Showing papers by "Laboratory of Molecular Biology published in 2006"

Scaling and assessment of data quality

Abstract: The various physical factors affecting measured diffraction intensities are discussed, as are the scaling models which may be used to put the data on a consistent scale. After scaling, the intensities can be analysed to set the real resolution of the data set, to detect bad regions (e.g. bad images), to analyse radiation damage and to assess the overall quality of the data set. The significance of any anomalous signal may be assessed by probability and correlation analysis. The algorithms used by the CCP4 scaling program SCALA are described. A requirement for the scaling and merging of intensities is knowledge of the Laue group and point-group symmetries: the possible symmetry of the diffraction pattern may be determined from scores such as correlation coefficients between observations which might be symmetry-related. These scoring functions are implemented in a new program POINTLESS.

A Century of Alzheimer's Disease

Abstract: One hundred years ago a small group of psychiatrists described the abnormal protein deposits in the brain that define the most common neurodegenerative diseases Over the past 25 years, it has become clear that the proteins forming the deposits are central to the disease process Amyloid-β and tau make up the plaques and tangles of Alzheimer's disease, where these normally soluble proteins assemble into amyloid-like filaments Tau inclusions are also found in a number of related disorders Genetic studies have shown that dysfunction of amyloid-β or tau is sufficient to cause dementia The ongoing molecular dissection of the neurodegenerative pathways is expected to lead to a true understanding of disease pathogenesis

Structure of the 70S Ribosome Complexed with mRNA and tRNA

Abstract: The crystal structure of the bacterial 70S ribosome refined to 2.8 angstrom resolution reveals atomic details of its interactions with messenger RNA (mRNA) and transfer RNA (tRNA). A metal ion stabilizes a kink in the mRNA that demarcates the boundary between A and P sites, which is potentially important to prevent slippage of mRNA. Metal ions also stabilize the intersubunit interface. The interactions of E-site tRNA with the 50S subunit have both similarities and differences compared to those in the archaeal ribosome. The structure also rationalizes much biochemical and genetic data on translation.

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology

Abstract: Background The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database.

Chronic inflammation: importance of NOD2 and NALP3 in interleukin-1beta generation

Abstract: Inflammation is part of the non-specific immune response that occurs in reaction to any type of bodily injury. In some disorders, the inflammatory process - which under normal conditions is self-limiting - becomes continuous and chronic inflammatory diseases might develop subsequently. Pattern recognition molecules (PRMs) represent a diverse collection of molecules responsible for sensing danger signals, and together with other immune components they are involved in the first line of defence. NALP3 and NOD2, which belong to a cytosolic subgroup of PRMs, dubbed Nod-like-receptors (NLRs), have been associated recently with inflammatory diseases, specifically Crohn's disease and Blau syndrome (NOD2) and familial cold autoinflammatory syndrome, Muckle-Wells syndrome and chronic infantile neurological cutaneous and articular syndrome (NALP3). The exact effects of the defective proteins are not fully understood, but activation of nuclear factor (NF)-kappaB, transcription, production and secretion of interleukin (IL)-1beta and activation of the inflammasome are some of the processes that might hold clues, and the present review will provide a thorough update in this area.

Notch signalling regulates stem cell numbers in vitro and in vivo.

Abstract: One barrier to using stem cells for therapies is the difficulty in controlling their growth and differentiation. Androutsellis-Theotokis et al. show that the Notch receptor plays a pivotal role in a pathway that controls the survival of embryonic, fetal and adult stem cells, and that administration of Notch ligands to the brains of adult rats can help the animals recover movement after a simulated stroke. The finding paves the way towards therapies that aim to encourage the body's endogenous stem cells to repair damage, rather than trying to grow and transplant new cells. The Notch receptor plays a pivotal role in a pathway that controls the survival of embryonic, fetal and adult stem cells, as it activates the phosphatidylinositol-3-OH kinase/Akt signalling pathway and identifies a key serine on STAT3. The hope of developing new transplantation therapies for degenerative diseases is limited by inefficient stem cell growth and immunological incompatibility with the host1,2. Here we show that Notch receptor activation induces the expression of the specific target genes hairy and enhancer of split 3 (Hes3) and Sonic hedgehog (Shh) through rapid activation of cytoplasmic signals, including the serine/threonine kinase Akt, the transcription factor STAT3 and mammalian target of rapamycin, and thereby promotes the survival of neural stem cells. In both murine somatic and human embryonic stem cells, these positive signals are opposed by a control mechanism that involves the p38 mitogen-activated protein kinase. Transient administration of Notch ligands to the brain of adult rats increases the numbers of newly generated precursor cells and improves motor skills after ischaemic injury. These data indicate that stem cell expansion in vitro and in vivo, two central goals of regenerative medicine, may be achieved by Notch ligands through a pathway that is fundamental to development and cancer3,4,5.

