Hydration of a hydrophobic cavity and its functional role: a simulation study of human interleukin-1beta

Molecular dynamics simulations reveal that the hydrophobic cavity in human cytokine Interleukin-1beta is hydrated and can dynamically accommodate between one and four water molecules. These waters have residence times > 500 ps and can give rise to detectable NOEs, in agreement with NMR observations of Ernst et al. (Science 1995; 267:1813-1817). The waters also display high positional disorder within the cavity, which explains why they have not been resolved crystallographically. The average distribution of water molecules over time within the cavity matches well the low resolution electron density extracted by Yu et al. (Proc Natl Acad Sci 1999; 96:103-108). The water molecules hydrate the hydrophobic cavity preferentially as complex clusters. These clusters result from a combination of hydrogen bonds between the waters and stabilizing interactions between the waters and aromatic rings forming the cavity. Free energy estimates suggest that it takes 4-waters to hydrate the cavity in a thermodynamically stable manner leading to a gain in free energy of transfer from bulk of approximately approximately 3.6 kcal/mol. This arises from the existence of the water clusters in multiple hydrogen bonded states. In addition, the waters are found to migrate either individually or as clusters out of the cavity through several pathways. The upper limit for one-dimensional diffusion of the waters within the protein matrix is 4 A/ps (relative to 6 A/ps for bulk). Simulations reveal pathways in addition to those identified crystallographically, with motions controlled by the rotations of sidechains. We find that only when the hydrophobic cavity is hydrated, do correlated motions couple distant sites with the sites that make contact with the receptor and this data partly offers an explanation of experimental mutagenesis data. Simulations, together with recent observations based on mutagenesis by Heidary et al. (J Mol Biol 2005; 353:1187-1198) that hydrogen bond networks couple motions across long distances in interleukin-1beta, lead us to hypothesize that the hydration of the cavity (conserved across mammals) can thermodynamically enhance hydrogen bond networks to enable coupling across long distances by acting as a plug and this in turn enables a kinetic control of the rate of transmission of signals.     

Specific adeno-associated virus serotypes facilitate efficient gene transfer into human and non-human primate mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) show great promise for ex vivo gene and cell-mediated therapies. The immunophenotype and in vitro differentiation capacity of primary baboon MSCs was demonstrated to be near-identical to that observed in human MSCs. To optimize gene transfer efficiency, we compared the efficiency of serotypes 1, 2, 3, 4, 5, 6, and 8 of adeno-associated virus (AAV) vectors for their ability to mediate transduction of human and baboon MSCs. AAV serotype 2 vectors were the most efficient in transducing MSCs from humans and baboons. As a reference, human Ad293 cells were transduced with these seven AAV serotypes, and were found to have the highest transduction levels followed by baboon MSCs, and then human MSCs. The order of increasing transduction efficiency for the serotypes tested was similar for human and baboon MSCs, but was different for human Ad293 cells. The transduction efficiency of MSCs isolated from different individuals was comparable within the same species. We also demonstrated that baboon MSCs transduced with AAV serotype 2 vectors retain their potential to differentiate into adipocytes in vitro, and can incorporate into injured muscle tissue of NODSCID mice in vivo. We detected beta-galactosidase reporter gene expression in host muscle tissue for up to 9 weeks in this study, indicating engraftment of transduced baboon MSCs and sustained transgene expression in vivo.     

Expression and purification of the recombinant His-tagged GST-CD38 fusion protein using the baculovirus/insect cell expression system

CD38 is a type II transmembrane glycoprotein found in myriad mammalian tissues and cell types. It is known for its involvement in the metabolism of cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, two nucleotides with calcium mobilizing activity independent of inositol trisphosphate. CD38 itself has been shown to have clinical significance in certain diseases with possible utilization in diagnostic and prognostic applications. Previous studies on several autoimmune diseases have shown the usefulness of recombinant CD38 protein expressed from Escherichia coli and Pichia pastoris in the detection of autoantibodies to CD38 via Western blot and ELISA. In this study, we produced a 6 x His-tagged GST-CD38 fusion protein using a recombinant baculovirus/insect cell expression technique that was purified as a soluble protein. The fusion protein was purified to homogeneity by affinity and gel filtration chromatography steps. It has an apparent molecular mass of 56 kDa on SDS-PAGE gel stained with Coomassie blue and was recognized on Western blots by antibodies against human CD38 as well as the polyhistidine tag. Peptide mass fingerprinting analysis confirmed the identity of human CD38 in the fusion protein.     

