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Truncation
Kinds of Truncation Terms modified by Truncation Selected AbstractsMeasurement of Left Ventricular Ejection Fraction by Real Time 3D Echocardiography in Patients with Severe Systolic Dysfunction: Comparison with Radionuclide AngiographyECHOCARDIOGRAPHY, Issue 1 2010Hajo Müller M.D. Aim: Measurement of left ventricular ejection fraction (LVEF) using real time 3D echocardiography (3DE) has been performed in subjects with preserved or modestly reduced systolic function. Our aim was to evaluate this technique in the subset of patients with severe systolic dysfunction. Methods and results: Consecutive patients with LVEF less than 0.35 at two-dimensional echocardiography were included. LVEF obtained by 3DE was compared to the value measured by radionuclide angiography (RNA). Real time full-volume 3DE was performed, with offline semiautomated measurement of LVEF using dedicated software (Cardioview RT, Tomtec) by a single observer blinded to the results of RNA. A total of 50 patients were evaluated, of whom 38 (76%, 27 males, age 69 ± 13 years) had a 3DE of sufficient quality for analysis. LVEF for this group was 0.21 ± 0.07 using 3DE and 0.27 ± 0.08 using RNA. The agreement between the two techniques was rather poor (r = 0.49; P < 0.001; 95% limits of agreements of ,0.20 to 0.09). Truncation of the apex was observed in 6 of 38 (16%) patients. Conclusion: In patients with severe systolic dysfunction, 3DE shows poor agreement for measurement of LVEF as compared to RNA. There may be underestimation of up to 20% in absolute terms by 3DE. Accordingly, the two methods are not interchangeable for the follow-up of LV function. A limitation of 3DE may, at least in part, be related to the incomplete incorporation of the apical region into the pyramidal image sector in patients with dilated hearts. (Echocardiography 2010;27:58-63) [source] Heteroskedasticity,Autocorrelation Robust Standard Errors Using The Bartlett Kernel Without TruncationECONOMETRICA, Issue 5 2002Nicholas M. Kiefer No abstract is available for this article. [source] Identification of a novel region of the GABAB2 C-terminus that regulates surface expression and neuronal targeting of the GABAB receptorEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2009A. M. Pooler Abstract GABAB is a G protein-coupled receptor composed of two subunits, GABAB1 and GABAB2. GABAB1 contains an endoplasmic reticulum-retention sequence and is trafficked to the cell surface only in association with GABAB2. To determine whether the C-terminus of GABAB2 regulates GABAB trafficking, we constructed forms of GABAB2 with various C-terminal truncations and examined their surface expression. Truncation of GABAB2 after residue 841 significantly reduced surface expression of both the subunit and the heterodimerized receptor. Turnover of the ,841 construct, however, did not differ from that of full-length GABAB2. To determine whether the C-terminus of GABAB2 might target GABAB to neurites, cultured hippocampal neurons were transfected with the truncated GABAB2 constructs. Truncation of GABAB2 at residue 841 resulted in primarily somatic localization; furthermore, axonal trafficking of this construct was significantly more restricted than dendritic trafficking. Finally, to biochemically assess trafficking of the truncated GABAB2 constructs, we digested transfected HEK293 cell lysates with endoglycosidase H. When GABAB2 was truncated at residue 841, it became sensitive to digestion by this enzyme, indicating incomplete trafficking. Taken together, these data show that the region of the GABAB2 C-terminus between residues 841 and 862 is important for regulating forward trafficking and neuronal targeting of the GABAB receptor. [source] Functional analysis of the C-terminal cytoplasmic region of the M-factor receptor in fission yeastGENES TO CELLS, Issue 3 2001Kouji Hirota Background Yeast mating-pheromone receptors facilitate the study of G protein-coupled signal transduction. To date, molecular dissection of the budding yeast ,-factor receptor has been done extensively, but little analysis has been performed with pheromone receptors of fission yeast, another genetically tractable yeast species. Results We analysed the fission yeast M-factor receptor Map3p. Truncation of the C-terminal 54 amino acids made Map3p dominant-negative over the wild-type. This form, called Map3-dn9p, was competent in the induction of pheromone-dependent gene expression, although it could not direct proper conjugation. Map3-dn9p failed both to provoke the orientated projection of conjugation tubes and to induce adaptation to the pheromone signal associated with endocytosis of the receptor. Deletion and substitution analyses suggested that the integrity of the C-terminal region, rather than a specific subgroup of amino acid residues therein, was vital for the respective Map3p activities. Ubiquitination of the C-terminus was not absolutely essential for Map3p function. Conclusions The C-terminal region of Map3p is dispensable for the pheromone signalling per se, but is pivotal for adaptation and pheromone-induced conjugation tube formation, as is true with the budding yeast ,-factor receptor. However, the mechanisms which induce adaptation appear to differ between fission and budding yeast concerning the necessity of ubiquitination. [source] Truncation of the MLL gene in