P2X receptors as cell-surface ATP sensors in health and disease

Abstract: P2X receptors are membrane ion channels activated by the binding of extracellular adenosine triphosphate (ATP). For years their functional significance was consigned to distant regions of the autonomic nervous system, but recent work indicates several further key roles, such as afferent signalling, chronic pain, and in autocrine loops of endothelial and epithelial cells. P2X receptors have a molecular architecture distinct from other ion channel protein families, and have several unique functional properties.

Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi's sarcoma

Abstract: Emerging evidence suggests that both human stem cells and mature stromal cells can play an important role in the development and growth of human malignancies. In contrast to these tumor-promoting properties, we observed that in an in vivo model of Kaposi's sarcoma (KS), intravenously (i.v.) injected human mesenchymal stem cells (MSCs) home to sites of tumorigenesis and potently inhibit tumor growth. We further show that human MSCs can inhibit the in vitro activation of the Akt protein kinase within some but not all tumor and primary cell lines. The inhibition of Akt activity requires the MSCs to make direct cell–cell contact and can be inhibited by a neutralizing antibody against E-cadherin. We further demonstrate that in vivo, Akt activation within KS cells is potently down-regulated in areas adjacent to MSC infiltration. Finally, the in vivo tumor-suppressive effects of MSCs correlates with their ability to inhibit target cell Akt activity, and KS tumors engineered to express a constitutively activated Akt construct are no longer sensitive to i.v. MSC administration. These results suggest that in contrast to other stem cells or normal stromal cells, MSCs possess intrinsic antineoplastic properties and that this stem cell population might be of particular utility for treating those human malignancies characterized by dysregulated Akt.

Integration of macromolecular diffraction data

Abstract: In this chapter the integration of macromolecular diffraction data from two-dimensional area detectors is described. Data integration refers to the process of obtaining estimates of diffracted intensities (and their standard deviations) from the raw images recorded by an X-ray detector. When collecting data, a decision has to be taken about the magnitude of the angular rotation of the crystal during the recording of each image: the rotation per image can be comparable to, or greater than, the angular reflection range of a typical reflection (coarse ϕ slicing), or it can be much less than the reflection width (fine ϕ slicing). The latter approach allows the use of three-dimensional profile fitting and, providing that the detector is relatively noise-free, improves the quality of the resulting data by minimizing the contribution of the X-ray background to the total measured intensity. Methods of integration are described and integration by simple summation and by profile fitting is discussed. Keywords: background; data integration; detector overloads; errors; integration of diffraction data; outliers; overloads; partially recorded reflections; profile fitting; standard profiles; summation integration

Identification of an interleukin (IL)-25–dependent cell population that provides IL-4, IL-5, and IL-13 at the onset of helminth expulsion

Abstract: Type 2 immunity, which involves coordinated regulation of innate and adaptive immune responses, can protect against helminth parasite infection, but may lead to allergy and asthma after inappropriate activation. We demonstrate that il25 −/− mice display inefficient Nippostrongylus brasiliensis expulsion and delayed cytokine production by T helper 2 cells. We further establish a key role for interleukin (IL)-25 in regulating a novel population of IL-4–, IL-5–, IL-13–producing non–B/non–T (NBNT), c-kit + , FceR1 − cells during helminth infection. A deficit in this population in il25 −/− mice correlates with inefficient N. brasiliensis expulsion. In contrast, administration of recombinant IL-25 in vivo induces the appearance of NBNT, c-kit + , FceR1 − cells and leads to rapid worm expulsion that is T and B cell independent, but type 2 cytokine dependent. We demonstrate that these IL-25–regulated cells appear rapidly in the draining lymph nodes, implicating them as a source of type 2 cytokines during initiation of worm expulsion.

