bluuebunny/crossref_metadata_2025 huggingface dataset:
Activities of mixed NOP and μ‐opioid receptor ligands
Authors: Spagnolo B , Calo G , Polgar W E , Jiang F , Olsen C M , Berzetei‐Gurske I , Khroyan T V , Husbands S M , Lewis J W , Toll L , Zaveri N T
Published: December 2007

Background and purpose:

Compounds that activate both NOP and μ‐opioid receptors might be useful as analgesics and drug abuse medications. Studies were carried out to better understand the biological activity of such compounds.

Experimental approach:

Binding affinities were determined on membranes from cells transfected with NOP and opioid receptors. Functional activity was determined by [35S]GTPγS binding on cell membranes and using the mouse vas deferens preparationin vitroand the tail flick antinociception assayin vivo.

Key results:

Compounds ranged in affinity from SR14150, 20‐fold selective for NOP receptors, to buprenorphine, 50‐fold selective for μ‐opioid receptors. In the [35S]GTPγS assay, SR compounds ranged from full agonist to antagonist at NOP receptors and most were partial agonists at μ‐opioid receptors. Buprenorphine was a low efficacy partial agonist at μ‐opioid receptors, but did not stimulate [35S]GTPγS binding through NOP. In the mouse vas deferens, each compound, except for SR16430, inhibited electrically induced contractions. In each case, except for N/OFQ itself, the inhibition was due to μ‐opioid receptor activation, as determined by equivalent results in NOP receptor knockout tissues. SR14150 showed antinociceptive activity in the tail flick test, which was reversed by the opioid antagonist naloxone.

Conclusions and implications:

Compounds that bind to both μ‐opioid and NOP receptors have antinociceptive activity but the relative contribution of each receptor is unclear. These experiments help characterize compounds that bind to both receptors, to better understand the mechanism behind their biological activities, and identify new pharmacological tools to characterize NOP and opioid receptors.

British Journal of Pharmacology(2008)153, 609–619; doi:10.1038/sj.bjp.0707598; published online 3 December 2007
Interleukin‐1‐induced interleukin‐6 synthesis is mediated by the neutral sphingomyelinase/Src kinase pathway in neurones
Authors: Tsakiri N , Kimber I , Rothwell N J , Pinteaux E
Published: December 2007

Background and purpose:

Interleukin (IL)‐1 is a key mediator of inflammatory and host defence responses and its effects in the brain are mediated primarily via effects on glia. IL‐1 induces release of inflammatory mediators such as IL‐6 from glia via the type‐1 receptor (IL‐1R1) and established signalling mechanisms including mitogen‐activated protein kinases and nuclear factor kappa‐B. IL‐1 also modifies physiological functions via actions on neurones, through activation of the neutral sphingomyelinase (nSMase)/Src kinase signalling pathway, although the mechanism of IL‐1‐induced IL‐6 synthesis in neurones remains unknown.

Experimental approach:

Primary mouse neuronal cell cultures, ELISA, Western blot and immunocytochemistry techniques were used.

Key results:

We show here that IL‐1β induces the synthesis of IL‐6 in primary mouse neuronal cultures, and this is dependent on the activation of IL‐1R1, nSMase and Src kinase. We demonstrate that IL‐1β‐induced Src kinase activation triggers the phosphorylation of the NMDA receptor NR2B subunit, leading to activation of Ca2+/calmodulin‐dependent protein kinase II (CamKII) and the nuclear transcription factor CREB. We also show that NR2B, CamKII and CREB are essential signalling elements involved in IL‐1β‐induced IL‐6 synthesis in neurones.

Conclusions and implications:

These results demonstrate that IL‐1 interacts with the same receptors on neurones and glia to elicit IL‐6 release, but does so via distinct signalling pathways. The mechanism by which IL‐1β induces IL‐6 synthesis in neurones could be critical in both physiological and pathophysiological actions of IL‐1β, and may provide a new therapeutic target for the treatment of acute CNS injury.

British Journal of Pharmacology (2008) 153, 775–783; doi:10.1038/sj.bjp.0707610; published online 3 December 2007
Conformational changes in G‐protein‐coupled receptors—the quest for functionally selective conformations is open
Authors: Hoffmann C , Zürn A , Bünemann M , Lohse M J
Published: December 2007

The G‐protein‐coupled receptors (GPCRs) represent one the largest families of drug targets. Upon agonist binding a receptor undergoes conformational rearrangements that lead to a novel protein conformation which in turn can interact with effector proteins. During the last decade significant progress has been made to prove that different conformational changes occur. Today it is mostly accepted that individual ligands can induce different receptor conformations. However, the nature or molecular identity of the different conformations is still ill‐known. Knowledge of the potential functionally selective conformations will help to develop drugs that select specific conformations of a given GPCR which couple to specific signalling pathways and may, ultimately, lead to reduced side effects. In this review we will summarize recent progress in biophysical approaches that have led to the current understanding of conformational changes that occur during GPCR activation.

