The TEL/platelet-derived growth factor β receptor (PDGFβR) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGFβR kinase-dependent signaling pathways

The TEL/PDGFβR fusion protein is the product of the t(5;12) translocation in patients with chronic myelomonocytic leukemia. The TEL/PDGFβR is an unusual fusion of a putative transcription factor, TEL, to a receptor tyrosine kinase. The translocation fuses the amino terminus of TEL, containing the helix-loop-helix (HLH) domain, to the transmembrane and cytoplasmic domain of the PDGFβR. We hypothesized that TEL/PDGFβR self-association, mediated by the HLH domain of TEL, would lead to constitutive activation of the PDGFβR tyrosine kinase domain and cellular transformation. Analysis of in vitro-translated TEL/PDGFβR confirmed that the protein self-associated and that self-association was abrogated by deletion of 51 aa within the TEL HLH domain. In vivo, TEL/PDGFβR was detected as a 100-kDa protein that was constitutively phosphorylated on tyrosine and transformed the murine hematopoietic cell line Ba/F3 to interleukin 3 growth factor independence. Transformation of Ba/F3 cells required the HLH domain of TEL and the kinase activity of the PDGFβR portion of the fusion protein. Immunoblotting demonstrated that TEL/PDGFβR associated with multiple signaling molecules known to associate with the activated PDGFβR, including phospholipase C γ1, SHP2, and phosphoinositol-3-kinase. TEL/PDGFβR is a novel transforming protein that self-associates and activates PDGFβR-dependent signaling pathways. Oligomerization of TEL/PDGFβR that is dependent on the TEL HLH domain provides further evidence that the HLH domain, highly conserved among ETS family members, is a self-association motif.

Magnesium-dependent folding of self-splicing RNA: Exploring the link between cooperativity, thermodynamics, and kinetics

Folding of the Tetrahymena self-splicing RNA into its active conformation involves a set of discrete intermediate states. The Mg2+-dependent equilibrium transition from the intermediates to the native structure is more cooperative than the formation of the intermediates from the unfolded states. We show that the degree of cooperativity is linked to the free energy of each transition and that the rate of the slow transition from the intermediates to the native state decreases exponentially with increasing Mg2+ concentration. Monovalent salts, which stabilize the folded RNA nonspecifically, induce states that fold in less than 30 s after Mg2+ is added to the RNA. A simple model is proposed that predicts the folding kinetics from the Mg2+-dependent change in the relative stabilities of the intermediate and native states.

Evidence for balancing selection operating at the het-c heterokaryon incompatibility locus in a group of filamentous fungi

In filamentous fungi, het loci (for heterokaryon incompatibility) are believed to regulate self/nonself-recognition during vegetative growth. As filamentous fungi grow, hyphal fusion occurs within an individual colony to form a network. Hyphal fusion can occur also between different individuals to form a heterokaryon, in which genetically distinct nuclei occupy a common cytoplasm. However, heterokaryotic cells are viable only if the individuals involved have identical alleles at all het loci. One het locus, het-c, has been characterized at the molecular level in Neurospora crassa and encodes a glycine-rich protein. In an effort to understand the role of this locus in filamentous fungi, we chose to study its evolution by analyzing het-c sequence variability in species within Neurospora and related genera. We determined that the het-c locus was polymorphic in a field population of N. crassa with close to equal frequency of each of the three allelic types. Different species and even genera within the Sordariaceae shared het-c polymorphisms, indicating that these polymorphisms originated in an ancestral species. Finally, an analysis of the het-c specificity region shows a high occurrence of nonsynonymous substitution. The persistence of allelic lineages, the nearly equal allelic distribution within populations, and the high frequency of nonsynonymous substitutions in the het-c specificity region suggest that balancing selection has operated to maintain allelic diversity at het-c. Het-c shares this particular evolutionary characteristic of departing from neutrality with other self/nonself-recognition systems such as major histocompatibility complex loci in mammals and the S (self-incompatibility) locus in angiosperms.