Periostin: A novel component of subepithelial fibrosis of bronchial asthma downstream of IL-4 and IL-13 signals

Abstract: Background Subepithelial fibrosis is a cardinal feature of bronchial asthma. Collagen I, III, and V; fibronectin; and tenascin-C are deposited in the lamina reticularis. Extensive evidence supports the pivotal role of IL-4 and IL-13 in subepithelial fibrosis; however, the precise mechanism remains unclear. We have previously identified the POSTN gene encoding periostin as an IL-4/IL-13–inducible gene in bronchial epithelial cells. Periostin is thought to be an adhesion molecule because it possesses 4 fasciclin I domains. Objective We explore the possibility that periostin is involved in subepithelial fibrosis in bronchial asthma. Methods We analyzed induction of periostin in lung fibroblasts by IL-4 or IL-13. We next analyzed expression of periostin in patients with asthma and in ovalbumin-sensitized and ovalbumin-inhaled mice. Furthermore, we examined the binding ability of periostin to other extracellular matrix proteins. Results Both IL-4 and IL-13 induced secretion of periostin in lung fibroblasts independently of TGF-β. Periostin colocalized with other extracellular matrix proteins involved in subepithelial fibrosis in both asthma patients and ovalbumin-sensitized and ovalbumin-inhaled wild-type mice, but not in either IL-4 or IL-13 knockout mice. Periostin had an ability to bind to fibronectin, tenascin-C, collagen V, and periostin itself. Conclusion Periostin secreted by lung fibroblasts in response to IL-4 and/or IL-13 is a novel component of subepithelial fibrosis in bronchial asthma. Periostin may contribute to this process by binding to other extracellular matrix proteins. Clinical implications Periostin induced by IL-4/IL-13 shows promise in inhibiting subepithelial fibrosis in bronchial asthma.

Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells

Abstract: Previous studies provide evidence for an endocytic mechanism in mammalian cells that is distinct from both clathrin-coated pits and caveolae1,2,3,4,5, and is not inhibited by overexpression of GTPase-null dynamin mutants1,2,3,4,6. This mechanism, however, has been defined largely in these negative terms. We applied a ferro-fluid-based purification of endosomes to identify endosomal proteins. One of the proteins identified in this way was flotillin-1 (also called reggie-2)7,8. Here, we show that flotillin-1 resides in punctate structures within the plasma membrane and in a specific population of endocytic intermediates. These intermediates accumulate both glycosylphosphatidylinositol (GPI)-linked proteins and cholera toxin B subunit4,9. Endocytosis in flotillin-1-containing intermediates is clathrin-independent. Total internal reflection microscopy and immuno-electron microscopy revealed that flotillin-1-containing regions of the plasma membrane seem to bud into the cell, and are distinct from clathrin-coated pits and caveolin-1-positive caveolae10. Flotillin-1 small interfering RNA (siRNA) inhibited both clathrin-independent uptake of cholera toxin and endocytosis of a GPI-linked protein. We propose that flotillin-1 is one determinant of a clathrin-independent endocytic pathway in mammalian cells.

Mother's little helpers: mechanisms of maternal-fetal tolerance

Abstract: The evolutionary adaptation in mammals that allows implantation of their embryos in the mother's womb creates an immunological problem. Although it ensures optimal nourishment and protection of the fetus throughout its early development, intimate contact with the mother's uterine tissue makes the fetus a potential target for her immune system. As half the fetal genes are derived from the father, the developing embryo and placenta must be considered a 'semi-allograft'. Such a mismatched organ transplant would be readily rejected without powerful immune suppression. During pregnancy, however, the semi-allogeneic fetus is protected from assault by the maternal immune system over an extended period of time. The mother's immune system seems to recognize the fetus as 'temporary self'. How this feat is managed is key to understanding immunological tolerance and intervention in treating disease.

