Adenovirus-mediated gene transfer of Lp-PLA2 reduces LDL degradation and foam cell formation in vitro

Oxidation of LDL generates biologically active platelet-activating factor (PAF)-like phospholipid derivatives, which have potent proinflammatory activity. These products are inactivated by lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme capable of hydrolyzing PAF-like phospholipids. In this study, we generated an adenovirus (Ad) encoding human Lp-PLA2 and injected 10(8), 10(9), and 10(10) plaque-forming unit doses of Adlp-PLA2 and control AdlacZ intra-arterially into rabbits to achieve overexpression of Lp-PLA2 in liver and in vivo production of Lp-PLA2-enriched LDL. As a result, LDL particles with 3-fold increased Lp-PLA2 activity were produced with the highest virus dose. Increased Lp-PLA2 activity in LDL particles decreased the degradation rate in RAW 264 macrophages after standard in vitro oxidation to 60-80% compared with LDL isolated from LacZ-transduced control rabbits. The decrease was proportional to the virus dose and Lp-PLA2 activity. Lipid accumulation and foam cell formation in RAW 264 macrophages were also decreased when incubated with oxidized LDL containing the highest Lp-PLA2 activity. Inhibition of the Lp-PLA2 activity in the LDL particles led to an increase in lipid accumulation and foam cell formation. It is concluded that increased Lp-PLA2 activity in LDL attenuates foam cell formation and decreases LDL oxidation and subsequent degradation in macrophages.     

Responses of Strawberry (Fragaria × ananassa) to Supplemental UV-B Radiation and Selenium Under Field Conditions

In greenhouse experiments, selenium (Se) has been shown to defend plants against detrimental effects of heavy UV-B radiation stress. The aim of this study was to investigate whether this positive effect can be found in open-field conditions with enhancement of UV-B radiation. In the experiment, conducted with strawberry (Fragaria×ananassa, cultivars “Jonsok” and “Polka”) over two growing seasons, plants were exposed to UV-B radiation (including UV-A) and cultivated without Se or supplied with Se added at two levels (0.1 and 1.0 mg kg⁻¹). The plants were monitored for growth, flavonoids, chlorophyll fluorescence, net photosynthesis as well as tissue and cell structure. Photosystem II was observed to be sensitive to UV-B stress under field conditions. In the leaves, a decrease in F_v/F_m was seen at the end of the growing season, implying a cumulative effect of UV-B stress. Several parameters, especially cell and tissue structures, were affected by UV-B and UV-A treatments, which proves the need for UV-A control in outdoor UV-B supplementation studies. Addition of Se did not ameliorate the harmful effects of UV-B but the lower Se-increment level increased leaf growth. The effects of UV-B and Se differed during the two experimental years, indicating the need to repeat experiments during several growing seasons.     

NMR and crystal structures of the Pyrococcus horikoshii RadA intein guide a strategy for engineering a highly efficient and promiscuous intein

In protein splicing, an intervening protein sequence (intein) in the host protein excises itself out and ligates two split host protein sequences (exteins) to produce a mature host protein. Inteins require the involvement for the splicing of the first residue of the extein that follows the intein (which is Cys, Ser, or Thr). Other extein residues near the splicing junctions could modulate splicing efficiency even when they are not directly involved in catalysis. Mutual interdependence between this molecular parasite (intein) and its host protein (exteins) is not beneficial for intein spread but could be advantageous for intein survival during evolution. Elucidating extein-intein dependency has increasingly become important since inteins are recognized as useful biotechnological tools for protein ligation. We determined the structures of one of inteins with high splicing efficiency, the RadA intein from Pyrococcus horikoshii (PhoRadA). The solution NMR structure and the crystal structures elucidated the structural basis for its high efficiency and directed our efforts of engineering that led to rational design of a functional minimized RadA intein. The crystal structure of the minimized RadA intein also revealed the precise interactions between N-extein and the intein. We systematically analyzed the effects at the -1 position of N-extein and were able to significantly improve the splicing efficiency of a less robust splicing variant by eliminating the unfavorable extein-intein interactions observed in the structure. This work provides an example of how unveiling structure-function relationships of inteins offer a promising way of improving their properties as better tools for protein engineering.     

No effects of short-term GSM mobile phone radiation on cerebral blood flow measured using positron emission tomography

The present study investigated the effects of 902.4 MHz Global System for Mobile Communications (GSM) mobile phone radiation on cerebral blood flow using positron emission tomography (PET) with the ¹⁵O‐water tracer. Fifteen young, healthy, right‐handed male subjects were exposed to phone radiation from three different locations (left ear, right ear, forehead) and to sham exposure to test for possible exposure effects on brain regions close to the exposure source. Whole‐brain [¹⁵O]H₂O–PET images were acquired 12 times, 3 for each condition, in a counterbalanced order. Subjects were exposed for 5 min in each scan while performing a simple visual vigilance task. Temperature was also measured in the head region (forehead, eyes, cheeks, ear canals) during exposure. The exposure induced a slight temperature rise in the ear canals but did not affect brain hemodynamics and task performance. The results provided no evidence for acute effects of short‐term mobile phone radiation on cerebral blood flow. Bioelectromagnetics 33:247–256, 2012. © 2011 Wiley Periodicals, Inc.     

