On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum--neurotensin, opiorphin, B27-KK10 epitope, NPY

The terminal homologation by CH(2) insertion into the peptides mentioned in the title is described. This involves replacement of the N-terminal amino acid residue by a β(2) - and of the C-terminal amino acid residue by a β(3) -homo-amino acid moiety (β(2) hXaa and β(3) hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N-terminal to the C-terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1) and its C-terminal fragment NT(8-13) are ligands of the G-protein-coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b-2e, for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5-1.3 vs. 0.6 nM). At the same time, one of the homologated NT analogs, 2c, survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8-13) (Tables 2 and 4, and Fig. 8) reveals that this N-terminal NT fragment folds to a turn in CD(3) OH. - In the case of the human analgesic opiorphin (3a), a pentapeptide, and of the HIV-derived B27-KK10 (4a), a decapeptide, terminal homologation (→3b and 4b, resp.) led to a 7- and 70-fold half-life increase in plasma (Fig. 9). With N-terminally homologated NPY, 5c, we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c, and 5c, were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability-increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed.

Thymic peptides for treatment of cancer patients

Background Purified thymus extracts (pTE) and synthetic thymic peptides (sTP) are thought to enhance the immune system of cancer patients in order to fight the growth of tumour cells and to resist infections due to immunosuppression induced by the disease and antineoplastic therapy. Objectives To evaluate the effectiveness of pTE and sTP for the management of cancer. Search methods We searched CENTRAL (The Cochrane Library 2010, Issue 3), MEDLINE, EMBASE, AMED, BIOETHICSLINE, BIOSIS, CATLINE, CISCOM, HEALTHSTAR, HTA, SOMED and LILACS (to February 2010). Selection criteria Randomised trials of pTE or sTP in addition to chemotherapy or radiotherapy, or both, compared to the same regimen with placebo or no additional treatment in adult cancer patients. Data collection and analysis Two authors independently extracted data from published trials. We derived odds ratios (OR) from overall survival (OS) and disease-free survival (DFS) rates, tumour response (TR) rates, and rates of adverse effects (AE) related to antineoplastic treatments. We used a random-effects model for meta-analysis. Main results We identified 26 trials (2736 patients). Twenty trials investigated pTE (thymostimulin or thymosin fraction 5) and six trials investigated sTP (thymopentin or thymosin α1). Twenty-one trials reported results for OS, six for DFS, 14 for TR, nine for AE and 10 for safety of pTE and sTP. Addition of pTE conferred no benefit on OS (RR 1.00, 95% CI 0.79 to 1.25); DFS (RR 0.97, 95% CI 0.82 to 1.16); or TR (RR 1.07, 95% CI 0.92 to 1.25). Heterogeneity was moderate to high for all these outcomes. For thymosin α1 the pooled RR for OS was 1.21 (95% CI 0.94 to 1.56, P = 0.14), with low heterogeneity; and 3.37 (95% CI 0.66 to 17.30, P = 0.15) for DFS, with moderate heterogeneity. The pTE reduced the risk of severe infectious complications (RR 0.54, 95% CI 0.38 to 0.78, P = 0.0008; I² = 0%). The RR for severe neutropenia in patients treated with thymostimulin was 0.55 (95% CI 0.25 to 1.23, P = 0.15). Tolerability of pTE and sTP was good. Most of the trials had at least a moderate risk of bias. Authors' conclusions Overall, we found neither evidence that the addition of pTE to antineoplastic treatment reduced the risk of death or disease progression nor that it improved the rate of tumour responses to antineoplastic treatment. For thymosin α1, there was a trend for a reduced risk of dying and of improved DFS. There was preliminary evidence that pTE lowered the risk of severe infectious complications in patients undergoing chemotherapy or radiotherapy.

Plant volatiles: production, function and pharmacology

Plant volatiles typically occur as a complex mixture of low-molecular weight lipophilic compounds derived from different biosynthetic pathways, and are seemingly produced as part of a defense strategy against biotic and abiotic stress, as well as contributing to various physiological functions of the producer organism. The biochemistry and molecular biology of plant volatiles is complex, and involves the interplay of several biochemical pathways and hundreds of genes. All plants are able to store and emit volatile organic compounds (VOCs), but the process shows remarkable genotypic variation and phenotypic plasticity. From a physiological standpoint, plant volatiles are involved in three critical processes, namely plant–plant interaction, the signaling between symbiotic organisms, and the attraction of pollinating insects. Their role in these ‘‘housekeeping’’ activities underlies agricultural applications that range from the search for sustainable methods for pest control to the production of flavors and fragrances. On the other hand, there is also growing evidence that VOCs are endowed with a range of biological activities in mammals, and that they represent a substantially under-exploited and still largely untapped source of novel drugs and drug leads. This review summarizes recent major developments in the study of biosynthesis, ecological functions and medicinal applications of plant VOCs.

