Although divergent in residues of the peptide binding site, conserved chimpanzee Patr-AL and polymorphic human HLA-A*02 have overlapping peptide-binding repertoires

Patr-AL is an expressed, non-polymorphic MHC class I gene carried by ～50% of chimpanzee MHC haplotypes. Comparing Patr-AL(+) and Patr-AL(-) haplotypes showed Patr-AL defines a unique 125-kb genomic block flanked by blocks containing classical Patr-A and pseudogene Patr-H. Orthologous to Patr-AL are polymorphic orangutan Popy-A and the 5' part of human pseudogene HLA-Y, carried by ～10% of HLA haplotypes. Thus, the AL gene alternatively evolved in these closely related species to become classical, nonclassical, and nonfunctional. Although differing by 30 aa substitutions in the peptide-binding α(1) and α(2) domains, Patr-AL and HLA-A*0201 bind overlapping repertoires of peptides; the overlap being comparable with that between the A*0201 and A*0207 subtypes differing by one substitution. Patr-AL thus has the A02 supertypic peptide-binding specificity. Patr-AL and HLA-A*0201 have similar three-dimensional structures, binding peptides in similar conformation. Although comparable in size and shape, the B and F specificity pockets of Patr-AL and HLA-A*0201 differ in both their constituent residues and contacts with peptide anchors. Uniquely shared by Patr-AL, HLA-A*0201, and other members of the A02 supertype are the absence of serine at position 9 in the B pocket and the presence of tyrosine at position 116 in the F pocket. Distinguishing Patr-AL from HLA-A*02 is an unusually electropositive upper face on the α(2) helix. Stimulating PBMCs from Patr-AL(-) chimpanzees with B cells expressing Patr-AL produced potent alloreactive CD8 T cells with specificity for Patr-AL and no cross-reactivity toward other MHC class I molecules, including HLA-A*02. In contrast, PBMCs from Patr-AL(+) chimpanzees are tolerant of Patr-AL.     

Changing patterns of acute phase proteins and inflammatory mediators in experimental caprine coccidiosis

This experiment was conducted to assess the changing patterns and relative values of acute phase proteins and inflammatory cytokines in experimental caprine coccidiosis. Eighteen newborn kids were allocated to 3 equal groups. Two groups, A and B, were inoculated with a single dose of 1×10(3) and1×10(5) sporulated oocysts of Eimeria arloingi, respectively. The third group, C, received distilled water as the control. Blood samples were collected from the jugular vein of each kid in both groups before inoculation and at days 7, 14, 21, 28, 35, and 42 post-inoculation (PI), and the levels of haptoglobin (Hp), serum amyloid A (SAA), TNF-α, and IFN-γ were measured. For histopathological examinations, 2 kids were selected from each group, euthanized, and necropsied on day 42 PI. Mean Hp concentrations in groups A and B (0.34 and 0.68 g/L) at day 7 PI were 3.2 and 6.3 times higher than the levels before inoculation. The mean SAA concentrations in groups A and B (25.6 and 83.5 μg/ml) at day 7 PI were 4.2 and 13.7 times higher than the levels before inoculation. The magnitude and duration of the Hp and SAA responses correlated well with the inoculation doses and the severity of the clinical signs and diarrhea in kids. These results were consistent with the histopathological features, which showed advanced widespread lesions in group B. In both groups, significant correlations were observed for TNF-α and IFN-γ with SAA and Hp, respectively. In conclusion, Hp and SAA can be useful non-specific diagnostic indicators in caprine coccidiosis.     

Bound water at protein-protein interfaces: partners, roles and hydrophobic bubbles as a conserved motif

Background

There is a great interest in understanding and exploiting protein-protein associations as new routes for treating human disease. However, these associations are difficult to structurally characterize or model although the number of X-ray structures for protein-protein complexes is expanding. One feature of these complexes that has received little attention is the role of water molecules in the interfacial region.

