Supramolecular assembly of collagen triblock peptides

The relationship between primary sequence and collagen triple-helix formation is relatively well characterized, while higher levels of structural assembly from these sequences is poorly understood. To address this gap, a new collagen-like triblock peptide design was used to study the relationship between amino acid sequence and supramolecular assembly. Four collagen-like peptides with the sequence (Glu)(5)(Gly-Xaa-Hyp-Gly-Pro-Hyp)(6)(Glu)(5) and corresponding to Xaa = alanine, proline, serine, or valine, and an analogous peptide without the glutamic acid end blocks, were solubilized in water at high concentrations (20-150 mg/mL) and analyzed in optical polarizing microscopy and transmission electron microscopy. Some of the peptides self-assembled into supramolecular structures, the nature of which was determined by the core collagen-like sequence. The globular end blocks appeared necessary for these short triple-helix-forming peptides to spontaneously organize into supramolecular structures in solution and also provided enhanced thermal stability based on CD analysis. The results indicate a strong dependence of the peptide triblock assembly behavior on the identity of the guest residue Xaa; nematic order when Xaa was valine, no organization when Xaa was serine, and banded spherulites displaying a cholesteric-like twist when Xaa was proline or alanine. According to these results, the identity of the amino acid in position Xaa of the triplet Gly-Xaa-Yaa dramatically determined the type of supramolecular assembly formed by short triple helices based on collagen-triblock like sequences. Moreover, the structural organization observed for these collagen-triblock peptides was analogous to some assemblies observed for native collagen in vivo and in vitro. The amino acid sequence in the native collagen proteins may therefore be a direct determinant of the different supramolecular architectures found in connective tissues.     

Inbreeding depression in a rare plant,Scabiosa canescens (Dipsacaceae)

Plants from a population of Scabiosa canescens, a locally rare species with a narrow ecological amplitude, were raised under uniform growth conditions to examine the phenotypic effects of one generation selfing and outcrossing. Particular attention was given to direct components of fitness (seedling biomass, rosette leaf number, head number, flower number per head), but two morphological characters (plant height, flower size) were also considered. Estimates of inbreeding depression (delta), adjusted for maternal effects and lack of balance, were compared and tested for significance using randomization and boostrap procedures. Inbreeding significantly depressed several characters during both early and late stages of the life cycle, with delta ranging from 0.14 (flower size) to 0.37 (seedling biomass). Based on these and other results, we propose that S. canescens is susceptible to inbreeding and that the genetic basis of inbreeding depression varies across life stages.     

Modifications in synthesis strategy improve the yield and efficacy of geldanamycin-herceptin immunoconjugates

Geldanamycin (GA) was modified with N-tert-butyloxycarbonyl-1,3-diaminopropane to introduce a latent primary amine. After deprotection, this primary amine provided a site for introduction of a maleimide group that enabled linkage to proteins. This maleimido derivative of geldanamycin (GMB-APA-GA) was linked to the monoclonal antibody Herceptin after the antibody had been modified with Traut's reagent to introduce thiol groups. By this sequence, a new immunoconjugate (H:APA-GA) was generated that showed greater antiproliferative activity than the previously reported analogous immunoconjugate created with a 1,4-diaminobutane spacer derivative of geldanamycin to form an immunoconjugate, H:ABA-GA. Both immunoconjugates inhibited in vitro the growth of MDA-361/DYT2 cells, a cell line overexpressing the HER2 antigen, while Herceptin alone was ineffective. However, H:APA-GA showed better efficacy than H:ABA-GA (IC(50) = 0.2 vs 0.58 mg/mL and cell doubling time >12 vs 6 days, respectively). Results of the in vivo therapy experiments in a xenograft model were consistent with the in vitro findings. Treatment with Herceptin prolonged the survival of the tumor-bearing mice when compared with the control group, but H:ABA-GA and H:APA-GA were each more efficacious than unmodified Herceptin. However, unlike H:ABA-GA, the immunoconjugate H:APA-GA caused stable tumor regression (in 25% of the recipients), showing a qualitative improvement with potential clinical relevance.     