Combining NMR ensembles and molecular dynamics simulations provides more realistic models of protein structures in solution and leads to better chemical shift prediction

While chemical shifts are invaluable for obtaining structural information from proteins, they also offer one of the rare ways to obtain information about protein dynamics. A necessary tool in transforming chemical shifts into structural and dynamic information is chemical shift prediction. In our previous work we developed a method for 4D prediction of protein ¹H chemical shifts in which molecular motions, the 4th dimension, were modeled using molecular dynamics (MD) simulations. Although the approach clearly improved the prediction, the X-ray structures and single NMR conformers used in the model cannot be considered fully realistic models of protein in solution. In this work, NMR ensembles (NMRE) were used to expand the conformational space of proteins (e.g. side chains, flexible loops, termini), followed by MD simulations for each conformer to map the local fluctuations. Compared with the non-dynamic model, the NMRE+MD model gave 6–17% lower root-mean-square (RMS) errors for different backbone nuclei. The improved prediction indicates that NMR ensembles with MD simulations can be used to obtain a more realistic picture of protein structures in solutions and moreover underlines the importance of short and long time-scale dynamics for the prediction. The RMS errors of the NMRE+MD model were 0.24, 0.43, 0.98, 1.03, 1.16 and 2.39 ppm for ¹Hα, ¹HN, ¹³Cα, ¹³Cβ, ¹³CO and backbone ¹⁵N chemical shifts, respectively. The model is implemented in the prediction program 4DSPOT, available at .     

Phylogenetics and evolution of host-plant use in leaf-mining sawflies (Hymenoptera: Tenthredinidae: Heterarthrinae)

The habit of mining within leaves has evolved convergently in numerous plant-feeding insect taxa. Many leaf-mining groups contain a large number of species with distinct feeding preferences, which makes them highly suitable for studies on the evolutionary history of host-plant use and on the role of niche shifts in speciation. We aimed to clarify the origin, classification, and ecological evolution of the tenthredinid sawfly subfamily Heterarthrinae, which contains c. 150 leaf-mining species that collectively feed on over 20 plant genera around the world. For this, we reconstructed the phylogeny of representative heterarthrine species and diverse outgroups from the superfamily Tenthredinoidea on the basis of DNA sequence data collected from two mitochondrial (CoI and Cytb) and two nuclear (EF-1α and NaK) genes. Thereafter, we inferred the history of niche diversification within Heterarthrinae by plotting larval host-plant associations on the trees, and by contrasting a time-calibrated leaf-miner phylogeny with the phylogeny of their host plants. The results show that: (1) heterarthrine leaf-miners constitute a monophyletic group that arose from external-feeding blennocampine lineages within the Tenthredinidae c. 110-80 million years ago; (2) heterarthrines generally radiated well after their host taxa, and extant host-plant associations therefore result from a combination of host conservatism and occasional shifts among available plant taxa; and (3) diversification in Heterarthrinae apparently occurs by multiple mechanisms, including sympatric or allopatric ecological speciation, non-ecological allopatric speciation, and possibly allochronic speciation. Overall, both present and historical host-use patterns within the Heterarthrinae exhibit striking similarities to patterns found in co-occurring herbivore taxa.     

Structural MRI in Frontotemporal Dementia: Comparisons between Hippocampal Volumetry,Tensor-Based Morphometry and Voxel-Based Morphometry

Background

MRI is an important clinical tool for diagnosing dementia-like diseases such as Frontemporal Dementia (FTD). However there is a need to develop more accurate and standardized MRI analysis methods.

Objective

To compare FTD with Alzheimer’s Disease (AD) and Mild Cognitive Impairment (MCI) with three automatic MRI analysis methods - Hippocampal Volumetry (HV), Tensor-based Morphometry (TBM) and Voxel-based Morphometry (VBM), in specific regions of interest in order to determine the highest classification accuracy.

Methods

Thirty-seven patients with FTD, 46 patients with AD, 26 control subjects, 16 patients with progressive MCI (PMCI) and 48 patients with stable MCI (SMCI) were examined with HV, TBM for shape change, and VBM for gray matter density. We calculated the Correct Classification Rate (CCR), sensitivity (SS) and specificity (SP) between the study groups.

Results

We found unequivocal results differentiating controls from FTD with HV (hippocampus left side) (CCR = 0.83; SS = 0.84; SP = 0.80), with TBM (hippocampus and amygdala (CCR = 0.80/SS = 0.71/SP = 0.94), and with VBM (all the regions studied, especially in lateral ventricle frontal horn, central part and occipital horn) (CCR = 0.87/SS = 0.81/SP = 0.96). VBM achieved the highest accuracy in differentiating AD and FTD (CCR = 0.72/SS = 0.67/SP = 0.76), particularly in lateral ventricle (frontal horn, central part and occipital horn) (CCR = 0.73), whereas TBM in superior frontal gyrus also achieved a high accuracy (CCR = 0.71/SS = 0.68/SP = 0.73). TBM resulted in low accuracy (CCR = 0.62) in the differentiation of AD from FTD using all regions of interest, with similar results for HV (CCR = 0.55).