Mechanism of endophilin N-BAR domain-mediated membrane curvature.

Abstract: Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain and an N-terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N-BAR domain) to promote dimerisation and membrane curvature generation. By electron paramagnetic resonance spectroscopy, we show that this amphipathic helix is peripherally bound in the plane of the membrane, with the midpoint of insertion aligned with the phosphate level of headgroups. This places the helix in an optimal position to effect membrane curvature generation. We solved the crystal structure of rat endophilin-A1 BAR domain and examined a distinctive insert protruding from the membrane interaction face. This insert is predicted to form an additional amphipathic helix and is important for curvature generation. Its presence defines an endophilin/nadrin subclass of BAR domains. We propose that N-BAR domains function as low-affinity dimers regulating binding partner recruitment to areas of high membrane curvature.

EM measurements define the dimensions of the "30-nm" chromatin fiber: evidence for a compact, interdigitated structure.

Abstract: Chromatin structure plays a fundamental role in the regulation of nuclear processes such as DNA transcription, replication, recombination, and repair. Despite considerable efforts during three decades, the structure of the 30-nm chromatin fiber remains controversial. To define fiber dimensions accurately, we have produced very long and regularly folded 30-nm fibers from in vitro reconstituted nucleosome arrays containing the linker histone and with increasing nucleosome repeat lengths (10 to 70 bp of linker DNA). EM measurements show that the dimensions of these fully folded fibers do not increase linearly with increasing linker length, a finding that is inconsistent with two-start helix models. Instead, we find that there are two distinct classes of fiber structure, both with unexpectedly high nucleosome density: arrays with 10 to 40 bp of linker DNA all produce fibers with a diameter of 33 nm and 11 nucleosomes per 11 nm, whereas arrays with 50 to 70 bp of linker DNA all produce 44-nm-wide fibers with 15 nucleosomes per 11 nm. Using the physical constraints imposed by these measurements, we have built a model in which tight nucleosome packing is achieved through the interdigitation of nucleosomes from adjacent helical gyres. Importantly, the model closely matches raw image projections of folded chromatin arrays recorded in the solution state by using electron cryo-microscopy.

Amplification of complex gene libraries by emulsion PCR

Abstract: 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK. 2Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel. 3Institut de Science et d’Ingénierie Supramoléculaires (ISIS), 8 allée Gaspard Monge, BP 70028, 67083 Strasbourg Cedex, France. 4Casali Institute of Applied Chemistry, The Hebrew University of Jerusalem, Givat Ram, 91904, Jerusalem, Israel. Correspondence should be addressed to A.D.G. (griffiths@isis.u-strasbg.fr) or D.S.T. (tawfik@weizmann.ac.il).

Immunotoxin Therapy of Cancer

Abstract: Rationally designed anticancer agents that target cell-surface antigens or receptors represent a promising approach for treating cancer patients. However, antibodies that bind these targets are often, by themselves, non-cytotoxic. By attaching potent toxins we can dramatically improve the clinical utility of some anti-tumour antibodies. Here we describe the construction and clinical utility of several recombinant immunotoxins; each of which is composed of antibody Fv fragments fused to powerful bacterial toxins. Results from clinical trials indicate that recombinant immunotoxins and similar agents that are designed to combine antibody selectivity with toxin cell-killing potency will be useful additions to cancer therapy.

The α-Synucleinopathies: Parkinson's Disease, Dementia with Lewy Bodies, and Multiple System Atrophy

Abstract: Parkinson's disease is the second most common neurodegenerative disease, after Alzheimer's disease. Neuropathologically, it is characterized by the degeneration of populations of nerve cells that develop filamentous inclusions in the form of Lewy bodies and Lewy neurites. Recent work has shown that the filamentous inclusions of Parkinson's disease are made of the protein alpha-synuclein and that rare, familial forms of Parkinson's disease are caused by missense mutations in the alpha-synuclein gene. Besides Parkinson's disease, the filamentous inclusions of two additional neurodegenerative diseases, namely, dementia with Lewy bodies and multiple system atrophy, have also been found to be made of alpha-synuclein. Recombinant alpha-synuclein has been shown to assemble into filaments with similar morphologies to those found in the human diseases and with a cross-beta fiber diffraction pattern. The new work has established the alpha-synucleinopathies as a major class of neurodegenerative disease.