Chrysanthemum chlorotic mottle viroid: Unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes

The causal agent of chrysanthemum chlorotic mottle (CChM) disease has been identified, cloned, and sequenced. It is a viroid RNA (CChMVd) of 398–399 nucleotides. In vitro transcripts with the complete CChMVd sequence were infectious and induced the typical symptoms of the CChM disease. CChMVd can form hammerhead structures in both polarity strands. Plus and minus monomeric CChMVd RNAs self-cleaved during in vitro transcription and after purification as predicted by these structures, which are stable and most probably act as single hammerhead structures as in peach latent mosaic viroid (PLMVd), but not in avocado sunblotch viroid (ASBVd). Moreover, the plus CChMVd hammerhead structure also appears to be active in vivo, because the 5′ terminus of the linear plus CChMVd RNA isolated from infected tissue is that predicted by the corresponding hammerhead ribozyme. Both hammerhead structures of CChMVd display some peculiarities: the plus self-cleaving domain has an unpaired A after the conserved A9 residue, and the minus one has an unusually long helix II. The most stable secondary structure predicted for CChMVd is a branched conformation that does not fulfill the rod-like or quasi-rod-like model proposed for the in vitro structure of most viroids with the exception of PLMVd, whose proposed secondary structure of lowest free energy also is branched. The unusual conformation of CChMVd and PLMVd is supported by their insolubility in 2 M LiCl, in contrast to ASBVd and a series of representative non-self-cleaving viroids that are soluble under the same high salt conditions. These results support the classification of self-cleaving viroids into two subgroups, one formed by ASBVd and the other one by PLMVd and CChMVd.

Crossreactive recognition of viral, self, and bacterial peptide ligands by human class I-restricted cytotoxic T lymphocyte clonotypes: Implications for molecular mimicry in autoimmune disease

The immunodominant, CD8+ cytotoxic T lymphocyte (CTL) response to the HLA-B8-restricted peptide, RAKFKQLL, located in the Epstein–Barr virus immediate-early antigen, BZLF1, is characterized by a diverse T cell receptor (TCR) repertoire. Here, we show that this diversity can be partitioned on the basis of crossreactive cytotoxicity patterns involving the recognition of a self peptide—RSKFRQIV—located in a serine/threonine kinase and a bacterial peptide—RRKYKQII—located in Staphylococcus aureus replication initiation protein. Thus CTL clones that recognized the viral, self, and bacterial peptides expressed a highly restricted αβ TCR phenotype. The CTL clones that recognized viral and self peptides were more oligoclonal, whereas clones that strictly recognized the viral peptide displayed a diverse TCR profile. Interestingly, the self and bacterial peptides equally were substantially less effective than the cognate viral peptide in sensitizing target cell lysis, and also resulted only in a weak reactivation of memory CTLs in limiting dilution assays, whereas the cognate peptide was highly immunogenic. The described crossreactions show that human antiviral, CD8+ CTL responses can be shaped by peptide ligands derived from autoantigens and environmental bacterial antigens, thereby providing a firm structural basis for molecular mimicry involving class I-restricted CTLs in the pathogenesis of autoimmune disease.

The effect of 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate on CO2 permeability of the red blood cell membrane

It has long been assumed that the red cell membrane is highly permeable to gases because the molecules of gases are small, uncharged, and soluble in lipids, such as those of a bilayer. The disappearance of 12C18O16O from a red cell suspension as the 18O exchanges between labeled CO2 + HCO3− and unlabeled HOH provides a measure of the carbonic anhydrase (CA) activity (acceleration, or A) inside the cell and of the membrane self-exchange permeability to HCO3− (Pm,HCO−3). To test this technique, we added sufficient 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate (DIDS) to inhibit all the HCO3−/Cl− transport protein (Band III or capnophorin) in a red cell suspension. We found that DIDS reduced Pm,HCO−3 as expected, but also appeared to reduce intracellular A, although separate experiments showed it has no effect on CA activity in homogenous solution. A decrease in Pm,CO2 would explain this finding. With a more advanced computational model, which solves for CA activity and membrane permeabilities to both CO2 and HCO3−, we found that DIDS inhibited both Pm,HCO−3 and Pm,CO2, whereas intracellular CA activity remained unchanged. The mechanism by which DIDS reduces CO2 permeability may not be through an action on the lipid bilayer itself, but rather on a membrane transport protein, implying that this is a normal route for at least part of red cell CO2 exchange.

