Evolution, and functional analysis of Natural Resistance-Associated Macrophage proteins (NRAMPs) from Theobroma cacao and their role in cadmium accumulation

The presence of the toxic metal cadmium (Cd2+) in certain foodstuffs is recognised as a global problem, and there is increasing legislative pressure to reduce the content of Cd in food. The present study was conducted on cacao (Theobroma cacao), the source of chocolate, and one of the crops known to accumulate Cd in certain conditions. There are a range of possible genetic and agronomic methods being tested as a route to such reduction. As part of a gene-based approach, we focused on the Natural Resistance-Associated Macrophage Proteins (NRAMPS), a family of proton/metal transporter proteins that are evolutionarily conserved across all species from bacteria to humans. The plant NRAMP gene family are of particular importance as they are responsible for uptake of the nutritionally vital divalent cations Fe2+, Mn2+, Zn2+, as well as Cd2+. We identified the five NRAMP genes in cacao, sequenced these genes and studied their expression in various organs. We then confirmed the expression patterns in response to variation in nutrient cation availability and addition of Cd2+. Functional analysis by expression in yeast provided evidence that NRAMP5 encoded a protein capable of Cd2+ transport, and suggested this gene as a target for genetic selection/modification

Conservation of the Human Integrin-Type Beta-Propeller Domain in Bacteria

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca2+-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca2+-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem copies are found

Automated flow enzyme-linked immunosorbent assay (ELISA) system for analysis of methyl parathion

sensitive detection of pesticides is of utmost importance in environment pesticides in agricultural and environmental samples wherein antibodies are employed against the target molecules. Accurate diagnosis depends on the affinity and specificity of the antibody preparation used, and high affinity antibodies are essential for the detection of very small amounts of pesticides. Enzyme linked immuno sorbent assay (ELISA) coupled with flow injection analysis (FIA) technique provides a very high sensitivity with high throughput of analyses. Automation of this analysis scheme ensures precise detection with high accuracy. The present development aims at providing a user-friendly system for achieving this objective. It employs a 8952 microcontroller for precise flow of reagents, samples, substrate and conjugates used for analysis to be passed through an immobilized antibody column at predetermined time. With the sequence and flow control of buffers used, it also provides the option for reuse of the immobilized antibody column. The system is flexible to accommodate multiple sequences up to a maximum of 99 steps. It is customizable for different flow ELISA applications. It can control up to eight solenoid valves (dc 24 V) and two peristaltic pumps and has one 12 bit analog channel for data acquisition. With the serial interface port, the system provides convenient means for data acquisition into the computer. The system has been successfully tested for immuno analysis of organophosphorous pesticide methyl parathion. (c) 2005 Elsevier B.V. All rights reserved