Evolution by recombination and transspecies polymorphism in the MHC class I gene of Xenopus laevis

The patterns of major histocompatibility complex (MHC) evolution involve duplications, deletions, and independent divergence of loci during episodes punctuated by natural selection. Major differences in MHC evolution among taxa have previously been attributed to variation in linkage patterns of class I and class II MHC genes. Here we characterize patterns of evolution in the MHC class Ia gene of Xenopus laevis in terms of polymorphism, recombination, and extent of transspecies polymorphism. We also compare these patterns to see if a correlation exists with linkage or separation of the MHC class I and class II regions as seen in amphibians and teleost fishes. In X. laevis, we find high levels of polymorphism. Also, genetic exchange is relatively frequent and occurs in intron II, reshuffling allelic forms of exons 2 and 3. Evolutionary relationships among class I alleles show an intermingling of alleles from divergent Xenopus species rather than a species-specific clustering. Results indicate that the patterns of evolution are similar to those found in salmonid fishes and are different from the mode of evolution seen in primates. Similar patterns of class Ia evolution in salmonid fishes and X. laevis suggest that nonlinkage of class I and class II regions alone is insufficient to explain some patterns of MHC evolution in salmonids.     

Polymorphism, natural selection, and structural modeling of class Ia MHC in the African clawed frog (Xenopus laevis)

In the African clawed frog (Xenopus laevis), two deeply divergent allelic lineages of multiple genes of the class I MHC region have been discovered. For the MHC class I UAA locus, functional differences and the molecular basis for lineages maintenance are unknown. Alleles of linked class I region genes also exhibit strong disequilibrium with specific MHC alleles, but the underlying cause is not clear. We use MHC class Ia sequence data to estimate substitution rates and investigate structural differences between allelic lineages from protein models. Results indicate the operation of natural selection, and differences in the steric properties in the F pocket of the peptide-binding region among lineages. Variability in this pocket likely enables allelic lineages to bind very different sets of peptides and to interact differently with MHC chaperones in the endoplasmic reticulum. These results constitute evidence of the molecular evolutionary basis for 1) the maintenance of allelic lineages, 2) functional differences among lineages, and 3) strong linkage disequilibrium of allelic variants of class I region genes in X. laevis.Electronic Supplementary Material Supplementary material is available for this article at     

Identification of a novel class of inhibitor of human and Escherichia coli thymidine phosphorylase by in silico screening

Structure-based computational screening of the National Cancer Institute database of anticancer compounds identified novel non-nucleobase-derived inhibitors of human thymidine phosphorylase as candidates for lead optimization. The hierarchical in silico screening strategy predicted potentially strong low molecular weight ligands exhibiting a range of molecular scaffolds. Of the thirteen ligands assayed for activity, all displayed inhibitory activity against Escherichia coli thymidine phosphorylase. One compound, hydrazine carboxamide 2-[(1-methyl-2,5-dioxo-4-pentyl-4-imidazolidinyl)methylene], was found to inhibit E. coli thymidine phosphorylase with an IC(50) value of 20 microM and an IC(50) value of 77 microM against human thymidine phosphorylase. As this hydantoin derivative lacks the undesirable ionic sites of existing tight-binding nucleobase-derived inhibitors, such as 5-chloro-6-[(2-iminopyrrolidin-1-yl)methyl]uracil hydrochloride, it provides an opportunity for the design of potent thymidine phosphorylase inhibitors with improved pharmacokinetic properties.     

