4 Q0: Rgk \/\/FREE\\\\
Ventilation - To improve breathability and keep your hands cool, dry, and playing their best, all Renegade GK gloves come with different types of ventilation technology to ensure your gloves perform during any condition.
Goalkeepers usually face the problem of glove sizing when they are in the market to find a new goalie gloves. Different brands have different glove sizing. There are a lot of variability across the goalkeeper gloves available today. Through extensive research, we were able to simplify the process of finding the right goalie glove size for you.
Simply measure these two parts of your hand and then compare it with the goalie glove size chart of your brand choice.Ideally, you will want to use a tape measure for this activity, but any string or yarn can be a substitute. Just measure it after getting the circumference and length of your palm and hand.
Lastly, don't panic when you feel a space at the end of your glove fingertips. This is a good thing. Since you want to have a little bit of space at the tips of your fingers, both for performance and the wear and care of the gloves.
Citation: Puhl HL III, Lu VB, Won Y-J, Sasson Y, Hirsch JA, Ono F, et al. (2014) Ancient Origins of RGK Protein Function: Modulation of Voltage-Gated Calcium Channels Preceded the Protostome and Deuterostome Split. PLoS ONE 9(7): e100694.
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: The authors confirm that all data underlying the findings are freely available without restriction. The genetic analysis is based on public databases and the search paradigm and accession numbers of items analyzed are in the manuscript. Data are available at Genbank under the accessions: KJ826437, KJ826438, KJ826439, KJ826440, KJ826441, KJ826442, KJ826443, KJ826444. The Electrophysiological data including the program, plugins, and custom software, are available upon request. Requests for this data may be sent to the corresponding author.
Funding: This work was supported by the intramural program at the National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism. YS and JAH were supported by the Israel Science Foundation (grant 1519/12). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Additional functions for RGK proteins have emerged from large-scale genomic screens. For example, Rem2 emerged from an RNAi screen designed to identify proteins involved in CNS synaptogenesis . Subsequent work confirmed that Rem2 impacts synapse development and dendritic morphology , . Likewise, Gem may impact dendritic arborization and participate in certain forms of autism . In non-mammalian vertebrates, RGK proteins also influence neuronal development. Inhibition of Rem2 expression in embryonic Danio rerio (zebrafish) interfered with midbrain development at 36 hours post fertilization . Finally, a differential display screen in the urodele amphibian Cynops pyrrhogaster (Japanese fire belly newt) to identify proteins involved in limb regeneration revealed that Rad was highly up-regulated in skeletal muscle at the site of amputation . Together, these indicate that: 1) RGK proteins serve important functions during development or regeneration of excitable cells, and 2) orthologs of RGK proteins are found throughout the vertebrate lineage.
Pipette solution contained (in mM) 120 N-methyl-d-glucamine, 20 tetraethylammonium hydroxide (TEA-OH), 11 EGTA, 10 HEPES, 10 sucrose, 1 CaCl2, 14 Tris-creatine phosphate, 4 MgATP and 0.3 Na2GTP, pH 7.2 with methanesulfonic acid. External ICa recording solution consisted of (in mM) 140 methanesulfonic acid, 145 TEA-OH, 10 HEPES, 10 glucose, 10 CaCl2 and 0.0003 tetrodotoxin (TTX; Alomone Laboratories, Jerusalem, Israel), pH 7.4 with TEA-OH.
Statistical tests were performed with Prism 6 for Mac OS X (GraphPad Software, La Jolla, CA). All data were expressed as mean ± SEM. Statistical significance between two groups was determined using an unpaired Student's t tests. Comparisons of multiple groups against a pooled control was done using one-way analysis of variance (ANOVA) followed by a Dunnett's post-test. P
Zebrafish RGK protein orthologs were cloned from cDNA prepared from an adult male fish using the RNeasy kit (Qiagen, Germantown, MD). Briefly, after anesthesia with MS-222 (tricaine methanesulfonate), a section (approximately 50 mg) was dissected from the trunk immediately caudal to the gills. The section was ground with a small ground glass pestle in a 1.5 ml microcentrifuge tube containing 600 µl of RTL buffer. The lysate was applied to a QiaShredder column to shear genomic DNA. The resulting lysate (approximately 450 µl) was used to isolate total RNA as per the RNeasy kit instructions. Total RNA concentration was determined from absorbance at 260 nm. First strand cDNA was generated from 1 µg of total RNA using the RT for PCR kit (PT1107-1) from Clontech and oligo dT primer.