PlasmoDB: An integrative database of the Plasmodium falciparum genome. Tools for accessing and analyzing finished and unfinished sequence data

The Plasmodium falciparum Genome Database (http://PlasmoDB.org) integrates sequence information, automated analyses and annotation data emerging from the P.falciparum genome sequencing consortium. To date, raw sequence coverage is available for >90% of the genome, and two chromosomes have been finished and annotated. Data in PlasmoDB are organized by chromosome (1–14), and can be accessed using a variety of tools for graphical and text-based browsing or downloaded in various file formats. The GUS (Genomics Unified Schema) implementation of PlasmoDB provides a multi-species genomic relational database, incorporating data from human and mouse, as well as P.falciparum. The relational schema uses a highly structured format to accommodate diverse data sets related to genomic sequence and gene expression. Tools have been designed to facilitate complex biological queries, including many that are specific to Plasmodium parasites and malaria as a disease. Additional projects seek to integrate genomic information with the rich data sets now becoming available for RNA transcription, protein expression, metabolic pathways, genetic and physical mapping, antigenic and population diversity, and phylogenetic relationships with other apicomplexan parasites. The overall goal of PlasmoDB is to facilitate Internet- and CD-ROM-based access to both finished and unfinished sequence information by the global malaria research community.

Medial prefrontal cortex and self-referential mental activity: Relation to a default mode of brain function

Medial prefrontal cortex (MPFC) is among those brain regions having the highest baseline metabolic activity at rest and one that exhibits decreases from this baseline across a wide variety of goal-directed behaviors in functional imaging studies. This high metabolic rate and this behavior suggest the existence of an organized mode of default brain function, elements of which may be either attenuated or enhanced. Extant data suggest that these MPFC regions may contribute to the neural instantiation of aspects of the multifaceted “self.” We explore this important concept by targeting and manipulating elements of MPFC default state activity. In this functional magnetic resonance imaging (fMRI) study, subjects made two judgments, one self-referential, the other not, in response to affectively normed pictures: pleasant vs. unpleasant (an internally cued condition, ICC) and indoors vs. outdoors (an externally cued condition, ECC). The ICC was preferentially associated with activity increases along the dorsal MPFC. These increases were accompanied by decreases in both active task conditions in ventral MPFC. These results support the view that dorsal and ventral MPFC are differentially influenced by attentiondemanding tasks and explicitly self-referential tasks. The presence of self-referential mental activity appears to be associated with increases from the baseline in dorsal MPFC. Reductions in ventral MPFC occurred consistent with the fact that attention-demanding tasks attenuate emotional processing. We posit that both self-referential mental activity and emotional processing represent elements of the default state as represented by activity in MPFC. We suggest that a useful way to explore the neurobiology of the self is to explore the nature of default state activity.

The natural killer cell receptor Ly-49A recognizes a peptide-induced conformational determinant on its major histocompatibility complex class I ligand.

Natural killer (NK) cells are inhibited from killing cellular targets by major histocompatibility complex (MHC) class I molecules. In the mouse, this can be mediated by the Ly-49A NK cell receptor that specifically binds the H-2Dd MHC class I molecule, then inhibits NK cell activity. Previous experiments have indicated that Ly-49A recognizes the alpha 1/alpha 2 domains of MHC class I and that no specific MHC-bound peptide appeared to be involved. We demonstrate here that alanine-substituted peptides, having only the minimal anchor motifs, stabilized H-2Dd expression and provided resistance to H-2Dd-transfected, transporter associated with processing (TAP)-deficient cells from lysis by Ly-49A+ NK cells. Peptide-induced resistance was blocked only by an mAb that binds a conformational determinant on H-2Dd. Moreover, stabilization of "empty" H-2Dd heavy chains by exogenous beta 2-microglobulin did not confer resistance. In contrast to data for MHC class I-restricted T cells that are specific for peptides displayed MHC molecules, these data indicate that NK cells are specific for a peptide-induced conformational determinant, independent of specific peptide. This fundamental distinction between NK cells and T cells further implies that NK cells are sensitive only to global changes in MHC class I conformation or expression, rather than to specific pathogen-encoded peptides. This is consistent with the "missing self" hypothesis, which postulates that NK cells survey tissues for normal expression of MHC class I.

A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA.

RNA-RNA interactions govern a number of biological processes. Several RNAs, including natural sense and antisense RNAs, interact by means of a two-step mechanism: recognition is mediated by a loop-loop complex, which is then stabilized by formation of an extended intermolecular duplex. It was proposed that the same mechanism holds for dimerization of the genomic RNA of human immunodeficiency virus type 1 (HIV-1), an event thought to control crucial steps of HIV-1 replication. However, whereas interaction between the partially self-complementary loop of the dimerization initiation site (DIS) of each monomer is well established, formation of the extended duplex remained speculative. Here we first show that in vitro dimerization of HIV-1 RNA is a specific process, not resulting from simple annealing of denatured molecules. Next we used mutants of the DIS to test the formation of the extended duplex. Four pairs of transcomplementary mutants were designed in such a way that all pairs can form the loop-loop "kissing" complex, but only two of them can potentially form the extended duplex. All pairs of mutants form heterodimers whose thermal stability, dissociation constant, and dynamics were analyzed. Taken together, our results indicate that, in contrast with the interactions between natural sense and antisense RNAs, no extended duplex is formed during dimerization of HIV-1 RNA. We also showed that 55-mer sense RNAs containing the DIS are able to interfere with the preformed HIV-1 RNA dimer.

Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.

Structure-activity relationships derived by machine learning: the use of atoms and their bond connectivities to predict mutagenicity by inductive logic programming.

We present a general approach to forming structure-activity relationships (SARs). This approach is based on representing chemical structure by atoms and their bond connectivities in combination with the inductive logic programming (ILP) algorithm PROGOL. Existing SAR methods describe chemical structure by using attributes which are general properties of an object. It is not possible to map chemical structure directly to attribute-based descriptions, as such descriptions have no internal organization. A more natural and general way to describe chemical structure is to use a relational description, where the internal construction of the description maps that of the object described. Our atom and bond connectivities representation is a relational description. ILP algorithms can form SARs with relational descriptions. We have tested the relational approach by investigating the SARs of 230 aromatic and heteroaromatic nitro compounds. These compounds had been split previously into two subsets, 188 compounds that were amenable to regression and 42 that were not. For the 188 compounds, a SAR was found that was as accurate as the best statistical or neural network-generated SARs. The PROGOL SAR has the advantages that it did not need the use of any indicator variables handcrafted by an expert, and the generated rules were easily comprehensible. For the 42 compounds, PROGOL formed a SAR that was significantly (P < 0.025) more accurate than linear regression, quadratic regression, and back-propagation. This SAR is based on an automatically generated structural alert for mutagenicity.

The putative actin-binding role of hydrophobic residues Trp546 and Phe547 in chicken gizzard heavy meromyosin.

In the course of myosin-catalyzed ATP hydrolysis, certain amino acid residues in myosin interact with counterparts in actin to produce the relational changes that underlie muscle contraction; some of these interactions are ionic, but the stronger interactions are hydrophobic. In an effort to identify myosin residues participating in hydrophobic interactions, myosin (from smooth muscle) fragments with mutations at suspected sites were engineered and compared with wild-type fragments. It was found that the ATPase of doubly mutated (Trp546Ser and Phe547His) fragments was minimally activated by actin and did not decorate actin well to form the regular arrowhead pattern characteristic of myosin binding to actin filaments. Thus, we suggest that Trp546 and Phe547 are important participants in the hydrophobic actin-myosin interaction.

Recoverin, a photoreceptor-specific calcium-binding protein, is expressed by the tumor of a patient with cancer-associated retinopathy.

Recoverin is a member of the EF-hand family of calcium-binding proteins involved in the transduction of light by vertebrate photoreceptors. Recoverin also was identified as an autoantigen in the degenerative disease of the retina known as cancer-associated retinopathy (CAR), a paraneoplastic syndrome whereby immunological events lead to the degeneration of photoreceptors in some individuals with cancer. In this study, we demonstrate that recoverin is expressed in the lung tumor of a CAR patient but not in similar tumors obtained from individuals without the associated retinopathy. Recoverin was identified intially by Western blot analysis of the CAR patient's biopsy tissue by using anti-recoverin antibodies generated against different regions of the recoverin molecule. In addition, cultured cells from the biopsy tissue expressed recoverin, as demonstrated by reverse transcription-PCR using RNA extracted from the cells. The immunodominant region of recoverin also was determined in this study by a solid-phase immunoassay employing overlapping heptapeptides encompassing the entire recoverin sequence. Two linear stretches of amino acids (residues 64-70, Lys-Ala-Tyr-Ala-Gln-His-Val; and 48-52, Gln-Phe-Gln-Ser-Ile) made up the major determinants. One of the same regions of the recoverin molecule (residues 64-70) also was uniquely immunopathogenic, causing photoreceptor degeneration upon immunization of Lewis rats with the corresponding peptide. These data demonstrate that the neural antigen recoverin more than likely is responsible for the immunological events associated with vision loss in some patients with cancer. These data also establish CAR as one of the few autoimmune-mediated diseases for which the specific self-antigen is known.