Showing papers by "Howard Hughes Medical Institute published in 2003"
TL;DR: This issue of Pharmacological Reviews includes a new venture in the collaboration between the International Union of Pharmacology (IUPHAR) and the American Society for Pharmacology and Experimental Therapeutics (ASPET), in that a new classification of voltage-gated ion channels is outlined.
Abstract: This issue of Pharmacological Reviews includes a new venture in the collaboration between the International Union of Pharmacology (IUPHAR) and the American Society for Pharmacology and Experimental Therapeutics (ASPET), in that a new classification of voltage-gated ion channels is outlined in this
7,389 citations
TL;DR: It is reported that the forkhead transcription factor Foxp3 is specifically expressed in CD4+CD25+ regulatory T cells and is required for their development and function and ectopic expression ofFoxp3 confers suppressor function on peripheral CD4-CD25− T cells.
Abstract: CD4+CD25+ regulatory T cells are essential for the active suppression of autoimmunity. Here we report that the forkhead transcription factor Foxp3 is specifically expressed in CD4+CD25+ regulatory T cells and is required for their development. The lethal autoimmune syndrome observed in Foxp3-mutant scurfy mice and Foxp3-null mice results from a CD4+CD25+ regulatory T cell deficiency and not from a cell-intrinsic defect of CD4+CD25- T cells. CD4+CD25+ regulatory T cells rescue disease development and preferentially expand when transferred into neonatal Foxp3-deficient mice. Furthermore, ectopic expression of Foxp3 confers suppressor function on peripheral CD4+CD25- T cells. Thus, Foxp3 is a critical regulator of CD4+CD25+ regulatory T cell development and function.
7,321 citations
TL;DR: It is demonstrated that human pre-miRNA nuclear export, and miRNA function, are dependent on Exportin-5, an additional cellular cofactor required for miRNA biogenesis and function.
Abstract: MicroRNAs (miRNAs) are initially expressed as long transcripts that are processed in the nucleus to yield approximately 65-nucleotide (nt) RNA hairpin intermediates, termed pre-miRNAs, that are exported to the cytoplasm for additional processing to yield mature, approximately 22-nt miRNAs. Here, we demonstrate that human pre-miRNA nuclear export, and miRNA function, are dependent on Exportin-5. Exportin-5 can bind pre-miRNAs specifically in vitro, but only in the presence of the Ran-GTP cofactor. Short hairpin RNAs, artificial pre-miRNA analogs used to express small interfering RNAs, also depend on Exportin-5 for nuclear export. Together, these findings define an additional cellular cofactor required for miRNA biogenesis and function.
3,049 citations
TL;DR: Using crystal structures, a detailed catalytic cycle has been derived for typical 2-Cys Prxs, including a model for the redox-regulated oligomeric state proposed to control enzyme activity.
2,388 citations
TL;DR: It is demonstrated that beclin 1 is a critical component of mammalian autophagy and a role forAutophagy in tumor suppression is established, and mutations in other genes operating in this pathway may contribute to tumor formation through deregulation of Autophagy.
Abstract: The biochemical properties of beclin 1 suggest a role in two fundamentally important cell biological pathways: autophagy and apoptosis. We show here that beclin 1-/- mutant mice die early in embryogenesis and beclin 1+/- mutant mice suffer from a high incidence of spontaneous tumors. These tumors continue to express wild-type beclin 1 mRNA and protein, establishing that beclin 1 is a haploinsufficient tumor suppressor gene. Beclin 1-/- embryonic stem cells have a severely altered autophagic response, whereas their apoptotic response to serum withdrawal or UV light is normal. These results demonstrate that beclin 1 is a critical component of mammalian autophagy and establish a role for autophagy in tumor suppression. They both provide a biological explanation for recent evidence implicating beclin 1 in human cancer and suggest that mutations in other genes operating in this pathway may contribute to tumor formation through deregulation of autophagy.
2,017 citations
TL;DR: A large-scale screen is described to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways.
Abstract: The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community.
1,989 citations
TL;DR: A role for Dicer, and, by implication, the RNAi machinery, in maintaining the stem cell population during early mouse development is suggested.
