Selective translation of mRNAs at synapses

Synaptic efficacy, a phenomenon that may underlie long-term memory storage, is controlled in part by the regulated translation of mRNAs stored in dendrites. The molecular basis by which specific mRNAs are selected for translation is beginning to emerge and appears to involve at least one mechanism that helps program early metazoan development. Because different neural transmitters elicit different synaptic responses that rely on local protein synthesis, a number of sequence-specific mRNA translational regulatory mechanisms are likely to function in neurons. Such mechanisms may be inferred from those operating in early development and in cognitive disease.     

Raf kinase inhibitor protein (RKIP) dimer formation controls its target switch from Raf1 to G protein-coupled receptor kinase (GRK) 2

Proteins controlling cellular networks have evolved distinct mechanisms to ensure specificity in protein-protein interactions. Raf kinase inhibitor protein (RKIP) is a multifaceted kinase modulator, but it is not well understood how this small protein (21 kDa) can coordinate its diverse signaling functions. Raf1 and G protein-coupled receptor kinase (GRK) 2 are direct interaction partners of RKIP and thus provide the possibility to untangle the mechanism of its target specificity. Here, we identify RKIP dimer formation as an important mechanistic feature in the target switch from Raf1 to GRK2. Co-immunoprecipitation and cross-linking experiments revealed RKIP dimerization upon phosphorylation of RKIP at serine 153 utilizing purified proteins as well as in cells overexpressing RKIP. A functional phosphomimetic RKIP mutant had a high propensity for dimerization and reproduced the switch from Raf1 to GRK2. RKIP dimerization and GRK2 binding, but not Raf1 interaction, were prevented by a peptide comprising amino acids 127-146 of RKIP, which suggests that this region is critical for dimer formation. Furthermore, a dimeric RKIP mutant displayed a higher affinity to GRK2, but a lower affinity to Raf1. Functional analyses of phosphomimetic as well as dimeric RKIP demonstrated that enhanced dimerization of RKIP translates into decreased Raf1 and increased GRK2 inhibition. The detection of RKIP dimers in a complex with GRK2 in murine hearts implies their physiological relevance. These findings represent a novel mechanistic feature how RKIP can discriminate between its different interaction partners and thus advances our understanding how specific inhibition of kinases can be achieved.     

A shortcut from plasma to chromatographic analysis: Straightforward and fast sample preparation for analysis of green tea catechins in human plasma

This paper describes a new and straightforward method for determination of the green tea catechins epicatechin, epicatechin gallate, epigallocatechin, and epigallocatechin gallate in human plasma. Sample preparation includes addition only of dimethylformamide and trichloroacetic acid. After centrifugation, the supernatant can be injected into the HPLC. If required, the glucuronides and sulphates of the catechins can be enzymatically hydrolysed before extraction. Recovery ranges from 92.9 to 98.2%; limits of detection, from 2.4 to 5.0 ng/mL; and relative standard deviations, from 3.1 to 8.6%. Twelve samples can be processed within 45 min, and are then ready to be injected into the HPLC. The method was successfully applied to human plasma. This method is suitable for studies on absorption, bioavailability, and kinetics of green tea catechins in plasma. Since manual work and time consumption are minimal, the procedure is especially useful for large numbers of samples.     

Analysis of myosin heavy chain functionality in the heart

Comparison of mammalian cardiac alpha- and beta-myosin heavy chain isoforms reveals 93% identity. To date, genetic methodologies have effected only minor switches in the mammalian cardiac myosin isoforms. Using cardiac-specific transgenesis, we have now obtained major myosin isoform shifts and/or replacements. Clusters of non-identical amino acids are found in functionally important regions, i.e. the surface loops 1 and 2, suggesting that these structures may regulate isoform-specific characteristics. Loop 1 alters filament sliding velocity, whereas Loop 2 modulates actin-activated ATPase rate in Dictyostelium myosin, but this remains untested in mammalian cardiac myosins. Alpha --> beta isoform switches were engineered into mouse hearts via transgenesis. To assess the structural basis of isoform diversity, chimeric myosins in which the sequences of either Loop 1+Loop 2 or Loop 2 of alpha-myosin were exchanged for those of beta-myosin were expressed in vivo. 2-fold differences in filament sliding velocity and ATPase activity were found between the two isoforms. Filament sliding velocity of the Loop 1+Loop 2 chimera and the ATPase activities of both loop chimeras were not significantly different compared with alpha-myosin. In mouse cardiac isoforms, myosin functionality does not depend on Loop 1 or Loop 2 sequences and must lie partially in other non-homologous residues.     

