High pressure macromolecular crystallography: the 140-MPa crystal structure at 2.3 A resolution of urate oxidase, a 135-kDa tetrameric assembly

We report the three-dimensional structure determined by high-pressure macromolecular crystallography (HPMX) of a 135-kDa homo-tetrameric enzyme, urate oxidase from Aspergillus flavus complexed with its potent inhibitor 8-azaxanthin. Urate oxidase crystals are quite sensitive to pressure, as three-dimensional order is lost at about 180 MPa. A highly complete 2.3 A resolution data set was collected at 140 MPa, close to the critical pressure. Crystal structures at atmospheric pressure and at high pressure were refined in the orthorhombic space group I222 with final crystallographic R factors 14.1% and 16.1%, respectively. The effect of pressure on temperature factors, ordered water molecules, hydrogen bond lengths, contacts, buried surface areas as well as cavity volume was investigated. Results suggest that the onset of disruption of the tetrameric assembly by pressure has been captured in the crystalline state.     

First cell cycles of sea urchin Paracentrotus lividus are dramatically impaired by exposure to extremely low-frequency electromagnetic field

Exposure of fertilized eggs of the sea urchin Paracentrotus lividus to an electromagnetic field of 75-Hz frequency and low amplitudes (from 0.75 to 2.20 mT of magnetic component) leads to a dramatic loss of synchronization of the first cell cycle, with formation of anomalous embryos linked to irregular separation of chromatids during the mitotic events. Because acetylcholinesterase (ACHE) is thought to regulate the embryonic first developmental events of the sea urchin, its enzymatic activity was assayed in embryo homogenates and decreased by 48% when the homogenates were exposed to the same pulsed field. This enzymatic inactivation had a threshold of about 0.75 +/- 0.01 mT. The same field threshold was found for the effect on the formation of anomalous embryos of P. lividus. Moreover, ACHE inhibitors seem to induce the same teratological effects as those caused by the field, while blockers of acetylcholine (ACh) receptors are able to antagonize those effects. We conclude that one of the main causes of these dramatic effects on the early development of the sea urchin by field exposure could be the accumulation of ACh due to ACHE inactivation. The crucial role of the membrane in determining the conditions for enzyme inactivation is discussed.     

Evolutionary Dynamics of Multiple Sublineages of H5N1 Influenza Viruses in Nigeria from 2006 to 2008