Conclusion

Hippocampal atrophy is present not only in AD but also in FTD. Of the methods used, VBM achieved the highest accuracy in its ability to differentiate between FTD and AD.     

Enhanced behavioral sensitivity to the competitive GABA agonist, gaboxadol, in transgenic mice over-expressing hippocampal extrasynaptic alpha6beta GABA(A) receptors

The behavioral and functional significance of the extrasynaptic inhibitory GABA(A) receptors in the brain is still poorly known. We used a transgenic mouse line expressing the GABA(A) receptor alpha6 subunit gene in the forebrain under the Thy-1.2 promoter (Thy1alpha6) mice ectopically expressing alpha6 subunits especially in the hippocampus to study how extrasynaptically enriched alphabeta(gamma2)-type receptors alter animal behavior and receptor responses. In these mice extrasynaptic alpha6beta receptors make up about 10% of the hippocampal GABA(A) receptors resulting in imbalance between synaptic and extrasynaptic inhibition. The synthetic GABA-site competitive agonist gaboxadol (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; 3 mg/kg) induced remarkable anxiolytic-like response in the light : dark exploration and elevated plus-maze tests in Thy1alpha6 mice, while being almost inactive in wild-type mice. The transgenic mice also lost quicker and for longer time their righting reflex after 25 mg/kg gaboxadol than wild-type mice. In hippocampal sections of Thy1alpha6 mice, the alpha6beta receptors could be visualized autoradiographically by interactions between gaboxadol and GABA via [(35)S]TBPS binding to the GABA(A) receptor ionophore. Gaboxadol inhibition of the binding could be partially prevented by GABA. Electrophysiology of recombinant GABA(A) receptors revealed that GABA was a partial agonist at alpha6beta3 and alpha6beta3delta receptors, but a full agonist at alpha6beta3gamma2 receptors when compared with gaboxadol. The results suggest strong behavioral effects via selective pharmacological activation of enriched extrasynaptic alphabeta GABA(A) receptors, and the mouse model represents an example of the functional consequences of altered balance between extrasynaptic and synaptic inhibition.     

Setup and dosimetry for exposing anaesthetised pigs in vivo to 900 MHz GSM mobile phone fields

The aim of this study was a dosimetrical analysis of the setup used in the exposure of the heads of domestic pigs to GSM-modulated radio frequency electromagnetic fields (RF-EMF) at 900 MHz. The heads of pigs were irradiated with a half wave dipole using three different exposure routines; short bursts of 1-3 s at two different exposure levels and a continuous 10-min exposure. The electroencephalogram (EEG) was registered continuously during the exposures to search for RF-EMF originated changes. The dosimetry was based on simulations with the anatomical heterogeneous numerical model of the pig head. The simulation results were validated by experimental measurements with the exposure dipole and a homogeneous liquid phantom resembling the pig head. The specific absorption rate (SAR), defined as a maximum average over 10 g tissue mass (SAR(10g)), was 7.3 W/kg for the first set of short bursts and 31 W/kg for the second set of short bursts. The SAR(10g) in the continuous 10-min exposure was 31 W/kg. The estimated uncertainty for the dosimetry was +/-25% (K = 2).     

Gender influence on vasoactive hormones at rest and during a 70 degrees head-up tilt in healthy humans.

To evaluate the influence of age and gender on the neuroendocrine control of blood pressure in normal subjects, a 13-min 70 degrees head-up tilt (HUT) was applied after 3 h of recumbency to 109 healthy men and women aged 23-50 yr (age group I) and 51-77 yr (age group II). We found that age and gender had a significant influence on plasma norepinephrine (PNE) concentration at baseline and in the upright position. PNE was significantly higher in older men compared with the younger men and women of both age groups, suggesting a divergent age-related activation of the sympathetic nervous system between genders at baseline as well as during a sustained orthostatic challenge. There was no significant influence of age or gender on plasma epinephrine at baseline or during HUT. Plasma renin activity was significantly higher at baseline as well as in the upright position during HUT in elderly men than in women. Age or gender had no influence on plasma vasopressin (PAVP), and, regardless of age, nonhypotensive HUT induced an extremely modest increase in PAVP. The syncopal subjects displayed a hormonal pattern associating increased PNE and a surge in plasma epinephrine and PAVP minutes before syncope during HUT. The orthostatic intolerance appears not to be a feature of healthy aging per se. In healthy subjects, both age and gender modulate markedly the cardiovascular and neuroendocrine responses to an orthostatic challenge and must be taken into consideration, particularly when catecholamine responses are studied.