A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities

CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.

Community mean traits as additional indicators to monitor effects of land-use intensity on grassland plant diversity

Semi-natural grasslands, biodiversity hotspots in Central-Europe, suffer from the cessation of traditional land-use. Amount and intensity of these changes challenge current monitoring frameworks typically based on classic indicators such as selected target species or diversity indices. Indicators based on plant functional traits provide an interesting extension since they reflect ecological strategies at individual and ecological processes at community levels. They typically show convergent responses to gradients of land-use intensity over scales and regions, are more directly related to environmental drivers than diversity components themselves and enable detecting directional changes in whole community dynamics. However, probably due to their labor- and cost intensive assessment in the field, they have been rarely applied as indicators so far. Here we suggest overcoming these limitations by calculating indicators with plant traits derived from online accessible databases. Aiming to provide a minimal trait set to monitor effects of land-use intensification on plant diversity we investigated relationships between 12 community mean traits, 2 diversity indices and 6 predictors of land-use intensity within grassland communities of 3 different regions in Germany (part of the German ‘Biodiversity Exploratory’ research network). By standardization of traits and diversity measures, use of null models and linear mixed models we confirmed (i) strong links between functional community composition and plant diversity, (ii) that traits are closely related to land-use intensity, and (iii) that functional indicators are equally, or even more sensitive to land-use intensity than traditional diversity indices. The deduced trait set consisted of 5 traits, i.e., specific leaf area (SLA), leaf dry matter content (LDMC), seed release height, leaf distribution, and onset of flowering. These database derived traits enable the early detection of changes in community structure indicative for future diversity loss. As an addition to current monitoring measures they allow to better link environmental drivers to processes controlling community dynamics.

Central European plant species from more productive habitats are more invasive at a global scale

Aim: Accumulating evidence indicates that species may be pre-adapted for invasion success in new ranges. In the light of increasing global nutrient accumulation, an important candidate pre-adaptation for invasiveness is the ability to grow in nutrient-rich habitats. Therefore we tested whether globally invasive species originating from Central Europe have come from more productive rather than less productive habitats. A further important candidate pre-adaptation for invasiveness is large niche width. Therefore, we also tested whether species able to grow across habitats with a wider range of productivity are more invasive. Location: Global with respect to invasiveness, and Central European with respect to origin of study species. Methods  We examined whether average habitat productivity and its width across habitats are significant predictors of the success of Central European species as aliens and as weeds elsewhere in the world based on data in the Global Compendium of Weeds. The two habitat productivity measures were derived from nutrient indicator values (after Ellenberg) of accompanying species present in vegetation records of the comprehensive Czech National Phytosociological Database. In the analyses, we accounted for phylogenetic relatedness among species and for size of the native distribution ranges. Results: Species from more productive habitats and with a wider native habitat-productivity niche in Central Europe have higher alien success elsewhere in the world. Weediness of species increased with mean habitat productivity. Niche width was also an important determinant of weediness for species with their main occurrence in nutrient-poor habitats, but not for those from nutrient-rich habitats. Main conclusions: Our results indicate that Central European plant species from productive habitats and those species from nutrient-poor habitat with wide productivity-niche are pre-adapted to become invasive. These results suggest that the world-wide invasion success of many Central European species is likely to have been promoted by the global increase of resource-rich habitats.

Use of Genetically Modified Glial Cells Overexpressing Laminin α1-Chain Peptides in Neurite Outgrowth Studies

1. C6 glioma cells were transfected with two constructs carrying C-terminal laminin alpha1-chain sequences of 117 and 114 bp length, respectively. These sequences are specifically known to code for peptides which have neurite-promoting activity. 2. The stable expression and secretion of the two peptides was detected by Northern and Western blot analysis. 3. Primary neuronal cultures derived from embryonic mouse forebrain were cocultured with these transfected cells and exhibited a substantial increase in neurite outgrowth and in survival time. Conditioned media from the transfected cells generated similar effects. 4. Organotypic cultures from embryonic mouse brain were used as a second system as being closer to the in vivo situation. Again, coculture of brain slices with transfected cells or treatment with laminin peptide-containing media increased neuronal outgrowth.

