Efficient production of a folded and functional, highly disulfide-bonded beta-helix antifreeze protein in bacteria

The Tenebrio molitor thermal hysteresis protein has a cysteine content of 19%. This 84-residue protein folds as a compact beta-helix, with eight disulfide bonds buried in its core. Exposed on one face of the protein is an array of threonine residues, which constitutes the ice-binding face. Previous protocols for expression of this protein in recombinant expression systems resulted in inclusion bodies or soluble but largely inactive material. A long and laborious refolding procedure was performed to increase the fraction of active protein and isolate it from inactive fractions. We present a new protocol for production of fully folded and active T. molitor thermal hysteresis protein in bacteria, without the need for in vitro refolding. The protein coding sequence was fused to those of various carrier proteins and expressed at low temperature in a bacterial strain specially suited for production of disulfide-bonded proteins. The product, after a simple and robust purification procedure, was analyzed spectroscopically and functionally and was found to compare favorably to previously published data on refolded protein and protein obtained from its native source.     

Expression and purification of the intact cytoplasmic domain of the human ephrin receptor A2 tyrosine kinase in Escherichia coli

The ephrin receptor A2 (EphA2) is an integral membrane protein tyrosine kinase and a member of the Eph family, the largest known family of receptor tyrosine kinases. EphA2 overexpression is sufficient to transform normal epithelial cells into an aggressive, metastatic phenotype. In normal cells, EphA2 negatively regulates cell growth and invasiveness. Here we report expression of the intact cytoplasmic domain (juxtamembrane linker, tyrosine kinase, and sterile alpha motif domains) of the human EphA2 receptor in an Escherichia coli system. The expressed protein was purified to near homogeneity by use of metal chelation chromatography combined with removal of vector-encoded tags by specific proteolysis. The cytoplasmic domains of EphA2 are expressed as an active kinase, with the expressed protein found to contain phosphorylated tyrosine residues. In addition, protein tyrosine phosphorylation appears only after EphA2 expression is induced and is removable with alkaline phosphatase treatment. The enzyme was purified 5-fold in yields that average 10-30 mg/L of active EphA2 cytoplasmic domains, which will now be used for further biophysical and structural characterization.     

EC5S ubiquitin complex is recruited by KSHV latent antigen LANA for degradation of the VHL and p53 tumor suppressors

Cellular protein degradation pathways can be utilized by viruses to establish an environment that favors their propagation. Here we report that the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded latency-associated nuclear antigen (LANA) directly functions as a component of the EC5S ubiquitin complex targeting the tumor suppressors von Hippel-Lindau (VHL) and p53 for degradation. We have characterized a suppressor of cytokine signaling box-like motif within LANA composed of an Elongin B and C box and a Cullin box, which is spatially located at its amino and carboxyl termini. This motif is necessary for LANA interaction with the Cul5-Elongin BC complex, to promote polyubiquitylation of cellular substrates VHL and p53 in vitro via its amino- and carboxyl-terminal binding domain, respectively. In transfected cells as well as KSHV-infected B lymphoma cells, LANA expression stimulates degradation of VHL and p53. Additionally, specific RNA interference-mediated LANA knockdown stabilized VHL and p53 in primary effusion lymphoma cells. Thus, manipulation of tumor suppressors by LANA potentially provides a favorable environment for progression of KSHV-infected tumor cells.     

Balancing selection and its effects on sequences in nearby genome regions

Our understanding of balancing selection is currently becoming greatly clarified by new sequence data being gathered from genes in which polymorphisms are known to be maintained by selection. The data can be interpreted in conjunction with results from population genetics models that include recombination between selected sites and nearby neutral marker variants. This understanding is making possible tests for balancing selection using molecular evolutionary approaches. Such tests do not necessarily require knowledge of the functional types of the different alleles at a locus, but such information, as well as information about the geographic distribution of alleles and markers near the genes, can potentially help towards understanding what form of balancing selection is acting, and how long alleles have been maintained.     