Methodology

A data set of 4741 water molecules abstracted from 179 high-resolution (≤ 2.30 Å) X-ray crystal structures of protein-protein complexes was analyzed with a suite of modeling tools based on the HINT forcefield and hydrogen-bonding geometry. A metric termed Relevance was used to classify the general roles of the water molecules.

Results

The water molecules were found to be involved in: a) (bridging) interactions with both proteins (21%), b) favorable interactions with only one protein (53%), and c) no interactions with either protein (26%). This trend is shown to be independent of the crystallographic resolution. Interactions with residue backbones are consistent for all classes and account for 21.5% of all interactions. Interactions with polar residues are significantly more common for the first group and interactions with non-polar residues dominate the last group. Waters interacting with both proteins stabilize on average the proteins'' interaction (−0.46 kcal mol⁻¹), but the overall average contribution of a single water to the protein-protein interaction energy is unfavorable (+0.03 kcal mol⁻¹). Analysis of the waters without favorable interactions with either protein suggests that this is a conserved phenomenon: 42% of these waters have SASA ≤ 10 Ų and are thus largely buried, and 69% of these are within predominantly hydrophobic environments or “hydrophobic bubbles”. Such water molecules may have an important biological purpose in mediating protein-protein interactions.     

Hemoglobin-ligand binding: understanding Hb function and allostery on atomic level

The major physiological function of hemoglobin (Hb) is to bind oxygen in the lungs and deliver it to the tissues. This function is regulated and/or made efficient by endogenous heterotropic effectors. A number of synthetic molecules also bind to Hb to alter its allosteric activity. Our purpose is to review the current state of Hb structure and function that involves ensemble of tense and relaxed hemoglobin states and the dynamic equilibrium of the multistate due to the binding of endogenous heterotropic or synthetic allosteric effectors. The review also discusses the atomic interactions of synthetic ligands with the function or altered allosteric function of Hb that could be potentially harnessed for the treatment of diseases. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.     

Highly parallel oligonucleotide purification and functionalization using reversible chemistry

We have developed a cost-effective, highly parallel method for purification and functionalization of 5'-labeled oligonucleotides. The approach is based on 5'-hexa-His phase tag purification, followed by exchange of the hexa-His tag for a functional group using reversible reaction chemistry. These methods are suitable for large-scale (micromole to millimole) production of oligonucleotides and are amenable to highly parallel processing of many oligonucleotides individually or in high complexity pools. Examples of the preparation of 5'-biotin, 95-mer, oligonucleotide pools of >40K complexity at micromole scale are shown. These pools are prepared in up to ~16% yield and 90-99% purity. Approaches for using this method in other applications are also discussed.     

Developing species-specific primers to identify Bulinus truncatus and Bulinus beccari, the intermediate hosts of Schistosoma haematobium in Saudi Arabia

This work aimed to determine the inter- and intra-specific variations in populations of Bulinus truncatus and Bulinus beccari, the intermediate hosts of Schistosoma haematobium in Saudi Arabia, and to develop species-specific primers to identify these snails as a first step in the development of multiplex PCR for simultaneously identifying the snails and diagnosing its infections in a single step. Two populations of B. truncatus were collected from Asser and Bisha (A and B), and two B. beccari populations were collected from Mahial Asser and Merba (C and D). The snails' genomic DNA was extracted and amplified using 5 different primers. The primers displayed variable intra- and inter-specific differences across the populations. The largest RAPD-PCR fragments were cloned into a vector as a preparatory step for sequencing. Similarity searches for the sequenced cloned inserts revealed no similar sequences in the GenBank database or its associated databases. Specific primers used to target the B. truncatus and B. beccari genomes were designed using the Gene Runner program and based on the DNA sequences obtained from RAPD fragment sequence analyses. Using these primers for specific PCRs resulted in expected single-band PCR products of 536 bp for B. beccari and 478 bp for B. truncatus. These results will be helpful for simultaneously identifying B. truncatus and B. beccari snails and diagnosing S. haematobium infections within the snails using single step multiplex PCR.     