Synthesis and evaluation of antiproliferative activity of a geldanamycin-Herceptin immunoconjugate

Geldanamycin was modified with 1,4-diaminobutane to introduce a primary amine that was subsequently employed to provide a maleimide for protein linkage. Monoclonal antibody Herceptin was then derivatized to generate thiol groups that reacted with the maleimide derivative to produce the immunoconjugate. The product showed antiproliferative activity greater than native Herceptin.     

Synthesis of a triply phosphorylated pentapeptide from human tau-protein

Two different strategies for the synthesis of a triply phosphorylated pentapeptide are described. In both cases a monophosphorylated selectively N-deprotected tripeptide is employed as C-terminal fragment. Coupling of this building block with a C-terminally unmasked bis-phosphorylated seryl-dipeptide unexpectedly failed due to decomposition of this peptide upon activation with different coupling reagents. Instead stepwise N-terminal elongation of the peptide chain with serine derivatives and subsequent O-phosphorylation of the serine OH-groups was successful. These results indicate that assembly of multiply phosphorylated peptides from preformed multiply phosphorylated phosphopeptide building blocks in general may be problematic and that a stepwise elongation of the amino acid chain may be preferable.     

Multiple, non-allelic, intein-coding sequences in eukaryotic RNA polymerase genes

Background  

Inteins are self-splicing protein elements. They are translated as inserts within host proteins that excise themselves and ligate the flanking portions of the host protein (exteins) with a peptide bond. They are encoded as in-frame insertions within the genes for the host proteins. Inteins are found in all three domains of life and in viruses, but have a very sporadic distribution. Only a small number of intein coding sequences have been identified in eukaryotic nuclear genes, and all of these are from ascomycete or basidiomycete fungi.     

Optimum contribution selection in large general tree breeding populations with an application to Scots pine

Development of selection methods that optimises selection differential subject to a constraint on the increase of inbreeding (or coancestry) in a population is an important part of breeding programmes. One such method that has received much attention in animal breeding is the optimum contribution (OC) dynamic selection method. We implemented the OC algorithm and applied it to a diallel progeny trial of Pinus sylvestris L. (Scots pine) focussing on two traits (total tree height and stem diameter). The OC method resulted in a higher increase in genetic gain (8–30%) compared to the genetic gain achieved using standard restricted selection method at the same level of coancestry constraint. Genetic merit obtained at two different levels of restriction on coancestry showed that the benefit of OC was highest when restriction was strict. At the same level of genetic merit, OC decreased coancestry with 56 and 39% for diameter and height, respectively, compared to the level of coancestry obtained using unrestricted truncation selection. Inclusion of a dominance term in the statistical model resulted in changes in contribution rank of trees with 7 and 13% for diameter and height, respectively, compared to results achieved by using a pure additive model. However, the genetic gain was higher for the pure additive model than for the model including dominance for both traits.     

Bayesian Inference of Genetic Parameters Based on Conditional Decompositions of Multivariate Normal Distributions

