Dexamethasone regulates expression of BRUCE/Apollon and the proliferation of neural progenitor cells

Glucocorticoid hormones (GHs) regulate cell proliferation of neural progenitor cells (NPCs) contributing to reduction of neurogenesis after stress. We show here that dexamethasone (Dex) decreases BRUCE/Apollon (BRUCE) in cultured NPCs in a GH-receptor-dependent manner. Downregulation of BRUCE by Dex or using silencing RNA reduced the number of proliferating NPCs, whilst overexpression of BRUCE counteracted the effect of Dex. Dex also elevated the deubiquitinating enzyme, Usp8/Ubpy, which via Nrdp1 decreases BRUCE. The results show that BRUCE is a target for GHs in the NPCs, and that BRUCE controls cell division of NPCs and possibly of other stem cells.

Structured summary

MINT-7148564: Nrdp1 (uniprotkb:Q8BH75) physically interacts (MI:0914) with BRUCE (uniprotkb:O88738) by anti bait co-immunoprecipitation (MI:0006)MINT-7148555: Nrdp1 (uniprotkb:Q8BH75) physically interacts (MI:0914) with Usp8 (uniprotkb:Q80U87) by anti bait co-immunoprecipitation (MI:0006)     

The porphyrin TMPyP4 inhibits elongation during the noncanonical translation of the FTLD/ALS-associated GGGGCC repeat in the C9orf72 gene

GGGGCC (G₄C₂) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G₄C₂ repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G₄C₂ repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G₄C₂ repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G₄C₂ repeat RNA. Urea-resistant interaction between G₄C₂ repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G₄C₂ repeat translation elongation.     

Mapping of a Wheat Resistance Gene to Yellow Mosaic Disease by Amplified Fragment Length Polymorphism and Simple Sequence Repeat Markers

Wheat （Triticum aestivum L.） yellow mosaic virus （WYMV） is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted plants. In the present study, the amplified fragment length polymorphisms （AFLP） and simple sequence repeat （SSR） were used to identify the molecular linkages with the resistance gene against WYMV. Bulked segregant analysis was performed with an F2 population derived from the cross of cultivar Ningmai 9 （resistant） × cultivar Yangmai 10 （susceptible）. By screening among the resistant or susceptible parents, the F2 pools and the individuals in the F2 population with 64 combined selective AFLP primers （EcoRI/MseI） or 290 reported SSR primers, a polymorphic DNA segment （approximately 120 bp） was amplified using the primer pair E2/M5, and an SSR marker （approximately 180 bp） was located on wheat chromosome 2A using the primer Xgwm328. Analysis with MAPMAKER/Exp Version 3.0b （Whitehead institute for Biomedical Research, Cambridge, MA, USA） indicated that these two markers were dominantly associated with the resistance gene at distances of 5.4 cM or 17.6 cM, respectively. The resistance gene to WYMV derived from Ningmai 9, is temporarily named YmNM, and was mapped to wheat chromosome 2A.     

Characterization of trinucleotide SSR motifs in wheat

Length differences among trinucleotide-based microsatellite alleles can be more easily detected and frequently produce fewer ”stutter bands” as compared to dinucleotide-based microsatellite markers. Our objective was to determine which trinucleotide motif(s) would be the most-polymorphic and abundant source of trinucleotide microsatellite markers in wheat (Triticum aestivum L.). Four genomic libraries of cultivar ’Chinese Spring’ were screened with nine trinucleotide probes. Based on the screening of 28550 clones, the occurrences of (CTT/GAA) _n , (GGA/CCT) _n , (TAA/ATT) _n , (CAA/GTT) _n , (GGT/CCA) _n , (CAT/GTA) _n , (CGA/GCT) _n , (CTA/GAT) _n , and (CGT/GCA) _n repeats were estimated to be 5.4×10⁴, 3.5×10⁴, 3.2×10⁴, 1.2×10⁴, 6.3×10³, 4.9×10³, 4.5×10³, 4.5×10³ and 3.6×10³, i.e., once every 293 kbp, 456 kbp, 500 kbp, 1.3 Mbp, 2.6 Mbp, 3.2 Mbp, 3.6 Mbp, 3.6 Mbp and 4.5 Mbp in the wheat genome, respectively. Of 236 clones selected for sequencing, 38 (93%) (TAA/ATT) _n , 30 (43%) (CTT/GAA) _n , 16 (59%) (CAA/GTT) _n , 3 (27%) (CAT/GTA) _n and 2 (4%) (GGA/CCT) _n clones contained microsatellites with eight or more perfect repeats. From these data, 29, 27 and 16 PCR primer sets were designed and tested to the (TAA/ATT) _n , (CTT/GAA) _n and (CAA/GTT) _n microsatellites, respectively. A total of 12 (41.4%) primers designed to (TAA/ATT) _n , four (14.8%) to (CTT/GAA) _n , and two (12.5%) to (CAA/GTT) _n resulted in polymorphic markers. The results indicated that (TAA/ATT) _n microsatellites would provide the most-abundant and the most-polymorphic source of trinucleotide microsatellite markers in wheat. Received: 17 February 2001 / Accepted: 31 May 2001     

