Genetic Variability in Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) Estimated by Morphological Data and Amplified Fragment Length Polymorphism Markers

Data on genetic similarity among crop cultivars is of vital importance for the plant breeder. The objectives of this study were to group pepper (Capsicum annuum L.) genotypes into clusters according to their distances as estimated by morphological traits and amplified fragment length polymorphism (AFLP) markers and to assess the relationships between the two. Thirty-nine pepper genotypes obtained from different countries were grown in the greenhouse at University of the Free State, South Africa, during 2001 and 2002 in a randomized complete block design with three replications. A total of 20 different morphological traits were measured and six AFLP primer pairs were used to estimate pairwise genetic distances. Both datasets showed high genetic distances among the different genotypes, indicating high genetic diversity among them. The mean genetic distance among Ethiopian pungent elongated-fruit genotypes, was lower than that between them and the introduced ones. Morphological and AFLP distance estimations generally clustered together genotypes with similar fruit sizes. Significant, positive correlation was observed between morphological and AFLP diversity estimations. The narrow genetic basis among the Ethiopian pungent elongated-fruit cultivars suggests that the pepper breeding program of Ethiopia should focus on enriching its germplasm through local collection and introductions from other parts of the world.     

Identifying Plasmodium falciparum cytoadherence-linked asexual protein 3 (CLAG 3) sequences that specifically bind to C32 cells and erythrocytes

Adhesion of mature asexual stage Plasmodium falciparum parasite-infected erythrocytes (iRBC) to the vascular endothelium is a critical event in the pathology of Plasmodium falciparum malaria. It has been suggested that the clag gene family is essential in cytoadherence to endothelial receptors. Primers used in PCR and RT-PCR assays allowed us to determine that the gene encoding CLAG 3 (GenBank accession no. NP_473155) is transcribed in the Plasmodium falciparum FCB2 strain. Western blot showed that antisera produced against polymerized synthetic peptides from this protein recognized a 142-kDa band in P. falciparum schizont lysate. Seventy-one 20-amino-acid-long nonoverlapping peptides, spanning the CLAG 3 (cytoadherence-linked asexual protein on chromosome 3) sequence were tested in C32 cell and erythrocyte binding assays. Twelve CLAG peptides specifically bound to C32 cells (which mainly express CD36) with high affinity, hereafter referred to as high-affinity binding peptides (HABPs). Five of them also bound to erythrocytes. HABP binding to C32 cells and erythrocytes was independent of peptide charge or peptide structure. Affinity constants were between 100 nM and 800 nM. Cross-linking and SDS-PAGE analysis allowed two erythrocyte binding proteins of around 26 kDa and 59 kDa to be identified, while proteins of around 53 kDa were identified as possible receptor sites for C-32 cells. The HABPs' role in Plasmodium falciparum invasion inhibition was determined. Such an approach analyzing various CLAG 3 regions may elucidate their functions and may help in the search for new antigens important for developing antimalarial vaccines.     

Linking two immuno-suppressive molecules: indoleamine 2,3 dioxygenase can modify HLA-G cell-surface expression

Nonclassical human leukocyte antigen (HLA) class I molecule HLA-G and indoleamine 2,3 dioxygenase (INDO) in humans and mice, respectively, have been shown to play crucial immunosuppressive roles in fetal-maternal tolerance. HLA-G inhibits natural killer and T cell function by high-affinity interaction with inhibitory receptors, and INDO acts by depleting the surrounding microenvironment of the essential amino acid tryptophan, thus inhibiting T cell proliferation. We investigated whether HLA-G expression and INDO function were linked. Working with antigen-presenting cell (APC) lines and monocytes, we found that functional inhibition of INDO by 1-methyl-tryptophan induced cell surface expression of HLA-G1 by HLA-G1-negative APCs that were originally cell-surface negative, and that in reverse, the functional boost of INDO by high concentrations of tryptophan induced a complete loss of HLA-G1 cell surface expression by APCs that were originally cell-surface HLA-G1-positive. This mechanism was shown to be posttranslational because HLA-G protein cell contents remained unaffected by the treatments used. Furthermore, HLA-G cell surface expression regulation by INDO seems to relate to INDO function, but not to tryptophan catabolism itself. Potential implications in fetal-maternal tolerance are discussed.     

Ferns and Melastomataceae as indicators of vascular plant composition in rain forests of Colombian Amazonia

In a case-study from Colombian Amazonia, species information from ferns and Melastomataceae was used to explain the compositional patterns of other vascular plant species in 40 widely distributed 0.1-ha plots. Canonical correspondence analysis was applied to regress vascular plant species composition in the forests against information from these two indicator groups (summarized as axes of principal coordinate analyses), together with that from soils, landscape, and the spatial sampling design. In total, 53,941 individuals of 2480 vascular plant species were recorded. Of these, 17,473 individuals and 132 species were from ferns and Melastomataceae. In 19 well-drained upland (tierra firme) plots 19,622 vascular plant individuals and 1716 species were found, with 3793 plants and 91 species from ferns and Melastomataceae. In both the set of all landscapes and the subset of tierra firme forests the principal PCoA axes of the two indicator groups were highly related to the main patterns of forest species composition. In principle, therefore, ferns and Melastomataceae can be used to detect and forecast changes in the forest composition of the study area. However, evidence was not obtained that ferns and Melastomataceae show more potential to predict the main patterns in species composition of forests than soil, landscape, and spatial variables. The partioning of the total variation in forest composition showed that the correlation of ferns and Melastomataceae with other forest plants was quite independent from that of soil, landscape, and space. Direct effects of ferns and Melastomataceae on other plants might be obtained from experimental studies of between-plant interactions, concentrating on the seedling or juvenile stages of trees and lianas, both above-ground as well as in the rooting environment.     

