Showing papers by "Laboratory of Molecular Biology published in 2001"
TL;DR: It is indicated that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease.
Abstract: Myocardial infarction leads to loss of tissue and impairment of cardiac performance The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time1 Injury to a target organ is sensed by distant stem cells, which migrate to the site of damage and undergo alternate stem cell differentiation2,3,4,5; these events promote structural and functional repair6,7,8 This high degree of stem cell plasticity prompted us to test whether dead myocardium could be restored by transplanting bone marrow cells in infarcted mice We sorted lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein9 by fluorescence-activated cell sorting on the basis of c-kit expression10 Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells The developing tissue comprised proliferating myocytes and vascular structures Our studies indicate that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease
5,331 citations
TL;DR: This work generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells that self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons.
Abstract: Although the source of embryonic stem (ES) cells presents ethical concerns, their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas, insulin is produced and secreted by specialized structures, islets of Langerhans. Diabetes, which affects 16 million people in the United States, results from abnormal function of pancreatic islets. We have generated cells expressing insulin and other pancreatic endocrine hormones from mouse ES cells. The cells self-assemble to form three-dimensional clusters similar in topology to normal pancreatic islets where pancreatic cell types are in close association with neurons. Glucose triggers insulin release from these cell clusters by mechanisms similar to those employed in vivo. When injected into diabetic mice, the insulin-producing cells undergo rapid vascularization and maintain a clustered, islet-like organization.
1,634 citations
TL;DR: A high dose of BrdU (300 mg/kg) is shown to be a specific, quantitative, and nontoxic marker of dividing cells in the adult rat dentate gyrus, whereas lower doses label only a fraction of the S‐phase cells.
Abstract: Knowing the rate of addition of new granule cells to the adult dentate gyrus is critical to understanding the function of adult neurogenesis. Despite the large number of studies of neurogenesis in the adult dentate gyrus, basic questions about the magnitude of this phenomenon have never been addressed. The S-phase marker bromodeoxyuridine (BrdU) has been extensively used in recent studies of adult neurogenesis, but it has been carefully tested only in the embryonic brain. Here, we show that a high dose of BrdU (300 mg/kg) is a specific, quantitative, and nontoxic marker of dividing cells in the adult rat dentate gyrus, whereas lower doses label only a fraction of the S-phase cells. By using this high dose of BrdU along with a second S-phase marker, [(3)H]thymidine, we found that young adult rats have 9,400 dividing cells proliferating with a cell cycle time of 25 hours, which would generate 9,000 new cells each day, or more than 250,000 per month. Within 5-12 days of BrdU injection, a substantial pool of immature granule neurons, 50% of all BrdU-labeled cells in the dentate gyrus, could be identified with neuron-specific antibodies TuJ1 and TUC-4. This number of new granule neurons generated each month is 6% of the total size of the granule cell population and 30-60% of the size of the afferent and efferent populations (West et al. [1991] Anat Rec 231:482-497; Mulders et al. [1997] J Comp Neurol 385:83-94). The large number of the adult-generated granule cells supports the idea that these new neurons play an important role in hippocampal function.
1,611 citations
TL;DR: It is reported that expression of wild-type but not of mutated SIP1 downregulates mammalian E-cadherin transcription via binding to both conserved E2 boxes of the minimal E- cadheringin promoter.
1,369 citations
TL;DR: The molecular properties of the synucleins, the different diseases characterized by the accumulation of α-synuclein, and the possible mechanisms by which dysfunction ofα- synuclein might lead to neurodegeneration are reviewed.
Abstract: In recent years, two developments have imparted a new direction to research on the aetiology and pathogenesis of Parkinson's disease. First, the discovery that a missense mutation in the α-synuclein gene is a rare genetic cause of Parkinson's disease. Second, the identification of the α-synuclein protein as the main component of Lewy bodies and Lewy neurites, the defining neuropathological characteristics of all cases of Parkinson's and several other diseases. The filamentous inclusions of multiple system atrophy are also made of α-synuclein. These findings have placed α-synuclein dysfunction at the centre of several common neurodegenerative diseases. Here, I review the molecular properties of the synucleins, the different diseases characterized by the accumulation of α-synuclein, and the possible mechanisms by which dysfunction of α-synuclein might lead to neurodegeneration.
1,288 citations
TL;DR: A new procedure is described for detecting and correcting those errors that arise at the model-building stage of the procedure and a good procedure for creating HMMs for sequences of proteins of known structure are determined.
