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111.
Neurofibromatosis type 1 (NF1) is a common inherited disease affecting one in 3,500 individuals. The mutation rate in the NF1 gene is one of the highest known for human genes. Compared to other methods, the protein truncation test (PTT) and subsequent sequence analysis of cloned cDNA provides improved efficiency in detecting NF1 mutations that are dispersed throughout the gene spanning 350 kb of genomic DNA. Sequencing of cDNA of patients affected with NF1 mutations revealed multiple splicing errors. Since similar missplicings were also found in "aged" blood of healthy individuals, they are most likely attributable to a general decrease in splice site selection in aged blood. We show that restoring viability of lymphocytes before RNA extraction by cultivation and PHA stimulation diminishes aberrant splicing in aged blood and is thus useful to circumvent splicing alterations which are frequently compromising mutation detection in patient samples and mimic mutation-induced alterations of mRNA. 相似文献
112.
A substantial amount of the human genome is composed of human endogenous retroviruses (HERVs). Manifold HERV families have been identified, among them several so-called HERV-K(HML) families. Although the HERV-K(HML-2) family has been studied in detail, other HERV-K families are not as well characterized. We describe here the HERV-K HML-3 family in more detail. We estimate that there are about 140 proviral loci or remains of such per haploid genome. Most loci are severely mutated. Proviruses displaying larger deletions in gag and pol are common. A multiple alignment of 73 HERV-K(HML-3) sequences displays several potentially important differences compared with the HERVK9I sequence in Repbase. A consensus sequence with open reading frames for all retroviral genes was generated, for which intact dUTPase motifs and env gene variants with different coding capacities are observed. Phylogenetic analysis shows near-monophyly with distinction of two closely related subgroups. Proviruses formed about 36 million years ago. However, no continuous activity through primate evolution is indicated. 相似文献
113.
Engineering the protein N-glycosylation pathway in insect cells for production of biantennary,complex N-glycans 总被引:4,自引:0,他引:4
Insect cells, like other eucaryotic cells, modify many of their proteins by N-glycosylation. However, the endogenous insect cell N-glycan processing machinery generally does not produce complex, terminally sialylated N-glycans such as those found in mammalian systems. This difference in the N-glycan processing pathways of insect cells and higher eucaryotes imposes a significant limitation on their use as hosts for baculovirus-mediated recombinant glycoprotein production. To address this problem, we previously isolated two transgenic insect cell lines that have mammalian beta1,4-galactosyltransferase or beta1,4-galactosyltransferase and alpha2,6-sialyltransferase genes. Unlike the parental insect cell line, both transgenic cell lines expressed the mammalian glycosyltransferases and were able to produce terminally galactosylated or sialylated N-glycans. The purpose of the present study was to investigate the structures of the N-glycans produced by these transgenic insect cell lines in further detail. Direct structural analyses revealed that the most extensively processed N-glycans produced by the transgenic insect cell lines were novel, monoantennary structures with elongation of only the alpha1,3 branch. This led to the hypothesis that the transgenic insect cell lines lacked adequate endogenous N-acetylglucosaminyltransferase II activity for biantennary N-glycan production. To test this hypothesis and further extend the N-glycan processing pathway in Sf9 cells, we produced a new transgenic line designed to constitutively express a more complete array of mammalian glycosyltransferases, including N-acetylglucosaminyltransferase II. This new transgenic insect cell line, designated SfSWT-1, has higher levels of five glycosyltransferase activities than the parental cells and supports baculovirus replication at normal levels. In addition, direct structural analyses showed that SfSWT-1 cells could produce biantennary, terminally sialylated N-glycans. Thus, this study provides new insight on the glycobiology of insect cells and describes a new transgenic insect cell line that will be widely useful for the production of more authentic recombinant glycoproteins by baculovirus expression vectors. 相似文献
114.
Functional and protein chemical characterization of the N-terminal domain of the rat corticotropin-releasing factor receptor 1 总被引:1,自引:0,他引:1 下载免费PDF全文
Hofmann BA Sydow S Jahn O van Werven L Liepold T Eckart K Spiess J 《Protein science : a publication of the Protein Society》2001,10(10):2050-2062
Rat corticotropin-releasing factor receptor 1 (rCRFR1) was produced either in transfected HEK 293 cells as a complex glycosylated protein or in the presence of the mannosidase I inhibitor kifunensine as a high mannose glycosylated protein. The altered glycosylation did not influence the biological function of rCRFR1 as demonstrated by competitive binding of rat urocortin (rUcn) or human/rat corticotropin-releasing factor (h/rCRF) and agonist-induced cAMP accumulation. The low production rate of the N-terminal domain of rCRFR1 (rCRFR1-NT) by transfected HEK 293 cells, was increased by a factor of 100 in the presence of kifunensine. The product, rCRFR1-NT-Kif, bound rUcn specifically (K(D) = 27 nM) and astressin (K(I) = 60 nM). This affinity was 10-fold lower than the affinity of full length rCRFR1. However, it was sufficiently high for rCRFR1-NT-Kif to serve as a model for the N-terminal domain of rCRFR1. With protein fragmentation, Edman degradation, and mass spectrometric analysis, evidence was found for the signal peptide cleavage site C-terminally to Thr(23) and three disulfide bridges between precursor residues 30 and 54, 44 and 87, and 68 and 102. Of all putative N-glycosylation sites in positions 32, 38, 45, 78, 90, and 98, all Asn residues except for Asn(32) were glycosylated to a significant extent. No O-glycosylation was observed. 相似文献
115.
