Mutations in the activation loop tyrosine of the oncoprotein v-Fps

Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalysis. Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and purified as fusion proteins of glutathione-S-transferase from Escherichia coli and their catalytic properties were evaluated. Y1073E, Y1073Q, and Y1073F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to the wild-type enzyme, respectively. For all mutant enzymes, the Km values for ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compared to the wild-type enzyme. The slopes for the plots of relative turnover versus solvent viscosity [(k(cat))eta] are 0.71 +/- 0.08, 0.10 +/- 0.06, and approximately 0 for wild type, Y1073Q, and Y1073E, respectively. These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme. The dissociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type. The inhibition constant for EAEIFEAIE, a competitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q compared to wild type. Y1073E and Y1073Q are strongly activated by free magnesium to the same extent and the apparent affinity constant for the metal is similar to that for the wild-type enzyme. The data indicate that the major role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-site accessibility or the local environment of the activating metal. Finally, the similar rate enhancements for phosphoryl transfer in v-Fps compared to protein kinase A [Adams et al. (1995) Biochemistry 34, 2447-2454] upon autophosphorylation suggest a conserved mechanism for communication between the activation loop and the catalytic residues of these two enzymes.     

Tumor regression in mice following vaccination with human papillomavirus E7 recombinant protein in PROVAX

Induction of CD8+ cytotoxic T lymphocytes (CTLs) specific for human papillomavirus (HPV) antigens provides an attractive strategy for immunotherapy of HPV-related cancers in humans. In this study, we investigated the potential of utilizing soluble E7 protein of HPV 16 in an adjuvant formulation, PROVAX as a vaccine against a progressively growing E7 transfected K1735-X21 (H-2k) metastatic melanoma cells (HOPE2) in a mouse model. Vaccination of HOPE2 tumor bearing mice (C3H) with E7 protein in PROVAX resulted in significant inhibition of tumor growth, compared to mice vaccinated with E7 in Alum or saline. In vivo depletion of CD8+ or CD4+ cells indicated that CD8+ cells are the major effector cells in mediating the anti-tumor activity in this model. Furthermore, E7-specific CTL activity in vitro was detected in tumor bearing mice vaccinated with E7-PROVAX. Our studies suggest that recombinant HPV antigens in combination with PROVAX could serve as an effective subunit vaccine to stimulate tumor specific CD8+ T cell mediated immunity against HPV-related cancers.     

Seroepidemiology of invasive pneumococcal disease in Queensland, 1990 to 1997

Serotypes responsible for 842 cases of invasive pneumococcal disease in Queensland between February 1990 and October 1997 were identified. Type 14 caused 37.5% of episodes in children aged 0-4 years and 19.2% of adult cases. Types 6A, 6B, 14, 18C, and 19F were significantly more frequent in young children while types 3, 4, 7F, 9V and 23F predominated in adults. The regional incidence of type 14 and 7F disease differed significantly in Southeast and Far North Queensland. Coverage for 87% of children aged less than 5 years in this study would be provided by a recently advocated polysaccharide-protein conjugate vaccine containing capsular antigens of types 4, 6B, 9V, 14, 18C, 19F and 23F. Similarly, more than 90% of adults would be covered by the currently available 23- valent polysaccaride vaccine.     

Both hypothyroidism and hyperthyroidism enhance low density lipoprotein oxidation

Hypothyroidism is frequently associated with hypercholesterolemia and an increased risk for atherosclerosis, whereas hyperthyroidism is known to precipitate angina or myocardial infarction in patients with underlying coronary heart disease. We have shown previously that L-T4 functions as an antioxidant in vitro and inhibits low density lipoprotein (LDL) oxidation in a dose-dependent fashion. The present study was designed to evaluate the changes in LDL oxidation in subjects with hypothyroidism and hyperthyroidism. Fasting blood samples for LDL oxidation analyses, lipoprotein determinations, and thyroid function tests were collected at baseline and after the patients were rendered euthyroid. The lag phase (mean +/- SEM hours) of the Cu+2-catalyzed LDL oxidation in the hypothyroid state and the subsequent euthyroid states were 4 +/- 0.0.65 and 14 +/- 0.68 h, respectively (P < 0.05). The lag phase during the hyperthyroid phase was 6 +/- 0.55 h, and that during the euthyroid phase was 12 +/- 0.66 h (P < 0.05). The total and LDL cholesterol levels were higher in hypothyroidism than in euthyroidism and were lower in hyperthyroidism than in the euthyroid state. We conclude that LDL has more susceptibility to oxidation in both the hypothyroid and hyperthyroid states. Thus, the enhanced LDL oxidation may play a role in the cardiac disease process in both hypothyroidism and hyperthyroidism.     

True multimedia presentations: A case study

This paper describes work undertaken as part of a three-stranded project. The main deliverable will be a suite of software tools, classes and components, as well as teaching resources and methodologies to facilitate the production of intelligent multimedia tutoring systems.

The three strands of this project explore differing aspects of multimedia, and while they stand as separate pieces of work in their own right, they also form the building blocks of the final deliverable, which is an intelligent tutoring system. Intermediate outcomes include a multimedia shell and the Manley Group Demonstrator.

This paper focuses on the design and development of the “true multimedia” demonstrator that has now reached the stage of being a fairly mature prototype. It is used commercially by a communications company to illustrate the varying levels of sophistication which can be achieved in presentation systems and provides an exemplar demonstrating the varying degrees of functionality possible using current technology. The main requirement in the design and implementation has been usability both for end users and content developers.

Real-time control of the multimedia devices is achieved using sophisticated software which is tightly integrated with dedicated control hardware. The software can be used to produce canned presentations or highly interactive systems.

The system is installed in a dedicated presentation suite, thus allowing the true nature of multimedia to be demonstrated. It is scaleable in that it can be used to produce stand-alone portable solutions, highly interactive touch-screen controlled applications or complete multi-device high end presentations. This is achieved using the appropriate combination of software tools and hardware for a specific application.

Macromedia Director is used to produce visual and creative material for the system, while Macromedia Authorware can be used to provide interactivity. © 1997 Elsevier Science B.V.     

Comparison of Cuban and Anglo-American cultural values in a clinical population.

Attempted to replicate an earlier study investigating cultural differences between Cuban immigrants and Anglo-Americans. Whereas the earlier study used a nonclinical adolescent population, the current study used 52 adults in outpatient treatment. Ss were given a biographical questionnaire and the Value Orientation scales. Results indicate that the groups differed in relational, temporal, and person–nature orientations, confirming previous findings and clinical observations. (3 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Starch granulomas after transurethral resection of bladder tumors

Cerebrovascular reactivity to CO2 inhalation and voluntary hyperventilation was studied in seven normotensive subjects and nine hypertensive patients without clinical or angiographical signs of arteriosclerosis. Cerebral blood flow (CBF) was measured by the intracarotid 133Xe clearance method and calculated as the initial slope index. Three to five CBF measurements were made in each patient in the PaCO2 range of 20 to 55 mm Hg. No difference was observed in reactivity between hypertensive and normotensive patients, either during CO2 inhalation or during hyperventilation. The shape of the CBF:PaCO2 curve suggested a decrease in reactivity below a PaCO2 of 30 to 35 mm Hg in both groups. Above a PaCO2 of 35 mm Hg, exponential regression analysis yielded a mean reactivity of 6 +/- 2%, whereas below a PaCO2 of 30 mm Hg it was about 2%. The rise in CBF during CO2 inhalation was not influenced by the intravenous infusion of a small dose of trimethaphan which blocked the concomitant rise in blood pressure.