Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems

AbstractWe have developed a new method for the C-terminus-specific fluorescence labeling of proteins. This method is based on the experimental finding that a fluorescent puromycin analogue at lower concentrations bonds efficiently to the C-terminus of mature proteins in cell-free translation systems using mRNA without a stop codon. This labeling is performed under moderate conditions and its labeling efficiency is in the range of 50–95%. Here we demonstrate a protein-protein interaction assay using fluorescence polarization measurement. This labeling method should also be useful for other rapid molecular interaction assays without purification of the labeled proteins, such as fluorescence correlation spectroscopy

In vitro virus: Bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro

AbstractAdequate means for genotype assignment to phenotype is essential in evolutionary molecular engineering. In this study, construction of ‘in vitro virus’ was carried out in which a genotype molecule (mRNA) covalently binds to the phenotype molecule (protein) through puromycin on the ribosome in a cell-free translation system. Bonding efficiency was ∼10%, thus indicating a population of the in vitro virus to have ∼1012 protein variants, this number being 104 that in the phage display. The in vitro virus is useful for examining protein evolution in a test tube and the results may possibly serve as basis for a general method for selecting proteins possessing the most desirable functions

Dampak Struktur Pasar dan Efsiensi terhadap Kinerja Bank Pembangunan Daerah

Conditions Regional Development Bank (BPD) in terms of Business Growth, Markets and Competitiveness within the last three years shows an alarming condition. The study aims to assess the impact of market structure and efficiency of the performance of the BPD in Indonesia. The data used is secondary data in the form of financial statements of banks BPD in 2004-2013. The analytical tool used in this research is panel data regression. The results found that the banking industry in particular BPD in Indonesia supports the traditional hypothesis that assumes that the concentration is a proxy of market power (market power). Variable efficiency is not significant positive effect on the performance

Affinity selection of DNA-binding protein complexes using mRNA display

Comprehensive analysis of DNA–protein interactions is important for mapping transcriptional regulatory networks on a genome-wide level. Here we present a new application of mRNA display for in vitro selection of DNA-binding protein heterodimeric complexes. Under improved selection conditions using a TPA-responsive element (TRE) as a bait DNA, known interactors c-fos and c-jun were simultaneously enriched about 100-fold from a model library (a 1:1:20 000 mixture of c-fos, c-jun and gst genes) after one round of selection. Furthermore, almost all kinds of the AP-1 family genes including c-jun, c-fos, junD, junB, atf2 and b-atf were successfully selected from an mRNA display library constructed from a mouse brain poly A(+) RNA after six rounds of selection. These results indicate that the mRNA display selection system can identify a variety of DNA-binding protein complexes in a single experiment. Since almost all transcription factors form heterooligomeric complexes to bind with their target DNA, this method should be most useful to search for DNA-binding transcription factor complexes

Protein complex prediction via verifying and reconstructing the topology of domain-domain interactions

<p>Abstract</p> <p>Background</p> <p>High-throughput methods for detecting protein-protein interactions enable us to obtain large interaction networks, and also allow us to computationally identify the associations of proteins as protein complexes. Although there are methods to extract protein complexes as sets of proteins from interaction networks, the extracted complexes may include false positives because they do not account for the structural limitations of the proteins and thus do not check that the proteins in the extracted complex can simultaneously bind to each other. In addition, there have been few searches for deeper insights into the protein complexes, such as of the topology of the protein-protein interactions or into the domain-domain interactions that mediate the protein interactions.</p> <p>Results</p> <p>Here, we introduce a combinatorial approach for prediction of protein complexes focusing not only on determining member proteins in complexes but also on the DDI/PPI organization of the complexes. Our method analyzes complex candidates predicted by the existing methods. It searches for optimal combinations of domain-domain interactions in the candidates based on an assumption that the proteins in a candidate can form a true protein complex if each of the domains is used by a single protein interaction. This optimization problem was mathematically formulated and solved using binary integer linear programming. By using publicly available sets of yeast protein-protein interactions and domain-domain interactions, we succeeded in extracting protein complex candidates with an accuracy that is twice the average accuracy of the existing methods, MCL, MCODE, or clustering coefficient. Although the configuring parameters for each algorithm resulted in slightly improved precisions, our method always showed better precision for most values of the parameters.</p> <p>Conclusions</p> <p>Our combinatorial approach can provide better accuracy for prediction of protein complexes and also enables to identify both direct PPIs and DDIs that mediate them in complexes.</p