Toxicity of diclofenac to Gyps vultures

Three endemic vulture species Gyps bengalensis, Gyps indicus and Gyps tenuirostris are critically endangered following dramatic declines in South Asia resulting from exposure to diclofenac, a veterinary drug present in the livestock carcasses that they scavenge. Diclofenac is widely used globally and could present a risk to Gyps species from other regions. In this study, we test the toxicity of diclofenac to a Eurasian (Gyps fulvus) and an African (Gyps africanus) species, neither of which is threatened. A dose of 0.8 mg kg(-1) of diclofenac was highly toxic to both species, indicating that they are at least as sensitive to diclofenac as G. bengalensis, for which we estimate an LD50 of 0.1-0.2 mg kg(-1). We suggest that diclofenac is likely to be toxic to all eight Gyps species, and that G. africanus, which is phylogenetically close to G. bengalensis, would be a suitable surrogate for the safety testing of alternative drugs to diclofenac.     

Fat-body remodeling in Drosophila melanogaster

The remodeling of the larval fat body is observed in many insects during metamorphosis, but little is known about the physiological importance or the regulation of this process. In Drosophila melanogaster, fat-body remodeling involves the dissociation of the fat body into individual fat cells, which persist throughout pupal development but are later removed by cell death in the young adult. Inhibition of fat-body dissociation is associated with pharate adult lethality and thus is likely to be an essential developmental event. As a start toward understanding the role of fat-body remodeling in the life history of insects, we carried out a detailed study of fat-body disassociation in D. melanogaster using fluorescent microscopy, and tested whether this process is mediated by hemocytes as proposed for fat-body remodeling in Sarcophaga peregrina. We identified and correlated stereotypic events in fat-body dissociation with developmental changes during metamorphosis, and have demonstrated by cell ablation studies that fat-body remodeling in D. melanogaster is a hemocyte independent process.     

Systematic characterization of nuclear proteome during apoptosis: a quantitative proteomic study by differential extraction and stable isotope labeling

Identification and characterization of the nuclear proteome is important for detailed understanding of multiple signaling events in eukaryotic cells. Toward this goal, we extensively characterized the nuclear proteome of human T leukemia cells by sequential extraction of nuclear proteins with different physicochemical properties using three buffer conditions. This large scale proteomic study also tested the feasibility and technical challenges associated with stable isotope labeling by amino acids in cell culture (SILAC) to uncover quantitative changes during apoptosis. Analyzing proteins from three nuclear fractions extracted from naive and apoptotic cells generated 780,530 MS/MS spectra that were used for database searching using the SEQUEST algorithm. This analysis resulted in the identification and quantification of 1,174 putative nuclear proteins. A number of known nuclear proteins involved in apoptosis as well as novel proteins not known to be part of the nuclear apoptotic machinery were identified and quantified. Consistent with SILAC-based quantifications, immunofluorescence staining of nucleus, mitochondria, and some associated proteins from both organelles revealed a dynamic recruitment of mitochondria into nuclear invaginations during apoptosis.     

The muscular Dystrophy Surveillance Tracking and Research Network (MD STARnet): surveillance methodology

BACKGROUND: This report focuses on the common protocol developed by the Muscular Dystrophy Surveillance Tracking and Research Network (MD STARnet) for population-based surveillance of Duchenne and Becker muscular dystrophy (DBMD) among 4 states (Arizona, Colorado, Iowa, and New York). METHODS: The network sites have developed a case definition and surveillance protocol along with software applications for medical record abstraction, clinical review, and pooled data. Neuromuscular specialists at each site review the pooled data to determine if a case meets the case criteria. Sources of potential cases of DBMD include neuromuscular specialty clinics, service sites for children with special healthcare needs, and hospital discharge databases. Each site also adheres to a common information assurance protocol. RESULTS: A population-based surveillance system for DBMD was created and implemented in participating states. CONCLUSIONS: The development and implementation of the population-based system will allow for the collection of information that is intended to provide a greater understanding of DBMD prevalence and health outcomes.