exon 5 by gene targeting leads to early preimplantation lethality of homozygous embryosGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 4 2001Paul Ayton Abstract Summary: The mixed lineage leukemia gene (MLL) was originally identified through its involvement in reciprocal translocations in leukemias. MLL codes for a large multidomain protein and bears homology to the Drosophila developmental control gene trithorax in two small domains in the amino terminal region, the central zinc finger domain and the carboxy SET domain. Like the Drosophila trx, MLL has also been shown to be a positive regulator of Hox gene expression. We have targeted Mll (the murine homologue of MLL) in exon 5 causing expression of three truncated in-frame Mll transcripts. These transcripts retain all or some of the AT hook motifs and the DMT domain. This mutant allele causes early in vivo preimplantation lethality of homozygous embryos prior to the 2-cell stage. Embryos cultured in vitro progress to the 2-cell stage, but further development is arrested. The heterozygotes exhibit mild skeletal defects as well as defects in some neuroectodermal derivatives. genesis 30:201,212, 2001. © 2001 Wiley-Liss, Inc. [source] Canine COL1A2 Mutation Resulting in C-Terminal Truncation of Pro-,2(I) and Severe Osteogenesis ImperfectaJOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2001Bonnie G. Campbell Abstract RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 ("CTAG") were replaced with "TGTCATTGG." The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-,2(I) suggested comigration with the similarly sized pro-,2(I) derived from the mutant allele. Furthermore, ,-chains were overhydroxylated and the ratio of ,1(I):,2(I) was 3.2:1, consistent with the presence of ,1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-,2(I) C-propeptide and confirmed a diagnosis of OI. [source] Pseudophosphorylation of tau at serine 422 inhibits caspase cleavage: in vitro evidence and implications for tangle formation in vivoJOURNAL OF NEUROCHEMISTRY, Issue 4 2006Angela L. Guillozet-Bongaarts Abstract The tangles of Alzheimer's disease (AD) are comprised of the tau protein displaying numerous alterations, including phosphorylation at serine 422 (S422) and truncation at aspartic acid 421 (D421). Truncation at the latter site appears to result from activation of caspases, a class of proteases that cleave specifically at aspartic acid residues. It has been proposed that phosphorylation at or near caspase cleavage sites could regulate the ability of the protease to cleave at those sites. Here, we use tau pseudophosphorylated at S422 (S422E) to examine the effects of tau phosphorylation on its cleavage by caspase 3. We find that S422E tau is more resistant to proteolysis by caspase 3 than non-pseudophosphorylated tau. Additionally, we use antibodies directed against the phosphorylation site and against the truncation epitope to assess the presence of these epitopes in neurofibrillary tangles in the aged human brain. We show that phosphorylation precedes truncation during tangle maturation. Moreover, the distribution of the two epitopes suggests that a significant length of time (perhaps as much as two decades) elapses between S422 phosphorylation and cleavage at D421. We further conclude that tau phosphorylation at S422 may be a protective mechanism that inhibits cleavage in vivo. [source] DLGdifferentially localizes Shaker K+ -channels in the central nervous system and retina of DrosophilaJOURNAL OF NEUROCHEMISTRY, Issue 6 2002C. Ruiz-Cañada Abstract Subcellular localization of ion channels is crucial for the transmission of electrical signals in the nervous system. Here we show that Discs-Large (DLG), a member of the MAGUK (membrane-associated guanylate kinases) family in Drosophila, co-localizes with Shaker potassium channels (Sh Kch) in most synaptic areas of the adult brain and in the outer membrane of photoreceptors. However, DLG is absent from axonal tracts in which Sh channels are concentrated. Truncation of the C-terminal of Sh (including the PDZ binding site) disturbs its pattern of distribution in both CNS and retina, while truncation of the guanylate kinase/C-terminal domain of DLG induces ectopic localization of these channels to neuronal somata in the CNS, but does not alter the distribution of channels in photoreceptors. Immunocytochemical, membrane fractionation and detergent solubilization analysis indicate that the C-terminal of Sh Kch is required for proper trafficking to its final destination. Thus, several major conclusions emerge from this study. First, DLG plays a major role in the localization of Shchannels in the CNS and retina. Second, localization of DLG in photoreceptors but not in the CNS seems to depend on its interaction with Sh. Third, the guanylate kinase/C-terminal domain of DLG is involved in the trafficking of Shaker channels but not of DLG in the CNS. Fourth, different mechanisms for the localization of Sh Kch operate in different cell types. [source] C-terminal domains within human MT1 and MT2 melatonin receptors are involved in internalization processesJOURNAL OF PINEAL RESEARCH, Issue 2 2008Shalini Sethi Abstract:, Melatonin, a molecule implicated in a variety of diseases, including cancer, often exerts its effects through G-protein-coupled melatonin receptors, MT1 and MT2. In this study, we sought to understand further the domains