Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation

Conventional drug discovery approaches require a priori selection of an appropriate molecular target, but it is often not obvious which biological pathways must be targeted to reverse a disease phenotype. Phenotype-based screens offer the potential to identify pathways and potential therapies that influence disease processes. The zebrafish mutation gridlock (grl, affecting the gene hey2) disrupts aortic blood flow in a region and physiological manner akin to aortic coarctation in humans. Here we use a whole-organism, phenotype-based, small-molecule screen to discover a class of compounds that suppress the coarctation phenotype and permit survival to adulthood. These compounds function during the specification and migration of angioblasts. They act to upregulate expression of vascular endothelial growth factor (VEGF), and the activation of the VEGF pathway is sufficient to suppress the gridlock phenotype. Thus, organism-based screens allow the discovery of small molecules that ameliorate complex dysmorphic syndromes even without targeting the affected gene directly.     

Activation of the kallikrein-kinin system and release of new kinins through alternative cleavage of kininogens by microbial and human cell proteinases

Kinins are released from kininogens through the activation of the Hageman factor-prekallikrein system or by tissue kallikrein. These peptides exert various biological activities, such as vascular permeability increase, smooth muscle contraction, pain sensation and induction of hypotension. In many instances kinins are thought to be involved in the pathophysiology of various diseases. Recent studies have revealed that microbial and human cell proteinases activate Hageman factor and/or prekallikrein, or directly release kinin from kininogens. This review discusses the activation of the kinin-release system by mast-cell tryptase and microbial proteinases, including gingipains, which are cysteine proteinases from Porphyromonas gingivalis , the major pathogen of periodontal disease. Each enzyme is evaluated in the context of its association to allergy and infectious diseases, respectively. Furthermore, a novel system of kinin generation directly from kininogens by the concerted action of two proteinases is described. An interesting example of this system with implications to bacterial pathogenicity is the release of kinins from kininogens by neutrophil elastase and a synergistic action of cysteine proteinases from Staphylococcus aureus . This alternative production of kinins by proteinases present in diseased sites indicates a significant contribution of proteinases other than kallikreins in kinin generation. Therefore kinin receptor antagonists and proteinase inhibitors may be useful as therapeutic agents.     

Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways

In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Here, we show that apoptotic cells express the secretory factors wingless (wg) and decapentaplegic (dpp). When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation was observed. Significantly, wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, we provide evidence that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation.     

Anion binding to a protein-protein complex lacks dependence on net charge

The binding of anions to proteins occurs in numerous physiological and metabolic processes. In an effort to understand the factors important in these interactions, we have studied the weak binding of phosphate and sulfate to a protein-protein complex using isothermal titration calorimetry. To our knowledge, this is the first system in which the thermodynamics of anion binding have been determined calorimetrically. By studying both phosphate and sulfate binding and using a range of pH values, the charge on the anion was varied from approximately -1 to -2. Surprisingly, no dependence of the binding energetics on the charge of the anion was observed. This result indicates that charge-charge interactions are not the dominant factor in binding and suggests the importance of hydrogen bonding in specifically recognizing and coordinating anions.     

Functional epitope on human neutrophil flavocytochrome b558

mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) region of gp91(phox). In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (K(m)). mAb NL7 did not inhibit translocation of p47(phox), p67(phox), or Rac to the plasma membrane, and bound its epitope on gp91(phox) independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91(phox) on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47(phox) binding sites. We conclude that the (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding.     