Peptides, blocked either at the N or C terminus, and thus unsuited for Edman degradation, and those containing N-alkylated amino acids, which are not detectable when using conventional amino acid analysis, can be easily sequenced by applying a method in which fast atom bombardment (FAB) is combined with tandem mass spectrometry (MSMS). Moreover, the structure of the N-alkylated amino acid constituents is provided by this approach. A widely applicable strategy will be presented, and to demonstrate its scope and limitations eighteen analogues of sequences related to the C terminus of substance P, a biologically active neuropeptide, were investigated. The power and reliability of the approach will be demonstrated by analyzing an 'unknown' peptide. Moreover, the detection and structure elucidation of N-alkylated amino acids which usually escape amino acid analysis will be described, as will be the unequivocal differentiation and identification of isomeric MeLeu/MeIle. The influence of the N-alkylation on the mass spectrometric fragmentation behaviour will be discussed. Furthermore, the sequencing of two adipokinetic hormones by using the combined FAB-MSMS approach is described. Analysis of peptides can be achieved with sample sizes less than 0.1 mumol and be completed within 2-4 h. 相似文献
116.
Citizen participation in the conservation and use of rural landscapes in Britain: the Alport Valley case study 总被引:2,自引:0,他引:2
With the shift from the formerly dominating primary production sector to the secondary and now the tertiary sector, the vast
majority of the population has lost direct influence on shaping our landscape. However, public interest in landscape and environmental
decision making remains active. Approaches to public participation were introduced some decades ago, but only sporadically.
International declarations and conventions of strategic importance, such as the Rio Declaration on Environment and Development
and the AARHUS Convention on Access to Information, Public Participation in Decision-making and Access to Justice in Environmental
Matters, provide the foundation for integration in national regulations addressing public involvement in decision making.
Using the Alport Valley in the Peak District National Park, UK, this case study illustrates how planning proposals that did
not involve stakeholders and their opinions came to a halt and had to be changed. Through stakeholder involvement, there is
now a broad consensus about the future of this landscape. As part of an iterative consultation and participation process,
a long-term vision for the landscape and land management of the Alport Valley has been developed to improve the valley’s special
landscape character, enhance its visual and recreational attractiveness and regenerate the woodlands in ways that maximise
the long-term benefit for ecology, wildlife and landscape. 相似文献
117.
Clarissa L Waites Sergio A Leal‐Ortiz Nathan Okerlund Hannah Dalke Anna Fejtova Wilko D Altrock Eckart D Gundelfinger Craig C Garner 《The EMBO journal》2013,32(7):954-969
The presynaptic active zone (AZ) is a specialized microdomain designed for the efficient and repetitive release of neurotransmitter. Bassoon and Piccolo are two high molecular weight components of the AZ, with hypothesized roles in its assembly and structural maintenance. However, glutamatergic synapses lacking either protein exhibit relatively minor defects, presumably due to their significant functional redundancy. In the present study, we have used interference RNAs to eliminate both proteins from glutamatergic synapses, and find that they are essential for maintaining synaptic integrity. Loss of Bassoon and Piccolo leads to the aberrant degradation of multiple presynaptic proteins, culminating in synapse degeneration. This phenotype is mediated in part by the E3 ubiquitin ligase Siah1, an interacting partner of Bassoon and Piccolo whose activity is negatively regulated by their conserved zinc finger domains. Our findings demonstrate a novel role for Bassoon and Piccolo as critical regulators of presynaptic ubiquitination and proteostasis. 相似文献
118.
Julià L. Amengual Nuria Rojo Misericordia Veciana de las Heras Josep Marco-Pallarés Jennifer Grau-Sánchez Sabine Schneider Lucía Vaquero Montserrat Juncadella Jordi Montero Bahram Mohammadi Francisco Rubio Nohora Rueda Esther Duarte Carles Grau Eckart Altenmüller Thomas F. Münte Antoni Rodríguez-Fornells 《PloS one》2013,8(4)
Background
Several recently developed therapies targeting motor disabilities in stroke sufferers have shown to be more effective than standard neurorehabilitation approaches. In this context, several basic studies demonstrated that music training produces rapid neuroplastic changes in motor-related brain areas. Music-supported therapy has been recently developed as a new motor rehabilitation intervention.Methods and Results
In order to explore the plasticity effects of music-supported therapy, this therapeutic intervention was applied to twenty chronic stroke patients. Before and after the music-supported therapy, transcranial magnetic stimulation was applied for the assessment of excitability changes in the motor cortex and a 3D movement analyzer was used for the assessment of motor performance parameters such as velocity, acceleration and smoothness in a set of diadochokinetic movement tasks. Our results suggest that the music-supported therapy produces changes in cortical plasticity leading the improvement of the subjects'' motor performance.Conclusion
Our findings represent the first evidence of the neurophysiological changes induced by this therapy in chronic stroke patients, and their link with the amelioration of motor performance. Further studies are needed to confirm our observations. 相似文献119.
120.
Maria Joy Daigo Schulte Rainer Matyssek Sebastian Gayler Eckart Priesack Thorsten E. E. Grams 《Trees - Structure and Function》2013,27(6):1763-1773