involved in the function and desensitization patterns of these receptors through site-directed mutagenesis. Two mutations were constructed in the cytoplasmic C-terminal tail of each receptor subtype: (i) a cysteine residue in the C-terminal tail was mutated to alanine, thus removing a putative palmitoylation site, and a site possibly required for normal receptor function (MT1C7.72A and MT2C7.77A) and (ii) the C-terminal tail in the MT1 and MT2 receptors was truncated, removing the putative phosphorylation and ,-arrestin binding sites (MT1Y7.64 and MT2Y7.64). These mutations did not alter the affinity of 2-[125I]-iodomelatonin binding to the MT1 or MT2 receptors. Using confocal microscopy, it was determined that the putative palmitoylation site (cysteine residue) did not play a role in receptor internalization; however, this residue was essential for receptor function, as determined by 3,,5,-cyclic adenosine monophosphate (cAMP) accumulation assays. Truncation of the C-terminal tail of both receptors (MT1Y7.64 and MT2Y7.64) inhibited internalization as well as the cAMP response, suggesting the importance of the C-terminal tail in these receptor functions. [source] FIB-Nanotomography of Particulate Systems,Part II: Particle Recognition and Effect of Boundary TruncationJOURNAL OF THE AMERICAN CERAMIC SOCIETY, Issue 8 2006Beat Münch The focused ion beam-nanotomography (FIB-nt) technique presented in Part I of this article is a novel high-resolution three-dimensional (3D) microscopy method that opens new possibilities for the microstructural investigation of fine-grained granular materials. Specifically, FIB-nt data volumes allow particle size distributions (PSD) to be determined, and the current paper discusses all the processing steps required to obtain the PSD from 3D data. This includes particle recognition and the subsequent PSD estimation. A refined watershed approach for 3D particle recognition that tolerates concavities on the particle surfaces is presented. Particles at the edge of the 3D data volume are invariably clipped, and because the data volume is of a very limited size, this effect of boundary truncation seriously affects the PSD and needs to be corrected. Therefore, two basic approaches for the stereological correction of the truncation effects are proposed and validated on artificially modeled particle data. Finally, the suggested techniques are applied to real 3D-particle data from ordinary portland cement and the resulting PSDs compared with data from laser granulometry. [source] A novel NO-responding regulator controls the reduction of nitric oxide in Ralstonia eutrophaMOLECULAR MICROBIOLOGY, Issue 3 2000Anne Pohlmann Ralstonia eutropha H16 mediates the reduction of nitric oxide (NO) to nitrous oxide (N2O) with two isofunctional single component membrane-bound NO reductases (NorB1 and NorB2). This reaction is integrated into the denitrification pathway that involves the successive reduction of nitrate to dinitrogen. The norB1 gene is co-transcribed with norA1 from a ,54 (RpoN)-dependent promoter, located upstream of norA1. With the aid of norA1,,lacZ transcriptional fusions and the generation of regulatory mutants, it was shown that norB1 gene transcription requires a functional rpoN gene and the regulator NorR, a novel member of the NtrC family of response regulators. The regulator gene maps adjacent to norAB, is divergently transcribed and present in two copies on the megaplasmid pHG1 (norR1) and the chromosome (norR2). Transcription activation by NorR responds to the availability of NO. A nitrite reductase-deficient mutant that is incapable of producing NO endogenously, showed a 70% decrease of norA1 expression. Addition of the NO-donating agent sodium nitroprusside caused induction of norA1,,lacZ transcription. Truncation of the N-terminal receiver domain of NorR1 interrupted the NO signal transduction and led to a constitutive expression of norA1,,lacZ. The results indicate that NorR controls the reductive conversion of NO in R. eutropha. This reaction is not strictly co-ordinated on the regulatory level with the other nitrogen oxide-reducing steps of the denitrification chain that are independent of NorR. [source] The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain bindingPROTEIN SCIENCE, Issue 4 2007Shugui Chen Abstract The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis -interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain. [source] Substructure solution with SHELXDACTA CRYSTALLOGRAPHICA SECTION D, Issue 10-2 2002Thomas R. Schneider Iterative dual-space direct methods based on phase refinement in reciprocal space and peak picking in real space are able to locate relatively large numbers of anomalous scatterers efficiently from MAD or SAD data. Truncation of the data at a particular resolution, typically in the range 3.0,3.5,Å, can be critical to success. The efficiency can be improved by roughly an order of magnitude by Patterson-based seeding instead of starting from random phases or sites; Patterson superposition methods also provide useful validation. The program SHELXD implementing this approach is available as part of the SHELX package. [source] Small-Sample Inference for Incomplete Longitudinal Data with Truncation and Censoring in Tumor Xenograft ModelsBIOMETRICS, Issue 3 2002Ming Tan Summary. In cancer drug development, demonstrating activity in xenograft models, where