Temperature dependent gene expression induced by PNIPAM-based copolymers: potential of hyperthermia in gene transfer

The objective of this work was to obtain gene delivery vectors with high efficiency induced by application of local hyperthermia. As a building construct for the polyplex particles, block copolymers were used, in which one block represents poly(ethyleneimine) (PEI) and another block a statistical copolymer of poly(N-isopropylacryamide) (PNIPAM) and different hydrophilic monomers (acrylamide or vinylpyrrolidinone). The block copolymers were synthesizized by radical polymerization of the corresponding monomers directly onto PEI. The complexation of DNA with these copolymers led to small, charge neutral particles, which aggregated upon increasing the temperature from 37 degrees C to 42 degrees C. This aggregation was found to be responsible for the enhanced transfection efficiency of these formulations under hyperthermic conditions. Gene expression in cells treated by hyperthermia was found to be nearly 2 orders of magnitude higher in comparison to cells transfected at physiological temperature. The mechanism by which hyperthermia influences the gene transfection efficiency is proposed.     

Bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase. Comparison of pre-steady state and single turnover methylation of 40-mer duplexes containing two (un)modified target sites

We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5'-GATC/5'-GATC) or some modified variant(s). The results extend a model from studies with single-site 20-mer duplexes. Under pre-steady state conditions, monomeric T4Dam methyltransferase-AdoMet complexes were capable of rapid methylation of adenine residues in 40-mer duplexes containing two sites. During processive movement of T4Dam to the next site, the rate-limiting step was the exchange of the product S-adenosyl-l-homocysteine (AdoHcy) for AdoMet without T4Dam dissociating from the duplex. Consequently, instead of a single exponential rate dependence, complex methylation curves were obtained with at least two pre-steady state steps. With 40-mer duplexes containing a single target site, the kinetics were simpler, fitting a single exponential followed by a linear steady state phase. Single turnover methylation of 40-mer duplexes also proceeded in two stages. First, two dimeric T4Dam-AdoMet molecules bound, and each catalyzed a two-step methylation. Instead of processive movement of T4Dam, a conformational adaptation occurred. We propose that following methyl transfer to one strand, dimeric (T4Dam-AdoMet)-(T4Dam-AdoHcy) was capable of rapidly reorienting itself and catalyzing methyl transfer to the target adenine on the complementary, unmethylated strand. This second stage methyl transfer occurred at a rate about 25-fold slower than in the first step; it was rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. Under single turnover conditions, there was complete methylation of all target adenine residues with each of the two-site 40-mer duplexes.     

Temporal pattern of locomotor activity in Drosophila melanogaster

The temporal pattern of locomotor activity of single Drosophila melanogaster flies freely walking in small tubes is described. Locomotor activity monitored by a light gate has a characteristic time-course that depends upon age and the environmental conditions. Several methods are applied to assess the complexity of the temporal pattern. The pattern varies according to sex, genotype, age and environmental conditions (food; light). Activity occurs clustered in bouts. The intrinsic bout structure is quantified by four parameters: number of light gate passages (counts) per bout, duration of a bout, pause between two successive bouts and mean bout period. In addition, the distribution of the periods between light-gate crossings (inter-count intervals) as function of inter-count interval duration reveals a power law, suggesting that the overall distribution of episodes of activity and inactivity has a fractal structure. In the dark without food, the fractal dimension which represents a measure of the complexity of the pattern is sex, genotype and age specific. Fractality is abolished by additional sensory stimulation (food; light). We propose that time-course, bout structure and fractal dimension of the temporal pattern of locomotor activity describe different aspects of the fly's central pattern generator for locomotion and its motivational control. Accepted: 10 October 1998     

Preparation of a monolithic capillary column with immobilized alpha-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for alpha-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)-N,N -diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, alpha-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the alpha-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.     

Life cycle energy and GHG emissions of PET recycling: change-oriented effects

Purpose  

The demand of PET bottles has increased rapidly in the past decades. The purpose of this study is to understand the environmental impact of PET recycling system, in which used bottles are recycled into both fibre and bottles, and to compare the recycling system with single-use PET.     

Design,synthesis, biological evaluation,and modeling of a non-carbohydrate antagonist of the myelin-associated glycoprotein

Broad modifications of various positions of the minimal natural epitope recognized by the myelin-associated glycoprotein (MAG), a blocker of regeneration of neurite injuries, produced sialosides with nanomolar affinities. However, important pharmacokinetic issues, for example, the metabolic stability of these sialosides, remain to be addressed. For this reason, the novel non-carbohydrate mimic 3 was designed and synthesized from (?)-quinic acid. For the design of 3, previously identified beneficial modifications of side chains of Neu5Ac were combined with the replacement of the ring oxygen by a methylene group and the substitution of the C(4)-OH by an acetamide. Although docking experiments to a homology model of MAG revealed that mimic 3 forms all but one of the essential hydrogen bonds identified for the earlier reported lead 2, its affinity was substantially reduced. Extensive molecular-dynamics simulation disclosed that the missing hydrogen bond of the former C(8)-OH leads to a change of the orientation of the side chain. As a consequence, an important hydrophobic contact is compromised leading to a loss of affinity.     

Simultaneous EEG-fMRI during a Working Memory Task: Modulations in Low and High Frequency Bands

Background

EEG studies of working memory (WM) have demonstrated load dependent frequency band modulations. FMRI studies have localized load modulated activity to the dorsolateral prefrontal cortex (DLPFC), medial prefrontal cortex (MPFC), and posterior parietal cortex (PPC). Recently, an EEG-fMRI study found that low frequency band (theta and alpha) activity negatively correlated with the BOLD signal during the retention phase of a WM task. However, the coupling of higher (beta and gamma) frequencies with the BOLD signal during WM is unknown.