The shape of disposable diaper affects spontaneous movements of lower limbs in young infants

This study examined the characteristics of young infants\u27 lower limb spontaneous movements based on differences in shape of diapers. Twenty-seven healthy infants (103 +/- 16.3 days old) were enrolled in this study. We measured the spontaneous movements of their lower limbs in four conditions (Naked, wearing Normal type diapers, wearing Type A diapers, and wearing Type B diapers). The Normal diaper has a wider waist belt than the Type A diaper, and the Type B diaper has a narrower crotch area than the Type A diaper. We observed them in seven indices (the velocity of lower limb movements, the trajectory area of knee movement in the sagittal plane and the frontal plane, the distance between both knees and between side of abdomen and knee, and correlation of velocities between side of abdomen and knee and between left and right ankles). The results showed that the velocity of the lower limb movements in the Naked condition was higher than when wearing Normal diapers. The value for the trajectory area of knee movement in sagittal plane, which reflects the range of lower leg lifting movements and closeness of such movements to the trunk, for the Type B diaper condition was higher than that for the Normal diaper condition. This result indicates that the shape of the diaper affects the spontaneous movements of the lower limbs of young infants

Evaluation of both perfusion and atrophy in multiple system atrophy of the cerebellar type using brain SPECT alone

BACKGROUND: Partial volume effects in atrophied areas should be taken into account when interpreting brain perfusion single photon emission computed tomography (SPECT) images of neurodegenerative diseases. To evaluate both perfusion and atrophy using brain SPECT alone, we developed a new technique applying tensor-based morphometry (TBM) to SPECT. METHODS: After linear spatial normalization of brain perfusion SPECT using (99m)Tc-ethyl cysteinate dimer ((99m)Tc-ECD) to a Talairach space, high-dimension-warping was done using an original (99m)Tc-ECD template. Contraction map images calculated from Jacobian determinants and spatially normalized SPECT images using this high-dimension-warping were compared using statistical parametric mapping (SPM2) between two groups of 16 multiple system atrophy of the cerebellar type (MSA-C) patients and 73 age-matched normal controls. This comparison was also performed in conventionally warped SPECT images. RESULTS: SPM2 demonstrated statistically significant contraction indicating local atrophy and decreased perfusion in the whole cerebellum and pons of MSA-C patients as compared to normal controls. Higher significance for decreased perfusion in these areas was obtained in high-dimension-warping than in conventional warping, possibly due to sufficient spatial normalization to a (99m)Tc-ECD template in high-dimensional warping of severely atrophied cerebellum and pons. In the present high-dimension-warping, modification of tracer activity remained within 3% of the original tracer distribution. CONCLUSIONS: The present new technique applying TBM to brain SPECT provides information on both perfusion and atrophy at the same time thereby enhancing the role of brain perfusion SPEC

The dual origin of the peripheral olfactory system: placode and neural crest

<p>Abstract</p> <p>Background</p> <p>The olfactory epithelium (OE) has a unique capacity for continuous neurogenesis, extending axons to the olfactory bulb with the assistance of olfactory ensheathing cells (OECs). The OE and OECs have been believed to develop solely from the olfactory placode, while the neural crest (NC) cells have been believed to contribute only the underlying structural elements of the olfactory system. In order to further elucidate the role of NC cells in olfactory development, we examined the olfactory system in the transgenic mice Wnt1-Cre/Floxed-EGFP and P0-Cre/Floxed-EGFP, in which migrating NC cells and its descendents permanently express GFP, and conducted transposon-mediated cell lineage tracing studies in chick embryos.</p> <p>Results</p> <p>Examination of these transgenic mice revealed GFP-positive cells in the OE, demonstrating that NC-derived cells give rise to OE cells with morphologic and antigenic properties identical to placode-derived cells. OECs were also positive for GFP, confirming their NC origin. Cell lineage tracing studies performed in chick embryos confirmed the migration of NC cells into the OE. Furthermore, spheres cultured from the dissociated cells of the olfactory mucosa demonstrated self-renewal and trilineage differentiation capacities (neurons, glial cells, and myofibroblasts), demonstrating the presence of NC progenitors in the olfactory mucosa.</p> <p>Conclusion</p> <p>Our data demonstrates that the NC plays a larger role in the development of the olfactory system than previously believed, and suggests that NC-derived cells may in part be responsible for the remarkable capacity of the OE for neurogenesis and regeneration.</p