The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function

Brain-derived neurotrophic factor (BDNF) modulates hippocampal plasticity and hippocampal-dependent memory in cell models and in animals. We examined the effects of a valine (val) to methionine (met) substitution in the 5' pro-region of the human BDNF protein. In human subjects, the met allele was associated with poorer episodic memory, abnormal hippocampal activation assayed with fMRI, and lower hippocampal n-acetyl aspartate (NAA), assayed with MRI spectroscopy. Neurons transfected with met-BDNF-GFP showed lower depolarization-induced secretion, while constitutive secretion was unchanged. Furthermore, met-BDNF-GFP failed to localize to secretory granules or synapses. These results demonstrate a role for BDNF and its val/met polymorphism in human memory and hippocampal function and suggest val/met exerts these effects by impacting intracellular trafficking and activity-dependent secretion of BDNF.     

Highly efficient "tight fit" immobilization of alpha-chymotrypsin in mesoporous MCM-41: a novel approach using precursor immobilization and activation

The zymogen alpha-chymotrypsinogen A is bound to mesoporous silica MCM-41 with a protein loading of 170 mg/g solid (MCM-Z) by a simple stirring in aqueous tris-HCl buffer (pH 7.2). The bound zymogen is then activated with trypsin to obtain alpha-chymotrypsin immobilized on MCM-41 (MCM-E.I) that displays an effective enzyme activity corresponding to 65 mg protein/g of solid support (3250 BTEE units/g). A direct immobilization of commercially available alpha-chymotrypsin (MCM-E.II) gives lower loading (1250 BTEE units/g). Protein content of the solid support after immobilization is confirmed by thermogravimetric analysis (TGA). The enzyme is tightly bound to the support and can be used over 100 recycles over 1 week in aqueous as well as reverse micellar media. The immobilized enzyme (MCM-E.I) has been used for resolution of N-acetyl-dl-amino acid esters and racemic trans-4-methoxy-3-phenylglycidic acid (PGA) methyl ester.     

Preparation and characterization of flurbiprofen beads by melt solidification technique

Amelt solidification technique has been developed to obtain sustained-release waxy beads of flurbiprofen. Low glass transition temperature (t _g) and shear-induced crystallization of flurbiprofen made it a suitable candidate for melt solidification technique. The process involved emulsification and solidification of flurbiprofen-cetyl alcohol melt at significantly low temperature (5°C). The effect of variables, namely, the amount of cetyl alcohol and the speed of agitation, was studied using 3² factorial design. The technique and the beads were evaluated on the basis of process and desired yield, surface topography, Fourier-transform infrared (FT-IR), differential scanning calorimetry (DSC), particle size distribution, crushing strength, and drug release. Average values for process and desired yields were 97% wt/wt and 26% wt/wt, respectively. No interaction was observed between drug and excipient. Multiple regression analysis was carried out, and response surfaces were obtained. A curvilinear relationship was observed between percentage of desired yield and the amount of cetyl alcohol. Linear decrease in crushing strength was observed with increase in the amount of cetyl alcohol. Drug released from the beads followed zero order kinetics. Burst release was shown to a greater extent in beads containing a lower amount of cetyl alcohol. Response surfaces of time required for certain percentage of drug (t _D%) showed that after critical concentration of about 20% of cetyl alcohol (400 mg/batch), no significant release retardant effect was observed.     

A study on the effect of operating parameters on the cross-flow microfiltration of yeasts

The effects of pump speed, cumulative permeate volume and concentration of feed (yeast cells) on the permeate flux have been studied on a batch cross-flow microfiltration process. The experiments were conducted for two different cellulose acetate membrane modules of 0.2 m and 0.45 m pore size. A three factor experiment was designed for this purpose and the effect of the operating parameters on the filtration rate was studied by the analysis of variance (ANOVA). It is concluded from the analysis of the experimental data that pump speed has the maximum bearing upon the permeate rate within the operating range of parameters. Fouling conditions were examined in the light of colloids deposition on membranes due to surface interactions. However this paper looks into the relationship and sensitivity of the operating parameters in a cross-flow microfiltration unit rather than exploring the theoretical principles behind the observed phenomena.This revised version was published online in October 2005 with corrections to the Cover Date.     

Enhanced stability of cis Pro-Pro peptide bond in Pro-Pro-Phe sequence motif

Identification of sequence motifs that favor cis peptide bonds in proteins is important for understanding and designing proteins containing turns mediated by cis peptide conformations. From (1)H NMR solution studies on short peptides, we show that the Pro-Pro peptide bond in Pro-Pro-Phe almost equally populates the cis and trans isomers, with the cis isomer stabilized by a CHc...pi interaction involving the terminal Pro and Phe. We also show that Phe is over-represented at sequence positions immediately following cis Pro-Pro motifs in known protein structures. Our results demonstrate that the Pro-Pro cis conformer in Pro-Pro-Phe sequence motifs is as important as the trans conformer, both in short peptides as well as in natively folded proteins.     

Evolution of hydraulic traits in closely related species pairs from Mediterranean and nonMediterranean environments of North America

Chaparral shrubs in California experience cool, wet winters and hot, dry summers characteristic of mediterranean-type climates; by contrast, morphologically similar close relatives in central Mexico experience summer rainfall. A comparison of closely related species pairs was conducted to examine whether evolutionary divergences in plant hydraulic conductivity were associated with contrasting seasonality of precipitation. Six species pairs in Santa Barbara, California and Tehuacan, Mexico were chosen to test for repeated directional divergences across the habitat contrast. Additionally, evolutionary correlations were examined using phylogenetically independent contrasts (PICs) among a suite of hydraulic traits, including stem- and leaf-specific conductivity, resistance to embolism, wood density, inverse Huber value, and minimum seasonal water potential. Leaf-specific conductivity was generally higher in California, but for most hydraulic traits the species pairs exhibited varied evolutionary trajectories across the climate contrast. A significant correlation was found between divergences in xylem resistance to embolism and minimum seasonal water potential, but no evolutionary trade-off was found between resistance and stem conductivity. Higher leaf-specific conductivity may be adaptive in California, where soil and atmospheric droughts coincide during summer months. This response is consistent with a hydraulic strategy of high leaf water supply under high evaporative demand to prevent excessive drops in water potential.     

Integration specificity of phage phiC31 integrase in the human genome

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of phiC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed approximately 101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a approximately 30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that phiC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. phiC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from phiC31 integrase have great potential utility.