mice are grafted with human cancer cells, is an important step in bringing a promising compound to humans. A key outcome variable is the tumor volume measured in a given period of time for groups of mice given different doses of a single or combination anticancer regimen. However, a mouse may die before the end of a study or may be sacrificed when its tumor volume quadruples, and its tumor may be suppressed for some time and then grow back. Thus, incomplete repeated measurements arise. The incompleteness or missingness is also caused by drastic tumor shrinkage (<0.01 cm3) or random truncation. Because of the small sample sizes in these models, asymptotic inferences are usually not appropriate. We propose two parametric test procedures based on the EM algorithm and the Bayesian method to compare treatment effects among different groups while accounting for informative censoring. A real xenograft study on a new antitumor agent, temozolomide, combined with irinotecan is analyzed using the proposed methods. [source] Identification of key residues involved in mediating the in vivo anti-tumor/anti-endothelial activity of AlphastatinJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2007C. A. STATON Summary., Background :,We have recently shown that Alphastatin, a 24-amino-acid peptide (ADSGEGDFLAEGGGVRGPRVVERH) derived from human fibrinogen has anti-endothelial properties in vitro and in vivo. Objectives:, The aim of this study was to determine the activity of a terminally modified (stabilized) form of Alphastatin in vitro and in vivo and to identify the key residues required for this activity. Methods:, The in vitro activity of modified Alphastatin, truncates and mutants was determined by endothelial cell (HuDMEC) tubule formation and migration. Active peptides were then assessed in vivo using syngeneic murine subcutaneous 4T1 mammary carcinomas. Results:, Modified Alphastatin-inhibited HuDMEC migration and tubule formation in response to multiple growth factors and caused a 45% inhibition in tumor growth when administered intravenously at 0.25 mg kg,1 (three times per week). Intravenous (i.v.) administration proved non-toxic at all doses investigated, whereas oral and intraperitoneal (i.p.) administration demonstrated neither anti-tumor activity nor toxicity. Truncations of Alphastatin revealed an 11-amino-acid peptide (DFLAEGGGVRG), termed AHN419, which inhibited endothelial cell activity in vitro; however, intravenous AHN419 caused a non-significant growth inhibition in vivo. Single amino acid substitutions to alanine along the entire length of Alphastatin indicated that additional residues outside the AHN419 sequence were required for full activity. Conclusions:, Terminal modification of Alphastatin altered the in vivo efficacy and these studies suggest that a hydrophobic cluster (Phe8, Leu9, Ala10 and Val15) is essential for the biological activity, but additional residues, including Ser3-Gly14, Pro18-Val20 and Arg23 are required for full inhibitory activity of Alphastatin. [source] Vector fragmentation: Characterizing vector integrity in transfected clones by Southern blottingBIOTECHNOLOGY PROGRESS, Issue 1 2010Say Kong Ng Abstract The Chinese Hamster Ovary production cell line development process using methotrexate (MTX) amplification is well studied and commonly used for biopharmaceutical processes. However, successful MTX amplification varies from clone to clone and suggested reasons include vector fragmentation during the transfection process and genomic rearrangement of the Chinese Hamster Ovary chromosomes. Here, we elucidated the vector integration patterns of 40 transfected single-cell clones by Southern blotting and showed that vector fragmentation occurs at a significant level in our experiment. This concurs with MTX amplification studies implying that single-cell cloning is necessary to ensure a successful amplification process. Truncations at the ends of the integrated vectors were also observed, whereas gross DNA insertions were not detected in our data. This suggests that end deletions are common, whereas insertion events are rare in animal cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Labor productivity of small and large manufacturing firms: the case of TaiwanCONTEMPORARY ECONOMIC POLICY, Issue 3 2000M. Hsu This work studies the factors influencing the labor productivity of small and medium-sized enterprises (SMEs) and large firms using Taiwan as a case study. A special emphasis is placed on two possible international channels: exports and foreign direct investment (FDI). Different from conventional studies, we employ the two-stage switching regressions to correct the firm-size effect on labor productivity and estimate labor productivity for SMEs and large firms. The main findings are as follows. First, the estimates of the selectivity variable are statistically significant for both SMEs and large firms, supporting the hypothesis of correcting the effect of firm-size truncation. Second, while a larger trade intensity significantly increases the labor productivity of SMEs, it deteriorates significantly that of large firms. Third, FDI enhances the labor productivity of SMEs internally, whereas it has a negative spillover on that of other small and large firms in the industry. While the first outcome lends supports to the role of self-selection, the remaining stands in sharp contrast to conventional