Abstract: To address the biological function of RNA interference (RNAi)-related pathways in mammals, we disrupted the gene Dicer1 in mice. Loss of Dicer1 lead to lethality early in development, with Dicer1-null embryos depleted of stem cells. Coupled with our inability to generate viable Dicer1-null embryonic stem (ES) cells, this suggests a role for Dicer, and, by implication, the RNAi machinery, in maintaining the stem cell population during early mouse development.
1,966 citations
TL;DR: The prevalence of self-reactive antibody formation and its regulation in human B cells is determined and a majority (55 to 75%) of all antibodies expressed by early immature B cells displayedSelf-reactivity, including polyreactive and anti-nuclear specificities.
Abstract: During B lymphocyte development, antibodies are assembled by random gene segment reassortment to produce a vast number of specificities. A potential disadvantage of this process is that some of the antibodies produced are self-reactive. We determined the prevalence of self-reactive antibody formation and its regulation in human B cells. A majority (55 to 75%) of all antibodies expressed by early immature B cells displayed self-reactivity, including polyreactive and anti-nuclear specificities. Most of these autoantibodies were removed from the population at two discrete checkpoints during B cell development. Inefficient checkpoint regulation would lead to substantial increases in circulating autoantibodies.
1,863 citations
TL;DR: The structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix, is presented and a crystal structure of the full-length channel at a resolution of 3.2 Å is determined, which suggests that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.
Abstract: Voltage-dependent K+ channels are members of the family of voltage-dependent cation (K+, Na+ and Ca2+) channels that open and allow ion conduction in response to changes in cell membrane voltage. This form of gating underlies the generation of nerve and muscle action potentials, among other processes. Here we present the structure of KvAP, a voltage-dependent K+ channel from Aeropyrum pernix. We have determined a crystal structure of the full-length channel at a resolution of 3.2 A, and of the isolated voltage-sensor domain at 1.9 A, both in complex with monoclonal Fab fragments. The channel contains a central ion-conduction pore surrounded by voltage sensors, which form what we call 'voltage-sensor paddles'-hydrophobic, cationic, helix-turn-helix structures on the channel's outer perimeter. Flexible hinges suggest that the voltage-sensor paddles move in response to membrane voltage changes, carrying their positive charge across the membrane.
1,758 citations
TL;DR: It seems that some tumours arise from small populations of 'cancer stem cells' that give rise to phenotypically diverse cancer cells, with less proliferative potential.
Abstract: Why are tumours heterogeneous, in terms of cell phenotype and proliferative potential, even in cases in which all cells are derived from a single clone? Ongoing mutagenesis can partially explain this heterogeneity, but it also seems that some tumours arise from small populations of 'cancer stem cells' that give rise to phenotypically diverse cancer cells, with less proliferative potential. These cancer stem cells are likely to arise from mutations that dysregulate normal stem-cell self-renewal. Using this information, it might be possible to devise more effective therapies.
1,603 citations
TL;DR: Two computer programs are presented, CTFFIND3 and CTFTILT, which determine defocus parameters from images of untilted specimens, as well as defocus and tilt parameters from image of tilted specimens, respectively, using a simple algorithm.
TL;DR: Genetic analysis shows that Wim, Polaris and the IFT motor protein Kif3a are required for Hedgehog signalling at a step downstream of Patched1 (the Hedgehog receptor) and upstream of direct targets of hedgehog signalling.
Abstract: Intraflagellar transport (IFT) proteins were first identified as essential factors for the growth and maintenance of flagella in the single-celled alga Chlamydomonas reinhardtii. In a screen for embryonic patterning mutations induced by ethylnitrosourea, here we identify two mouse mutants, wimple (wim) and flexo (fxo), that lack ventral neural cell types and show other phenotypes characteristic of defects in Sonic hedgehog signalling. Both mutations disrupt IFT proteins: the wim mutation is an allele of the previously uncharacterized mouse homologue of IFT172; and fxo is a new hypomorphic allele of polaris, the mouse homologue of IFT88. Genetic analysis shows that Wim, Polaris and the IFT motor protein Kif3a are required for Hedgehog signalling at a step downstream of Patched1 (the Hedgehog receptor) and upstream of direct targets of Hedgehog signalling. Our data show that IFT machinery has an essential and vertebrate-specific role in Hedgehog signal transduction.
