Mamba dendrotoxins, 7K M(r) polypeptides with three disulfide bonds, selectively inhibit certain fast-activating, voltage-sensitive neuronal K+ channels and have been instrumental in their identification, localization, and purification. However, derivatives with more refined specificity are essential to define the structural and functional properties of the multiple subtypes known to reside in the nervous system. Hence, utilizing a constructed cDNA library from the venom glands of the black mamba (Dendroaspis polylepis), the gene encoding dendrotoxin K was isolated, amplified, and expressed as a maltose-binding fusion protein in the periplasmic space of Escherichia coli. After cleavage of the chaperone from the affinity-purified product, a recombinant protein was isolated and shown to be identical to native dendrotoxin K in its N-terminal sequence, chromatographic behavior, convulsive-inducing activity, and binding to voltage-activated K+ channels in bovine synaptic membranes. This successful expression of refolded active toxin, in adequate yield, makes possible for the first time the preparation of mutants with specificity tailored for each K+ channel subtype, based both on the recently derived three-dimensional structure of alpha-dendrotoxin and the identified binding site on cloned K+ channels.

We have identified conserved autoantigenic cellular proteins that bind to G-rich sequence motifs in recombinogenic regions of Epstein-Barr virus (EBV) DNA. This binding activity, called TRBP, recognizes the EBV terminal repeats, a locus responsible for interconversion of linear and circular EBV DNA. We found that TRBP also binds to EBV DNA sequences involved in deletion of EBNA2, a gene product required for immoralization. We show that TRBP binds sequences present in repetitive cellular DNA, such as variablenumber tandem repeats (VNTR) and immunoglobulin heavychain class switch regions. We propose that EBV utilizes cellular DNA recombination systems to mediate several types of viral genome alterations. These findings may lead to an understanding of the mechanism of rearrangements of EBV DNA that are a central feature of the biology of the virus.In this report we address the hypothesis that recombination events in Epstein-Barr virus (EBV) DNA resemble comparable cellular processes and are mediated by similar machinery. We have sought and identified an activity in cellular nuclear extracts that recognizes recombinogenic sequences in EBV and cellular DNA.The genome of EBV assumes several forms. In virions, EBV DNA is linear; in cells, during latent infection, the viral genome becomes a circular plasmid (1-3). After induction of lytic viral DNA replication a linear EBV DNA molecule is once again produced for packaging into virions (4, 5). Interconversion between the linear and circular forms of EBV DNA, an essential process of the EBV life cycle, takes place in the 538-bp terminal repeats (TRs) (see Fig. 1A). The occasional integration of EBV DNA into the cellular genome also occurs at the TRs (6, 7).Internal rearrangements and deletions are frequently present in EBV DNA molecules. Rearrangements ofEBV DNA, called heterogeneous (het) DNA are found in defective viral genomes and may lead to activation of the EBV lytic replication cycle (8). A region of the EBV genome spanning the coding regions for a portion ofthe leader protein (LP) and the entire EBV-encoded nuclear antigen 2 (EBNA2) protein is prone to deletion: viruses with LP/EBNA2 deletions are unable to immortalize lymphocytes (9). het DNA and deletions that remove the EBNA2 gene are detected in the tongue of patients with oral hairy leukoplakia, a disease characterized by unbridled EBV replication in epithelial cells (10, 11). Thus, het DNA and the EBNA2 deletions, paraphenomena of lytic EBV replication in vitro, may be important in EBV pathogenesis (12). Generation of EBV genomes with het DNA and EBNA2 deletions occurs in sequences of IR1, the 3072-bp large internal repeat (IR) (Fig. 1B) (9, 13-17 (TGGGTGG). The portion of EBV IR1 involved in the recombination with retrovirus is identical to that which participates in the EBNA2 deletion in the P3J-HR-1 EBV strain (13). Furthermore, sequences at the junction between EBV and the retrovirus were similar to G-rich sequences found in the EBV TR (20).G-rich sequences similar to those present in recombinog...