Highly pathogenic A/H5N1 avian influenza (HPAI H5N1) viruses have seriously affected the Nigerian poultry industry since early 2006. Previous studies have identified multiple introductions of the virus into Nigeria and several reassortment events between cocirculating lineages. To determine the spatial, evolutionary, and population dynamics of the multiple H5N1 lineages cocirculating in Nigeria, we conducted a phylogenetic analysis of whole-genome sequences from 106 HPAI H5N1 viruses isolated between 2006 and 2008 and representing all 25 Nigerian states and the Federal Capital Territory (FCT) reporting outbreaks. We identified a major new subclade in Nigeria that is phylogenetically distinguishable from all previously identified sublineages, as well as two novel reassortment events. A detailed analysis of viral phylogeography identified two major source populations for the HPAI H5N1 virus in Nigeria, one in a major commercial poultry area (southwest region) and one in northern Nigeria, where contact between wild birds and backyard poultry is frequent. These findings suggested that migratory birds from Eastern Europe or Russia may serve an important role in the introduction of HPAI H5N1 viruses into Nigeria, although virus spread through the movement of poultry and poultry products cannot be excluded. Our study provides new insight into the genesis and evolution of H5N1 influenza viruses in Nigeria and has important implications for targeting surveillance efforts to rapidly identify the spread of the virus into and within Nigeria.Since its emergence in 1996 in Guangdong, China, highly pathogenic avian influenza virus of the H5N1 subtype (HPAI H5N1 virus) has disseminated widely across Asia, Europe, and Africa, infecting a range of domestic and wild avian species and sporadically spilling over into humans and other mammals (4, 35). Over time, the HPAI H5N1 virus has diversified into multiple phylogenetically distinct lineages, classified as clades 0 to 9 according to the unified nomenclature system (33). The H5N1 lineage currently circulating in central Asia, the Middle East, Europe, and Africa is referred to as clade 2.2 (33) and has also been described as “EMA” or Qinghai-like in previous publications (4, 17, 27). This clade originated in April 2005 during a large outbreak of a phylogenetically distinct H5N1 virus among wild bird populations at Qinghai Lake in western China (4, 17) and rapidly spread west through central Asia and Europe, eventually reaching Africa in 2006 (27). Clade 2.2 has further diversified, forming the genetic third-order clade 2.2.1 (32) and three genetically distinct sublineages (I, II, and III) (2, 19, 28), all of which are found in Africa.Since 2006 HPAI H5N1 viruses belonging to clade 2.2 have disseminated across multiple countries in western, eastern, and northern Africa: Egypt, Niger, Cameroon, Sudan, Burkina Faso, Djibouti, Ivory Coast, Ghana, Togo, Benin, and Nigeria (2). With a large poultry industry, estimated at 140 million birds (11), Nigeria has experienced several major outbreaks of HPAI H5N1 virus, posing a serious threat to food security and public health in Africa. The first case of HPAI H5N1 virus in Nigeria (sublineage I) occurred in January 2006 in the state of Kaduna, and the virus subsequently was detected in Ghana, Burkina Faso, Ivory Coast, and Sudan (2). In February 2006 sublineage II was reported in Nigeria, and it disseminated widely across the country during 2006 and 2007, also appearing in Togo (2). Clade 2.2.1, which has been prevalent in Egypt, Israel, and the Gaza Strip from 2006 to 2008, was also detected in Nigeria in 2006 (10).By the end of 2007, outbreaks of HPAI H5N1 virus in Nigeria appeared to have been successfully controlled by measures such as “stamping out with compensation,” restrictions on movement of poultry, and enhanced surveillance (13). However, in July 2008 new cases of HPAI H5N1 from a sublineage never previously detected in Africa (sublineage III) were registered in the Nigerian states of Kano and Katsina and in live bird markets in Gombe and Kebbi states (13, 21). Hence, Nigeria is the only African country where viruses belonging to clade 2.2.1 and to three different sublineages (I, II, and III) of clade 2.2 have all been detected. At least three different reassortment events between sublineages have been documented in Nigeria. Salzberg et al. identified the first reassortant strain (which we refer to as “R1”), in which four genome segments (hemagglutinin [HA], NP, NS, and PB1) belong to sublineage I and the other four segments (NA, MP, PA, and PB2) are derived from sublineage II (27). Subsequently, phylogenetic analysis showed that a 2007 reassortant strain (which we refer to as “R3”) contained the HA and NS segments from sublineage I and the other six segments from sublineage II (19, 22). Another reassortant virus (which we refer to as “R5”) contained only the NS gene segment from sublineage I, while the other seven segments were derived from sublineage II (22).Although the genetic diversity of the Nigerian HPAI H5N1 virus population has been well characterized, including multiple introductions of the virus into Nigeria and several reassortment events, little is known about the evolutionary and population growth dynamics of the virus within Nigeria. Particularly understudied are the spatial movements of individual sublineages among Nigeria''s vast poultry population. To explore the spatial, evolutionary, and population dynamics of the multiple H5N1 lineages cocirculating in Nigeria, we conducted a phylogenetic analysis of whole-genome sequences from 106 HPAI H5N1 viruses isolated between 2006 and 2008 and representing all 25 Nigerian states and the Federal Capital Territory (FCT) reporting outbreaks. Using the exact date and location of collection for each viral isolate, we inferred from their phylogenetic relationships the directionality of viral gene flow among Nigerian states and identified critical regions that are likely to serve as key sources for the H5N1 virus in Nigeria.     

Production of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from Schistosoma mansoni

The number of sequences generated by genome projects has increased exponentially, but gene characterization has not followed at the same rate. Sequencing and analysis of full-length cDNAs is an important step in gene characterization that has been used nowadays by several research groups. In this work, we have selected Schistosoma mansoni clones for full-length sequencing, using an algorithm that investigates the presence of the initial methionine in the parasite sequence based on the positions of alignment start between two sequences. BLAST searches to produce such alignments have been performed using parasite expressed sequence tags produced by Minas Gerais Genome Network against sequences from the database Eukaryotic Cluster of Orthologous Groups (KOG). This procedure has allowed the selection of clones representing 398 proteins which have not been deposited as S. mansoni complete CDS in any public database. Dedicated sequencing of 96 of such clones with reads from both 5' and 3' ends has been performed. These reads have been assembled using PHRAP, resulting in the production of 33 full-length sequences that represent novel S. mansoni proteins. These results shall contribute to construct a more complete view of the biology of this important parasite.     

