An approximately 400 kDa membrane-associated complex that contains one molecule of the resistance protein Cf-4

Despite sharing more than 91% sequence identity, the tomato Cf-4 and Cf-9 proteins discriminate between two Cladosporium-encoded avirulence determinants, Avr4 and Avr9. Comparative studies between Cf-4 and Cf-9 are thus of particular interest. To investigate Cf-4 protein function in initiating defence signalling, we established transgenic tobacco lines and derived cell suspension cultures expressing c-myc-tagged Cf-4. Cf-4:myc encodes a membrane-localized glycoprotein of approximately 145 kDa, which confers recognition of Avr4. Elicitation of Cf-4:myc and Cf-9:myc tobacco cell cultures with Avr4 and Avr9, respectively, triggered the synthesis of active oxygen species and MAP kinase activation. Additionally, an Agrobacterium-mediated transient assay was used to express Cf-4:myc and a newly engineered fusion protein Cf-4:TAP. Both transiently expressed proteins were found to be functional in an in vivo assay, conferring a hypersensitive response (HR) to Avr4. Consistent with previous observations that Cf-9 is present in a protein complex, gel filtration analysis of microsomal fractions solubilized with octylglucoside revealed that epitope-tagged Cf-4 proteins migrated at a molecular mass of 350-475 kDa. Using blue native gel electrophoresis, the molecular size was confirmed to be approximately 400 kDa. Significantly, this complex appeared to contain only one Cf-4 molecule, supporting the idea that, as previously described for Cf-9, additional glycoprotein partners participate with Cf-4 in the perception of the Avr4 protein. Intriguingly, Cf proteins and Clavata2 (CLV2) of Arabidopsis are highly similar in structure, and the molecular mass of Cf-4 and CLV complexes is also very similar (400 and 450 kDa, respectively). However, extensive characterization of the Cf-4 complex revealed essentially identical characteristics to the Cf-9 complex and significant differences from the CLV2 complex.     

Detection of microsatellite instability during somatic embryogenesis of oak (Quercus robur L.)

Five microsatellite loci (QpZAG1/5, QpZAG9, QpZAG36, MSQ4, MSQ13) were used to test for genetic stability of three somatic embryogenic culture lines of Quercus robur L. and plantlets derived therefrom. DNA variation was detected among somatic embryos within all embryogenic lines, whereas no genetic instability was found among the regenerated plants. Two microsatellite loci revealed variation, and a locus-dependent instability was observed. The most polymorphic and useful microsatellite locus for detecting genetic variation was QpZAG9, with 28.5% of the investigated loci being variable.     

Design and evaluation of a novel flow chamber for measuring cell adhesion to absorbable polymer films

There is great interest in improving cellular attachment to synthetic materials, particularly for developing small diameter tissue-engineered vascular grafts. However, limited research has been conducted to evaluate the adhesion characteristics of different cell types to absorbable substrates. Tissue engineered vessels typically fail as a result of delamination of the endothelial cell layer when exposed to fluid or blood flow. The focus of this research was to design and evaluate a flow chamber, using fibroblasts, smooth muscle cells, and endothelial cells, to probe the bounds of the system. A flow chamber was designed and fabricated to compare the relative adhesion characteristics of cells to absorbable polymer films. A preliminary investigation of mouse fibroblast (3T3M) adhesion to semicrystalline poly-L-lactide (PLL) films was conducted to determine general operating specifications. Cell coverage on films was evaluated using a live-dead assay and image analysis; following exposure to flow, tests were similarly conducted. Based on these results, additional studies were conducted to compare the adhesion of rat aortic smooth muscle cells (SMC) and endothelial cells (EC) on PLL films.     

Performance of serum-supplemented and serum-free media in IFNγ Elispot Assays for human T cells