1,211 citations
TL;DR: Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution.
Abstract: Crystal structures of the 30S ribosomal subunit in complex with messenger RNA and cognate transfer RNA in the A site, both in the presence and absence of the antibiotic paromomycin, have been solved at between 3.1 and 3.3 angstroms resolution. Cognate transfer RNA (tRNA) binding induces global domain movements of the 30S subunit and changes in the conformation of the universally conserved and essential bases A1492, A1493, and G530 of 16S RNA. These bases interact intimately with the minor groove of the first two base pairs between the codon and anticodon, thus sensing Watson-Crick base-pairing geometry and discriminating against near-cognate tRNA. The third, or “wobble,” position of the codon is free to accommodate certain noncanonical base pairs. By partially inducing these structural changes, paromomycin facilitates binding of near-cognate tRNAs. During protein synthesis, the ribosome catalyzes the sequential addition of amino acids to a growing polypeptide chain, using mRNA as a template and aminoacylated tRNAs (aatRNAs) as substrates. Correct base pairing between the three bases of the codon on mRNA and those of the anticodon of the cognate aatRNA dictates the sequence of the polypeptide
1,177 citations
TL;DR: In this paper, a refined model of the alpha-beta-tubulin dimer was presented, which includes residues alpha: 2-34, alpha:61-439, beta:2-437, one molecule of GTP, one of GDP, and one of taxol, as well as one magnesium ion near the M-loop in the alpha subunit.
1,104 citations
TL;DR: It is confirmed that mPER1 influences rhythmicity primarily through interaction with other clock proteins, while mPER2 positively regulates rhythmic gene expression, and there is partial compensation between products of these two genes.
858 citations
TL;DR: It is concluded that the binding of Axin to LRP-5 is an important part of the Wnt signal transduction pathway, and the L RP-5 sequences involved in interactions with Axin are required for LEF-1 activation.
846 citations
TL;DR: It is demonstrated that the bacterial MreB protein assembles into filaments with a subunit repeat similar to that of F-actin—the physiological polymer of eukaryotic actin, demonstrating that M reB and actin are very similar in three dimensions.
Abstract: It was thought until recently that bacteria lack the actin or tubulin filament networks that organize eukaryotic cytoplasm. However, we show here that the bacterial MreB protein assembles into filaments with a subunit repeat similar to that of F-actin-the physiological polymer of eukaryotic actin. By elucidating the MreB crystal structure we demonstrate that MreB and actin are very similar in three dimensions. Moreover, the crystals contain protofilaments, allowing visualization of actin-like strands at atomic resolution. The structure of the MreB protofilament is in remarkably good agreement with the model for F-actin, showing that the proteins assemble in identical orientations. The actin-like properties of MreB explain the finding that MreB forms large fibrous spirals under the cell membrane of rod-shaped cells, where they are involved in cell-shape determination. Thus, prokaryotes are now known to possess homologues both of tubulin, namely FtsZ, and of actin.
TL;DR: The structure of the NH2-terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate [PtdIns( 4,5)P2] via a lysine-rich motif is presented, and it is shown that AP180 may serve to tether clathrin to the membrane simultaneously, and was shown by using purified components and a budding assay on preformed lipid monolayers.
Abstract: Adaptor protein 180 (AP180) and its homolog, clathrin assembly lymphoid myeloid leukemia protein (CALM), are closely related proteins that play important roles in clathrin-mediated endocytosis. Here, we present the structure of the NH2-terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate [PtdIns(4,5)P2] via a lysine-rich motif. This motif is found in other proteins predicted to have domains of similar structure (for example, Huntingtin interacting protein 1). The structure is in part similar to the epsin NH2-terminal (ENTH) domain, but epsin lacks the PtdIns(4,5)P2-binding site. Because AP180 could bind to PtdIns(4,5)P2 and clathrin simultaneously, it may serve to tether clathrin to the membrane. This was shown by using purified components and a budding assay on preformed lipid monolayers. In the presence of AP180, clathrin lattices formed on the monolayer. When AP2 was also present, coated pits were formed.
TL;DR: A role is demonstrated for the Ets1 and Ets2 transcription factors based on their ability to activate the p16INK4a promoter through an ETS-binding site and their patterns of expression during the lifespan of human diploid fibroblasts.
