Effect of diaminopropionic acid (Dap) on the biophysical properties of a modified synthetic channel-forming peptide.

Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK4-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, incorporated into cell membranes and was nonimmunogenic, but lacked the structural properties for high conductance and anion selectivity when assembled into a pore. Further studies suggested that the threonine residues at positions 13, 17, and 20 line the pore of assembled p22-T19R, S22W, and here we used 2,3-diaminopropionic acid (Dap) substitutions to introduce positive charges to the pore-lining interface of the predicted p22-T19R, S22W channel. Dap-substituted p22-T19R, S22W peptides retained the α-helical secondary structure characteristic of their parent peptide, and induced short-circuit transepithelial currents when exposed to the apical membrane of Madin-Darby canine kidney (MDCK) cells; the sequences containing multiple Dap-substituted residues induced larger currents than the peptides with single or no Dap substitutions. To gain further insights into the effects of Dap residues on the properties of the putative pore, we performed two-electrode voltage clamp electrophysiology on Xenopus oocytes exposed to p22-T19R, S22W or its Dap-modified analogues. We observed that Dap-substituted peptides also induced significantly larger voltage-dependent currents than the parent compound, but there was no apparent change in reversal potential upon replacement of external Na+, Cl- or K+, indicating that these currents remained nonselective. These results suggest that the introduction of positively charged side chains in predicted pore-lining residues does not improve anion-to-cation selectivity, but results in higher conductance, perhaps due to higher oligomerization numbers.

Small ß2-glycoprotein I peptides protect from intestinal ischemia reperfusion injury.

Intestinal ischemic events, which are followed by reperfusion, induce significant tissue damage and frequently result in multiple organ failure, with >70% mortality. Upon reperfusion, excessive inflammation leads to exacerbated tissue damage. Previous studies indicated that binding of the serum protein, ß2-glycoprotein I, to the endothelium initiates a cascade of inflammatory molecules that is required for damage. We hypothesized that peptides derived from the binding domain (domain V) of ß2-glycoprotein I would attenuate ischemia/reperfusion-induced damage and inflammation in a therapeutic manner. Using a mouse model of intestinal ischemia/reperfusion, we administered peptides either prior to ischemia or at clinically relevant time points during reperfusion and evaluated intestinal tissue damage and inflammation after 2 h of reperfusion. We demonstrate that multiple peptides attenuate injury and inflammation in a dose-dependent manner and, perhaps more significantly, are efficacious when administered up to 30 min after the onset of reperfusion. In addition, an all D-amino acid retro-inverso peptide was biologically active. Thus, the ß2-glycoprotein I-derived peptides attenuate injury and inflammation when administered in a therapeutic manner in intestinal ischemia/reperfusion injury.

Domain V peptides inhibit beta2-glycoprotein I-mediated mesenteric ischemia/reperfusion-induced tissue damage and inflammation.

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, ß2-glycoprotein I as an initiating Ag for Ab recognition and ß2-glycoprotein I (ß2-GPI) peptides as a therapeutic for mesenteric IR. The time course of ß2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of ß2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine-rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In addition, infusion of wild-type serum containing reduced levels of anti-ß2-GPI Abs into Rag-1(-/-) mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein ß2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.

Rubisco activase and wheat productivity under heat-stress conditions.

Rubisco activase (RCA) constrains the photosynthetic potential of plants at high temperatures (heat stress). Endogenous levels of RCA could serve as an important determinant of plant productivity under heat-stress conditions. Thus, in this study, the possible relationship between expression levels of RCA and plant yield in 11 European cultivars of winter wheat following prolonged exposure to heat stress was investigated. In addition, the effect of a short-term heat stress on RCA expression in four genotypes of wheat, five genotypes of maize, and one genotype of Arabidopsis thaliana was examined. Immunoblots prepared from leaf protein extracts from control plants showed three RCA cross-reacting bands in wheat and two RCA cross-reacting bands in maize and Arabidopsis. The molecular mass of the observed bands was in the range between 40 kDa and 46 kDa. Heat stress affected RCA expression in a few genotypes of wheat and maize but not in Arabidopsis. In wheat, heat stress slightly modulated the relative amounts of RCA in some cultivars. In maize, heat stress did not seem to affect the existing RCA isoforms (40 kDa and 43 kDa) but induced the accumulation of a new putative RCA of 45-46 kDa. The new putative 45-46 kDa RCA was not seen in a genotype of maize (ZPL 389) that has been shown to display an exceptional sensitivity to heat stress. A significant, positive, linear correlation was found between the expression of wheat 45-46 kDa RCA and plant productivity under heat-stress conditions. Results support the hypothesis that endogenous levels of RCA could play an important role in plant productivity under supraoptimal temperature conditions.