The choice of serum for supplementation of media for T cell assays and in particular, Elispot has been a major challenge for assay performance, standardization, optimization, and reproducibility. The Assay Working Group of the Cancer Vaccine Consortium (CVC-CRI) has recently identified the choice of serum to be the leading cause for variability and suboptimal performance in large international Elispot proficiency panels. Therefore, a serum task force was initiated to compare the performance of commercially available serum-free media to laboratories’ own medium/serum combinations. The objective of this project was to investigate whether a serum-free medium exists that performs as well as lab-own serum/media combinations with regard to antigen-specific responses and background reactivity in Elispot. In this way, a straightforward solution could be provided to address the serum challenge. Eleven laboratories tested peripheral blood mononuclear cells (PBMC) from four donors for their reactivity against two peptide pools, following their own Standard Operating Procedure (SOP). Each laboratory performed five simultaneous experiments with the same SOP, the only difference between the experiments was the medium used. The five media were lab-own serum-supplemented medium, AIM-V, CTL, Optmizer, and X-Vivo. The serum task force results demonstrate compellingly that serum-free media perform as well as qualified medium/serum combinations, independent of the applied SOP. Recovery and viability of cells are largely unaffected by serum-free conditions even after overnight resting. Furthermore, one serum-free medium was identified that appears to enhance antigen-specific IFNγ-secretion.     

Comparison of the effectivity of different snare types for collecting and retaining hair from Eurasian Lynx (Lynx lynx)

Genetic methods are increasingly being used as noninvasive tools to survey populations of wild animals. One challenge of these methods is the sampling of genetic material from the target species. Genetic material of various predators, such as bears, canids, and felids, has been successfully obtained from both hair trapped in snares and scat. However, there is currently no standard procedure for sampling genetic material from the Eurasian Lynx (Lynx lynx). We tested established and newly developed hair snares in two near-natural lynx enclosures in the Bavarian Forest National Park. All snares consisted of a wooden post; they differed in the type of material attached to the post for snaring hair: carpet (velour with 40 nails), wildcat (spruce wood with 2–3?mm deep, horizontal and diagonal ridges), wire brush, doormat, or rubber bands (250?g of rubber bands wrapped around the post). We determined the acceptance of the hair snares by the animals by observing their behavior with the aid of video cameras. The number of rubbing events on the different trap types did not significantly differ, but the rubbing duration was longer for the doormat hair snare. The wire brush hair snare collected the highest total amount of hair and — beside the carpet — the highest amount of hair per unit of time. Almost all hair trapped on the wire brush snare were retained during a 2-week exposure to the elements outside of the enclosures. The results of our study may hold for other felid species with hair characteristics similar to those of lynx.     

Genetic covariation between effectiveness and cost of defence in aphids

Ecological immunology distinguishes between the long-term evolutionary costs of possessing defences against parasites and the short-term costs of using them. Evolutionary biologists have typically focused on the former in the search for constraints on the evolution of resistance. Here, we show in the peach-potato aphid, Myzus persicae, that short-term costs may be of equal evolutionary importance. Survivors of more resistant aphid clones suffered a higher reduction of fecundity upon parasitoid attack than survivors of more susceptible clones. This genetically based trade-off between benefits and costs of defence may limit the evolution of increased resistance and explain the maintenance of genetic variation for resistance under environmental variation in parasitism risk.     

Properties of photoreceptor-specific phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Mutations in the norpA gene drastically affect the phototransduction process in Drosophila. To study the biochemical characteristics of the norpA protein and its cellular and subcellular distributions, we have generated antisera against the major gene product of norpA. The antisera recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong norpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the norpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction.     

Peptidomimetic glutathione analogues as novel gammaGT stable GST inhibitors.

Elevated levels of glutathione-S-transferase (GST) isoenzymes are found in many tumor cells and are thought to play a role in the onset of multidrug resistance (MDR). To evaluate the contribution of GST to this process, inhibitors are needed. Glutathione (GSH) conjugates, although good GST inhibitors, cannot be used in vivo, because they are eliminated rapidly. In this paper, we describe the synthesis of a series of novel peptidomimetic glutathione analogues that are stabilized against peptidase mediated breakdown. The peptide bonds in GSH were replaced by isosteres, such as the 'reduced' amide (which was prepared using a novel method), N-methylamide, urethane, and methylene linkages. The in vitro evaluation of the compounds focuses on GST inhibition and stability towards gamma-glutamyl-transpeptidase (gammaGT), the main enzyme involved in GSH breakdown. The compounds were conjugated to the model electrophile ethacrynic acid (EA) to resemble GS-EA, an efficient GST inhibitor. All novel GSH-analogues were shown to inhibit rat liver cytosolic GSTs. Furthermore, peptidomimetic changes of the gamma-glutamyl-cysteine amide bond greatly improved stability towards gammaGT. These compounds may therefore be useful in the design of novel in vivo applicable GST inhibitors.