Sites of iodination in recombinant human brain-derived neurotrophic factor and its effect on neurotrophic activity.

Recombinant human brain-derived neurotrophic factor (BDNF) is now under extensive investigation because of its potential clinical applications. Radioactively labeled proteins are usually required to study receptor binding and pharmacokinetic properties of proteins. This study was undertaken to see if iodination affects the biological and conformational properties of a recombinant BDNF. BDNF was iodinated using a stoichiometric amount of nonradioactive cold NaI to minimize multiple iodinations. Of the four tyrosines present in BDNF--Tyr-52, Tyr-54, Tyr-63, and Tyr-86--only Tyr-63 and Tyr-86 were iodinated under the experimental conditions used. Iodination of Tyr-63 resulted in modification without alteration of the biological activity, whereas iodination of Tyr-86 resulted in a molecule with highly compromised biological activity. Similar inactivation was observed if both Tyr-63 and Tyr-86 were iodinated. These modified proteins exhibited conformation and dimerization apparently identical to those of the native protein, as demonstrated by analytical ultracentrifugation, gel filtration, light scattering, and circular dichroism. From these results, we concluded that Tyr-52 and Tyr-54 are not accessible to the reagent and are probably buried in the hydrophobic core, whereas Tyr-63 and Tyr-86 are exposed on the surface of the molecule; of the two exposed residues, only Tyr-86 contributes to the biological activity.     

Conformational analysis of protein structures derived from NMR data

A study is presented of the conformational characteristics of NMR-derived protein structures in the Protein Data Bank compared to X-ray structures. Both ensemble and energy-minimized average structures are analyzed. We have addressed the problem using the methods developed for crystal structures by examining the distribution of ?, Ψ, and χ angles as indicators of global conformational irregularity. All these features in NMR structures occur to varying degrees in multiple conformational states. Some measures of local geometry are very tightly constrained by the methods used to generate the structure, e.g., proline ? angles, α-helix ?, Ψ angles, ω angles, and C_α chirality. The more lightly restrained torsion angles do show increasead clustering as the number of overall experimental observations increases. ?, Ψ, and χ₁ angle conformational heterogeneity is strongly correlated with accessibility but shows additional differences which reflect the differing number of observations possible in NMR for the various side chains (e.g., many for Trp, few for Ser). In general, we find that the core is defined to a notional resolution of 2.0 to 2.3 Å. Of real interest is the behavior of surface residues and in particular the side chains where multiple rotameric states in different structures can vary from 10% to 88%. Later generation structures show a much tighter definition which correlates with increasing use of J-coupling information, stereospecific assignments, and heteronumclear techniques. A suite of programs is being developed to address the special needs of NMR-derived structures which will take into account the existence of increased mobility in solution. © 1993 Wiley-Liss, Inc.     

Bayesian estimation of two-part joint models for a longitudinal semicontinuous biomarker and a terminal event with INLA: Interests for cancer clinical trial evaluation

Two-part joint models for a longitudinal semicontinuous biomarker and a terminal event have been recently introduced based on frequentist estimation. The biomarker distribution is decomposed into a probability of positive value and the expected value among positive values. Shared random effects can represent the association structure between the biomarker and the terminal event. The computational burden increases compared to standard joint models with a single regression model for the biomarker. In this context, the frequentist estimation implemented in the R package frailtypack can be challenging for complex models (i.e., a large number of parameters and dimension of the random effects). As an alternative, we propose a Bayesian estimation of two-part joint models based on the Integrated Nested Laplace Approximation (INLA) algorithm to alleviate the computational burden and fit more complex models. Our simulation studies confirm that INLA provides accurate approximation of posterior estimates and to reduced computation time and variability of estimates compared to frailtypack in the situations considered. We contrast the Bayesian and frequentist approaches in the analysis of two randomized cancer clinical trials (GERCOR and PRIME studies), where INLA has a reduced variability for the association between the biomarker and the risk of event. Moreover, the Bayesian approach was able to characterize subgroups of patients associated with different responses to treatment in the PRIME study. Our study suggests that the Bayesian approach using the INLA algorithm enables to fit complex joint models that might be of interest in a wide range of clinical applications.     