Toward genomic identification of β-barrel membrane proteins: Composition and architecture of known structures

The amino acid composition and architecture of all beta-barrel membrane proteins of known three-dimensional structure have been examined to generate information that will be useful in identifying beta-barrels in genome databases. The database consists of 15 nonredundant structures, including several novel, recent structures. Known structures include monomeric, dimeric, and trimeric beta-barrels with between 8 and 22 membrane-spanning beta-strands each. For this analysis the membrane-interacting surfaces of the beta-barrels were identified with an experimentally derived, whole-residue hydrophobicity scale, and then the barrels were aligned normal to the bilayer and the position of the bilayer midplane was determined for each protein from the hydrophobicity profile. The abundance of each amino acid, relative to the genomic abundance, was calculated for the barrel exterior and interior. The architecture and diversity of known beta-barrels was also examined. For example, the distribution of rise-per-residue values perpendicular to the bilayer plane was found to be 2.7 +/- 0.25 A per residue, or about 10 +/- 1 residues across the membrane. Also, as noted by other authors, nearly every known membrane-spanning beta-barrel strand was found to have a short loop of seven residues or less connecting it to at least one adjacent strand. Using this information we have begun to generate rapid screening algorithms for the identification of beta-barrel membrane proteins in genomic databases. Application of one algorithm to the genomes of Escherichia coli and Pseudomonas aeruginosa confirms its ability to identify beta-barrels, and reveals dozens of unidentified open reading frames that potentially code for beta-barrel outer membrane proteins.     

应用红外荧光和加尾引物法进行向日葵SSR遗传分析中的多聚PCR和多聚凝胶电泳的优化

在向日葵(Helianthus annuus L.)自交系SSR遗传分析中,为了提高SSR荧光分析效率、简化分析程序和降低研究费用,我们进行了多聚PCR和多聚凝胶电泳两项技术的优化研究。结果表明,优化多聚PCR和多聚凝胶电泳的影响因子(如逐步降低的退火温度的循环数、各个多聚位点引物浓度的平衡、PCR缓冲液浓度以及Taq DNA多聚酶的浓度等)可以收到更好的实验效果。基于以上的优化研究,系统地提出了一套优化的加尾引物法的策略。同时,提出了在多聚PCR和多聚凝胶电泳中常常遇到的技术难题的有效防止和解决的方法。     

Use of microsatellite polymorphisms for paternity exclusion in rhesus macaques (Macaca multatta)

A total of 224 infant rhesus macaques and their dams and potential sires in 11 multimale groups in 2 different specific pathogen free breeding colonies were screened for up to 6 different microsatellite polymorphism using cross-species PCR amplification. Observed and expected success of paternity exclusion analysis (based on gene frequencies of sires and dams in each colony) were computed by individual locus and cumulatively. Greater or less success of PEA than expected was observed at most loci due to the nonrandom distribution of genotypes between sires and dams and among breeding groups at each colony and because genotypes at different loci did not provide completely independent information about parentage. The combined success of PEA using all loci, however, was slightly greater than predicted both with and without assuming knowledge of one parent's (i.e. the dam's) genotype and was far greater than that based on protein coding loci or DNA restriction fragment length polymorphisms.     

Biological insights from hydrogen exchange mass spectrometry

Over the past two decades, hydrogen exchange mass spectrometry (HXMS) has achieved the status of a widespread and routine approach in the structural biology toolbox. The ability of hydrogen exchange to detect a range of protein dynamics coupled with the accessibility of mass spectrometry to mixtures and large complexes at low concentrations result in an unmatched tool for investigating proteins challenging to many other structural techniques. Recent advances in methodology and data analysis are helping HXMS deliver on its potential to uncover the connection between conformation, dynamics and the biological function of proteins and complexes. This review provides a brief overview of the HXMS method and focuses on four recent reports to highlight applications that monitor structure and dynamics of proteins and complexes, track protein folding, and map the thermodynamics and kinetics of protein unfolding at equilibrium. These case studies illustrate typical data, analysis and results for each application and demonstrate a range of biological systems for which the interpretation of HXMS in terms of structure and conformational parameters provides unique insights into function. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.