Secretion is a fundamental process providing plants with the means for disposal of solutes, improvement of nutrient acquisition, and attraction of other organisms. Specific secretory organs, such as nectaries, hydathodes, and trichomes, use a combination of secretory and retrieval mechanisms, which are poorly understood at present. To study the mechanisms involved, an Arabidopsis thaliana activation tagged mutant, glutamine dumper1 (gdu1), was identified that accumulates salt crystals at the hydathodes. Chemical analysis demonstrated that, in contrast with the amino acid mixture normally present in guttation droplets, the crystals mainly contain Gln. GDU1 was cloned and found to encode a novel 17-kD protein containing a single putative transmembrane span. GDU1 is expressed in the vascular tissues and in hydathodes. Gln content is specifically increased in xylem sap and leaf apoplasm, whereas the content of several amino acids is increased in leaves and phloem sap. Selective secretion of Gln by the leaves may be explained by an enhanced release of this amino acid from cells. GDU1 study may help to shed light on the secretory mechanisms for amino acids in plants.

Selected channel-lining cysteine mutants from the M2 segment of rat ␣1 ␥-aminobutyric acid (GABA) type A receptor subunit, at positions 257, 261, 264, and 272 were co-expressed with ␤1 and ␥2 subunits in Xenopus oocytes. They generated functional receptors displaying conductance and response to both GABA and picrotoxinin similar to the wild type ␣1␤1␥2 receptor. Three chemically reactive affinity probes derived from noncompetitive blockers were synthesized to react with the engineered cysteines: 1) dithiane bis-sulfone derivative modified by an isothiocyanate function (probe A); 2) fiprole derivatives modified by an ␣-chloroketone (probe B) and ␣-bromoketone (probe C) moiety. These probes blocked the GABA-induced currents on all receptors. This blockade could be fully reversed by a washing procedure on the wild type, the ␣1T261C␤1␥2 and ␣1L264C␤1␥2 mutant receptors. In contrast, an irreversible effect was observed for all three probes on both ␣1V257C␤1␥2 and ␣1S272C␤1␥2 mutant receptors. This effect was probe concentration-dependent and could be abolished by picrotoxinin and/or t-butyl bicyclophosphorothionate. These data indicate a major interaction of non-competitive blockers at position 257 of the presumed M2 segment of rat ␣1 subunit but also suggest an interaction at the more extracellular position 272. ␥-Aminobutyric acid type A (GABA A ) 1 receptors exert their inhibitory effect in the central nervous system of vertebrates by regulating a chloride-sensitive channel which is very likely centered within a protein transmembrane heteropentameric subunits complex (1-5). The existence of 6␣, 4␤, 4␥, 1␦, and 2 subunits in addition to splicing variants, suggests a large diversity in the constitution of heteropentameric isoforms allowing a subtle tuning of the action of this neurotransmitter (6 -8). However, it has been proposed that a restricted number of combinations condition the functioning of this receptor and it is assumed that the ␣1␤2␥2 represents the major adult isoform (9). GABA A receptors serve as the target for several classes of molecules including important neuroactive drugs such as benzodiazepines, barbiturates, and neurosteroids. In contrast, only three receptor subunits have been cloned from insects up to now, RDL (10), ␤ (11), and GRD (12) leading to an apparently less complex situation for their structural assembly. Of particular interest are the action of non-competitive antagonists which are presumed to interact within the GABA receptor chloride channel leading to powerful insecticidal properties when presenting a selectivity for insect GABA receptor (13-16).To investigate, at a molecular level, the interaction of noncompetitive GABA antagonists with the chloride channel associated to the GABA A receptor, we defined an approach which uses chemically reactive non-competitive blockers (NCBs) as chemical sensors for cysteine mutants on the rat ␣1 GABA receptor subunit. This strategy was derived from the extensive work of Akabas and co-workers on several ionic channels including the chloride chan...

Lepidoptera have been reported to produce several antibacterial peptides in response to septic injury. However, in marked contrast to other insect groups, no inducible antifungal molecules had been described so far in this insect order. Surprisingly, also cysteine-rich antimicrobial peptides, which predominate in the antimicrobial defense of other insects, had not been discovered in Lepidoptera. Here we report the isolation from the hemolymph of immune induced larvae of the lepidopteran Heliothis virescens of a cysteine-rich molecule with exclusive antifungal activity. We have fully characterized this antifungal molecule, which has significant homology with the insect defensins, a large family of antibacterial peptides directed against Gram-positive strains. Interestingly, the novel peptide shows also similarities with the antifungal peptide drosomycin from Drosophila.Thus, Lepidoptera appear to have built their humoral immune response against bacteria on cecropins and attacins. In addition, we report that Lepidoptera have conferred antifungal properties to the well conserved structure of antibacterial insect defensins through amino acid replacements.

Genetically modified (GM) crops may contain newly expressed proteins that are described as "intractable". Safety assessment of these proteins may require some adaptations to the current assessment procedures. Intractable proteins are defined here as those proteins with properties that make it extremely difficult or impossible with current methods to express in heterologous systems; isolate, purify, or concentrate; quantify (due to low levels); demonstrate biological activity; or prove equivalency with plant proteins. Five classes of intractable proteins are discussed here: (1) membrane proteins, (2) signaling proteins, (3) transcription factors, (4) N-glycosylated proteins, and (5) resistance proteins (R-proteins, plant pathogen recognition proteins that activate innate immune responses). While the basic tiered weight-of-evidence approach for assessing the safety of GM crops proposed by the International Life Sciences Institute (ILSI) in 2008 is applicable to intractable proteins, new or modified methods may be required. For example, the first two steps in Tier I (hazard identification) analysis, gathering of applicable history of safe use (HOSU) information and bioinformatics analysis, do not require protein isolation. The extremely low level of expression of most intractable proteins should be taken into account while assessing safety of the intractable protein in GM crops. If Tier II (hazard characterization) analyses requiring animal feeding are judged to be necessary, alternatives to feeding high doses of pure protein may be needed. These alternatives are discussed here.

Bell in ref 1, Chapter 1.(7) The variation in rate with P and r could come from variations in electron distribution, electron energies, rates of interspecies energy transfer, residence time in the plasma region, or simply from the rate law for the specific reactions. Changes in product composition (ratios) could also arise from all of these factors. In addition, the possibility of converting primary products to other materials has often been suggested and a major change in mechanism with conditions is not unlikely.(8) The above derivation leaves unclear the reasons why ki Ht) is a linear function of Pip. One might suspect that knowing the number density of electrons (Ne') with energies greater than some limiting value for a quantized excitation and the average cross section for excitation by these electrons would be useful. Appropriate experimental data for reacting organic plasmas does not appear to be available. (9) H. Suhr and G. Kruppa, Z.