The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPAL1 and LYPLA2, two enzymes for which selective, in vivo-active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.

Mutations which allowed conjugation by Saccharomyces cerevisiae cells lacking a mating pheromone receptor gene were selected. One of the genes defined by such mutations was isolated from a yeast genomic library by complementation of a temperature-sensitive mutation and is identical to the gene GPAI (also known as SCG1), recently shown to be highly homologous to genes encoding the a subunits of mammalian G proteins. Physiological analysis of temperature-sensitive gpal mutations suggests that the encoded G protein is involved in signaling in response to mating pheromones. Mutational disruption of G-protein activity causes cell-cycle arrest in G1, deposition of mating-specific cell surface agglutinins, and induction of pheromone-specific mRNAs, all of which are responses to pheromone in wild-type cells. In addition, mutants can conjugate without the benefit of mating pheromone or pheromone receptor. A model is presented where the activated G protein has a negative impact on a constitutive signal which normally keeps the pheromone response repressed.G proteins represent a group of highly homologous proteins involved in receptor-mediated signal transduction (see reference 50 for a review). They apparently serve as informational transducers between a diverse group of cell surface receptors and an equally diverse assortment of second message effectors. In vertebrates, G proteins have been found to be ubiquitous in terms of their tissue and phylogenetic distribution.The a subunits of G proteins are part of a larger family of guanyl nucleotide-binding proteins, all of which show some primary structure homology, particularly in domains thought to bind GTP and to be responsible for GTP hydrolysis (30,51). Other members of this family include the ras proteins (10), discovered originally as the products of viral oncogenes, and the rho proteins (32), which were identified because of homology to members of the ras family. Although a role in signal transduction is suspected, the functions of members of the ras and rho protein families in higher organisms are not known. With the goal of applying genetic approaches to the resolution of this issue, homologs of both the ras (6) and rho (33) families have been identified in Saccharomyces cerevisiae. It has been determined that ras and rho homologs in S. cerevisiae perform essential functions (33,41) and that at least one of the functions of the yeast RAS proteins is to regulate adenylate cyclase (3, 55), consistent with a role for such proteins in signal transduction. Recently a gene encoding a protein highly homologous to the a subunit of mammalian G proteins, for which a role in signal transduction has already been established, was identified in S. cerevisiae (7,37 This study " For strains congenic with 381G. only markers different from the background are given. The same convention has been used with respect to the W303-1B background.b Strains carrying the ste3::LEU2 insertion mutation were constructed by fragment-mediated gene conversion (45) (MATa) was superimposed by replic...

A technique employing ferritin-conjugated antibody has been developed to visualize specific protein-DNA complexes in the electron microscope and has been used to demonstrate the preferential binding of simian virus 40 (SV40) T antigen at or near the origin of replication of SV40 DNA, 0.67 fractional length clockwise from the EcoRI restriction endonuclease cleavage site. Purified covalently closed supercoiled circles of SV40 DNA were treated with partially purified T antigen and the complex was stabilized by crosslinking with glutaraldehyde. Hamster anti-T antigen ry-globulin, ferritin-labeled goat anti-hamster 7y-globulin, and glutaraldehyde were then added sequentially. The location of the bound ferritin cores was measured with respect to the EcoRI cleavage site and the orientation of the cores relative to the ends of the DNA was determined with respect to the locations of Escherichia coli DNA unwinding protein, which binds to covalently closed supercoiled SV40 DNA at either of two preferred sites, 0.46 or 0.90 fractional length clockwise from the EcoRI cleavage site.

Beta-ig is a secretory protein embodied by fasciclin I-like repeats containing sequences that might bind integrins and glycosaminoglycans in vivo. Expression of Beta-ig is responsive to Transforming Growth Factor-beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating Beta-ig as an ECM adhesive protein of developmental processes. The spatiotemporal distribution of Beta-ig during various stages of murine development was examined and its ability to support adhesion of various cell types assessed. In situ hybridization of mouse embryos (E12.5-E18.5) indicated a prominent, distinct expression pattern for Beta-ig message in connective tissue. Beta-ig transcripts were abundantly expressed during mesenchymal cell condensation in areas of axial, craniofacial and appendicular primordial cartilage from E12.5-E14.5. Beginning at E15.5, Beta-ig transcripts appeared in collagen-rich tissues, including dura mater and corneal stroma. During E16.5-E18.5, Beta-ig transcripts were observed in proliferating chondrocytes and areas of endochondral ossification in joint and articular cartilage formation. Connective tissues expressed Beta-ig transcripts within the nasal septum and surrounding cartilage primordia, and in the pericardium, optic cup, kidney, ovary, esophagus, diaphragm, bronchi, trachea and corneal epithelium, and during cardiac valve formation. These patterns of expression indicate that Beta-ig may be involved in tissue morphogenesis. Cells derived from mesenchyme attached onto a substratum comprised of purified recombinant Beta-ig. Taken together, the results indicate that Beta-ig is expressed principally in collagen-rich tissues where it may interact with cells and ECM molecules, perhaps playing a role in tissue morphogenesis.

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.

We have shown previously that components of the extracellular matrix (ECM) modulate neuronal development. Here, we searched for additional ECM elements that might play roles in retinal histogenesis and identified a secreted glycoprotein that is heavily expressed in the retina. This molecule, named by others Wnt Inhibitory Factor-1 (WIF-1), is expressed during and after the period of rod photoreceptor morphogenesis in the mouse. We show that a potential WIF-1 ligand, Wnt4, as well as a potential Wnt4 receptor, fzd4, and a potential Wnt4 coreceptor, LRP6, are expressed in the region of, and at the time of, rod photoreceptor genesis. WIF-1 and Wnt4 are coexpressed during retinal development and bind to each other; therefore, they are likely to interact during rod production. WIF-1 protein inhibits rod production, and anti-WIF-1 antibodies increase rod production; in contrast, Wnt4 promotes rod production. Together, these data suggest that WIF-1 and Wnt4, both components of the ECM, regulate mammalian photoreceptor development.

An in vitro study was conducted to determine the susceptibility of the yeast Candida albicans to various intracanal irrigants and medications. The minimum inhibitory concentration (MIC) of sodium hypochlorite, hydrogen peroxide, chlorhexidine digluconate, and aqueous calcium hydroxide that is required to kill a standardized inoculum of C. albicans was determined. Growth of the yeast was measured by optical density. Sodium hypochlorite, hydrogen peroxide, and chlorhexidine digluconate were effective anticandidal agents with MICs of <10 microgram/ml, 234 microgram/ml, and <0.63 microgram/ml, respectively. Aqueous calcium hydroxide had no activity. A standardized inoculum of C. albicans cells was also placed in direct contact with either calcium hydroxide paste or camphorated para-monochlorophenol (CPMC), and candidal growth was assessed by colony counts on Sabouraud's dextrose agar. Calcium hydroxide paste and CPMC, when maintained in direct contact with C. albicans, were effective antifungal agents.

No abstract