Electrostatic control of the tryptophan radical in cytochrome c peroxidase

Previously a K(+)-binding site, analogous to that found in ascorbate peroxidase (APX), was engineered into cytochrome c peroxidase (CcP) to test the hypothesis that the bound K(+) influences the stability of the Trp191 cation radical formed during the CcP catalytic cycle (Bonagura et al., (1996) Biochemistry 35, 6107 and Bonagura et al., (1999) Biochemistry 38, 5528). Characterization of this mutant, designated CcPK2, showed that the stability of the Trp191 cation radical is dependent on the occupancy of the engineered K(+) site and that the Trp191 radical was much less stable in this mutant than in wild-type CcP. The mutations Met230Leu, Met231Gln, and Met172Ser have now been constructed on the CcPK2 mutant template to test if the Met residues also contribute to the stabilization of the Trp191 cation radical. Crystal structures show that the mutations affect only the local structure near the sites of mutation. Removal of these electronegative residues located less than 8 A from the Trp radical results in a further destabilization of the Trp radical. The characteristic EPR signal associated with the Trp radical is significantly narrowed and is characteristic of a tyrosine radical signal. Double-mixing stopped-flow experiments, where the delay time between the formation of CcP compound I and its mixing with horse heart ferrocytochrome c is varied, show that the stability of the Trp radical decreases as the Met residues are removed from the proximal cavity. When taken together, these results demonstrate a strong correlation between the experimentally determined stability of the Trp191 radical, the enzyme activity, and the calculated electrostatic stabilization of the Trp191 radical.     

High-Frequency Stimulation of the Subthalamic Nucleus Counteracts Cortical Expression of Major Histocompatibility Complex Genes in a Rat Model of Parkinson’s Disease

High-frequency stimulation of the subthalamic nucleus (STN-HFS) is widely used as therapeutic intervention in patients suffering from advanced Parkinson’s disease. STN-HFS exerts a powerful modulatory effect on cortical motor control by orthodromic modulation of basal ganglia outflow and via antidromic activation of corticofugal fibers. However, STN-HFS-induced changes of the sensorimotor cortex are hitherto unexplored. To address this question at a genomic level, we performed mRNA expression analyses using Affymetrix microarray gene chips and real-time RT-PCR in sensorimotor cortex of parkinsonian and control rats following STN-HFS. Experimental parkinsonism was induced in Brown Norway rats by bilateral nigral injections of 6-hydroxydopamine and was assessed histologically, behaviorally, and electrophysiologically. We applied prolonged (23h) unilateral STN-HFS in awake and freely moving animals, with the non-stimulated hemisphere serving as an internal control for gene expression analyses. Gene enrichment analysis revealed strongest regulation in major histocompatibility complex (MHC) related genes. STN-HFS led to a cortical downregulation of several MHC class II (RT1-Da, Db1, Ba, and Cd74) and MHC class I (RT1CE) encoding genes. The same set of genes showed increased expression levels in a comparison addressing the effect of 6-hydroxydopamine lesioning. Hence, our data suggest the possible association of altered microglial activity and synaptic transmission by STN-HFS within the sensorimotor cortex of 6-hydroxydopamine treated rats.     

Deep sequencing reveals clonal evolution patterns and mutation events associated with relapse in B-cell lymphomas

Background

Molecular mechanisms associated with frequent relapse of diffuse large B-cell lymphoma (DLBCL) are poorly defined. It is especially unclear how primary tumor clonal heterogeneity contributes to relapse. Here, we explore unique features of B-cell lymphomas - VDJ recombination and somatic hypermutation - to address this question.

Results

We performed high-throughput sequencing of rearranged VDJ junctions in 14 pairs of matched diagnosis-relapse tumors, among which 7 pairs were further characterized by exome sequencing. We identify two distinctive modes of clonal evolution of DLBCL relapse: an early-divergent mode in which clonally related diagnosis and relapse tumors diverged early and developed in parallel; and a late-divergent mode in which relapse tumors developed directly from diagnosis tumors with minor divergence. By examining mutation patterns in the context of phylogenetic information provided by VDJ junctions, we identified mutations in epigenetic modifiers such as KMT2D as potential early driving events in lymphomagenesis and immune escape alterations as relapse-associated events.

Conclusions

Altogether, our study for the first time provides important evidence that DLBCL relapse may result from multiple, distinct tumor evolutionary mechanisms, providing rationale for therapies for each mechanism. Moreover, this study highlights the urgent need to understand the driving roles of epigenetic modifier mutations in lymphomagenesis, and immune surveillance factor genetic lesions in relapse.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0432-0) contains supplementary material, which is available to authorized users.     

Comparative Study on Plant Latex Particles and Latex Coagulation in Ficus benjamina,Campanula glomerata and Three Euphorbia species

Among latex-producing plants, mainly the latex of Hevea brasiliensis has been studied in detail so far, while comprehensive comparative studies of latex coagulation mechanisms among the more than 20,000 latex-bearing plant species are lacking. In order to give new insights into the potential variety of coagulation mechanisms, the untreated natural latices of five latex-bearing plants from the families Euphorbiaceae, Moraceae and Campanulaceae were visualised using Cryo-SEM and their particle size compared using the laser diffraction method. Additionally, the laticifers of these plants species were examined in planta via Cryo-SEM. Similar latex particle sizes and shape were found in Ficus benjamina and Hevea brasiliensis. Hence, and due to other similarities, we hypothesize comparable, mainly chemical, coagulation mechanisms in these two species, whereas a physical coagulation mechanism is proposed for the latex of Euphorbia spp. The latter mechanism is based on the huge amount of densely packed particles that after evaporation of water build a large surface area, which accelerates the coagulation procedure.