Heat tolerance and expression of protein synthesis elongation factors, EF-Tu and EF-1α, in spring wheat.

Protein elongation factors, EF-Tu and EF-1α, have been implicated in cell response to heat stress. We investigated the expression (accumulation) of EF-Tu and EF-1α in mature plants of spring wheat cultivars Kukri and Excalibur, and tested the hypothesis that cultivars with contrasting tolerance to heat stress differ in the accumulation of these elongation factors under prolonged exposure to high temperature (16 days at 36/30°C). In addition, we investigated the expression of EF-Tu and EF-1α in young plants experiencing a 24-h heat shock (43°C). Excalibur showed better tolerance to heat stress than Kukri. Heat stress induced accumulation of EF-Tu and EF-1α in mature plants of both cultivars, but to a greater extent in Excalibur. Young plants did not show appreciable accumulation of EF-Tu in response to heat shock. However, these plants showed increased accumulation of EF-1α and the accumulation appeared greater in Excalibur than in Kukri. The results support the hypothesis that EF-Tu plays a role in heat tolerance in spring wheat. The results also suggest that EF-1α may be of importance to wheat response to heat stress.

Mycorrhizal status and diversity of fungal endophytes in roots of common buckwheat (Fagopyrum esculentum) and tartary buckwheat (F. tataricum).

To determine the mycorrhizal status and to identify the fungi colonising the roots of the plants, common buckwheat (Fagopyrum esculentum) and tartary buckwheat (F. tataricum) were inoculated with an indigenous fungal mixture from a buckwheat field. Root colonisation was characterised by the hyphae and distinct microsclerotia of dark septate endophytes, with occasional arbuscules and vesicles of arbuscular mycorrhizal fungi. Sequences of arbuscular mycorrhizal fungi colonising tartary buckwheat clustered close to the Glomus species group A. Sequences with similarity to the Ceratobasidium/Rhizoctonia complex, a putative dark septate endophyte fungus, were amplified from the roots of both common and tartary buckwheat. To the best of our knowledge, this is the first report of arbuscular mycorrhizal colonisation in tartary buckwheat and the first molecular characterisation of these fungi that can colonise both of these economically important plant species.

Heat-induced accumulation of chloroplast protein synthesis elongation factor, EF-Tu, in winter wheat.

Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize (Zea mays). Chloroplast EF-Tu is highly conserved, and it is possible that this protein may be of importance to heat tolerance in other species including wheat (Triticum aestivum). In this study, we assessed heat tolerance and determined the relative levels of EF-Tu in mature plants (at flowering stage) of 12 cultivars of winter wheat experiencing a 16-d-long heat treatment (36/30 degrees C, day/night temperature). In addition, we also investigated the expression of EF-Tu in young plants experiencing a short-term heat shock (4h at 43 degrees C). Heat tolerance was assessed by examining the stability of thylakoid membranes, measuring chlorophyll content, and assessing plant growth traits (shoot dry mass, plant height, tiller number, and ear number). In mature plants, relative levels of EF-Tu were determined after 7 d of heat stress. High temperature-induced accumulation of EF-Tu in mature plants of all cultivars, and a group of cultivars that showed greater accumulation of EF-Tu displayed better tolerance to heat stress. Young plants of all cultivars but one did not show significant increases in the relative levels of EF-Tu. The results of the study suggest that EF-Tu protein may play a role in heat tolerance in winter wheat.