Evolutionary Landscapes for the Acquisition of New Ligand Recognition by RNA Aptamers

The evolution of ligand specificity underlies many important problems in biology, from the appearance of drug resistant pathogens to the re-engineering of substrate specificity in enzymes. In studying biomolecules, however, the contributions of macromolecular sequence to binding specificity can be obscured by other selection pressures critical to bioactivity. Evolution of ligand specificity in vitro—unconstrained by confounding biological factors—is addressed here using variants of three flavin-binding RNA aptamers. Mutagenized pools based on the three aptamers were combined and allowed to compete during in vitro selection for GMP-binding activity. The sequences of the resulting selection isolates were diverse, even though most were derived from the same flavin-binding parent. Individual GMP aptamers differed from the parental flavin aptamers by 7 to 26 mutations (20 to 57% overall change). Acquisition of GMP recognition coincided with the loss of FAD (flavin-adenine dinucleotide) recognition in all isolates, despite the absence of a counter-selection to remove FAD-binding RNAs. To examine more precisely the proximity of these two activities within a defined sequence space, the complete set of all intermediate sequences between an FAD-binding aptamer and a GMP-binding aptamer were synthesized and assayed for activity. For this set of sequences, we observe a portion of a neutral network for FAD-binding function separated from GMP-binding function by a distance of three mutations. Furthermore, enzymatic probing of these aptamers revealed gross structural remodeling of the RNA coincident with the switch in ligand recognition. The capacity for neutral drift along an FAD-binding network in such close approach to RNAs with GMP-binding activity illustrates the degree of phenotypic buffering available to a set of closely related RNA sequences—defined as the sets functional tolerance for point mutations—and supports neutral evolutionary theory by demonstrating the facility with which a new phenotype becomes accessible as that buffering threshold is crossed.     

Differentiation between acetylcholinesterase and the organophosphate-inhibited form using antibodies and the correlation of antibody recognition with reactivation mechanism and rate

Two types of polyclonal antibodies were generated from (a) a decapeptide sequence that includes the active site serine of acetylcholinesterase (anti-AChE10S) and (b) the identical decapeptide sequence phosphorylated at the active site serine of acetylcholinesterase (anti-AChE10SP). The anti-AChE10S antiserum was found to specifically recognize native, control, and vehicle-treated recombinant mouse AChE (rMoAChE) but did not recognize rMoAChE that was phosphorylated by the four organophosphate (OP) compounds tested. Conversely the anti-AChE10SP antiserum recognized phosphoserine rMoAChE that resulted from reaction with phosphorous oxychloride (POCl3) but did not recognize native or vehicle-treated rMoAChE. Anti-AChE10SP also did not recognize OP-AChE conjugates that resulted from the reaction of rMoAChE with other OP compounds that afford neutral or monoanionic phosphoserine groups thereby indicating a high specificity for a precise OP conjugate. Antisera recognition correlated well with the rates of enzyme inhibition, aging, and oxime-induced reactivation indicating these antisera can both quantify the extent and type of inhibition and also differentiate between select mechanisms of inhibition. The ability to discern mechanistic differences between native AChE and OP-AChE conjugates suggests that these antisera can be used to identify biomarkers of OP exposure in a mechanism-based approach.     

Community outreach as an iterative dialogue among scientists and communities in the Texas gulf coast region

In response to significant environmental health challenges in Southeast Texas, a National Institute for Environmental Health Sciences Center at the University of Texas Medical Branch at Galveston was created to promote and conduct inter-disciplinary research in the areas of: (1) the molecular biology of DNA repair, replication and mutagenesis, (2) asthma pathogenesis in response to oxidative stress and viral exposures, and (3) environmental toxicant biotransformation. In addition, the NIEHS Center maintains close ties with neighboring communities through an active Community Outreach & Education Program (COEP) that develops and disseminates translational materials for use in environmental health awareness outreach, toxicology consultation, K-12 curriculum enrichment and in developing site-specific Community Partnership projects. The COEP core service divisions include: Environmental Arts & Sciences, Asthma Outreach & Education, Theater Outreach & Education, and Public Forum & Toxics Assistance. Public Forums focus on the use of Augusto Boal's Forum Theater dramaturgy to include the voices and local knowledge of communities within the process of Participatory Research. Forums create the preconditions for significant partnerships that link the hazardous risk perceptions and environmental health needs of communities with the expertise of NIEHS Center investigators and translational services provided through COEP outreach programs. The Forum process also creates leadership cores within environmentally challenged communities that facilitate the ongoing translational process and maintain the vital linkage between the health needs of communities and the analytic tools and the field and clinical technologies of the environmental sciences.