Translocation of Tomato bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing suppressor activity

The P19 protein of Tomato bushy stunt virus is a potent suppressor of RNA silencing and, depending on the host species, is required for short- and long-distance virus movement and symptom production. P19 interacts with plant ALY proteins and relocalizes a subset of these proteins from the nucleus to the cytoplasm. Here we showed that coexpression by agroinfiltration in Nicotiana benthamiana of P19 and the subset of ALY proteins that are not relocalized from the nucleus interfered with the ability of P19 to suppress RNA silencing. We demonstrated that this interference correlates with the relocation of P19 from the cytoplasm into the nucleus, and by constructing and analyzing chimeric ALY genes, we showed that the C-terminal part of the central, RNA recognition motif of ALY is responsible for interaction with P19, relocalization or nonrelocalization of ALY, and inhibition of silencing suppression by P19. We studied the interaction of ALY and P19 by using the technique of bimolecular fluorescence complementation to show that these proteins associate physically in the nucleus but not detectably in the cytoplasm, and we present a model to explain the dynamics of this interaction.     

Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes

A double-blind randomized phase I trial was conducted in human immunodeficiency virus type 1 (HIV-1)-negative subjects receiving vaccines vectored by plasmid DNA and modified vaccinia virus Ankara (MVA) expressing HIV-1 p24/p17 gag linked to a string of CD8(+) T-cell epitopes. The trial had two groups. One group received either two doses of MVA.HIVA (2x MVA.HIVA) (n=8) or two doses of placebo (2x placebo) (n=4). The second group received 2x pTHr.HIVA followed by one dose of MVA.HIVA (n=8) or 3x placebo (n=4). In the pTHr.HIVA-MVA.HIVA group, HIV-1-specific T-cell responses peaked 1 week after MVA.HIVA vaccination in both ex vivo gamma interferon (IFN-gamma) ELISPOT (group mean, 210 spot-forming cells/10(6) cells) and proliferation (group mean stimulation index, 37), with assays detecting positive responses in four out of eight and five out of eight subjects, respectively. No HIV-1-specific T-cell responses were detected in either assay in the 2x MVA.HIVA group or subjects receiving placebo. Using a highly sensitive and reproducible cultured IFN-gamma ELISPOT assay, positive responses mainly mediated by CD4(+) T cells were detected in eight out of eight vaccinees in the pTHr.HIVA-MVA.HIVA group and four out of eight vaccinees in the 2x MVA.HIVA group. Importantly, no false-positive responses were detected in the eight subjects receiving placebo. Of the 12 responders, 11 developed responses to previously identified immunodominant CD4(+) T-cell epitopes, with 6 volunteers having responses to more than one epitope. Five out of 12 responders also developed CD8(+) T-cell responses to the epitope string. Induced T cells produced a variety of anti-viral cytokines, including tumor necrosis factor alpha and macrophage inflammatory protein 1 beta. These data demonstrate that prime-boost vaccination with recombinant DNA and MVA vectors can induce multifunctional HIV-1-specific T cells in the majority of vaccinees.     

Free thyroxine, cognitive decline and depression in Alzheimer's disease

The role of thyroid function in Alzheimer's disease (AD) has been subject to a number of studies during the last years. We investigated the possible relationship between plasma levels of the biologically active free form of thyroxin (fT4) and cognitive function in 227 outpatients with mild to moderate Alzheimer s disease (AD) in a cross-sectional study design. A significant negative correlation was found between plasma fT4-levels and Mini-Mental state examination (MMSE) score (Spearman Rho = -0.14, p=0.04). When the lowest quartile of fT4-levels (<15.1 pmol/l) was compared to the highest quartile (>19.0 pmol/l), statistically significant lower mean MMSE-scores were seen in the group with the highest fT4-levels (p<0.05, ANOVA). The mean difference between the 1st and the 4th quartile of fT4 was 2.6 MMSE-score points. No correlations were found between plasma total T4-levels, plasma total T3-levels, plasma TSH-levels and the MMSE score (p>0.05). When fT4 quartile groups were compared for depression measured in the Geriatric Depression Score (GDS 15), a slightly higher score was seen in the 1s and 2nd compared to the 3rd and 4th quartile groups without reaching statistical significance (1st quartile of fT4: GDS 5.2 +/- 3.8; 2nd: 5.3 +/- 4.0; 3rd: 4.4 +/- 3.4; 4th: 4.5 +/- 3.8) pointing to a reverse correlation of fT4 levels and depressive mood. This study leads to the conclusion that high levels of plasma fT4 might result in a worsening of cognitive impairment and a positive effect on depressive mood in AD.     

Prodrug thiamine analogs as inhibitors of the enzyme transketolase

Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.     