wisdom. [source] Chemical-Picture-Based Modeling of Thermodynamic Properties of Dense Multicharged-Ion Plasmas Using the Superconfiguration ApproachCONTRIBUTIONS TO PLASMA PHYSICS, Issue 10 2009P.A. Loboda Abstract Using the chemical-picture representation of plasmas as a mixture of various ions and free electrons, a consistent description of thermodynamics of dense multicharged-ion plasmas is being developed that involves the effects of Coulomb non-ideality and degeneracy of plasma electrons; contribution of the excited ion states (on the base of the superconfiguration approach) that may exist under an appropriate truncation of ion energy spectra due to plasma effects; hard-sphere-model representation of the finite-volume effects of plasma ions with the model parameters (effective ion sizes) corresponding to superconfigurations yielding the greatest contribution to partition functions. We present the calculated data for average ionization, Grüneisen coefficient, and specific heat of aluminum and iron plasmas at temperatures of 0.03,3 keV and densities 10,3 , 10,5 of their normal material densities. Calculated thermodynamic functions and shock Hugoniots are compared with other theoretical and experimental data (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant-negative mechanismDEVELOPMENTAL DYNAMICS, Issue 4 2004Gary R. Hime Abstract Mutations in the human homolog of the patched gene are associated with the developmental (and cancer predisposition) condition Nevoid Basal Cell Carcinoma Syndrome (NBCCS), as well as with sporadic basal cell carcinomas. Most mutations that have been identified in the germline of NBCCS patients are truncating or frameshift mutations, with amino acid substitutions rarely found. We show that a missense mutation in the sterol-sensing domain G509V acts as a dominant negative when assayed in vivo in Drosophila. Ectopic expression of a Drosophila patched transgene, carrying the analogous mutation to G509V, causes ectopic activation of Hedgehog target genes and ectopic membrane stabilisation of Smoothened. The G509V transgene behaves in a manner similar, except in its subcellular distribution, to a C-terminal truncation that has been characterised previously as a dominant-negative protein. G509V exhibits vesicular localisation identical to the wild-type protein, but the C-terminal truncated Patched molecule is localised predominantly to the plasma membrane. This finding suggests that dominant-negative function can be conferred by interruption of different aspects of Patched protein behaviour. Another mutation at the same residue, G509R, did not exhibit dominant-negative activity, suggesting that simple removal of the glycine at 509 is not sufficient to impart dominant-negative function. Developmental Dynamics 229:780,790, 2004. © 2004 Wiley-Liss, Inc. [source] Phenotypic and genetic analysis of the cerebellar mutant tmgc26, a new ENU-induced ROR-alpha alleleEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2010Douglas J. Swanson Abstract ROR-alpha is an orphan nuclear receptor, inactivation of which cell-autonomously blocks differentiation of cerebellar Purkinje cells with a secondary loss of granule neurons. As part of our ENU mutagenesis screen we isolated the recessive tmgc26 mouse mutant, characterized by early-onset progressive ataxia, cerebellar degeneration and juvenile lethality. Detailed analysis of the tmgc26,/, cerebella revealed Purkinje cell and granule cell abnormalities, and defects in molecular layer interneurons and radial glia. Chimera studies suggested a cell-autonomous effect of the tmgc26 mutation in Purkinje cells and molecular layer interneurons, and a non-cell-autonomous effect in granule cells. The mutation was mapped to a 13-Mb interval on chromosome 9, a region that contains the ROR-alpha gene. Sequencing of genomic DNA revealed a T-to-A transition in exon 5 of the ROR-alpha gene, resulting in a nonsense mutation C257X and severe truncation of the ROR-alpha protein. Together, our data identify new roles for ROR-alpha in molecular layer interneurons and radial glia development and suggest tmgc26 as a novel ROR-alpha allele that may be used to further delineate the molecular mechanisms of ROR-alpha action. [source] Co-expression of C-terminal truncated alpha-synuclein enhances full-length alpha-synuclein-induced pathologyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2010Ayse Ulusoy Abstract Lewy bodies, which are a pathological hallmark of Parkinson's disease, contain insoluble polymers of alpha-synuclein (,syn). Among the different modifications that can promote the formation of toxic ,syn species, C-terminal truncation is among the most abundant alterations in patients with Parkinson's disease. In vitro, C-terminal truncated ,syn aggregates faster and sub-stoichiometric amounts of C-terminal truncated ,syn promote aggregation of the full-length ,syn (,synFL) and induce neuronal toxicity. To address in vivo the putative stimulation of ,syn-induced pathology by the presence of truncated ,syn, we used recombinant adeno-associated virus to express either ,synFL or a C-terminal truncated ,syn (1-110) in rats. We adjusted the recombinant adeno-associated virus vector concentrations so that either protein alone led to only mild to moderate axonal pathology in the terminals of nigrostriatal dopamine neurons without frank cell loss. When these two forms of ,syn were