Allele-specific activation of genetically engineered receptors.

The binding of agonists and antagonists to the beta-adrenergic receptor (beta AR) is postulated to involve an ionic interaction between the amine group of the ligand and the carboxylate side chain of Asp113 in the third hydrophobic domain of the receptor. To explore the importance of this interaction in the binding of ligands to the beta AR, a Ser residue was substituted for Asp113, and the ability of this mutant receptor to respond to compounds which could potentially interact with the hydroxyl side chain of the Ser residue was assessed. The mutant receptor was fully activated by catechol-containing esters and ketones, compounds which did not activate the wild-type beta AR. The demonstration that the molecular substitution of a single amino acid residue can alter the ligand binding specificity of the beta AR provides evidence that the chemical nature of this residue is a critical determinant in the recognition site of the receptor. Further, the ability to modify the specificity of a receptor by the replacement of amino acids at the binding site demonstrates the potential for the rational design of drugs which function specifically at genetically engineered receptors.     

Effects of isoproterenol treatment of isolated perfused rat hearts on myofibrillar phosphorylation and ATPase activity

Perfusion of isolated rat hearts with isoproterenol resulted in increases in the level of protein-bound phosphate of the myofibrils. After perfusion of the hearts with 32P, followed by SDS-polyacrylamide gel electrophoresis of the purified myofibrils, four major 32P-containing protein bands were identified. Most of the increased 32P incorporation produced by isoproterenol was localized on the troponin I and myosin light chain bands, and, to lesser extent, on the M-protein band. ATPase activity was tested in the purified myofibrils. No changes in Ca2+ requirement for activation were found after isoproterenol perfusion. However, maximal ATPase activity was markedly reduced in the myofibrils obtained from isoproterenol-treated hearts. It would appear that the myofibrillar protein phosphorylation induced by isoproterenol perfusion results in a decrease in actomyosin ATPase activity.     

p38 MAPK Signaling in Postnatal Tendon Growth and Remodeling

Tendon is a dynamic tissue whose structure and function is influenced by mechanical loading, but little is known about the fundamental mechanisms that regulate tendon growth and remodeling in vivo. Data from cultured tendon fibroblasts indicated that the p38 MAPK pathway plays an important role in tendon fibroblast proliferation and collagen synthesis in vitro. To gain greater insight into the mechanisms of tendon growth, and explore the role of p38 MAPK signaling in this process, we tested the hypotheses that inducing plantaris tendon growth through the ablation of the synergist Achilles tendon would result in rapid expansion of a neotendon matrix surrounding the original tendon, and that treatment with the p38 MAPK inhibitor SB203580 would prevent this growth. Rats were treated with vehicle or SB203580, and subjected to synergist ablation by bilateral tenectomy of the Achilles tendon. Changes in histological and biochemical properties of plantaris tendons were analyzed 3, 7, or 28 days after overload, and comparisons were made to non-overloaded animals. By 28 days after overload, tendon mass had increased by 30% compared to non-overloaded samples, and cross-sectional area (CSA) increased by around 50%, with most of the change occurring in the neotendon. The expansion in CSA initially occurred through the synthesis of a hyaluronic acid rich matrix that was progressively replaced with mature collagen. Pericytes were present in areas of active tendon growth, but never in the original tendon ECM. Inhibition of p38 MAPK resulted in a profound decrease in IL6 expression, and had a modest effect on the expression of other ECM and cell proliferation genes, but had a negligible impact on overall tendon growth. The combined results from this study provided novel insights into tendon mechanobiology, and suggest that p38 MAPK signaling does not appear to be necessary for tendon growth in vivo.     

Annexin-V promotes anti-tumor immunity and inhibits neuroblastoma growth in vivo

The goal of the current study is to determine the effects of blocking phosphatidylserine (PS) on the growth of neuroblastoma in mice. PS, an anionic phospholipid restricted to the cytoplasmic surface of plasma membranes in most cells, is externalized to the surface of apoptotic cells. PS has been shown to induce immune tolerance to self-antigens. PS can also be found on the surface of live cells and in particular tumor cells. Annexin-V (AnV) is a protein that specifically binds and blocks PS. To determine the effects of blocking PS with AnV on tumor growth and immunogenicity, mice were inoculated with AGN2a, a poorly immunogenic murine neuroblastoma that expresses high level of PS on the cell surface. Survival and anti-tumor T cell response were determined. AGN2a were engineered to secrete AnV. Secreted protein effectively blocked tumor PS. 40?% of mice inoculated with AnV-expressing AGN2a cells survived free of tumor, whereas none of the mice inoculated with control cells survived (p?=?0.0062). The benefits of AnV were lost when mice were depleted of T cells. The findings suggest that AnV could protect mice from tumor challenge through an immune mediated mechanism. Mice were then immunized with irradiated AnV-secreting or control cells, and challenged with wild-type AGN2a cells. AnV-secreting cell vaccine protected 80?% of mice from AGN2a challenge, while control cell vaccine prevented tumor growth in only 30?% of animals (p?=?0.012). ELISPOT analysis demonstrated that AnV-secreting cell vaccine induced a greater frequency of interferon-gamma producing splenic T cells. T cells isolated from mice immunized with AnV-secreting but not control vaccine lysed AGN2a. In summary, AnV blocked PS, enhanced T cell mediated tumor immunity, and inhibited tumor growth.     