Cutting edge: histamine is required for IL-4-driven eosinophilic allergic responses

Histamine is an important allergic mediator, and studies have defined roles for both histamine 1 and 4 receptors in allergic airway inflammation. In this study, we show that histamine is necessary to generate IL-4-driven eosinophilic inflammation, as histamine-deficient mice cannot generate eosinophilic lung inflammation in response to intratracheal IL-4 and exogenous histamine restores responsiveness. This is histamine 2 receptor (H2R) dependent because H2R knockout mice fail to respond to IL-4, and a H2R agonist restores inflammation in histidine decarboxylase knockout. Furthermore, alveolar epithelial cells require H2R to produce CCL24, an eosinophil recruitment factor, whereas H2R blockade reduces CCL24 production from wild-type cells. In an allergic inflammation model, H2R knockout mice show significantly reduced eosinophilic inflammation and CCL24 expression. These data demonstrate a previously unidentified role for H2R in allergic inflammation and establishes a synergy between endogenous histamine and IL-4 that supports eosinophilic recruitment to the lung.     

Assembly of the BclB glycoprotein into the exosporium and evidence for its role in the formation of the exosporium ‘cap’ structure in Bacillus anthracis

The outermost layer of the Bacillus anthracis spore consists of an exosporium comprised of an outer hair‐like nap layer and an internal basal layer. A major component of the hair‐like nap is the glycosylated collagen‐like protein BclA. A second collagen‐like protein, BclB, is also present in the exosporium. BclB possesses an N‐terminal sequence that targets it to the exosporium and is similar in sequence to a cognate targeting region in BclA. BclB lacks, however, sequence similarity to the region of BclA thought to mediate attachment to the basal layer via covalent interactions with the basal layer protein BxpB. Here we demonstrate that BxpB is critical for correct localization of BclB during spore formation and that the N‐terminal domains of the BclA and BclB proteins compete for BxpB‐controlled assembly sites. We found that BclB is located principally in a region of the exosporium that excludes a short arc on one side of the exosporium (the so‐called bottle‐cap region). We also found that in bclB mutant spores, the distribution of exosporium proteins CotY and BxpB is altered, suggesting that BclB has roles in exosporium assembly. In bclB mutant spores, the distance between the exosporium and the coat, the interspace, is reduced.     

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue^1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz^1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue^4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.     

Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2

The high level of immunogenicity of peptides displayed in dense repetitive arrays on virus-like particles makes recombinant VLPs promising vaccine carriers. Here, we describe a platform for vaccine development based on the VLPs of RNA bacteriophage MS2. It serves for the engineered display of specific peptide sequences, but will also allow the construction of random peptide libraries from which specific binding activities can be recovered by affinity selection. Peptides representing the V3 loop of HIV gp120 and the ECL2 loop of the HIV coreceptor, CCR5, were inserted into a surface loop of MS2 coat protein. Both insertions disrupted coat VLP assembly, apparently by interfering with protein folding, but these defects were suppressed efficiently by genetically fusing coat protein's two identical polypeptides into a single-chain dimer. The resulting VLPs displayed the V3 and ECL2 peptides on their surfaces where they showed the potent immunogenicity that is the hallmark of VLP-displayed antigens. Experiments with random-sequence peptide libraries show the single-chain dimer to be highly tolerant of six, eight and ten amino acid insertions. MS2 VLPs support the display of a wide diversity of peptides in a highly immunogenic format, and they encapsidate the mRNAs that direct their synthesis, thus establishing the genotype/phenotype linkage necessary for recovery of affinity-selected sequences. The single-chain MS2 VLP therefore unites in a single structural platform the selective power of phage display with the high immunogenicity of VLPs.     

Morphology of fibroblasts grown on substrates formed by dielectrophoretically aligned carbon nanotubes

Multiwall carbon nanotube templates formed on the surfaces of planar interdigitated microelectrode arrays by means of AC electric field-guided assembly are being explored as potential substrates for tissue engineering. The objective of the present study is to examine whether surface patterns of aligned multiwall carbon nanotubes can have an effect on cell growth, morphology, and alignment. Bovine fibroblasts grown on aligned carbon nanotubes for a period of 2 weeks were found to have raised bodies and pronounced cell extensions for anchoring themselves to the substrate similar to that of the cells found in native tissues. On the other hand, cells grown on various control surfaces had a flat, circular morphology. The cell cultures were visualized by means of SEM imaging and the resulting morphologies were statistically analyzed and compared.