A second RGK2 forward primer was designed to amplify from the second methionine residue after several attempts to generate a full length predicted product failed. This sequence is designated dm_RGK2t in the text.
The mouse DiRas2 open reading frame (NM_001024474) was amplified from mouse whole brain cDNA and cloned into the MluI and NotI sites of pCI (Promega). The open reading frame for the fluorescent protein variant Venus was amplified with MluI sites on both ends and cloned into the MluI site of the DiRas2 pCI clone. The insertion orientation was confirmed by sequencing. Open reading frames for human Rit1 (AF084462) and Rit2 (NM_002930) were amplified from human whole brain cDNA (Clontech) and cloned into pcDNA3.1 at the KpnI/XhoI sites for Rit1, or the BamHI/XhoI sites for Rit2. All clones were sequence verified.
Initial identification of RGK protein orthologs/homologs utilized standard tools such as pre-computed NCBI ( ) BlastP searches (BLink), annotated databases such as Ensembl ( ) gene trees and NCBI UniGene, and individual BlastP  searches using EnsemblMetazoa ( ) for non-vertebrate species using human RGK protein sequences as the query. After examining the sequence of several protostome RGK-like protein homologs, we generated a search protocol using the last eleven amino acids as PHI-BLAST pattern and human Gem or Rad as the query sequence: [KR][SAF][KR][SH][C][HNED][DNEV][LM]x[VSA][L]. The PHI-BLAST search set was the non-redundant protein sequences database (nr). The algorithm parameters were the defaults with the exception of the maximum target sequences which was increased to 1000. This was followed by a 2nd iteration PSI-BLAST. Protostome RGK orthologs were found together with vertebrate Ras superfamily members such as Rit1 and Rit2 in the hit list as sequence identity decreased.
Orthologs of RGK proteins within the vertebrate lineage were easily identified using the NCBI Basic Alignment Search Tool (BLAST) using human RGK proteins as the search query. At present, an Ensembl analysis of human Gem identifies 56 orthologs in vertebrates and expansion of the pre-computed genetic tree reveals 217 genes that represent the four known family members from mammals to ray-finned fish. We decided to clone and express RGK proteins identified in Danio rerio for several reasons. First, genes encoding putative orthologs of Gem, Rad, Rem1, and Rem2 were readily identified in the Danio rerio genome. Second, transcripts for some RGK proteins have been verified and their function inferred from knockdown or screening studies , , . Third, functional expression of zebrafish cDNAs in mammalian cell lines has been demonstrated . Finally, the popularity of zebrafish as a vertebrate model organism results in a more refined (high coverage and better annotation) genomic database and easily obtainable mRNA/cDNA for cloning.
Normalization of the I-V curve (Fig 1A) to the maximal ICa density (+10 mV) revealed that expression of RGK proteins appeared to reduce current density by a constant proportion at each voltage. An exemplar of dr_Rem2 vs. control normalized I-V curves is illustrated in Fig. 1C. Despite a reduction in mean current density of 93% by dr_Rem2, the normalized I-V curves come close to superimposing over the entire voltage range. Individual current traces for dr_Rem2 expressing neurons appeared similar when scaled to control currents (Fig. 1D) as previously shown for mammalian Rem2 expression  indicating no overt changes in activation or inactivation kinetics. However, this was not studied in detail as the large suppression of current amplitude in most cases made such comparisons difficult to interpret.
To examine whether expression of other Ras subfamily members affected SCG neuron ICa density, experiments were performed using human Rit1, Rit2, and diRas2. Mammalian Rit1 and Rit2 often showed up in BLAST search hit lists with scores similar to protostome RGK-like protein homologs. DiRas2 was examined because of similarities in the region distal to the G5 motif (α5-helix, see below). Following cDNA injection into rat SCG neurons, none of these constructs significantly reduced mean ICa density when compared with uninjected control neurons (Figure S2). Hence, the ability of RGK-like proteins to attenuate ICa density following expression appears specific, at least within this experimental context.
Taken together, the expression of RGK protein orthologs from Danio rerio and homologs from Drosophila melanogaster in rat sympathetic neurons recapitulates, at least superficially, the ICa phenotype resulting from rat Rem2 heterologous expression . Although the precise mechanism of Ca2+ channel inhibition by these RGK orthologs is unclear, the conservation of phenotype from diverse and evolutionarily distant species strengthens interpretations based on sequence comparisons. 2b1af7f3a8