Glycoprotein gIII of pseudorabies virus is multifunctional.

One of the major glycoproteins of pseudorabies virus, gIII, is nonessential for growth in cell culture. Mutants defective in gIII, however, consistently yield lower titers of infectious virus (3- to 20-fold) than does wild-type virus. The interactions of gIII- mutants with their host cells were compared with those of wild-type virus in an attempt to uncover the functions of gIII. We show that gIII plays a major role in the stable adsorption of the virus to its host cell; in the absence of gIII, the rate of adsorption is reduced and adsorption is easily reversed by washing. Thus, adsorption of pseudorabies virus can be said to occur in at least the following two ways: (i) a gIII-mediated rapid adsorption or (ii) a slower and more labile adsorption that is independent of gIII. After virions have been complexed with monoclonal antibodies against gIII (but not some monoclonal antibodies against other glycoproteins), both modes of adsorption were inhibited. Glycoprotein gIII affects virus stability and virus release, as well as adsorption. The effect on virus release is marked when the virus is defective in additional functions. Thus, although we found no obvious difference in the release of virus from gIII- or wild-type virus-infected rabbit kidney cells, release of a gIII-/gI- double mutant from the cells occurred less readily than did release of a gI- mutant. The gIII-/gI- and gIII- mutants, however, adsorbed to cells at a similar rate, indicating that the effects of gIII on adsorption and virus release constitute separate functions. The Bartha vaccine strain of pseudorabies virus has a defective gIII gene and is released poorly from rabbit kidney cells. After the resident Bartha gIII gene was replaced by the gIII gene of wild-type virus, virus release was enhanced considerably. Since inactivation of gIII in wild-type pseudorabies virus did not significantly affect virus release, the Bartha strain must be defective in another function which, in conjunction with gIII, significantly affects virus release. These results indicate again that gIII affects virus release in conjunction with other functions. Also, although the Bartha strain was functionally defective in virus release, it adsorbed to cells as well as wild-type virus did, showing that the effects of gIII on virus adsorption and release constitute separate functions. We conclude that gIII is a multifunctional glycoprotein.     

Aspergillus fumigatus chsE: A Gene Related to CHS3 of Saccharomyces cerevisiae and Important for Hyphal Growth and Conidiophore Development but Not Pathogenicity

The Aspergillus fumigatus chsE (AfchsE) gene was isolated from an A. fumigatus DNA library on the basis of hybridization to a segment of Saccharomyces cerevisiae CHS3 (ScCHS3). The amino acid sequence derived from AfchsE is 28% identical with ScCHS3 and 80% identical with the product of Aspergillus nidulans chsD (AnchsD). A mutant strain constructed by disruption of AfchsE has reduced levels of mycelial chitin, periodic swellings along the length of hyphae, and a block in conidiation that can be partially restored by growth in osmotic stabilizer. This phenotype is different from that reported for an AnchsD mutant, in which germinating conidia and hyphal tips undergo lysis and the colonial growth rate is significantly reduced. Despite the defects associated with the AfchsE- strain, its virulence was not significantly reduced when compared with the wild-type parental strain in a mouse model of pulmonary aspergillosis.     

Pre-counseling education materials for BRCA testing: does tailoring make a difference?

Although tailored print materials (TPMs) have been assessed for a variety of behavioral targets, their effectiveness as decision aids for genetic testing had not been evaluated at the time this study began. We compared TPMs and non-tailored print material (NPMs) that included similar content about genetic testing for breast and ovarian cancer susceptibility. TPMs were prepared especially for an individual based on information from and about her. We mailed baseline surveys to 461 women referred by physicians or identified through a tumor registry. All had personal and family histories of breast and/or ovarian cancer and, on the basis of these histories, an estimated > or =10% probability of carrying a mutation in the breast/ovarian cancer genes BRCA1 or BRCA2. The 325 (70%) who responded were randomly assigned to receive TPM or NPM. Followup surveys, mailed 2 weeks following receipt of print materials, were returned by 262 women (81% of baseline responders). Participants were predominately white (94%) and well-educated (50% college graduates). The mean age was 49 years. At follow-up, TPM recipients exhibited significantly greater improvement in percent of correct responses for the 13-item true/false measure of knowledge (24% increase for TPM vs. 16% for NPM; p < 0.0001) and significantly less over-estimation of risk of being a mutation carrier (40% TPM group overestimated vs. 70% NPM; p < 0.0001). Anxiety did not differ significantly between groups. Reactions to materials differed on two items: "seemed to be prepared just for me" (76% TPM vs. 52% NPM; p < 0.001) and "told me what I wanted to know about BRCA1 and 2 testing" (98% TPM vs. 91% NPM; p < 0.05). TPMs showed an advantage in increasing knowledge and enhancing accuracy of perceived risk. Both are critical components of informed decision making.