Showing papers by "Children's Medical Research Institute published in 2003"
TL;DR: It is shown that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo.
Abstract: Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.
306 citations
TL;DR: Current research into the factors influencing lineage differentiation in the mouse embryo are reviewed and the application of this knowledge to in vitro differentiation of ES cells is reviewed.
266 citations
TL;DR: This work proposes that H2A.Z functions to establish a specialized pericentric domain by assembling an architecturally distinct chromatin structure and by recruiting specific nuclear proteins in order to distinguish constitutive from facultative heterochromatin.
Abstract: Determining how chromatin is remodelled during early development, when totipotent cells begin to differentiate into specific cell types, is essential to understand how epigenetic states are established. An important mechanism by which chromatin can be remodelled is the replacement of major histones with specific histone variants. During early mammalian development H2A.Z plays an essential, but unknown, function(s). We show here that undifferentiated mouse cells of the inner cell mass lack H2A.Z, but upon differentiation H2A.Z expression is switched on. Strikingly, H2A.Z is first targeted to pericentric hetero chromatin and then to other regions of the nucleus, but is excluded from the inactive X chromosome and the nucleolus. This targeted incorporation of H2A.Z could provide a critical signal to distinguish constitutive from facultative heterochromatin. In support of this model, we demonstrate that H2A.Z can directly interact with the pericentric heterochromatin binding protein INCENP. We propose that H2A.Z functions to establish a specialized pericentric domain by assembling an architecturally distinct chromatin structure and by recruiting specific nuclear proteins.
248 citations
TL;DR: It is concluded that ALT is a prognostic indicator for patients with glioblastoma multiforme and Cox's regression analysis showed that this association is independent of age.
247 citations
TL;DR: It is concluded that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization ofActin filament function.
Abstract: The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5NM1), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5NM1 was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function.
237 citations
TL;DR: It is shown that human POT1 can act as a telomerase-dependent, positive regulator of telomere length and that a normal function of hPOT1 is to facilitate telomeres elongation by telomersase.
230 citations
TL;DR: Morphogenetic interactions between the endoderm and the other germ layer derivatives are critical for the morphogenesis of head structures and organogenesis of gut derivatives.
200 citations
TL;DR: By combining embryonic stem cell technology, molecularly tagged mutations and sensitive cell lineage markers, chimeras can provide invaluable insights into the tissue-specific requirement and the mode of action of many mouse genes.
Abstract: Embryonic chimeras of the mouse are well-established tools for studying cell lineage and cell potential. They are also a key part of the analysis of complex phenotypes of mutant mice. By combining embryonic stem cell technology, molecularly tagged mutations and sensitive cell lineage markers, chimeras can provide invaluable insights into the tissue-specific requirement and the mode of action of many mouse genes.
194 citations
TL;DR: It is hypothesized here that inherited abnormalities of telomerase activity and other aspects of telomere maintenance may contribute to cancer and ageing.
194 citations
TL;DR: There is a positive correlation between the level of expression of estrogen receptor and expression of both STC1 and STC2 in breast cancer, and the roles they may play in normal physiology and in breast and other cancers are discussed.
Abstract: Stanniocalcin (STC) is a glycoprotein hormone that is secreted by the corpuscle of Stannius, an endocrine gland of bony fish, and is involved in calcium and phosphate homeostasis. The related mammalian proteins, STC1 and STC2, are expressed in a wide variety of tissues. The ovaries have the highest level of STC1, and this increases during pregnancy and lactation. STC1 is present in breast ductal epithelium, and its expression is induced by BRCA1, a tumor suppressor gene that has an important role in breast and ovarian cancer. The expression of STC2 is induced by estrogen, and there is a positive correlation between the level of expression of estrogen receptor and expression of both STC1 and STC2 in breast cancer. This article reviews the data currently available regarding the mammalian STCs, and discusses the roles they may play in normal physiology and in breast and other cancers.
156 citations
TL;DR: Together, mot-2 and hTERT permitted bypass of senescence, a substantial extension of lifespan, and possibly immortalization, the first demonstration that mot- 2 and telomerase can cooperate in the immortalization process.
TL;DR: It is concluded that a primary function of Lim1 in the early embryo is to enable proper cell movements during gastrulation.
TL;DR: It is concluded that a synapsin-associated PI 3-kinase activity plays a role in synaptic vesicle delivery to the RRP, and suggests that PI 3 -kinase contributes to the maintenance of synaptic transmission during periods of high activity, indicating a possible role inaptic plasticity.
TL;DR: Unlike protein synthesis, skeletal muscle proteasome‐dependent proteolysis is not acutely responsive in vivo to insulin, AA, and/or nutrient intake in refed starved rats, which suggests that distinct and perhaps independent mechanisms are responsible for the nutrient‐dependent regulation of protein synthesis and ubiquitin‐proteasomesome‐ dependent proteolytic suppression following a prolonged period of catabolism.