TL;DR: The flux of mRNAs between polysomes and P bodies is defined as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.
Abstract: A major pathway of eukaryotic messenger RNA (mRNA) turnover begins with deadenylation, followed by decapping and 5' to 3' exonucleolytic decay. We provide evidence that mRNA decapping and 5' to 3' degradation occur in discrete cytoplasmic foci in yeast, which we call processing bodies (P bodies). First, proteins that activate or catalyze decapping are concentrated in P bodies. Second, inhibiting mRNA turnover before decapping leads to loss of P bodies; however, inhibiting turnover at, or after, decapping, increases the abundance and size of P bodies. Finally, mRNA degradation intermediates are localized to P bodies. These results define the flux of mRNAs between polysomes and P bodies as a critical aspect of cytoplasmic mRNA metabolism and a possible site for regulation of mRNA degradation.
TL;DR: It is shown here that targeted activation of PPARδ in adipose tissue specifically induces expression of genes required for fatty acid oxidation and energy dissipation, which in turn leads to improved lipid profiles and reduced adiposity.
TL;DR: This work describes a simple, software-based approach to operating a laser scanning microscope without the need for custom data acquisition hardware and quantifies the effectiveness of the data acquisition and signal conditioning algorithm under a variety of conditions.
Abstract: Background: Laser scanning microscopy is a powerful tool for analyzing the structure and function of biological specimens. Although numerous commercial laser scanning microscopes exist, some of the more interesting and challenging applications demand custom design. A major impediment to custom design is the difficulty of building custom data acquisition hardware and writing the complex software required to run the laser scanning microscope. Results: We describe a simple, software-based approach to operating a laser scanning microscope without the need for custom data acquisition hardware. Data acquisition and control of laser scanning are achieved through standard data acquisition boards. The entire burden of signal integration and image processing is placed on the CPU of the computer. We quantitate the effectiveness of our data acquisition and signal conditioning algorithm under a variety of conditions. We implement our approach in an open source software package (ScanImage) and describe its functionality. Conclusions: We present ScanImage, software to run a flexible laser scanning microscope that allows easy custom design.
TL;DR: This work aims to provide a systematic literature review and meta-analyses of the determinants of breast cancer recurrence and prognosis in mice using a simple, scalable, and scalable approach.
TL;DR: This approach is called BEAMing on the basis of four of its principal components (beads, emulsion, amplification, and magnetics) and can be used for the identification of rare mutations as well as to study variations in gene sequences or transcripts in specific populations or tissues.
Abstract: Many areas of biomedical research depend on the analysis of uncommon variations in individual genes or transcripts. Here we describe a method that can quantify such variation at a scale and ease heretofore unattainable. Each DNA molecule in a collection of such molecules is converted into a single magnetic particle to which thousands of copies of DNA identical in sequence to the original are bound. This population of beads then corresponds to a one-to-one representation of the starting DNA molecules. Variation within the original population of DNA molecules can then be simply assessed by counting fluorescently labeled particles via flow cytometry. This approach is called BEAMing on the basis of four of its principal components (beads, emulsion, amplification, and magnetics). Millions of individual DNA molecules can be assessed in this fashion with standard laboratory equipment. Moreover, specific variants can be isolated by flow sorting and used for further experimentation. BEAMing can be used for the identification and quantification of rare mutations as well as to study variations in gene sequences or transcripts in specific populations or tissues.
TL;DR: It is shown that therapeutic concentrations of lithium, a GSK-3 inhibitor, block the production of Aβ peptides by interfering with APP cleavage at the γ-secretase step, but do not inhibit Notch processing.