A major integral protein of the plant plasma membrane binds flavin

Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor.     

A novel mechanism by which hydrogen peroxide decreases calcium sensitivity in airway smooth muscle

The purpose of this study was to test the hypothesis that H(2)O(2) decreases the amount of force produced by a given intracellular Ca(2+) concentration (i.e., the Ca(2+) sensitivity) in airway smooth muscle (ASM) in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. A new preparation was developed and validated in which canine ASM strips were first exposed to H(2)O(2) and then permeabilized with 10% Triton X-100 to assess the persistent effects of H(2)O(2) on Ca(2+) sensitivity. Experiments in which H(2)O(2) was administered before permeabilization revealed a novel mechanism that contributed to reduced Ca(2+) sensitivity independently of changes in rMLC phosphorylation, in addition to an rMLC phosphorylation-dependent mechanism. The mechanism depended on factors not available in the permeabilized ASM strip or in the buffer to which the strip was exposed, since there was no effect when H(2)O(2) was added to permeabilized strips. H(2)O(2) treatment of a maximally thiophosphorylated purified myosin subfragment (heavy meromyosin) significantly reduced actomyosin ATPase activity, suggesting one mechanism by which the phosphorylation-independent reduction in Ca(2+) sensitivity may occur.     

ELISA: Structure-Function Inferences based on statistically significant and evolutionarily inspired observations

The problem of functional annotation based on homology modeling is primary to current bioinformatics research. Researchers have noted regularities in sequence, structure and even chromosome organization that allow valid functional cross-annotation. However, these methods provide a lot of false negatives due to limited specificity inherent in the system. We want to create an evolutionarily inspired organization of data that would approach the issue of structure-function correlation from a new, probabilistic perspective. Such organization has possible applications in phylogeny, modeling of functional evolution and structural determination. ELISA (Evolutionary Lineage Inferred from Structural Analysis, http://romi.bu.edu/elisa) is an online database that combines functional annotation with structure and sequence homology modeling to place proteins into sequence-structure-function "neighborhoods". The atomic unit of the database is a set of sequences and structural templates that those sequences encode. A graph that is built from the structural comparison of these templates is called PDUG (protein domain universe graph). We introduce a method of functional inference through a probabilistic calculation done on an arbitrary set of PDUG nodes. Further, all PDUG structures are mapped onto all fully sequenced proteomes allowing an easy interface for evolutionary analysis and research into comparative proteomics. ELISA is the first database with applicability to evolutionary structural genomics explicitly in mind.Availability: The database is available at http://romi.bu.edu/elisa.     

Strategy for the design of custom cDNA microarrays

DNA microarrays are valuable but expensive tools for expression profiling of cells, tissues, and organs. The design of custom microarrays leads to cost reduction without necessarily compromising their biological value. Here we present a strategy for designing custom cDNA microarrays and constructed a microarray for mouse immunology research (ImmunoChip). The strategy used interrogates expressed sequence tag databases available in the public domain but overcomes many of the problems encountered. Immunologically relevant clusters were selected based on the expression of expressed sequence tags in relevant libraries. Selected clusters were organized in modules, and the best representative clones were identified. When tested, this microarray was found to have minimal clone identity errors or phage contamination and identified molecular signatures of lymphoid cell lines. Our proposed design of custom microarrays avoids probe redundancy, allows the organization of the chip to optimize chip production, and reduces microarray production costs. The strategy described is also useful for the design of oligonucleotide microarrays.