EBV DNA contains G-rich, repeat regions that are involved in rearrangement and recombination events including terminal repeat (TR) processing and the EBNA-2 deletion in the EBV strain P3HR-1. Cellular proteins, called terminal or tandem repeat binding proteins (TRBPs), recognize sequences at the junctions of these recombination events. In this study, using antibody supershift assays and expression of recombinant proteins, we show that Sp1 and Sp3 are the sequence-specific components of TRBP and that Ku is the nonspecific binding component. Sp1 binds other recombinogenic regions of EBV DNA, but Sp3 does not bind to the large internal repeat. The sequence GGGGTGGGG, a low affinity site for Sp1 and Sp3, is the minimal binding site within terminal repeat binding site 1 (TRBS1). However, 3' flanking sequences in the sequence GGGGTGGGGCATGGGG augment binding of Sp1 and Sp3 so that their affinity of binding is increased approximately twofold relative to a classical high-affinity Sp1 site. EBV lytic cycle induction does not alter the abundance or binding activity of any of the three identified components of TRBP. Sp1 and Sp3 may act in trans to promote EBV terminal repeat processing and possibly other viral and cellular recombination events.

Ischemia and reperfusion injuries present major challenges for both military and civilian medicine. Improved methods for assessing the effects and predicting outcome could guide treatment decisions. Specific issues related to ischemia and reperfusion injury can include complications arising from tourniquet use, such as microvascular leakage in the limb, loss of muscle strength and systemic failures leading to hypotension and cardiac failure. Better methods for assessing the viability of limbs/tissues during ischemia and reducing complications arising from reperfusion are critical to improving clinical outcomes for at-risk patients. The purpose of this research is to develop and assess possible prediction models of outcome for acute limb ischemia using a pre-clinical model. Our model relies only on non-invasive imaging data acquired from an animal study. Outcome is measured by pathology and functional scores. We explore color, texture, and temporal features derived from both color and thermal motion imagery acquired during ischemia and reperfusion. The imagery features form the explanatory variables in a model for predicting outcome. Comparing model performance to outcome prediction based on direct observation of blood chemistry, blood gas, urinalysis, and physiological measurements provides a reference standard. Initial results show excellent performance for the imagery-base model, compared to predictions based direct measurements. This paper will present the models and supporting analysis, followed by recommendations for future investigations.

Objective. To identify one nuclear autoantigenic protein within a complex of DNA binding proteins that bind to GC-rich sequences in Epstein-Barr virus and cellular DNA, and to describe the clinical characteristics of patients whose sera contained autoantibodies to this novel autoantigen.Methods. Antibodies to autoantigen Spl were initially measured by an electrophoretic mobility shift assay to detect DNA binding proteins. Nuclear extracts and purified Spl protein were used in these assays. Recognition of the autoantigen by autoimmune sera was confirmed by immunoprecipitation and immunoblotting.Results. The autoantigen was identified as Spl. Anti-Spl was detected in sera from 8 (3%) of 230 patients. These sera contained antinuclear antibodies, but lacked antibodies to double-stranded DNA or to several extractable nuclear antigens. The patients whose sera contained antibodies to Spl were white women with fatigue, arthritis, Raynaud's phenomenon, malar rash, and photosensitivity.Conclusion. Spl is the first described example of an RNA polymerase I1 transcription activator as an autoantigen. The presence of Spl autoantibodies is associated with undifferentiated connective tissue disease.The present experiments were initiated as part of a study to identify proteins that might play a role in recombination events that are common to Epstein-Barr virus (EBV) DNA and cellular DNA. Circular EBV DNA is found during latency, and linear viral DNA is present during lytic replication and is found in virions (1-5). We previously identified a group of cellular Submitted for publication October 16, 1996; accepted in revised form December 31, 1996. proteins that recognized GC-rich motifs in the EBV terminal repeats, at a locus responsible for the interconversion of linear and circular viral DNA (6). These same proteins, called terminal repeat binding proteins (TRBP), also bound to GC-rich sequences in repetitive cellular DNA, within areas such as variable number tandem repeats and Ig heavy chain class switch regions.In efforts to further characterize TRBP, we found that it was a conserved novel autoantigen that was detected by serum antibodies from a group of patients with undifferentiated connective tissue disorders. The sera from these patients demonstrated antinuclear antibody (ANA) reactivity, but were nonreactive with double-stranded DNA (dsDNA) or with several extractable nuclear antigens, including Sm, Ro, La, U1 RNP, topoisomerase I, and Ku. Since the novel autoantigen was a DNA binding protein, electrophoretic mobility shift assay (EMSA) could be used to detect serum antibodies to the autoantigen (7). In this assay, the autoantibodies supershifted a DNA-protein complex. Human B cell nuclear extract was the source of antigen and duplex DNA probes were derived from GC-rich sequences in the EBV terminal repeats. TRBP was found to consist of 3 DNA-protein complexes, A, B, and C, in order of increasing electrophoretic mobility. The autoantisera supershifted TRBP complexes A and B, but not C. Therefore, we sought to character...