literature are overestimated. Cross-validation also indicated thatdominance cannot fully explain heterosis. Nevertheless, the dominance model hadconsiderable accuracy, clearly greater than that of a general/specific combiningability model. This work also showed that heterosis can be modelled even when pure-linephenotypes are unavailable. We concluded that SDAF is a useful predictor of heterosis incommercial layer breeding.