Odor supported place cell model and goal navigation in rodents

Experiments with rodents demonstrate that visual cues play an important role in the control of hippocampal place cells and spatial navigation. Nevertheless, rats may also rely on auditory, olfactory and somatosensory stimuli for orientation. It is also known that rats can track odors or self-generated scent marks to find a food source. Here we model odor supported place cells by using a simple feed-forward network and analyze the impact of olfactory cues on place cell formation and spatial navigation. The obtained place cells are used to solve a goal navigation task by a novel mechanism based on self-marking by odor patches combined with a Q-learning algorithm. We also analyze the impact of place cell remapping on goal directed behavior when switching between two environments. We emphasize the importance of olfactory cues in place cell formation and show that the utility of environmental and self-generated olfactory cues, together with a mixed navigation strategy, improves goal directed navigation.     

Path-finding in real and simulated rats: assessing the influence of path characteristics on navigation learning

A large body of experimental evidence suggests that the hippocampal place field system is involved in reward based navigation learning in rodents. Reinforcement learning (RL) mechanisms have been used to model this, associating the state space in an RL-algorithm to the place-field map in a rat. The convergence properties of RL-algorithms are affected by the exploration patterns of the learner. Therefore, we first analyzed the path characteristics of freely exploring rats in a test arena. We found that straight path segments with mean length 23 cm up to a maximal length of 80 cm take up a significant proportion of the total paths. Thus, rat paths are biased as compared to random exploration. Next we designed a RL system that reproduces these specific path characteristics. Our model arena is covered by overlapping, probabilistically firing place fields (PF) of realistic size and coverage. Because convergence of RL-algorithms is also influenced by the state space characteristics, different PF-sizes and densities, leading to a different degree of overlap, were also investigated. The model rat learns finding a reward opposite to its starting point. We observed that the combination of biased straight exploration, overlapping coverage and probabilistic firing will strongly impair the convergence of learning. When the degree of randomness in the exploration is increased, convergence improves, but the distribution of straight path segments becomes unrealistic and paths become 'wiggly'. To mend this situation without affecting the path characteristic two additional mechanisms are implemented: a gradual drop of the learned weights (weight decay) and path length limitation, which prevents learning if the reward is not found after some expected time. Both mechanisms limit the memory of the system and thereby counteract effects of getting trapped on a wrong path. When using these strategies individually divergent cases get substantially reduced and for some parameter settings no divergence was found anymore at all. Using weight decay and path length limitation at the same time, convergence is not much improved but instead time to convergence increases as the memory limiting effect is getting too strong. The degree of improvement relies also on the size and degree of overlap (coverage density) in the place field system. The used combination of these two parameters leads to a trade-off between convergence and speed to convergence. Thus, this study suggests that the role of the PF-system in navigation learning cannot be considered independently from the animals' exploration pattern.     

Element distribution is altered in a zone surrounding human glioblastoma multiforme

Recent data indicate that A₁ adenosine receptor (A₁AR) density is increased in a zone surrounding human and experimental gliomas. On the contrary, tumor tissue and adjacent brain tissue show low to intermediate A₁AR densities. In order to assess whether changes in A₁AR expression are indicating further processes of a chemical reorganization of the peritumoral zone, we investigated element concentrations and distribution patterns of copper and zinc in six human glioblastoma multiforme (GBM) specimens by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). Uranium and lead were used as external standards.

Copper and zinc levels were increased in a peritumoral zone corresponding to the region of elevated A₁AR density. They showed a lower density in the solid tumor in comparison to surrounding brain tissue, although the cellular density was higher within GBM.

Our findings suggest that the immediate vicinity of GBM is characterized by increased levels of copper and zinc supporting the view that higher A₁AR density surrounding GBM is not an isolated alteration of peritumoral tissue but an indicator of complex changes in the vicinity of infiltrative tumors. Further research is needed to explore the pathophysiological consequences of altered peritumoral element distribution.     

Early vertebrate chromosome duplications and the evolution of the neuropeptide Y receptor gene regions

Background  

One of the many gene families that expanded in early vertebrate evolution is the neuropeptide (NPY) receptor family of G-protein coupled receptors. Earlier work by our lab suggested that several of the NPY receptor genes found in extant vertebrates resulted from two genome duplications before the origin of jawed vertebrates (gnathostomes) and one additional genome duplication in the actinopterygian lineage, based on their location on chromosomes sharing several gene families. In this study we have investigated, in five vertebrate genomes, 45 gene families with members close to the NPY receptor genes in the compact genomes of the teleost fishes Tetraodon nigroviridis and Takifugu rubripes. These correspond to Homo sapiens chromosomes 4, 5, 8 and 10.