Senescence-related gene expression profiles of rosette leaves of Arabidopsis thaliana: Leaf age versus plant age

Senescence is a form of programmed cell death (PCD) which leads to the death of whole organs, e.g., leaves or flowers, and eventually to the death of entire plants. Like all forms of PCD, senescence is a highly regulated and energy consuming process. Senescence parameters, like protein content, chlorophyll content, expression of photosynthesis-associated genes or senescence-associated genes (SAGs), reveal that senescence occurs in old leaves derived from young plants (6 week old) as well as in young leaves derived from older plants (8 week old), indicating that it is governed by the actual age of the leaves. in order to analyse the differential gene expression profiles during leaf senescence, hybridizations of high-density genome arrays were performed with: i) individual leaves within the rosette of a 6-week-old plant and ii) leaves of the same position within the rosette but harvested from plants of different ages, ranging from 5 to 8 weeks. Cluster and genetree analyses, according to the expression pattern revealed that genes which are up-regulated with respect to the age of the entire plant, showed completely different expression profiles with respect to the age of the individual leaves within one rosette. This was observed even though the actual difference in leaf age was approximately the same. This indicates that gene expression appears to be governed by different parameters: i) the age of the individual leaf and ii) the age and developmental stage of the entire plant.

Influences of porcine circo-, arteriviruses and cathelicidins in plasmacytoid dendritic cell-derived interferon-alpha responses

Type I interferons (IFNs), mainly IFN-α/β play a crucial role in innate defense against viruses. In addition to their direct antiviral activity, type I IFNs have antitumoral and immunomodulatory effects. Although all cells are virtually able to induce IFN-α, the plasmacytoid dendritic cell (pDC) subset represents the ultimate producers of IFN-α as well as other proinflammatory cytokines. Due to the specific expression of TLR7 and TLR9 recognizing single-stranded (ss) RNA and unmethylated CpG motifs respectively, pDCs can secrete up to 1000 times more IFN-α than any cellular types. Additionally, it is well known that several cytokines including type I and II IFNs, Flt3-L, IL-4 and GM-CSF favor pDC-derived IFN-α responses to unmethylated CpG motifs. In a first step, we aimed to characterize and clarify the interactions of two porcine viruses with pDCs. The double-stranded DNA replicative forms of porcine circovirus type 2 (PCV2) were demonstrated to inhibit CpG-induced IFN- α by pDCs. Our study showed that none of the cytokines known to enhance pDC responsiveness can counter-regulate the PCV2-mediated inhibition of IFN-α induced by CpG, albeit IFN-γ significantly reduced the level of inhibition. Interestingly, the presence of IFN-γ enabled pDCs to induce IFN-α to low doses of PCV2. We also noted that after DNase treatment, PCV2 preparations were still able to stimulate pDCs. These data suggest that encapsulated viral ssDNA promotes the induction of IFN-α in pDCs treated with IFN-γ whereas free DNA, presumably as double-stranded forms, was responsible for inhibiting pDC responses. Regarding PRRSV, it has been reported that North American isolates did not induce and even inhibited IFN-α response in pDCs. However, PRRSV infection was also shown to lead to an induction of IFN-α in the serum and in the lungs suggesting that certain cells are responsive to the virus. Contrasting to previous reports we found that numerous PRRSV isolates directly induced IFN-α in pDCs. This response was still observed after UV-inactivation of viruses and required TLR7 signaling. The inhibition of CpG-induced IFN-α was weak and strain dependent, again contrasting with a previous report. We also observed that IFN-γ and IL-4 enhanced IFN-α response to two prototype strains, VR-2332 and LVP23. In summary, we demonstrated that both PCV2 and PRRSV promote IFN-α secretion in pDCs in vitro suggesting that IFN-α detected in PCV2- or PRRSV-infected animal might originate from pDCs. On the other hand, PRRSV replication is restricted to the macrophage (MΦ) lineage. These innate immune cells represent a heterogeneous population which can be induce to “classical” (M1) and “alternative” (M2) activated MΦ acquiring inflammatory or “wound-healing” functional properties, respectively. Nonetheless, little is known about the effect of polarization into M1 or M2 and the susceptibility of these cells to PRRSV. Thus, we examined the impact of cytokine on MΦ polarization into M1 or M2. Infections of these cells by several PRRSV isolates enabled the discrimination of PRRSV isolate in a genotype- and irulencedependent manner in M1 and IFN-β-activated MΦ. In contrast, the expression of PRRSV nucleocapsid in M2 or inactivated MΦ was indistinguishable among the PRRSV isolates tested. In the last part of my Thesis, we investigated the influence of three synthetic porcine cathelicidin peptides for their ability to deliver nucleic acid to pDCs. We reported that all cathelicidins tested can complex and quickly deliver nucleic acids resulting in IFN-α induction. Moreover, we show that the typical α- helical amphipathic conformation is required to mediate killing of bacteria but not for inducing IFN-α secretion by pDCs. Furthermore, we found that E.coli treated with one of these cathelicidins is able to induce significantly higher levels of IFN-α compared to a non-sense version of the peptide. These data suggest that cathelicidins could influence the immune response in a two-step process. First, these peptides target bacteria leading to cell lysis. In turn, cathelicidins form complexes and deliver extracellular microbial nucleic acids released into pDCs. These pDC-derived IFN-α responses could be of particular relevance in driving the adaptive immune responses against microbial infections.