Isolation and characterization of Stemphylium sedicola SBU-16 as a new endophytic taxol-producing fungus from Taxus baccata grown in Iran

In this study, a total of 25 endophytic fungi were successfully isolated from the inner bark of Taxus baccata grown in Iran by the aseptic technique. Genomic DNA was extracted from isolated endophytic fungi and subjected to polymerase chain reaction (PCR) analysis for the presence of the Taxus taxadiene synthase (ts) gene, which encodes the enzyme catalyzing the first committed step of taxol biosynthesis. Four of 25 isolated endophytic fungi isolates showed PCR positive for the ts gene. Subsequently, taxol and 10-deacetylbaccatin III (10-DAB III) were extracted from culture filtrates and mycelia of the PCR positive isolates and analyzed by high-performance liquid chromatography and mass spectrometry. The analysis showed that one isolate (SBU-16) produced taxol (6.9?±?0.2?μg?L(-1) ) and its intermediate compound, 10-DAB III (2.2?±?0.1?μg?L(-1) ). The isolate SBU-16 was identified as Stemphylium sedicola SBU-16, according to its morphological characteristics as well as the internal transcribed spacer nuclear rDNA gene sequence analysis. Interestingly, this is the first report of the genus Stemphylium as a taxol-producing taxon.     

Micropropagation of Araucaria excelsa R. Br. var. glauca Carrière from orthotropic stem explants

The objectives of the present work were in vitro propagation of Araucaria excelsa R. Br. var. glauca Carrière (Norfolk Island pine) with focus on the evaluation of the mean number of shoots per explant (MNS/E) and mean length of shoots per explants (MLS/E) produced by different parts of the orthotropic stem of A. excelsa R. Br. var. glauca in response to plant growth regulators. Norfolk Island pine axillary meristems responded very well to the 2-iso-pentenyl adenine (2iP) and thidiazuron (TDZ) levels. Explants taken from stem upper segments in the media containing 2iP had a higher MNS/E (3.47) and MLS/E (6.27 mm) in comparison to those taken from stem lower segments, which were 0.71 and 0.51 mm, respectively. Using 0.045 μM TDZ in the MS medium not only resulted in 4.60 MNS/E with 7.08 mm MLS/E but proliferated shoots showed a good performance as well. Investigating the best position of stem explant on mother plant as well as the best concentrations of growth regulators were performed which were useful for efficient micropropagation of this plant. Thirty three percent of explants were rooted in the MS medium containing 3 % sucrose, supplemented with 7.5 μM of both NAA and IBA for 2 weeks before transferring to a half strength MS medium without any growth regulator. Plantlets obtained were acclimatized and transferred to the greenhouse with less than 20 % mortality. This procedure considered the first successful report for regeneration and acclimatization of A. excelsa R. Br. var. glauca plantlet through main stem explants.     

Ants of the Genus Solenopsis Westwood 1840 (Hymenoptera: Formicidae) in the Arabian Peninsula with Description of a New Species,Solenopsis elhawagryi

Ants of the genus Solenopsis Westwood in the Arabian Peninsula are revised. Six species are treated: Solenopsis elhawagryi Sharaf & Aldawood sp. n., S. geminata (Fabricius, 1804), S. omana Collingwood & Agosti, 1996, S. saudiensis Sharaf & Aldawood, 2011, S. sumara Collingwood & Agosti, 1996, and S. zingibara Collingwood & Agosti, 1996. Solenopsis elhawagryi is described from Beljorashi Governorate, Al Baha Province, Saudi Arabia, based on worker castes and the queen with notes on this species biology and ecology. Solenopsis sumara workers are redescribed and illustrated for the first time and a lectotype is designated. An identification key to the Arabian and Egyptian species is provided with scanning electron micrographs to facilitate species recognition.