Sleep in Drosophila is regulated by adult mushroom bodies

Abstract: Sleep is one of the few major whole-organ phenomena for which no function and no underlying mechanism have been conclusively demonstrated. Sleep could result from global changes in the brain during wakefulness or it could be regulated by specific loci that recruit the rest of the brain into the electrical and metabolic states characteristic of sleep. Here we address this issue by exploiting the genetic tractability of the fruitfly, Drosophila melanogaster, which exhibits the hallmarks of vertebrate sleep. We show that large changes in sleep are achieved by spatial and temporal enhancement of cyclic-AMP-dependent protein kinase (PKA) activity specifically in the adult mushroom bodies of Drosophila. Other manipulations of the mushroom bodies, such as electrical silencing, increasing excitation or ablation, also alter sleep. These results link sleep regulation to an anatomical locus known to be involved in learning and memory.

TRPM2 activation by cyclic ADP‐ribose at body temperature is involved in insulin secretion

Abstract: There are eight thermosensitive TRP (transient receptor potential) channels in mammals, and there might be other TRP channels sensitive to temperature stimuli. Here, we demonstrate that TRPM2 can be activated by exposure to warm temperatures (>35°C) apparently via direct heat-evoked channel gating. β-NAD+- or ADP-ribose-evoked TRPM2 activity is robustly potentiated at elevated temperatures. We also show that, even though cyclic ADP-ribose (cADPR) does not activate TRPM2 at 25°C, co-application of heat and intracellular cADPR dramatically potentiates TRPM2 activity. Heat and cADPR evoke similar responses in rat insulinoma RIN-5F cells, which express TRPM2 endogenously. In pancreatic islets, TRPM2 is coexpressed with insulin, and mild heating of these cells evokes increases in both cytosolic Ca2+ and insulin release, which is KATP channel-independent and protein kinase A-mediated. Heat-evoked responses in both RIN-5F cells and pancreatic islets are significantly diminished by treatment with TRPM2-specific siRNA. These results identify TRPM2 as a potential molecular target for cADPR, and suggest that TRPM2 regulates Ca2+ entry into pancreatic β-cells at body temperature depending on the production of cADPR-related molecules, thereby regulating insulin secretion.

TRPC3 and TRPC6 are essential for angiotensin II-induced cardiac hypertrophy.

Abstract: Angiotensin (Ang) II participates in the pathogenesis of heart failure through induction of cardiac hypertrophy. Ang II-induced hypertrophic growth of cardiomyocytes is mediated by nuclear factor of activated T cells (NFAT), a Ca2+-responsive transcriptional factor. It is believed that phospholipase C (PLC)-mediated production of inositol-1,4,5-trisphosphate (IP3) is responsible for Ca2+ increase that is necessary for NFAT activation. However, we demonstrate that PLC-mediated production of diacylglycerol (DAG) but not IP3 is essential for Ang II-induced NFAT activation in rat cardiac myocytes. NFAT activation and hypertrophic responses by Ang II stimulation required the enhanced frequency of Ca2+ oscillation triggered by membrane depolarization through activation of DAG-sensitive TRPC channels, which leads to activation of L-type Ca2+ channel. Patch clamp recordings from single myocytes revealed that Ang II activated DAG-sensitive TRPC-like currents. Among DAG-activating TRPC channels (TRPC3, TRPC6, and TRPC7), the activities of TRPC3 and TRPC6 channels correlated with Ang II-induced NFAT activation and hypertrophic responses. These data suggest that DAG-induced Ca2+ signaling pathway through TRPC3 and TRPC6 is essential for Ang II-induced NFAT activation and cardiac hypertrophy.