In Vitro Characterization of VPS34 Lipid Kinase Inhibition by Small Molecules.

VPS34 is a class III phosphoinositide 3-kinase that acts on vesicle trafficking. This kinase has recently attracted significant attention because of the function it plays in the machinery involved in the early steps of autophagy. Moreover, because significant progress had been made in the optimization of specific kinase inhibitors, its potential to be targeted by catalytic inhibitors has been investigated by different groups. The aim of this review is to present the key in vitro assays necessary for characterizing inhibitors of the catalytic activity of VPS34. The review covers catalytic (IC50 on purified recombinant protein) and binding assays (KD, ka, kd on purified recombinant protein), and a cell-based assay (IC50 in GFP-FYVE expressing cell line). The methodology for crystallization of VPS34 protein is also presented as it can provide guidance for the design by medicinal chemistry of small molecular mass kinase inhibitor.

The effects of moderate exercise training on immune response.

The relationship between moderate exercise training (ET) (five 45-min sessions per week, brisk walking at 60 heart rate reserve for 15 wk) and changes in immune system variables and function was investigated in a group of 36 sedentary, mildly obese women. The study was conducted using a two (exercise (EX) and nonexercise (NEX) groups) by three (baseline, 6 wk, and 15 wk testing sessions) factorial design, with data analyzed using repeated measures ANOVA. The pattern of change over time between groups for number of peripheral blood lymphocytes (total), T cells (CD5), B cells (CD20), and serum IgG, IgA, and IgM levels was significantly different. This was not the case for spontaneous blastogenesis or number of T helper/inducer cells (CD4) or T cytotoxic/suppressor cells (CD8). Within-EX-group changes were characterized by significant decreases in percentage and number of total lymphocytes, and in T cell number after 6 wk, and significant increases in each of the serum immunoglobulins after both 6 and 15 wk of training. B cell number increased significantly in NEX subjects relative to baseline values at both 6 and 15 wk, with no significant changes experienced in EX subjects. In summary, these data suggest that moderate ET is not associated with an improvement in lymphocyte function but is associated with a 20% increase in serum immunoglobulins and several small changes in circulating numbers of immune system variables, highlighted by significant decreases in circulating numbers of lymphocytes, particularly the T cell subpopulation. These changes were especially apparent after 6 wk of training, with some attenuation by 15 wk.

The effects of moderate exercise training on natural killer cells and acute upper respiratory tract infections.

A randomly controlled 15-wk exercise training (ET) study (five 45-min sessions/wk, brisk walking at 60% heart rate reserve) with a group of 36 mildly obese, sedentary women was conducted to investigate the relationship between improvement in cardiorespiratory fitness, changes in natural killer (NK) cell number and activity, and acute upper respiratory tract infection (URI) symptomatology. The study was conducted using a 2 (exercise and nonexercise groups) x 3 (baseline, 6-, and 15-wk testing sessions) factorial design, with data analyzed using repeated measures ANOVA. No significant change in NK cell number occurred as a result of ET as measured by the CD16 and Leu-19 monoclonal antibodies. ET did have a significant effect on NK cell activity (E:T 50:1) especially during the initial 6-wk period [F(2.68) = 12.34, p less than 0.001]. Using data from daily logs kept by each subject, the exercise group was found to have significantly fewer URI symptom days/incident than the nonexercise group (3.6 +/- 0.7 vs 7.0 +/- 1.4 days, respectively, p = 0.049). Improvement in cardiorespiratory fitness was correlated significantly with a reduction in URI symptom days/incident (r = 0.37, p = 0.025) and a change in NK cell activity from baseline to six but not 15 wks (r = 0.35, p = 0.036). In summary, moderate ET is associated with elevated NK cell activity after six but not 15 weeks, and reduced URI symptomatology in comparison to a randomized, sedentary control group.