Abstract: The central role of the ubiquitin-proteasome system in the loss of skeletal muscle protein in many wasting conditions has been well established. However, it is unclear what factors are responsible for the suppression of this system during periods of protein gain. Thus, the aim of these studies was to examine the short-term effects of insulin release and nutrients on skeletal muscle protein turnover in young rats starved for 48 h, and then infused intravenously with amino acids (AA), or fed an oral diet. Forty-eight hours of starvation (i.e. prolonged starvation in young rats) decreased muscle protein synthesis and increased proteasome-dependent proteolysis. Four-hour AA infusion and 4 h of refeeding increased plasma insulin release and AA concentrations, and stimulated muscle protein synthesis, but had no effect on either total or proteasome-dependent proteolysis, despite decreased plasma corticosterone concentrations. Both muscle proteasome-dependent proteolysis and the rate of ubiquitination of muscle proteins were not suppressed until 10 h of refeeding. The temporal response of these two measurements correlated with the normalised expression of the 14-kDa E2 (a critical enzyme in substrate ubiquitination in muscle) and the expression of the MSS1 subunit of the 19S regulatory complex of the 26S proteasome. In contrast, the starvation-induced increase in mRNA levels for 20S proteasome subunits was normalised by refeeding within 24 h in muscle, and 6 h in jejunum, respectively. In conclusion, unlike protein synthesis, skeletal muscle proteasome-dependent proteolysis is not acutely responsive in vivo to insulin, AA, and/or nutrient intake in refed starved rats. This suggests that distinct and perhaps independent mechanisms are responsible for the nutrient-dependent regulation of protein synthesis and ubiquitin-proteasome-dependent proteolysis following a prolonged period of catabolism. Furthermore, factors other than the expression of ubiquitin-proteasome pathway components appear to be responsible for the suppression of skeletal muscle proteasome-dependent proteolysis by nutrition.
TL;DR: Tetrahymena telomerase does not need to dimerize to be active and processive, and a majority, if not all, of the recombinant Tetrahy mena telomersase in the reconstitution system is present as a monomeric complex.
Abstract: Telomerase is an enzyme that utilizes an internal RNA molecule as a template for the extension of chromosomal DNA ends. The catalytic core of telomerase consists of the RNA subunit and a protein reverse transcriptase subunit, known as telomerase reverse transcriptase (TERT). It has previously been shown that both yeast and human telomerase can form dimers or multimers in which one RNA in the complex can influence the activity of another. To test the proposal that dimerization might be essential for telomerase activity, we sought to determine whether Tetrahymena thermophila telomerase is active as a dimer or a monomer. Recombinant Tetrahymena telomerase eluted from a gel filtration column at the size of a monomeric complex (one RNA plus one TERT), and those fractions showed processive telomerase activity. We were unable to detect dimerization of Tetrahymena telomerase by coprecipitation experiments, by using tags on either the TERT protein or telomerase RNA. Therefore, a majority, if not all, of the recombinant Tetrahymena telomerase in our reconstitution system is present as a monomeric complex. We were also unable to detect dimerization of native telomerase from mating and vegetative Tetrahymena cell extracts. These results demonstrate that Tetrahymena telomerase does not need to dimerize to be active and processive.
TL;DR: O'Mahoney et al. as mentioned in this paper showed that both MEF2C, a known regulator of slow fiber-specific genes, and hMusTRD1α1 regulate hTnIslow through the Inrlike element.
TL;DR: The basis of this apparent neuron specificity of lentivirus-mediated transduction in murine DRG cultures is investigated, providing a basis for reevaluating data produced using early-generation U3-bearing lentiv virus vectors and for reconciling these with results obtained using more contemporary SIN lentiviral vectors carrying a U3 deletion.
Abstract: In a previous study using an early-generation VSV-G-pseudotyped lentivirus vector encoding enhanced green fluorescent protein (EGFP) under the transcriptional control of a human cytomegalovirus (CMV) immediate-early promoter, we examined transduction efficiency in dissociated dorsal root ganglia (DRG) cultures. In cultures of murine origin, transgene expression was observed solely in the sensory neurons with the stromal cell population failing to show evidence of transduction. In contrast, efficient and sustained transduction of both sensory neurons and the stromal cell population was observed in cultures of human origin. Given the widespread use of murine models in preclinical gene therapy studies, in the current study we investigated the basis of this apparent neuron specificity of lentivirus-mediated transduction in murine DRG cultures. The interspecies differences persisted at high multiplicities of infection, and irrespective of whether lentiviral vector stocks were packaged in the presence or absenc...
TL;DR: This work optimized the technique of electroporation for introducing genetic markers into cells of the gastrulating mouse embryo to follow cell fates, tissue movement, and lineage differentiation and proved the efficacy of marking cell lineages by CRE‐mediated activation of reporters proved to be inefficient.