co-expressed at these pre-determined levels, it resulted in a more aggressive pathology in fiber terminals as well as dopaminergic cell loss in the substantia nigra. Using an antibody that did not detect the C-terminal truncated ,syn (1-110) but only ,synFL, we demonstrated that the co-expressed truncated protein promoted the progressive accumulation of ,synFL and formation of larger pathological accumulations. Moreover, in the co-expression group, three of the eight animals showed apomorphine-induced turning, suggesting prominent post-synaptic alterations due to impairments in the dopamine release, whereas the mild pathology induced by either form alone did not cause motor abnormalities. Taken together these data suggest that C-terminal truncated ,syn can interact with and exacerbate the formation of pathological accumulations containing ,synFL in vivo. [source] Involvement of mitochondrial signaling pathways in the mechanism of Fas-mediated apoptosis after spinal cord injuryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2009Wen Ru Yu Abstract Activation of the Fas receptor has been recently linked to apoptotic cell death after spinal cord injury (SCI). Although it is generally considered that Fas activation mediates apoptosis predominantly through the extrinsic pathway, we hypothesized that intrinsic mitochondrial signaling could be involved in the underlying mechanism of Fas-induced apoptosis after SCI. In the present study, we utilized the FejotaTM clip compression model of SCI at T5,6 in C57BL/6 Fas-deficient (lpr) and wild-type mice. Complementary studies were conducted using an in vitro model of trauma or a Fas-activating antibody to induce apoptosis in primary neuronal,glial mixed spinal cord cultures. After in vivo SCI, lpr mice, in comparison with wild-type mice, exhibited reduced numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells at the lesion, reduced expression of truncation of Bid (tBid), apoptosis-inducing factor, activated caspase-9 and activated caspase-3, and increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. After in vitro neurotrauma or the induction of Fas signaling by the Jo2 activating antibody, lpr spinal cord cultures showed an increased proportion of cells retaining mitochondrial membrane integrity and a reduction of tBid expression, caspase-9 and caspase-3 activation, and TUNEL-positive cells as compared to wild-type spinal cord cultures. The neutralization of Fas ligand (FasL) protected against traumatically induced or Fas-mediated caspase-3 activation and the loss of mitochondrial membrane potential and tBid expression in wild-type spinal cord cultures. However, in lpr spinal cord cultures, FasL neutralization had no protective effects. In summary, these data provide direct evidence for the induction of intrinsic mitochondrial signaling pathways following Fas activation after SCI. [source] Synaptic glutamate receptor clustering in mice lacking the SH3 and GK domains of SAP97EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2002Nikolaj Klöcker Abstract Postsynaptic targeting of the Drosophila tumour suppressor discs-large (Dlg) critically depends on its SH3 and GK domains. Here, we asked whether these domains are also involved in subcellular targeting of the mammalian Dlg homolog SAP97 and its interacting partners in CNS cortical neurons by analysing a recently described mouse mutant lacking the SH3 and GK domains of SAP97. Both wildtype and truncated SAP97 were predominantly expressed in perinuclear regions, in a pattern suggesting association with the endoplasmic reticulum. Weaker immunoreactivity was found in neurites colocalizing with both dendritic and axonal markers. As SAP97 has been implicated in the early intracellular processing of the glutamate receptor GluR1, we studied biochemical maturation and subcellular localization of GluR1 in the mutants. Both the glycosylation pattern and synaptic clustering of GluR1 were indistinguishable from wildtype mice. Synaptic clustering of the guanylate kinase domain interacting protein GKAP was also intact. Our data demonstrate that truncation of the SH3 and GK domains of SAP97 in mice does neither change its subcellular distribution nor does it disrupt synaptic structure or protein clustering, as opposed to severe missorting of the respective mutant Dlg protein in Drosophila. [source] R120G ,B-crystallin promotes the unfolding of reduced ,-lactalbumin and is inherently unstableFEBS JOURNAL, Issue 3 2005Teresa M. Treweek ,-Crystallin is the principal lens protein which, in addition to its structural role, also acts as a molecular chaperone, to prevent aggregation and precipitation of other lens proteins. One of its two subunits, ,B-crystallin, is also expressed in many nonlenticular tissues, and a natural missense mutation, R120G, has been associated with cataract and desmin-related myopathy, a disorder of skeletal muscles [Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D, Chapon F, Tome F, Dupret JM, Paulin D & Fardeau M (1998) Nat Genet20, 92,95]. In the present study, real-time 1H-NMR spectroscopy showed that the ability of R120G ,B-crystallin to stabilize the partially folded, molten globule state of ,-lactalbumin was significantly reduced in comparison with wild-type ,B-crystallin. The mutant showed enhanced interaction with, and promoted unfolding of, reduced ,-lactalbumin, but showed limited chaperone activity