A Laser System for Counting Cloth Filaments

A simple high speed and high accuracy counter system for cloth filament has been constructed. The principle is based on the laser light attenuation through the cloth. The system is composed of semiconductor laser, objective lens, focusing lens PIN photodiode and processing system including amplifier, ΔV detector, counter, comparator, presser and marker. The counting accuracy is above 99% and the maximum counting speed is beyond 5 kHz, which will be enough for practical use

Using a Solid-Phase Ribozyme Aminoacylation System to Reprogram the Genetic Code

AbstractHere, we report a simple and economical tRNA aminoacylation system based upon a resin-immobilized ribozyme, referred to as Flexiresin. This catalytic system features a broad spectrum of activities toward various phenylalanine (Phe) analogs and suppressor tRNAs. Most importantly, it allows users to perform the tRNA aminoacylation reaction and isolate the aminoacylated tRNAs in a few hours. We coupled the Flexiresin system with a high-performance cell-free translation system and demonstrated protein mutagenesis with seven different Phe analogs in parallel. Thus, the technology developed herein provides a new tool that significantly simplifies the procedures for the synthesis of aminoacyl-tRNAs charged with nonnatural amino acids, which makes the nonnatural amino acid mutagenesis of proteins more user accessible

Evolution of the MAGUK protein gene family in premetazoan lineages

<p>Abstract</p> <p>Background</p> <p>Cell-to-cell communication is a key process in multicellular organisms. In multicellular animals, scaffolding proteins belonging to the family of membrane-associated guanylate kinases (MAGUK) are involved in the regulation and formation of cell junctions. These MAGUK proteins were believed to be exclusive to Metazoa. However, a MAGUK gene was recently identified in an EST survey of <it>Capsaspora owczarzaki</it>, an unicellular organism that branches off near the metazoan clade. To further investigate the evolutionary history of MAGUK, we have undertook a broader search for this gene family using available genomic sequences of different opisthokont taxa.</p> <p>Results</p> <p>Our survey and phylogenetic analyses show that MAGUK proteins are present not only in Metazoa, but also in the choanoflagellate <it>Monosiga brevicollis </it>and in the protist <it>Capsaspora owczarzaki</it>. However, MAGUKs are absent from fungi, amoebozoans or any other eukaryote. The repertoire of MAGUKs in Placozoa and eumetazoan taxa (Cnidaria + Bilateria) is quite similar, except for one class that is missing in <it>Trichoplax</it>, while Porifera have a simpler MAGUK repertoire. However, Vertebrata have undergone several independent duplications and exhibit two exclusive MAGUK classes. Three different MAGUK types are found in both <it>M. brevicollis </it>and <it>C. owczarzaki: DLG, MPP and MAGI</it>. Furthermore, <it>M. brevicollis </it>has suffered a lineage-specific diversification.</p> <p>Conclusions</p> <p>The diversification of the MAGUK protein gene family occurred, most probably, prior to the divergence between Metazoa+choanoflagellates and the <it>Capsaspora</it>+<it>Ministeria </it>clade. A MAGI-like, a DLG-like, and a MPP-like ancestral genes were already present in the unicellular ancestor of Metazoa, and new gene members have been incorporated through metazoan evolution within two major periods, one before the sponge-eumetazoan split and another within the vertebrate lineage. Moreover, choanoflagellates have suffered an independent MAGUK diversification. This study highlights the importance of generating enough genome data from the broadest possible taxonomic sampling, in order to fully understand the evolutionary history of major protein gene families.</p

Regulation of Src and Csk nonreceptor tyrosine kinases in the filasterean Ministeria vibrans

ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.The development of the phosphotyrosine-based signaling system predated the evolution of multicellular animals. Single-celled choanoflagellates, the closest living relatives to metazoans, possess numerous tyrosine kinases, including Src family nonreceptor tyrosine kinases. Choanoflagellates also have Csk (C-terminal Src kinase), the enzyme that regulates Src in metazoans; however, choanoflagellate Csk kinases fail to repress the cognate Src. Here, we have cloned and characterized Src and Csk kinases from Ministeria vibrans, a filasterean (the sister group to metazoans and choanoflagellates). The two Src kinases (MvSrc1 and MvSrc2) are enzymatically active Src kinases, although they have low activity toward mammalian cellular proteins. Unexpectedly, MvSrc2 has significant Ser/Thr kinase activity. The Csk homologue (MvCsk) is enzymatically inactive and fails to repress MvSrc activity. We suggest that the low activity of MvCsk is due to sequences in the SH2-kinase interface, and we show that a point mutation in this region partially restores MvCsk activity. The inactivity of filasterean Csk kinases is consistent with a model in which the stringent regulation of Src family kinases arose more recently in evolution, after the split between choanoflagellates and multicellular animals. © 2014 American Chemical Society.Peer Reviewe