Genetic mapping of a barley leaf rust resistance gene <Emphasis Type="Italic">Rph26</Emphasis> introgressed from <Emphasis Type="Italic">Hordeum bulbosum</Emphasis>

Key message

The quantitative barley leaf rust resistance gene, Rph26, was fine mapped within a H. bulbosum introgression on barley chromosome 1HL. This provides the tools for pyramiding with other resistance genes.

Abstract

A novel quantitative resistance gene, Rph26, effective against barley leaf rust (Puccinia hordei) was introgressed from Hordeum bulbosum into the barley (Hordeum vulgare) cultivar ‘Emir’. The effect of Rph26 was to reduce the observed symptoms of leaf rust infection (uredinium number and infection type). In addition, this resistance also increased the fungal latency period and reduced the fungal biomass within infected leaves. The resulting introgression line 200A12, containing Rph26, was backcrossed to its barley parental cultivar ‘Emir’ to create an F₂ population focused on detecting interspecific recombination within the introgressed segment. A total of 1368 individuals from this F₂ population were genotyped with flanking markers at either end of the 1HL introgression, resulting in the identification of 19 genotypes, which had undergone interspecific recombination within the original introgression. F₃ seeds that were homozygous for the introgressions of reduced size were selected from each F₂ recombinant and were used for subsequent genotyping and phenotyping. Rph26 was genetically mapped to the proximal end of the introgressed segment located at the distal end of chromosome 1HL. Molecular markers closely linked to Rph26 were identified and will enable this disease resistance gene to be combined with other sources of quantitative resistance to maximize the effectiveness and durability of leaf rust resistance in barley breeding. Heterozygous genotypes containing a single copy of Rph26 had an intermediate phenotype when compared with the homozygous resistant and susceptible genotypes, indicating an incompletely dominant inheritance.

     

Independent Mobility Achieved through a Wireless Brain-Machine Interface

Individuals with tetraplegia lack independent mobility, making them highly dependent on others to move from one place to another. Here, we describe how two macaques were able to use a wireless integrated system to control a robotic platform, over which they were sitting, to achieve independent mobility using the neuronal activity in their motor cortices. The activity of populations of single neurons was recorded using multiple electrode arrays implanted in the arm region of primary motor cortex, and decoded to achieve brain control of the platform. We found that free-running brain control of the platform (which was not equipped with any machine intelligence) was fast and accurate, resembling the performance achieved using joystick control. The decoding algorithms can be trained in the absence of joystick movements, as would be required for use by tetraplegic individuals, demonstrating that the non-human primate model is a good pre-clinical model for developing such a cortically-controlled movement prosthetic. Interestingly, we found that the response properties of some neurons differed greatly depending on the mode of control (joystick or brain control), suggesting different roles for these neurons in encoding movement intention and movement execution. These results demonstrate that independent mobility can be achieved without first training on prescribed motor movements, opening the door for the implementation of this technology in persons with tetraplegia.     

Molecular Characterization of Branchial aquaporin 1aa and Effects of Seawater Acclimation,Emersion or Ammonia Exposure on Its mRNA Expression in the Gills,Gut, Kidney and Skin of the Freshwater Climbing Perch,Anabas testudineus

We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa) from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH₃ transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus.     

Immunoprevalence and Immunodominance of HLA-Cw?0801-Restricted T Cell Response Targeting the Hepatitis B Virus Envelope Transmembrane Region

HLA-C-restricted T cells have been shown to play an important role in HIV control, but their impact on protection or pathogenesis in other viral infections remains elusive. Here, we characterized the hierarchy of HLA class I-restricted hepatitis B virus (HBV) epitopes targeted by CD8 T cells in HBV-infected subjects. The frequency of CD8 T cells specific for a panel of 18 HBV epitopes (restricted by HLA-A∗0201/03/07 [hereinafter HLA-A0201/03/07], -A1101, -A2402/07, -B5801, -B4001, -B1301, and -Cw0801) was quantified in a total of 59 subjects who resolved HBV infection. We found that the HLA-Cw0801-restricted epitope comprised of Env residues 171 to 180 (Env_171–180) is immunoprevalent in the Southeast Asian subjects (10/17 HLA-Cw0801-positive subjects) and immunodominant in the majority of HLA-Cw0801-positive subjects able to control HBV infection. HLA-Cw0801-restricted Env_171–180-specific CD8 T cells recognized endogenously produced HBV surface antigen (HBsAg) and tolerated amino acid variations within the epitope detected in HBV genotypes B and C. In conclusion, we demonstrate that the HLA-Cw0801-restricted Env_171–180 T cell response is an important component of the HBV-specific adaptive T cell immunity in Asians infected with HBV. Thus, HLA-C restricted T cells might play an important role in various viral infections.