It is widely recognized that the mixed linear model is an important tool for parameter estimation in the analysis of complex pedigrees, which includes both pedigree and genomic information, and where mutually dependent genetic factors are often assumed to follow multivariate normal distributions of high dimension. We have developed a Bayesian statistical method based on the decomposition of the multivariate normal prior distribution into products of conditional univariate distributions. This procedure permits computationally demanding genetic evaluations of complex pedigrees, within the user-friendly computer package WinBUGS. To demonstrate and evaluate the flexibility of the method, we analyzed two example pedigrees: a large noninbred pedigree of Scots pine (Pinus sylvestris L.) that includes additive and dominance polygenic relationships and a simulated pedigree where genomic relationships have been calculated on the basis of a dense marker map. The analysis showed that our method was fast and provided accurate estimates and that it should therefore be a helpful tool for estimating genetic parameters of complex pedigrees quickly and reliably.MUCH effort in genetics has been devoted to revealing the underlying genetic architecture of quantitative or complex traits. Traditionally, the polygenic model has been used extensively to estimate genetic variances and breeding values of natural and breeding populations, where an infinite number of genes is assumed to code for the trait of interest (Bulmer 1971; Falconer and Mackay 1996). The genetic variance of a quantitative trait can be decomposed into an additive part that corresponds to the effects of individual alleles and a part that is nonadditive because of interactions between alleles. Attention has generally been focused on the estimation of additive genetic variance (and heritability), since additive variation is directly proportional to the response of selection via the breeder''s equation (Falconer and Mackay 1996, Chap. 11). However, to estimate additive genetic variation and heritability accurately, it can be important to identify potential nonadditive sources in genetic evaluations (Misztal 1997; Ovaskainen et al. 2008; Waldmann et al. 2008), especially if the pedigree being analyzed contains a large proportion of full-sibs and clones, as these in particular give rise to nonadditive genetic relationships (Lynch and Walsh 1998, pp. 145). The polygenic model using pedigree and phenotypic information, i.e., the animal model (Henderson 1984), has been the model of choice for estimating genetic parameters in breeding and natural populations (Abney et al. 2000; Sorensen and Gianola 2002; O′Hara et al. 2008).Recent breakthroughs in molecular techniques have made it possible to create genome-wide, single nucleotide polymorphism (SNP) maps. These maps have helped to uncover a vast amount of new loci responsible for trait expression and have provided general insights into the genetic architecture of quantitative traits (e.g., Valdar et al. 2006; Visscher 2008; Flint and Mackay 2009). These insights can help when calculating disease risks in humans, when attempting to increase the yield from breeding programs, and when estimating relatedness in conservation programs. High-density SNPs of many species of importance to science and agriculture can now be scored quickly and relatively cheaply, for example, in mice (Valdar et al. 2006), chickens (Muir et al. 2008), and dairy cattle (VanRaden et al. 2009).In the analysis of populations of breeding stock, the inclusion of dense marker data has improved the predictive ability (i.e., reliability) of genetic evaluations compared to the traditional phenotype model, both in simulations (Meuwissen et al. 2001; Calus et al. 2008; Hayes et al. 2009) and when using real data (Legarra et al. 2008; VanRaden et al. 2009; González-Recio et al. 2009). Meuwissen et al. (2001) suggested that the effect of all markers should first be estimated, and then summed, to obtain genomic estimated breeding values (GEBVs). An alternative procedure, where all markers are used to compute the genomic relationship matrix (in place of the additive polygenic relationship matrix) has also been suggested (e.g., Villanueva et al. 2005; VanRaden 2008; Hayes et al. 2009); this matrix is then incorporated into the statistical analysis to estimate GEBVs. A comparison of both procedures (VanRaden 2008) yielded similar estimates of GEBVs in cases where the effect of an individual allele was small. In addition, if not all pedigree members have marker information, a combined relationship matrix derived from both genotyped and ungenotyped individuals could be computed; this has been shown to increase the accuracy of GEBVs (Legarra et al. 2009; Misztal et al. 2009). Another plausible option to incorporate marker information is to use low-density SNP panels within families and to trace the effect of SNPs from high-density genotyped ancestors, as suggested by Habier et al. (2009) and Weigel et al. (2009). However, fast and powerful computer algorithms, which can use the marker information as efficiently as possible in the analysis of quantitative traits, are needed to obtain accurate GEBVs from genome-wide marker data.This study describes the development of an efficient Bayesian method for incorporating general relationships into the genetic evaluation procedure. The method is based on expressing the multivariate normal prior distribution as a product of one-dimensional normal distributions, each conditioned on the descending variables. When evaluating the genetic parameters of natural and breeding populations, high-dimensional distributions are often used as prior distributions of various genetic effects, such as the additive polygenic effect (Wang et al. 1993), multivariate additive polygenic effects (Van Tassell and Van Vleck 1996), and quantitative trait loci (QTL) effects via the identical-by-decent matrix (Yi and Xu 2000). A Bayesian framework is adopted to obtain posterior distributions of all unknown parameters, estimated by using Markov chain Monte Carlo (MCMC) sampling algorithms in the software package WinBUGS (Lunn et al. 2000, 2009). By performing prior calculations in the form of the factorized product of simple univariate conditional distributions, the computational time of the MCMC estimation procedure is reduced considerably. This feature permits rapid inference for both the polygenic model and the genomic relationship model. Moreover, the decomposition allows for inbreeding of varying degree, since the correct genetic covariance structure can be inferred into the analysis. In this article, we test the method on two previously published pedigree data sets: phenotype data from a large pedigree of Scots pine, incorporation of information on both additive and dominance genetic relationships (Waldmann et al. 2008); and genomic information obtained from a genome-wide scan of a simulated animal population (Lund et al. 2009).     