Earliest holozoan expansion of phosphotyrosine signaling

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.Phosphotyrosine (pTyr) signaling is involved in development and maintenance of metazoans' multicellular body through cell-to-cell communication. Tyrosine kinases (TKs), tyrosine phosphatases, and other proteins relaying the signal compose the cascade. Domain architectures of the pTyr signaling proteins are diverse in metazoans, reflecting their complex intercellular communication. Previous studies had shown that the metazoan-type TKs, as well as other pTyr signaling proteins, were already diversified in the common ancestor of metazoans, choanoflagellates, and filastereans (which are together included in the clade Holozoa) whereas they are absent in fungi and other nonholozoan lineages. However, the earliest-branching holozoans Ichthyosporea and Corallochytrea, as well as the two fungi-related amoebae Fonticula and Nuclearia, have not been studied. Here, we analyze the complete genome sequences of two ichthyosporeans and Fonticula, and RNAseq data of three additional ichthyosporeans, one corallochytrean, and Nuclearia. Both the ichthyosporean and corallochytrean genomes encode a large variety of receptor TKs (RTKs) and cytoplasmic TKs (CTKs), as well as other pTyr signaling components showing highly complex domain architectures. However, Nuclearia and Fonticula have no TK, and show much less diversity in other pTyr signaling components. The CTK repertoires of both Ichthyosporea and Corallochytrea are similar to those of Metazoa, Choanoflagellida, and Filasterea, but the RTK sets are totally different from each other. The complex pTyr signaling equipped with positive/negative feedback mechanism likely emerged already at an early stage of holozoan evolution, yet keeping a high evolutionary plasticity in extracellular signal reception until the co-option of the system for cell-to-cell communication in metazoans. © 2013 The Author 2013. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.This work was supported by the European Research Council Starting grant ERC-2007-StG-206883 to I.R.-T; Ministerio de Ciencia e Innovación grant BFU2008-02839/BMC to I.R.-T.; and the Marie Curie Intra-European Fellowship (MMEMA) within the 7th European Community Framework Programme to H.S.Peer Reviewe

Src function in a unicellular relative of metazoans

Motivación: Tyrosine kinases play important roles in different cell signaling cascades in metazoans (1). The non-receptor Src family has been found to be involved in the regulation of cell-cell interaction, cell adhesion and migration (2). For this reason, its malfunctioning is implicated in diseases, such as cancer. Tyrosine kinases were thought to be specific to Metazoa, but recent comparative genomic analyses showed that they are also present in some of the closest unicellular relatives of metazoans (3). The role played by these kinases in these unicellular organisms remains unknown. The goal of this project is to decipher the function of Src in unicellular organisms.Métodos: The src gene was predicted in the genome of Creolimax fragrantissima. The gene was cloned from cDNA by PCR. A dominant negative (DN) version of the gene was designed introducing an amino acid change in the two conserved Tyr that are important for the regulation of the protein, blocking the downstream cascade (4). Both the wild-type version  and DN were cloned in a vector under the beta-tubulin promoter (b-tub) with constitutive expression, and fused to the mCherry fluorescent marker. Transformation was done by electroporation using the Neon® transfection system by Invitrogen (1μg each vector). The experiment consisted on transforming C. fragrantissima with either the normal Src or the DN  constructs plus b-tub::venus and b-tub::H2B-mCherry as controls for the transformation. Negative control had the two lasts constructs but none of the src constructs. The cells were examined using a Nikon fluorescence microscope, and time-lapse was performed when needed.Resultados: We know that the constitutive expression of src affects cell-cell interaction in cell culture experiments. C.fragrantissima has a colonial stage during its cell cycle that “explodes” giving rise to free-living amoebas (see http://www.youtube.com/watch?v=7Gvrg1I8jBA). We assume that cell-cell interaction may have some relevance during this stage. For this reason we expected to see a resulting phenotype either during the colony formation or during its development. The preliminary results showed some different phenotypes although they are not significant enough. Experiments on transformation and time-lapse need to be repeated, in order to be sure that differences are found between the overexpressed and the DN src transformants, in the colonies or amoebas

An optical system for monitoring torsion in a power transmission shaft in realtime

An optical system has been proposed and verified experimentally for monitoring the torsion of a power transmission shaft in realtime. The system consists of a pair of lasers, mirrors and light receivers as a sensor head, and logic circuit, high-frequency oscillator, and computer as a data processing system. The smallest measurable angle of torsion can be expressed by ω/ƒ, where ω is the rotational frequency of the power transmission shaft and ƒ is the frequency of the oscillator. The experimental error was found to be a few percentage points

Cloth Weft Densitometer Using a CCD Camera

A cloth weft densitometer has been proposed and its propriety verified by a preliminary experiment. The method is based on the counting of cloth wefts by a CCD camera with a shutter speed of 1 ms. The experiment shows that the densitometer can be used perfectly for many types of cloth if the cloth speed of less than approximately 10 m/min. The accuracy of the cloth-weft counting is above 99%, provided that the image contrast of the weft on the CCD camera is sufficiently high. Only 2-step digitations by the A/D converter is needed for 99% accuracy

Time-resolved studies of metalloproteins using X-ray free electron laser radiation at SACLA

Background: The invention of the X-ray free-electron laser (XFEL) has provided unprecedented new opportunities for structural biology. The advantage of XFEL is an intense pulse of X-rays and a very short pulse duration ( Scope of review: Recent time-resolved crystallographic analyses in XFEL facility SACLA are reviewed. Specifically, metalloproteins involved in the essential reactions of bioenergy conversion including photosystem II, cytochrome c oxidase and nitric oxide reductase are described. Major conclusions: XFEL with pump-probe techniques successfully visualized the process of the reaction and the dynamics of a protein. Since the active center of metalloproteins is very sensitive to the X-ray radiation, damage-free structures obtained by XFEL are essential to draw mechanistic conclusions. Methods and tools for sample delivery and reaction initiation are key for successful measurement of the time-resolved data. General significance: XFEL is at the center of approaches to gain insight into complex mechanism of structural dynamics and the reactions catalyzed by biological macromolecules. Further development has been carried out to expand the application of time-resolved X-ray crystallography. This article is part of a Special Issue entitled Novel measurement techniques for visualizing 'live' protein molecules