for other target proteins. Using NMR spectroscopy, gel electrophoresis, and MS, we observed that, unlike the wild-type protein, R120G ,B-crystallin is intrinsically unstable in solution, with unfolding of the protein over time leading to aggregation and progressive truncation from the C-terminus. Light scattering, MS, and size-exclusion chromatography data indicated that R120G ,B-crystallin exists as a larger oligomer than wild-type ,B-crystallin, and its size increases with time. It is likely that removal of the positive charge from R120 of ,B-crystallin causes partial unfolding, increased exposure of hydrophobic regions, and enhances its susceptibility to proteolysis, thus reducing its solubility and promoting its aggregation and complexation with other proteins. These characteristics may explain the involvement of R120G ,B-crystallin with human disease states. [source] The porcine trophoblastic interferon-,, secreted by a polarized epithelium, has specific structural and biochemical propertiesFEBS JOURNAL, Issue 11 2002Avrelija Cenci At the time of implantation in the maternal uterus, the trophectoderm of the pig blastocyst is the source of a massive secretion of interferon-gamma (IFN-,), together with lesser amounts of IFN-,, a unique species of type I IFN. This trophoblastic IFN-, (TrIFN-,) is an unprecedented example of IFN-, being produced spontaneously by an epithelium. We therefore studied some of its structural and biochemical properties, by comparison with pig IFN-, from other sources, either natural LeIFN-, (from adult leucocytes), or recombinant. Biologically active TrIFN-, is a dimeric molecule, of which monomers are mainly composed of a truncated polypeptide chain with two glycotypes, unlike LeIFN-, which is formed of at least two polypeptide chains and four glycotypes. TrIFN-, collected in the uterus lumen was enzymatically deglycosylated and analysed by mass spectrometry (MALDI-TOF). The data revealed that the more abundant polypeptide has a mass of 14.74 kDa, corresponding to a C-terminal cleavage of 17 residues from the expected 143-residue long mature sequence. A minor polypeptide, with a mass of 12.63 kDa, corresponds to a C-terminal truncation of 36 amino acids. MALDI-TOF analysis of tryptic peptides from the glycosylated molecule(s) identifies a single branched carbohydrate motif, with six N -acetylgalactosamines, and no sialic acid. The only glycan microheterogeneity seems to reside in the number of l -fucose residues (one to three). The lack of the C-terminal cluster of basic residues, and the presence of nonsialylated glycans, result in a very low net charge of TrIFN-, molecule. However, the 17-residue truncation does not affect the antiproliferative activity of TrIFN-, on different cells, among which is a porcine uterine epithelial cell line. It is suggested that these specific properties might confer on TrIFN-, a particular ability to invade the uterine mucosa and exert biological functions beyond the endometrial epithelium. [source] Calcineurin is implicated in the regulation of the septation initiation network in fission yeastGENES TO CELLS, Issue 10 2002Yabin Lu Background: In fission yeast, calcineurin has been implicated in cytokinesis because calcineurin-deleted cells form multiple septa and cell separation is impeded. However, this mechanism remains unclear. Results: We screened for mutations that confer syn-thetic lethality with calcineurin deletion and isolated a mutant, its10-1/cdc7-i10, a novel allele of the cdc7+ gene involved in the septation initiation network (SIN). The mutation created a termination codon, resulting in the truncation of Cdc7 by 162 amino acids, which is not localized in the spindle pole body. Following treatment with the immune suppressive drug FK506, cdc7-i10 and the original cdc7-24 mutant cells showed highly elongated multinuclear morphology with few visible septa, closely resembling the phenotype at the restrictive temperature. Other SIN mutants, cdc11, spg1, sid2 and mob1 showed similar phenotypes following FK506 treatment. Consistent with this, expression of the constitutively active calcineurin suppressed the growth defects and septum initiation deficiency of these SIN mutants at the restrictive temperature. Moreover, electron microscopy revealed that calcineurin-deleted cells had very thick multiple septa which were partially and ectopically formed. Conclusion: These results suggest that calcineurin is involved in the regulation of the SIN pathway, and is required for the proper formation and maturation of the septum in fission yeast. [source] Fusion gene-mediated truncation of RUNX1 as a potential mechanism underlying disease progression in the 8p11 myeloproliferative syndromeGENES, CHROMOSOMES AND CANCER, Issue 7 2007Helena Ågerstam The 8p11 myeloproliferative syndrome (EMS) is a chronic myeloproliferative disorder molecularly characterized by fusion of various 5, partner genes to the 3, part of the fibroblast growth factor receptor 1 (FGFR1) gene at 8p, resulting in constitutive activation of the tyrosine kinase activity contained within FGFR1. EMS is associated with a high risk of transformation to acute myeloid leukemia (AML), but the mechanisms underlying the disease progression are unknown. In the present study, we have investigated a case of EMS harboring a t(8;22)(p11;q11)/BCR-FGFR1 rearrangement as well as a t(9;21)(q34;q22) at the time of AML