Calcium regulates neuronal differentiation both directly and via co-cultured myocytes

Control of neuronal development by cellular interactions can be regulated by both extracellular and intracellular calcium. Removal of extracellular calcium affects the differentiation of amphibian spinal neurons in vitro by preventing neuronal calcium influx during the production of calcium-dependent action potentials (Holliday and Spitzer, Dev. Biol. 141:13-23, 1990). However, this culture condition affects differentiation through other mechanisms as well. We have investigated the interaction between neurons and myocytes to distinguish direct effects of low extracellular calcium on neuronal differentiation and indirect effects due to interference with neuron-myocyte interactions. We have examined the initiation of neurite outgrowth and the subsequent extension and orientation of processes. We find that (1) the number of neurons that initiate process outgrowth is reduced by the presence of myocytes in a standard medium containing calcium. Experiments with muscle-conditioned medium indicate that the production and/or secretion of inhibitory cues is calcium dependent. (2) When neurite initiation occurs, neuronal architecture in the absence of myocytes is similar to that in their presence, either in standard or in calcium-free medium, although neurite extension is enhanced by the absence of calcium. (3) Conditioned medium (CM) experiments additionally demonstrate that the orientation of neurite outgrowth to myocyte-derived cues is calcium dependent, although the production of directional cues by myocytes is calcium independent. © 1993 John Wiley & Sons, Inc.     

Intermittent altitude exposures improve muscular performance at 4,300 m.

Chronic altitude residence improves muscular performance at altitude, but the effect of intermittent altitude exposures (IAE) on muscular performance at altitude has not been defined. The purpose of this study was to determine the effects of 3 wk of IAE, in combination with rest and cycle training, on muscular performance at altitude. Six lowlanders (23 +/- 2 yr, 77 +/- 6 kg; means +/- SE) completed a cycle time trial and adductor pollicis endurance test at sea level and during a 30-h acute exposure to 4,300 m altitude equivalent (barometric pressure = 446 mmHg) once before (pre-IAE) and once after (post-IAE) a 3-wk period of IAE (4 h/day, 5 days/wk, 4,300 m). During each IAE, three subjects cycled for 45-60 min/day at 60%-70% of maximal O2 uptake and three subjects rested. Cycle training during each IAE did not appear to affect muscular performance at altitude. Thus data from all six subjects were combined. Three weeks of IAE resulted in 1) a 21 +/- 6% improvement (P < 0.05) in cycle time-trial performance (min) from pre-IAE (32.8 +/- 3.7) to post-IAE (24.8 +/- 1.2), 2) a 63 +/- 26% improvement (P < 0.05) in adductor pollicis endurance (min) from pre-IAE (9.2 +/- 2.8) to post-IAE (14.8 +/- 4.2), and 3) a 10 +/- 4% increase (P < 0.05) in resting arterial O2 saturation (%) from pre-IAE (82 +/- 2) to post-IAE (90 +/- 1). These improvements in muscular performance after IAE correlated strongly with increases in resting arterial O2 saturation and were comparable to those reported previously after chronic altitude residence. IAE may therefore be used as an alternative to chronic altitude residence to facilitate improvements in muscular performance in athletes, soldiers, mountaineers, shift workers, and others that are deployed to altitude.     

Nutrition and Physical Activity Program to Attenuate Obesity and Promote Physical and Metabolic Fitness in Elementary School Children

Obesity and low levels of physical and metabolic fitness are risk factors for cardiovascular disease and diabetes. The purpose of this investigation was to attenuate obesity and improve physical and metabolic fitness in elementary school children. Schools have the opportunity, mechanisms, and personnel in place to deliver nutrition education, fitness activities, and a school food service that is nutritious and healthy. Cohorts from grades 3 to 5 in two school districts in rural Nebraska (Intervention/Control) participated in a 2-year study of physical activity and modified school lunch program. Data collection for aerobic capacity, body composition, blood chemistry, nutrition knowledge, energy intake, and physical activity was at the beginning and end of each year. Int received enhanced physical activity, grade specific nutrition education, and a lower fat and sodium school lunch program. Con continued with a regular school lunch and team sports activity program. At year 2, Int lunches had significantly less energy (9%), fat (25%), sodium (21%), and more fiber (17%). However, measures of 24-hour energy intake for Int and Con showed significant differences for sodium only. Physical activity in the classroom was 6% greater for Int compared to Con (p < 0.05) but physical activity outside of school was ?16 % less for Int compared to Con (p < 0.05). Body weight and body fat were not different between schools for normal weight or obese children. No differences were found for cholesterol, insulin, and glucose; however, HDL cholesterol was significantly greater and cholesterol/HDL was significantly less for Int compared to Con (p < 0.05). It appears that compensation in both energy intake and physical activity outside of school may be responsible for the lack of differences between Int and Con.