Abstract: Alzheimer's disease is associated with increased production and aggregation of amyloid-β (Aβ) peptides1. Aβ peptides are derived from the amyloid precursor protein (APP) by sequential proteolysis, catalysed by the aspartyl protease BACE2, followed by presenilin-dependent γ-secretase cleavage3. Presenilin interacts with nicastrin4,5, APH-1 and PEN-2 (ref. 6), all of which are required for γ-secretase function. Presenilins also interact with α-catenin, β-catenin7,8 and glycogen synthase kinase-3β (GSK-3β)9,10,11, but a functional role for these proteins in γ-secretase activity has not been established. Here we show that therapeutic concentrations of lithium, a GSK-3 inhibitor12, block the production of Aβ peptides by interfering with APP cleavage at the γ-secretase step, but do not inhibit Notch processing. Importantly, lithium also blocks the accumulation of Aβ peptides in the brains of mice that overproduce APP. The target of lithium in this setting is GSK-3α, which is required for maximal processing of APP. Since GSK-3 also phosphorylates tau protein, the principal component of neurofibrillary tangles, inhibition of GSK-3α offers a new approach to reduce the formation of both amyloid plaques and neurofibrillary tangles, two pathological hallmarks of Alzheimer's disease.
TL;DR: Nonneuronal cells that do not express mutant SOD1 delay degeneration and significantly extend survival of mutant-expressing motor neurons.
Abstract: The most common inherited [correct] form of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting adult motor neurons, is caused by dominant mutations in the ubiquitously expressed Cu-Zn superoxide dismutase (SOD1) In chimeric mice that are mixtures of normal and SOD1 mutant-expressing cells, toxicity to motor neurons is shown to require damage from mutant SOD1 acting within nonneuronal cells Normal motor neurons in SOD1 mutant chimeras develop aspects of ALS pathology Most important, nonneuronal cells that do not express mutant SOD1 delay degeneration and significantly extend survival of mutant-expressing motor neurons
TL;DR: CLIP reveals that Nova coordinately regulates a biologically coherent set of RNAs encoding multiple components of the inhibitory synapse, an observation that may relate to the cause of abnormal motor inhibition in POMA.
Abstract: Nova proteins are neuron-specific antigens targeted in paraneoplastic opsoclonus myoclonus ataxia (POMA), an autoimmune neurologic disease characterized by abnormal motor inhibition Nova proteins regulate neuronal pre-messenger RNA splicing by directly binding to RNA To identify Nova RNA targets, we developed a method to purify protein-RNA complexes from mouse brain with the use of ultraviolet cross-linking and immunoprecipitation (CLIP)Thirty-four transcripts were identified multiple times by Nova CLIPThree-quarters of these encode proteins that function at the neuronal synapse, and one-third are involved in neuronal inhibitionSplicing targets confirmed in Nova-/- mice include c-Jun N-terminal kinase 2, neogenin, and gephyrin; the latter encodes a protein that clusters inhibitory gamma-aminobutyric acid and glycine receptors, two previously identified Nova splicing targetsThus, CLIP reveals that Nova coordinately regulates a biologically coherent set of RNAs encoding multiple components of the inhibitory synapse, an observation that may relate to the cause of abnormal motor inhibition in POMA
TL;DR: A new method to predict protein-protein complexes from the coordinates of the unbound monomer components using a low-resolution, rigid-body, Monte Carlo search followed by simultaneous optimization of backbone displacement and side-chain conformations using Monte Carlo minimization is presented.
TL;DR: The number of new proteins emerging with no prior connection to splicing was surprising and it would be premature to label these proteins as bona fide splicing factors, yet many were identified multiple times in complexes purified under diverse conditions or from different organisms.
TL;DR: A crystal structure of the soluble ectodomain of E from dengue virus type 2 reveals a hydrophobic pocket lined by residues that influence the pH threshold for fusion, which points to a structural pathway for the fusion-activating transition and suggests a strategy for finding small-molecule inhibitors of d Dengue and other flaviviruses.
Abstract: Dengue virus is an emerging global health threat. Its major envelope glycoprotein, E, mediates viral attachment and entry by membrane fusion. A crystal structure of the soluble ectodomain of E from dengue virus type 2 reveals a hydrophobic pocket lined by residues that influence the pH threshold for fusion. The pocket, which accepts a hydrophobic ligand, opens and closes through a conformational shift in a β-hairpin at the interface between two domains. These features point to a structural pathway for the fusion-activating transition and suggest a strategy for finding small-molecule inhibitors of dengue and other flaviviruses.