Spontaneous CD8 T cell responses against the melanocyte differentiation antigen RAB38/NY-MEL-1 in melanoma patients

The melanocyte differentiation Ag RAB38/NY-MEL-1 was identified by serological expression cloning (SEREX) and is expressed in the vast majority of melanoma lesions. The immunogenicity of RAB38/NY-MEL-1 has been corroborated previously by the frequent occurrence of specific Ab responses in melanoma patients. To elucidate potential CD8 T cell responses, we applied in vitro sensitization with overlapping peptides spanning the RAB38/NY-MEL-1 protein sequence and the reverse immunology approach. The identified peptide RAB38/NY-MEL-1(50-58) exhibited a marked response in ELISPOT assays after in vitro sensitization of CD8 T cells from HLA-A *0201(+) melanoma patients. In vitro digestion assays using purified proteasomes provided evidence of natural processing of RAB38/NY-MEL-1(50-58) peptide. Accordingly, monoclonal RAB38/NY-MEL-1(50-58)-specific T cell populations were capable of specifically recognizing HLA-A2(+) melanoma cell lines expressing RAB38/NY-MEL-1. Applying fluorescent HLA-A2/RAB38/NY-MEL-1(50-58) multimeric constructs, we were able to document a spontaneously developed memory/effector CD8 T cell response against this peptide in a melanoma patient. To elucidate the Ag-processing pathway, we demonstrate that RAB38/NY-MEL-1(50-58) is produced efficiently by the standard proteasome and the immunoproteasome. In addition to the identification of a RAB38/NY-MEL-1-derived immunogenic CD8 T cell epitope, this study is instrumental for both the onset and monitoring of future RAB38/NY-MEL-1-based vaccination trials.     

Expression and functions of the vascular endothelial growth factors and their receptors in human basophils

Angiogenesis is a multistep complex phenomenon critical for several inflammatory and neoplastic disorders. Basophils, normally confined to peripheral blood, can infiltrate the sites of chronic inflammation. In an attempt to obtain insights into the mechanism(s) underlying human basophil chemotaxis and its role in inflammation, we have characterized the expression and function of vascular endothelial growth factors (VEGFs) and their receptors in these cells. Basophils express mRNA for three isoforms of VEGF-A (121, 165, and 189) and two isoforms of VEGF-B (167 and 186). Peripheral blood and basophils in nasal polyps contain VEGF-A localized in secretory granules. The concentration of VEGF-A in basophils was 144.4 +/- 10.8 pg/10(6) cells. Immunologic activation of basophils induced the release of VEGF-A. VEGF-A (10-500 ng/ml) induced basophil chemotaxis. Supernatants of activated basophils induced an angiogenic response in the chick embryo chorioallantoic membrane that was inhibited by an anti-VEGF-A Ab. The tyrosine kinase VEGFR-2 (VEGFR-2/KDR) mRNA was expressed in basophils. These cells also expressed mRNA for the soluble form of VEGFR-1 and neuropilin (NRP)1 and NRP2. Flow cytometric analysis indicated that basophils express epitopes recognized by mAbs against the extracellular domains of VEGFR-2, NRP1, and NRP2. Our data suggest that basophils could play a role in angiogenesis and inflammation through the expression of several forms of VEGF and their receptors.     

Identification of amino acid residues important for assembly of GABA receptor alpha1 and gamma2 subunits

Comparative models of GABA(A) receptors composed of alpha1 beta3 gamma2 subunits were generated using the acetylcholine-binding protein (AChBP) as a template and were used for predicting putative engineered cross-link sites between the alpha1 and the gamma2 subunit. The respective amino acid residues were substituted by cysteines and disulfide bond formation between subunits was investigated on co-transfection into human embryonic kidney (HEK) cells. Although disulfide bond formation between subunits could not be observed, results indicated that mutations studied influenced assembly of GABA(A) receptors. Whereas residue alpha1A108 was important for the formation of assembly intermediates with beta3 and gamma2 subunits consistent with its proposed location at the alpha1(+) side of GABA(A) receptors, residues gamma2T125 and gamma2P127 were important for assembly with beta3 subunits. Mutation of each of these residues also caused an impaired expression of receptors at the cell surface. In contrast, mutated residues alpha1F99C, alpha1S106C or gamma2T126C only impaired the formation of receptors at the cell surface when co-expressed with subunits in which their predicted interaction partner was also mutated. These data are consistent with the prediction that the mutated residue pairs are located close to each other.     

Structure-function studies of the G-domain from human gem, a novel small G-protein

Gem, a member of the Rad,Gem/Kir subfamily of small G-proteins, has unique sequence features. We report here the crystallographic structure determination of the Gem G-domain in complex with nucleotide to 2.4 A resolution. Although the basic Ras protein fold is maintained, the Gem switch regions emphatically differ from the Ras paradigm. Our ensuing biochemical characterization indicates that Gem G-domain markedly prefers GDP over GTP. Two known functions of Gem are distinctly affected by spatially separated clusters of mutations.