Production and characterization of a noncytotoxic deletion variant of the Aspergillus fumigatus allergen Aspf1 displaying reduced IgE binding

Aspergillus fumigatus is responsible for many allergic respiratory diseases, the most notable of which - due to its severity - is allergic bronchopulmonary aspergillosis. Aspf1 is a major allergen of this fungus: this 149-amino acid protein belongs to the ribotoxin family, whose best characterized member is alpha-sarcin (EC 3.1.27.10). The proteins of this group are cytotoxic ribonucleases that degrade a unique bond in ribosomal RNA impairing protein biosynthesis. Aspf1 and its deletion mutant Aspf1Delta(7-22) have been produced as recombinant proteins; the deleted region corresponds to an exposed beta-hairpin. The conformation of these two proteins has been studied by CD and fluorescence spectroscopy. Their enzymatic activity and cytotoxicity against human rhabdomyosarcoma cells was also measured and their allergenic properties have been studied by using 58 individual sera of patients sensitized to Aspergillus. Aspf1Delta(7-22) lacks cytotoxicity and shows a remarkably reduced IgE reactivity. From these studies it can be concluded that the deleted beta-hairpin is involved in ribosome recognition and is a significant allergenic region.     

Structural characterization of NETNES, a novel glycoconjugate in Trypanosoma cruzi epimastigotes

The unicellular stercorarian protozoan parasite Trypanosoma cruzi is the etiological agent of Chagas' disease. The epimastigote form of the parasite is covered in a dense coat of glycoinositol phospholipids and short glycosylphosphatidylinositol (GPI)-anchored mucinlike molecules. Here, we describe the purification and structural characterization of NETNES, a relatively minor but unusually complex glycoprotein that coexists with these major surface components. The mature glycoprotein is only 13 amino acids in length, with the sequence AQENETNESGSID, and exists in two forms with either four or five post-translational modifications. These are either one or two asparagine-linked oligomannose glycans, two linear alpha-mannose glycans linked to serine residues via phosphodiester linkages, and a GPI membrane anchor attached to the C-terminal aspartic acid residue. The variety and density of post-translational modifications on an unusually small peptide core make NETNES a unique type of glycoprotein. The N-glycans are predominantly Manalpha1-6(Manalpha1-3) Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAcbeta1-Asn; the phosphate-linked glycans are a mixture of (Manalpha1-2)0-3Man1-P-Ser; and the GPI anchor has the structure Manalpha1-2(ethanolamine phosphate)Manalpha1-2Manalpha1-6Manalpha1-4(2-aminoethylphosphonate-6)GlcNalpha1-6-myo-inositol-1-P-3(sn-1-O-(C16:0)alkyl-2-O-(C16:0)acylglycerol). Four putative NETNES genes were found in the T. cruzi genome data base. These genes are predicted to encode 65-amino acid proteins with cleavable 26-amino acid N-terminal signal peptides and 26-amino acid C-terminal GPI addition signal peptides.     

New roles of myosin II during vesicle transport and fusion in chromaffin cells

Modified herpes virus (amplicons) were used to express myosin regulatory light chain (RLC) chimeras with green fluorescent protein (GFP) in cultured bovine chromaffin cells to study myosin II implication in secretion. After infection, RLC-GFP constructs were clearly identified in the cytoplasm and accumulated in the cortical region, forming a complex network that co-localized with cortical F-actin. Cells expressing wild type RLC-GFP maintained normal vesicle mobility, whereas cells expressing an unphosphorylatable form (T18A/S19A RLC-GFP) presented severe restrictions in granule movement as measured by individual tracking in dynamic confocal microscopy studies. Interestingly, the overexpression of this mutant form of RLC also affected the initial secretory burst elicited by either high K(+) or BaCl(2), as well as the secretion induced by fast release of calcium from caged compounds in individual cells. Moreover, T18A/S19A RLC-GFP-infected cells presented slower fusion kinetics of individual granules compared with controls as measured by analysis of amperometric spikes. Taken together, our results demonstrate the implication of myosin II in the transport of vesicles, and, surprisingly, in the final phases of exocytosis involving transitions affecting the activity of docked granules, and therefore uncovering a new role for this cytoskeletal element.     

Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cells

Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS-7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose-dependent and time-dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent-resistant cellular membranes showed that sticholysin II colocalizes with caveolin-1 in fractions corresponding to raft-like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane-associated protein. Toxin binding to raft-like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS-7 cells through interaction with membrane domains which behave like cellular rafts.