TL;DR: It is demonstrated that an endogenously encoded human miRNA is able to cleave an mRNA bearing fully complementary target sites, whereas an exogenously supplied siRNA can inhibit the expression of an RNA bearing partially complementary sequences without inducing detectable RNA cleavage.
Abstract: MicroRNAs (miRNAs) are endogenously encoded small noncoding RNAs, derived by processing of short RNA hairpins, that can inhibit the translation of mRNAs bearing partially complementary target sequences. In contrast, small interfering RNAs (siRNAs), which are derived by processing of long double-stranded RNAs and are often of exogenous origin, degrade mRNAs bearing fully complementary sequences. Here, we demonstrate that an endogenously encoded human miRNA is able to cleave an mRNA bearing fully complementary target sites, whereas an exogenously supplied siRNA can inhibit the expression of an mRNA bearing partially complementary sequences without inducing detectable RNA cleavage. These data suggest that miRNAs and siRNAs can use similar mechanisms to repress mRNA expression and that the choice of mechanism may be largely or entirely determined by the degree of complementary of the RNA target.
TL;DR: The results indicate that circulating activated platelets and platelet–leukocyte/monocyte aggregates promote formation of atherosclerotic lesions.
Abstract: We studied whether circulating activated platelets and platelet-leukocyte aggregates cause the development of atherosclerotic lesions in apolipoprotein-E-deficient (Apoe(-/-)) mice. Circulating activated platelets bound to leukocytes, preferentially monocytes, to form platelet-monocyte/leukocyte aggregates. Activated platelets and platelet-leukocyte aggregates interacted with atherosclerotic lesions. The interactions of activated platelets with monocytes and atherosclerotic arteries led to delivery of the platelet-derived chemokines CCL5 (regulated on activation, normal T cell expressed and secreted, RANTES) and CXCL4 (platelet factor 4) to the monocyte surface and endothelium of atherosclerotic arteries. The presence of activated platelets promoted leukocyte binding of vascular cell adhesion molecule-1 (VCAM-1) and increased their adhesiveness to inflamed or atherosclerotic endothelium. Injection of activated wild-type, but not P-selectin-deficient, platelets increased monocyte arrest on the surface of atherosclerotic lesions and the size of atherosclerotic lesions in Apoe(-/-) mice. Our results indicate that circulating activated platelets and platelet-leukocyte/monocyte aggregates promote formation of atherosclerotic lesions. This role of activated platelets in atherosclerosis is attributed to platelet P-selectin-mediated delivery of platelet-derived proinflammatory factors to monocytes/leukocytes and the vessel wall.
TL;DR: It is reported that a loss-of-function (E1425G) mutation in ankyrin-B, a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans.
Abstract: Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.
TL;DR: The classical biogenic amine neurotransmitters — dopamine, noradrenaline, and 5-hydroxytryptamine — control a variety of functions including locomotion, autonomic function, hormone secretion, and the complex behaviours that are associated with affect, emotion and reward.
Abstract: The classical biogenic amine neurotransmitters — dopamine, noradrenaline, and 5-hydroxytryptamine — control a variety of functions including locomotion, autonomic function, hormone secretion, and the complex behaviours that are associated with affect, emotion and reward. A key step that determines the intensity and duration of monoamine signalling at synapses is the reuptake of the released transmitter into nerve terminals through high-affinity plasma membrane transporters. In recent years, molecular, pharmacological and genetic approaches have established the importance of monoamine transporters in the control of monoamine homeostasis and have provided insights into their regulation.
TL;DR: The results support the idea that a vertebrate PCP pathway regulates gastrulation movements and suggest that there is overlap between the PCP and Wnt/calcium pathways.
TL;DR: Paraneoplastic neurologic syndromes are incited by a tumor outside the nervous system that produces a characteristic antigen of the nervous System that damages nerve tissue and inhibits growth of the tumor.
Abstract: Paraneoplastic neurologic syndromes are incited by a tumor outside the nervous system that produces a characteristic antigen of the nervous system. An immune response against the ectopic tumor antigen not only damages nerve tissue but also inhibits growth of the tumor.