Abstract: The p16INK4a cyclin-dependent kinase inhibitor is implicated in replicative senescence, the state of permanent growth arrest provoked by cumulative cell divisions or as a response to constitutive Ras-Raf-MEK signalling in somatic cells Some contribution to senescence presumably underlies the importance of p16INK4a as a tumour suppressor but the mechanisms regulating its expression in these different contexts remain unknown Here we demonstrate a role for the Ets1 and Ets2 transcription factors based on their ability to activate the p16INK4a promoter through an ETS-binding site and their patterns of expression during the lifespan of human diploid fibroblasts The induction of p16INK4a by Ets2, which is abundant in young human diploid fibroblasts, is potentiated by signalling through the Ras-Raf-MEK kinase cascade and inhibited by a direct interaction with the helix-loop-helix protein Id1 (ref 11) In senescent cells, where the Ets2 levels and MEK signalling decline, the marked increase in p16INK4a expression is consistent with the reciprocal reduction of Id1 and accumulation of Ets1
TL;DR: Current data on the population genetics and molecular mechanisms of DSD illustrate how the details of developmental processes are constantly changing within evolutionary lineages, indicating that developmental systems may possess a great deal of plasticity in their responses to natural selection.
Abstract: The comparative analysis of homologous characters is a staple of evolutionary developmental biology and often involves extrapolating from experimental data in model organisms to infer developmental events in non-model organisms. In order to determine the general importance of data obtained in model organisms, it is critical to know how often and to what degree similar phenotypes expressed in different taxa are formed by divergent developmental processes. Both comparative studies of distantly related species and genetic analysis of closely related species indicate that many characters known to be homologous between taxa have diverged in their morphogenetic or gene regulatory underpinnings. This process, which we call "developmental system drift" (DSD), is apparently ubiquitous and has significant implications for the flexibility of developmental evolution of both conserved and evolving characters. Current data on the population genetics and molecular mechanisms of DSD illustrate how the details of developmental processes are constantly changing within evolutionary lineages, indicating that developmental systems may possess a great deal of plasticity in their responses to natural selection.
TL;DR: An increased AtSERK1 level is sufficient to confer embryogenic competence in culture and demonstrate its role during establishment of somatic embryogenesis in culture.
Abstract: We report here the isolation of the Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (AtSERK1) gene and we demonstrate its role during establishment of somatic embryogenesis in culture. The AtSERK1 gene is highly expressed during embryogenic cell formation in culture and during early embryogenesis. The AtSERK1 gene is first expressed in planta during megasporogenesis in the nucleus of developing ovules, in the functional megaspore, and in all cells of the embryo sac up to fertilization. After fertilization, AtSERK1 expression is seen in all cells of the developing embryo until the heart stage. After this stage, AtSERK1 expression is no longer detectable in the embryo or in any part of the developing seed. Low expression is detected in adult vascular tissue. Ectopic expression of the full-length AtSERK1 cDNA under the control of the cauliflower mosaic virus 35S promoter did not result in any altered plant phenotype. However, seedlings that overexpressed the AtSERK1 mRNA exhibited a 3- to 4-fold increase in efficiency for initiation of somatic embryogenesis. Thus, an increased AtSERK1 level is sufficient to confer embryogenic competence in culture.
TL;DR: It is shown that B-cell interaction with antigens that are immobilized on the surface of a target cell leads to the formation of a synapse and the acquisition, even, of membrane-integral antigen from the target.
Abstract: Soluble antigen binds to the B-cell antigen receptor and is internalized for subsequent processing and the presentation of antigen-derived peptides to T cells. Many antigens are not soluble, however, but are integral components of membrane; furthermore, soluble antigens will usually be encountered in vivo in a membrane-anchored form, tethered by Fc or complement receptors. Here we show that B-cell interaction with antigens that are immobilized on the surface of a target cell leads to the formation of a synapse and the acquisition, even, of membrane-integral antigens from the target. B-cell antigen receptor accumulates at the synapse, segregated from the CD45 co-receptor which is excluded from the synapse, and there is a corresponding polarization of cytoplasmic effectors in the B cell. B-cell antigen receptor mediates the gathering of antigen into the synapse and its subsequent acquisition, thereby potentiating antigen processing and presentation to T cells with high efficacy. Synapse formation and antigen acquisition will probably enhance the activation of B cells at low antigen concentration, allow context-dependent antigen recognition and enhance the linking of B- and T-cell epitopes.
TL;DR: The HMG-box proteins, one of the three classes of high mobility group (HMG) chromosomal proteins, bend DNA and bind preferentially to distorted DNA structures and might be targeted to particular DNA sites in chromatin by either protein-protein interactions or recognition of specific DNA structures.