Abstract: Summary: We have optimized the technique of electroporation for introducing genetic markers into cells of the gastrulating mouse embryo to follow cell fates, tissue movement, and lineage differentiation. Using a plate-needle electrode combination and specific route of plasmid delivery, labeling could be targeted to discrete regions of the epiblast or the endoderm of the late gastrula. Among the various types of fluorescent and chromogenic reporter constructs tested, those driven by CMV promoter are efficient and strong expression can be detected as soon as 2–3 h after electroporation. The efficacy of marking cell lineages by CRE-mediated activation of reporters proved to be inefficient for tracking cell lineages due to an obligatory 8–9-h lag from the electroporation of constructs to the expression of reporter. This significant time lag also raises concern of the temporal precision at which tissue- or stage-specific knock-out or activation of genetic activity may be achieved by the Cre-loxP mechanism. genesis 35:57–62, 2003. © 2002 Wiley-Liss, Inc.
TL;DR: Results from this study support the idea that the expansion of type I IFN gene complexity in mammals coincides with a concomitant expansion in the functionality of these molecules, which may have facilitated the evolution of viviparity in marsupials and eutherians.
TL;DR: Co-transfection studies in C2C12 muscle cell cultures reveal that isoforms differentially regulate muscle fibre-type-specific promoters, indicating that the presence of different domains of MusTRD influences the activity exerted by this molecule on multiple promoters active in skeletal muscle.
Abstract: A human MusTRD (muscle TFII-I repeat domain (RD)-containing protein) isoform was originally identified in a yeast one-hybrid screen as a protein that binds the slow fibre-specific enhancer of the muscle gene troponin I slow [O'Mahoney, Guven, Lin, Joya, Robinson, Wade and Hardeman (1998) Mol. Cell. Biol. 18, 6641-6652]. MusTRD shares homology with the general transcription factor TFII-I by the presence of diagnostic I-RDs [Roy (2001) Gene 274, 1-13]. The human gene encoding MusTRD, GTF2IRD1 ( WBSCR11 / GTF3 ), was subsequently located on chromosome 7q11.23, a region deleted in the neurodegenerative disease, Williams-Beuren Syndrome [Osborne, Campbell, Daradich, Scherer, Tsui, Franke, Peoples, Francke, Voit, Kramer et al. (1999) Genomics 57, 279-284; Franke, Peoples and Francke (1999) Cytogenet. Cell. Genet. 86, 296-304; Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737-747]. The haploinsufficiency of MusTRD has been implicated in the myopathic aspect of this disease, which manifests itself in symptoms such as lowered resistance to fatigue, kyphoscoliosis, an abnormal gait and joint contractures [Tassabehji, Carette, Wilmot, Donnai, Read and Metcalfe (1999) Eur. J. Hum. Genet. 7, 737-747]. Here, we report the identification of 11 isoforms of MusTRD in mouse skeletal muscles. These isoforms were isolated from a mouse skeletal muscle cDNA library and reverse transcription-PCR on RNA from various adult and embryonic muscles. The variability in these isoforms arises from alternative splicing of a combination of four cassettes and two mutually exclusive exons, all in the 3' region of the primary transcript of Gtf2ird1, the homologous mouse gene. The expression of some of these isoforms is differentially regulated spatially, suggesting individual regulation of the expression of these isoforms. Co-transfection studies in C2C12 muscle cell cultures reveal that isoforms differentially regulate muscle fibre-type-specific promoters. This indicates that the presence of different domains of MusTRD influences the activity exerted by this molecule on multiple promoters active in skeletal muscle.
TL;DR: The coculture system presented here provides an excellent system for investigating the morphological, immunocytochemical, and electrophysiological differentiation of Purkinje neurons under controlled conditions and for studying cell-cell interactions and extrinsic factors, e.g., glutamate in normal and neuropathological conditions.
Abstract: An in vitro coculture system is described to study the avian Purkinje neuron and the interactions occurring with astrocytes and granule cells during development in the cerebellum. Astrocytes initially and granule cells later regulate Purkinje neuron morphology. The coculture system presented here provides an excellent system for investigating the morphological, immunocytochemical, and electrophysiological differentiation of Purkinje neurons under controlled conditions and for studying cell-cell interactions and extrinsic factors, e.g., glutamate in normal and neuropathological conditions.
01 Jan 2003
TL;DR: The in vitro senescence process has been widely studied as a model for in vivo cellular aging and is associated with a variety of morphological and biochemical changes in the cells.
Abstract: Human somatic cells undergo only a limited number of divisions in vitro before they withdraw from the cell division cycle and enter a permanent state of proliferation arrest. This arrest is accompanied by a variety of morphological and biochemical changes in the cells, and the arrest state is referred to as senescence. The in vitro senescence process has been widely studied as a model for in vivo cellular aging [1].