Loss of the TGFβ-Activating Integrin αvβ8 on Dendritic Cells Protects Mice from Chronic Intestinal Parasitic Infection via Control of Type 2 Immunity

Chronic intestinal parasite infection is a major global health problem, but mechanisms that promote chronicity are poorly understood. Here we describe a novel cellular and molecular pathway involved in the development of chronic intestinal parasite infection. We show that, early during development of chronic infection with the murine intestinal parasite Trichuris muris, TGFβ signalling in CD4+ T-cells is induced and that antibody-mediated inhibition of TGFβ function results in protection from infection. Mechanistically, we find that enhanced TGFβ signalling in CD4+ T-cells during infection involves expression of the TGFβ-activating integrin αvβ8 by dendritic cells (DCs), which we have previously shown is highly expressed by a subset of DCs in the intestine. Importantly, mice lacking integrin αvβ8 on DCs were completely resistant to chronic infection with T. muris, indicating an important functional role for integrin αvβ8-mediated TGFβ activation in promoting chronic infection. Protection from infection was dependent on CD4+ T-cells, but appeared independent of Foxp3+ Tregs. Instead, mice lacking integrin αvβ8 expression on DCs displayed an early increase in production of the protective type 2 cytokine IL-13 by CD4+ T-cells, and inhibition of this increase by crossing mice to IL-4 knockout mice restored parasite infection. Our results therefore provide novel insights into how type 2 immunity is controlled in the intestine, and may help contribute to development of new therapies aimed at promoting expulsion of gut helminths.     

IL-2-PE40 prevents the development of tumors in mice injected with IL-2 receptor expressing EL4 transfectant tumor cells

A number of different immunotherapeutic reagents are currently being developed to target IL-2R for the treatment of leukemia, graft rejection, and certain autoimmune diseases. Previously, we have shown that IL-2-PE40, a chimeric protein composed of human IL-2 linked to the N-terminus of a truncated form of Pseudomonas exotoxin (PE), could effectively kill a variety of cell lines in vitro expressing either low, intermediate, or high affinity IL-2R. Here, we demonstrate that IL-2-PE40 can successfully retard or prevent the growth of a lethal ascites tumor or a solid tumor composed of EL4J murine thymoma cells transfected with the p55 murine IL-2R. The transfected line, EL4J-3.4, expresses 1,000 to 3,000 high affinity IL-2R. Survival extension in the ascites model was achieved by initiating treatment either after 4 to 6 h or within 5 days post-tumor injection in both athymic nude and C57BL/6 mice. Similarly, the growth of an aggressive s.c. solid tumor could also be inhibited. Extension of survival was not achieved either by using the truncated toxin alone not attached to IL-2 or by using an IL-2-PE40Asp553 mutant lacking a functional toxin. Survival extension was not caused by IL-2 activated NK or other host effector mechanisms as IL-2-PE40 was unable to prevent the receptor-negative EL4J parental line from forming a lethal ascites or a solid tumor. Thus, IL-2-PE40 is a potent, specific cytolytic reagent that may prove useful in the arsenal of anti-IL-2R immunotherapeutics.