Seasonal Variation in the Capacity for Plant Trait Measures to Predict Grassland Carbon and Water Fluxes

There is a need for accurate predictions of ecosystem carbon (C) and water fluxes in field conditions. Previous research has shown that ecosystem properties can be predicted from community abundance-weighted means (CWM) of plant functional traits and measures of trait variability within a community (FDvar). The capacity for traits to predict carbon (C) and water fluxes, and the seasonal dependency of these trait-function relationships has not been fully explored. Here we measured daytime C and water fluxes over four seasons in grasslands of a range of successional ages in southern England. In a model selection procedure, we related these fluxes to environmental covariates and plant biomass measures before adding CWM and FDvar plant trait measures that were scaled up from measures of individual plants grown in greenhouse conditions. Models describing fluxes in periods of low biological activity contained few predictors, which were usually abiotic factors. In more biologically active periods, models contained more predictors, including plant trait measures. Field-based plant biomass measures were generally better predictors of fluxes than CWM and FDvar traits. However, when these measures were used in combination traits accounted for additional variation. Where traits were significant predictors their identity often reflected seasonal vegetation dynamics. These results suggest that database derived trait measures can improve the prediction of ecosystem C and water fluxes. Controlled studies and those involving more detailed flux measurements are required to validate and explore these findings, a worthwhile effort given the potential for using simple vegetation measures to help predict landscape-scale fluxes.

Immunologic response to the survivin-derived multi-epitope vaccine EMD640744 in patients with advanced solid tumors

PURPOSE Survivin is a member of the inhibitor-of-apoptosis family. Essential for tumor cell survival and overexpressed in most cancers, survivin is a promising target for anti-cancer immunotherapy. Immunogenicity has been demonstrated in multiple cancers. Nonetheless, few clinical trials have demonstrated survivin-vaccine-induced immune responses. EXPERIMENTAL DESIGN This phase I trial was conducted to test whether vaccine EMD640744, a cocktail of five HLA class I-binding survivin peptides in Montanide(®) ISA 51 VG, promotes anti-survivin T-cell responses in patients with solid cancers. The primary objective was to compare immunologic efficacy of EMD640744 at doses of 30, 100, and 300 μg. Secondary objectives included safety, tolerability, and clinical efficacy. RESULTS In total, 49 patients who received ≥2 EMD640744 injections with available baseline- and ≥1 post-vaccination samples [immunologic-diagnostic (ID)-intention-to-treat] were analyzed by ELISpot- and peptide/MHC-multimer staining, revealing vaccine-activated peptide-specific T-cell responses in 31 patients (63 %). This cohort included the per study protocol relevant ID population for the primary objective, i.e., T-cell responses by ELISpot in 17 weeks following first vaccination, as well as subjects who discontinued the study before week 17 but showed responses to the treatment. No dose-dependent effects were observed. In the majority of patients (61 %), anti-survivin responses were detected only after vaccination, providing evidence for de novo induction. Best overall tumor response was stable disease (28 %). EMD640744 was well tolerated; local injection-site reactions constituted the most frequent adverse event. CONCLUSIONS Vaccination with EMD640744 elicited T-cell responses against survivin peptides in the majority of patients, demonstrating the immunologic efficacy of EMD640744.

The maize lipoxygenase, ZmLOX10 , mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, majorregulatory genes and the signals that modulate these defense metabolites are vastly understudied, especiallyin important agro-economic monocot species. Here we show that products and signals derived from a singleZea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbiv-ory. We provide genetic evidence that two 13-LOXs, ZmLOX10 and ZmLOX8, specialize in providing substratefor the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the spe-cialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indi-cating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression ofJA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10-derived signaling isrequired for LOX8-mediated JA. The possible role of GLVs in JA signaling is supported by their ability to par-tially restore wound-induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produceGLVs and JA led to dramatic reductions in herbivore-induced plant volatiles (HIPVs) and attractiveness toparasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic linkto the diurnal regulation of GLVs and HIPVs. GLV-, JA- and HIPV-deficient lox10 mutants display compro-mised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence thatLOX10-dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene toagro-ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.