TL;DR: Rhomboid-1 is conserved throughout evolution from archaea to humans, and the results show that a human Rhomboid promotes Spitz cleavage by a similar mechanism, suggesting that this growth factor activation mechanism may therefore be widespread.
TL;DR: The results highlight the importance of tonic inhibition mediated by GABAA receptors, loss of which triggers a form of homeostatic plasticity leading to a change in the magnitude of a voltage-independent K + conductance that maintains normal neuronal behaviour.
Abstract: Many neurons receive a continuous, or 'tonic', synaptic input, which increases their membrane conductance, and so modifies the spatial and temporal integration of excitatory signals. In cerebellar granule cells, although the frequency of inhibitory synaptic currents is relatively low, the spillover of synaptically released GABA (gamma-aminobutyric acid) gives rise to a persistent conductance mediated by the GABA A receptor that also modifies the excitability of granule cells. Here we show that this tonic conductance is absent in granule cells that lack the alpha6 and delta-subunits of the GABAA receptor. The response of these granule cells to excitatory synaptic input remains unaltered, owing to an increase in a 'leak' conductance, which is present at rest, with properties characteristic of the two-pore-domain K+ channel TASK-1 (refs 9,10,11,12). Our results highlight the importance of tonic inhibition mediated by GABAA receptors, loss of which triggers a form of homeostatic plasticity leading to a change in the magnitude of a voltage-independent K + conductance that maintains normal neuronal behaviour.
TL;DR: The plethora of Gquadruplex DNA structures is explored, and their possible biological functions as well as the proteins that interact with them are discussed.
Abstract: To be functional, nucleic acids need to adopt particular three-dimensional structures. For a long time DNA was regarded as a rigid and passive molecule with the sole purpose to store genetic information, but experimental data has now accumulated that indicates the full dynamic repertoire of this macromolecule. During the last decade, four-stranded DNA structures known as G-quadruplexes, or DNA tetraplexes, have emerged as a three-dimensional structure of special interest. Motifs for the formation of G-quadruplex DNA structures are widely dispersed in eukaryotic genomes, and are abundant in regions of biological significance, for example, at telomeres, in the promoters of many important genes, and at recombination hotspots, to name but a few in man. Here I explore the plethora of G-quadruplex DNA structures, and discuss their possible biological functions as well as the proteins that interact with them.
TL;DR: The recent cloning of most of the FA-associated genes, and the characterization of their protein products, has provided tantalizing clues as to the molecular basis of this disease.
Abstract: The past few years have witnessed a considerable expansion in our understanding of the pathways that maintain chromosome stability in dividing cells through the identification of genes that are mutated in certain human chromosome instability disorders. Cells that are derived from patients with Fanconi anaemia (FA) show spontaneous chromosomal instability and mutagen hypersensitivity, but FA poses a unique challenge as the nature of the DNA-damage-response pathway thought to be affected by the disease has long been a mystery. However, the recent cloning of most of the FA-associated genes, and the characterization of their protein products, has provided tantalizing clues as to the molecular basis of this disease.
TL;DR: It is shown that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleosid diph phosphate kinase produces the substrates required for the replication of its own gene.
Abstract: We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication.
TL;DR: In hippocampal neurons, signaling to CREB can be activated by nuclear calcium alone and does not require import of cytoplasmic proteins into the nucleus, which is critical for CREB-mediated transcription by synaptic NMDA receptors.
Abstract: Information storage in the nervous system requires transcription triggered by synaptically evoked calcium signals. It has been suggested that translocation of calmodulin into the nucleus, initiated by submembranous calcium transients, relays synaptic signals to CREB. Here we show that in hippocampal neurons, signaling to CREB can be activated by nuclear calcium alone and does not require import of cytoplasmic proteins into the nucleus. The nucleus is particularly suited to integrate neuronal firing patterns, and specifies the transcriptional outputs through a burst frequency-to-nuclear calcium amplitude conversion. Calcium release from intracellular stores promotes calcium wave propagation into the nucleus, which is critical for CREB-mediated transcription by synaptic NMDA receptors. Pharmacological or genetic modulation of nuclear calcium may directly affect transcription-dependent memory and cognitive functions.
TL;DR: The C2 domain of synaptotagmin I, which binds to anionic phospholipids in cell membranes, was shown to bind to the plasma membrane of apoptotic cells by both flow cytometry and confocal microscopy.