Methodology

In 16 healthy adult subjects, we first investigated EEG-BOLD signal correlations for theta (5–7 Hz), alpha1 (8–10), alpha2 (10–12 Hz), beta1 (13–20), beta2 (20–30 Hz), and gamma (30–40 Hz) during the retention period of a WM task with set size 2 and 5. Secondly, we investigated whether load sensitive brain regions are characterised by effects that relate frequency bands to BOLD signals effects.

Principal Findings

We found negative theta-BOLD signal correlations in the MPFC, PPC, and cingulate cortex (ACC and PCC). For alpha1 positive correlations with the BOLD signal were found in ACC, MPFC, and PCC; negative correlations were observed in DLPFC, PPC, and inferior frontal gyrus (IFG). Negative alpha2-BOLD signal correlations were observed in parieto-occipital regions. Beta1-BOLD signal correlations were positive in ACC and negative in precentral and superior temporal gyrus. Beta2 and gamma showed only positive correlations with BOLD, e.g., in DLPFC, MPFC (gamma) and IFG (beta2/gamma). The load analysis revealed that theta and—with one exception—beta and gamma demonstrated exclusively positive load effects, while alpha1 showed only negative effects.

Conclusions

We conclude that the directions of EEG-BOLD signal correlations vary across brain regions and EEG frequency bands. In addition, some brain regions show both load sensitive BOLD and frequency band effects. Our data indicate that lower as well as higher frequency brain oscillations are linked to neurovascular processes during WM.     

Estimation of breed contributions to present and future genetic diversity of 44 North Eurasian cattle breeds using core set diversity measures

Extinction of breeds threatens genetic diversity of livestock species. The need to conserve genetic diversity is widely accepted but involves in general two questions: (i) is the expected loss of diversity in a set of breeds within a defined future time horizon large enough to establish a conservation plan, and if so (ii) which breeds should be prioritised for such a conservation plan? The present study uses a marker assisted methodology to address these questions. The methodology combines core set diversity measures with a stochastic method for the estimation of expected future diversity and breed marginal diversities. The latter is defined as the change in the total diversity of all breeds caused by a one unit decrease in extinction probability of a particular breed. The stochastic method was validated by means of simulations. A large field data set consisting of 44 North Eurasian cattle breeds was analysed using simplified determined extinction probabilities. The results show that the expected loss of diversity in this set within the next 20 to 50 years is between 1 and 3% of the actual diversity, provided that the extinction probabilities which were used are approximately valid. If this loss is to be reduced, it is sufficient to include those three to five breeds with the highest marginal diversity in a conservation scheme.     

Bovine oocyte maturation, fertilization and further development in vitro and after transfer into recipients

Bovine oocytes were aspirated from ovaries within 1.6 to 2 hours after slaughter. They were then matured in TCM-199 medium drops under oil in CO(2)/air incubator at 39 degrees C. Spermatozoa were capacitated in SP-TALP medium with heparin. The percentage of embryos that developed in vitro to the 4- and 6- cell stages 48 hours post insemination and then reached the morula or blastocyst stage was 64.3% and 59.2%, respectively, while only 3.6% of the embryos that reached the 2-cell stage became morula or blastocysts. An average of 6.3+/-3.2 total in vitro fertilized embryos per cow were obtained (range 2 to 11). Maturation of bovine oocytes in vitro for 18 or 24 hours did not influence the percentage of cleaved embryos (71.0 and 75.9%, respectively) or that developed to the blastocyst stage (25.6 and 24.2%, respectively). The use of reindeer blood serum for in vitro culture of immature bovine oocytes and of dividing of embryos gave the following results: 57.4% of the oocytes cleaved after fertilization and 16.2% developed further to the blastocyst stage. In contrast in the control group, where cow serum was used, the values were 73.4% and 24.8%, respectively. Rabbit oviduct epithelium cell monolayers were able to support the development of 16.3% of the cleaved bovine embryos to the blastocyst stage as compared with 24.0% of the embryos on cow oviduct epithelium cell monolayers. After nonsurgical transplantation, 12 calves were produced from 91 in vitro fertilized embryos.     

In vitro shoot regeneration of Populus deltoides: effect of cytokinin and genotype

Treatment differences were observed in the in vitro adventitious shoot regeneration response from internodal explants of three genotypes of Populus deltoides cultured on media supplemented with five concentrations each of the cytokinins 6-benzyladenine, 2-isopentyladenine, and zeatin. For each of the three genotypes, the greatest number of shoots were consistently regenerated on media containing the cytokinin zeatin. Tissue necrosis resulted when explants from any of the three genotypes were cultured on media supplemented with 6-benzyladenine. A zeatin concentration by genotype interaction was also observed. Genotypic differences in shoot regeneration were observed for 16 genotypes of Populus deltoides when cultured on medium supplemented with 0.5 mgL^–1 zeatin. Six genotypes were highly recalcitrant and failed to regenerate shoots. The percent of explants regenerating was greater than 50% for four genotypes.Abbreviations WNA modified Woody Plant Media - BA N⁶-benzyladenine - 2-iP 2-isopentenyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - MS Murashige Skoog (1962) medium - NAA 1-naphthaleneacetic acid - PAR photosynthetically active radiation Journal Series No. 8938, Agricultural Research Division, University of Nebraska