transformation. FISH and RT-PCR analyses revealed that the t(9;21) leads to a fusion gene consisting of the 5, part of RUNX1 (exons 1,4) fused to repetitive sequences of a gene with unknown function on chromosome 9, adding 70 amino acids to RUNX1 exon 4. The t(9;21) hence results in a truncation of RUNX1. No point mutations were found in the other RUNX1 allele. The most likely functional outcome of the rearrangement was haploinsufficiency of RUNX1, which thus may be one mechanism by which EMS transforms to AML. © 2007 Wiley-Liss, Inc. [source] A new complex rearrangement involving the ETV6, LOC115548, and MN1 genes in a case of acute myeloid leukemiaGENES, CHROMOSOMES AND CANCER, Issue 3 2004Elena Belloni A new complex rearrangement involving chromosome bands 5q13, 12p13, 22q11, and 3q12 was identified and characterized in a patient with acute myeloid leukemia. Fluorescence in situ hybridization showed the involvement of the ETV6 gene in 12p13. ETV6 primers were specifically designed for 3,- and 5,-RACE-PCR experiments, which led to the identification of the other two rearranged genes. The derivative chromosome 5 harbored a fusion of the ETV6 sequence with that of the LOC115548 gene. The two genes were placed in opposite orientation and did not encode a fusion protein. On the derivative chromosome 12, ETV6 was fused to the MN1 gene on chromosome 22. Also in this case, the insertion, within the MN1 sequence, of a portion of chromosome 3 prevented the formation of a fusion protein. Finally, the derivative chromosome 22 contained the 3, portions of both LOC115548 and MN1, and no fusion transcript with coding potential could be predicted. In conclusion, all chromosome breakpoints led to the truncation of the three involved genes in the absence of predicted fusion proteins. This study lends further support to the hypothesis that gene disruption resulting in either loss of function or haploinsufficiency may be relevant in acute myeloid leukemia pathogenesis. © 2004 Wiley-Liss, Inc. [source] TBid mediated activation of the mitochondrial death pathway leads to genetic ablation of the lens in Xenopus laevisGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 1 2007D. Du Pasquier Abstract Xenopus is a well proven model for a wide variety of developmental studies, including cell lineage. Cell lineage in Xenopus has largely been addressed by injection of tracer molecules or by micro-dissection elimination of blastomeres. Here we describe a genetic method for cell ablation based on the use of tBid, a direct activator of the mitochondrial apoptotic pathway. In mammalian cells, cross-talk between the main apoptotic pathways (the mitochondrial and the death domain protein pathways) involve the pro-death protein BID, the active form of which, tBID, results from protease truncation and translocation to mitochondria. In transgenic Xenopus, restricting tBID expression to the lens-forming cells enables the specific ablation of the lens without affecting the development of other eye structures. Thus, overexpression of tBid can be used in vivo as a tool to eliminate a defined cell population by apoptosis in a developing organism and to evaluate the degree of autonomy or the inductive effects of a specific tissue during embryonic development. genesis 45:1,10, 2007. © 2006 Wiley-Liss, Inc. [source] Paleoindian environmental change and landscape response in Barger Gulch, Middle Park, ColoradoGEOARCHAEOLOGY: AN INTERNATIONAL JOURNAL, Issue 6 2005James H. Mayer Middle Park, a high-altitude basin in the Southern Rocky Mountains of north-central Colorado, contains at least 59 known Paleoindian localities. At Barger Gulch Locality B, an extensive Folsom assemblage (,10,500 14C yr B.P.) occurs within a buried soil. Radiocarbon ages of charcoal and soil organic matter, as well as stratigraphic positions of artifacts, indicate the soil is a composite of a truncated, latest-Pleistocene soil and a younger mollic epipedon formed between ,6000 and 5200 14C yr B.P. and partially welded onto the older soil following erosion and truncation. Radiocarbon ages from an alluvial terrace adjacent to the excavation area indicate that erosion followed by aggradation occurred between ,10,200 and 9700 14C yr B.P., and that the erosion is likely related to truncation of the latest-Pleistocene soil. Erosion along the main axis of Barger Gulch occurring between ,10,000 and 9700 14C yr B.P. was followed by rapid aggradation between ,9700 and 9550 14C yr B.P., which, along with the erosion at Locality B, coincides with the abrupt onset of monsoonal precipitation following cooling in the region ,11,000,10,000 14C yr B.P. during the Younger Dryas oscillation. Buried soils dated between ,9500 and 8000 14C yr B.P. indicate relative landscape stability and soil formation throughout Middle Park. Morphological characteristics displayed by early Holocene soils suggest pedogenesis under parkland vegetation in areas currently characterized by sagebrush steppe. The expansion of forest cover into lower elevations during the early Holocene may have resulted in lower productivity in regards to mammalian fauna, and may partly explain the abundance of early Paleoindian sites (,11,000,10,000 14C yr B.P., 76%) relative to late Paleoindian sites (,10,000,8000 14C yr B.P., 24%) documented in Middle Park. © 2005 Wiley Periodicals, Inc. [source] | ||||||||||||||||||||||||||||||||||||||||||||||||||||