Abstract: The C2 domain of synaptotagmin I, which binds to anionic phospholipids in cell membranes, was shown to bind to the plasma membrane of apoptotic cells by both flow cytometry and confocal microscopy. Conjugation of the protein to superparamagnetic iron oxide nanoparticles allowed detection of this binding using magnetic resonance imaging. Detection of apoptotic cells, using this novel contrast agent, was demonstrated both in vitro, with isolated apoptotic tumor cells, and in vivo, in a tumor treated with chemotherapeutic drugs.
TL;DR: BL22 can induce complete remissions in patients with hairy-cell leukemia that is resistant to treatment with purine analogues, including cladribine.
Abstract: Background Hairy-cell leukemia that is resistant to treatment with purine analogues, including cladribine, has a poor prognosis. We tested the safety and efficacy of an immunotoxin directed against a surface antigen that is strongly expressed by leukemic hairy cells. Methods RFB4(dsFv)-PE38 (BL22), a recombinant immunotoxin containing an anti-CD22 variable domain (Fv) fused to truncated pseudomonas exotoxin, was administered in a dose-escalation trial by intravenous infusion every other day for a total of three doses. Results Of 16 patients who were resistant to cladribine, 11 had a complete remission and 2 had a partial remission with BL22. The three patients who did not have a response received low doses of BL22 or had preexisting toxin-neutralizing antibodies. Of the 11 patients in complete remission, 2 had minimal residual disease in the bone marrow or blood. During a median follow-up of 16 months (range, 10 to 23), 3 of the 11 patients who had a complete response relapsed and were retreated; all of t...
TL;DR: Using gene expression profiling, it is shown that activation of B cells and professional antigen-presenting cells (APCs) induces the expression of common chemokines, and CCL4 was the most potent chemoattractant of a CD4+CD25+ T cell population, which is a characteristic phenotype of regulatory T cells.
Abstract: Using gene expression profiling, we show here that activation of B cells and professional antigen-presenting cells (APCs) induces the expression of common chemokines Among these, CCL4 was the most potent chemoattractant of a CD4+CD25+ T cell population, which is a characteristic phenotype of regulatory T cells Depletion of either regulatory T cells or CCL4 resulted in a deregulated humoral response, which culminated in the production of autoantibodies This suggested that the recruitment of regulatory T cells to B cells and APCs by CCL4 plays a central role in the normal initiation of T cell and humoral responses, and failure to do this leads to autoimmune activation
TL;DR: It is shown that oligomerization and GTP binding alone, by dynamin, are not sufficient for endocytosis in vivo, and efficient GTP hydrolysis and an associated conformational change are also required.
Abstract: Dynamin is a large GTPase with a relative molecular mass of 96,000 (Mr 96K) that is involved in clathrin-mediated endocytosis and other vesicular trafficking processes. Although its function is apparently essential for scission of newly formed vesicles from the plasma membrane, the nature of dynamin's role in the scission process is still unclear. It has been proposed that dynamin is a regulator (similar to classical G proteins) of downstream effectors. Here we report the analysis of several point mutants of dynamin's GTPase effector (GED) and GTPase domains. We show that oligomerization and GTP binding alone, by dynamin, are not sufficient for endocytosis in vivo. Rather, efficient GTP hydrolysis and an associated conformational change are also required. These data argue that dynamin has a mechanochemical function in vesicle scission.
TL;DR: A steroid-inducible system is used to show that the transcription factor–type response regulator ARR1 directs transcriptional activation of the ARR6 gene, which responds to cytokinins without de novo protein synthesis, which indicates an intracellular signal transduction occurring immediately after cytokinin perception.
Abstract: Cytokinins are a class of phytohormones involved in various physiological events of plants. The Arabidopsis sensor histidine kinase CRE1 was recently reported to be a cytokinin receptor. We used a steroid-inducible system to show that the transcription factor-type response regulator ARR1 directs transcriptional activation of the ARR6 gene, which responds to cytokinins without de novo protein synthesis. This fact, together with characteristics of ARR1-overexpressing plants and arr1 mutant plants, indicates that the phosphorelay to ARR1, probably from CRE1, constitutes an intracellular signal transduction occurring immediately after cytokinin perception.
TL;DR: It is led to conclude that recombination between common families, as compared to the invention of new families and recombination among these, has also been a major contribution to the evolution of kingdom-specific and species-specific functions in organisms in all three kingdoms.
TL;DR: This work will discuss emerging new findings and their implications for the nature of lipid rafts themselves, as well as for the potential roles of non-clathrin endocytic pathways in remodeling of the plasma membrane and in regulating the membrane composition of specific intracellular organelles.