British Journal of Pharmacology (2008) 153, S358–S366; doi:10.1038/sj.bjp.0707615; published online 3 December 2007
Probing the role of AMPAR endocytosis and long‐term depression in behavioural sensitization: relevance to treatment of brain disorders, including drug addiction
Authors: Wang Y T
Published: December 2007

Modifying the function of postsynaptic α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid subtype glutamate receptors (AMPARs) is one of the most important mechanisms by which the efficacy of synaptic transmission at excitatory glutamatergic synapses in the mammalian brain is regulated. Traditionally these types of modifications have been thought to be achieved mainly by altering the channel gating properties or conductance of the receptors. A large body of evidence accumulated from recent studies strongly suggests that AMPARs, like most integral plasma membrane proteins, are continuously recycled between the plasma membrane and the intracellular compartments via vesicle‐mediated plasma membrane insertion and clathrin‐dependent endocytosis. Regulation of either receptor insertion or endocytosis results in a rapid change in the number of these receptors expressed on the plasma membrane surface and in the receptor‐mediated responses, thereby playing an important role in mediating certain forms of synaptic plasticity, such as long‐term potentiation (LTP) and depression (LTD). These studies have significantly advanced our understanding of the molecular mechanisms underlying LTP and LTD, and their potential contributions to learning and memory‐related behaviours. Here I provide a brief summary of the current state of knowledge concerning clathrin‐mediated AMPAR endocytosis and its relationship to the expression of certain forms of LTD in several brain areas. The potential impact of recent advancements on our efforts to probe the roles of synaptic plasticity in learning and memory‐related behaviours, and their relevance to some brain disorders, particularly drug addiction, are also discussed.

British Journal of Pharmacology (2008) 153, S389–S395; doi:10.1038/sj.bjp.0707616; published online 3 December 2007
Drugs and trafficking of ion channels: a new pro‐arrhythmic threat on the horizon?
Authors: van der Heyden M A G , Smits M E , Vos M A
Published: December 2007

Tuning of functional expression levels of the ion channels that make up the cardiac action potential (AP) is crucial for preserving correct AP duration (APD) and QTc times. Many compounds inhibit human ether‐à‐go‐go related gene (hERG)‐mediated delayed rectifier currents and thus prolong cardiac repolarization that may cause life‐threatening arrhythmias like Torsades de Pointes. An increasing number of drugs are found to inhibit hERG function by a dual mechanism of short‐term channel block and long‐term trafficking defects that operate over different time and concentration scales. In safety screens at present used by pharmaceutical companies, the short‐term effect of channel block is covered. In contrast, specific screening for long‐term trafficking defects is not common, with the consequent risk of drugs that disturb trafficking entering the market. Whether that poses another pro‐arrhythmic threat for the patients treated has to be determined, but is not unlikely.

British Journal of Pharmacology(2008)153, 406–409; doi:10.1038/sj.bjp.0707618; published online 3 December 2007
Interleukin 2: from immunostimulation to immunoregulation and back again
Authors: Bachmann Martin F , Oxenius Annette
Published: December 2007

Interleukin 2 (IL‐2) was one of the first cytokines to be discovered. However, the complex role of IL‐2 and its receptor in the regulation of immune responses is only now emerging. This review explores the various signals triggered by IL‐2 and discusses their translation into biological function. A model is outlined that accommodates the seemingly contradictory functions of IL‐2, and explains how one cytokine can be an essential T‐cell growth and differentiation factor and yet also be indispensable to maintain peripheral tolerance.
A variable topology for the 30‐nm chromatin fibre
Authors: Wu Chenyi , Bassett Andrew , Travers Andrew
Published: December 2007

The structure of the 30‐nm chromatin fibre is an important determinant of the regulation of eukaryotic transcription. A fundamental issue is whether the stacking of nucleosomes in this fibre is organized as a one‐start or two‐start helix. We argue that all recent experimental data are compatible with a two‐start helix and propose that the topology of the fibre, but not the mode of stacking the nucleosomes, is dependent on the length of the linker DNA. This arrangement conserves nucleosome stacking and thus the external morphology of the fibre, and also ensures that the fibre adopts the highest available packing density.
Towards completion of the Earth's proteome
Authors: Perez‐Iratxeta Carolina , Palidwor Gareth , Andrade‐Navarro Miguel A
Published: December 2007

New protein sequences are deposited in databases at an accelerating pace; however, many of these are homologous to known proteins and could be considered redundant. If all historical releases of the protein database are analysed using the original sequence‐clustering procedure described here, the fraction of newly sequenced proteins that are redundant is increasing. We interpret this as an indication that the sequencing of the Earth's proteome—the complete set of proteins on Earth—is approaching completion. We estimate the approximate size of the Earth's proteome to be 5 million sequences, most of which will be identified during the next 5 years. As the Earth's proteome nears completion, cluster analysis of the protein database will become essential to identify under‐explored taxa to which future sequencing efforts should be directed and to focus research on protein families without experimental characterization.
In vitro studies on ultrasmall superparamagnetic iron oxide nanoparticles coated with gummic acid for T2 MRI contrast agent
Authors: Rabias I. , Pratsinis H. , Drossopoulou G. , Fardis M. , Maris T. , Boukos N. , Tsotakos N. , Kletsas D. , Tsilibary E. , Papavassiliou G.
Published: December 2007

Ultrasmall superparamagnetic iron oxide nanoparticles coated with gummic acid have been investigated as possible constituents of aqueous ferrofluids for biomedical applications and especially for MRI contrast agent. The structural characteristics and the size of the nanoparticles have been analyzed as well as the magnetic properties. In order to evaluate any possible capabilities as a contrast agent, the relaxation time, T2, of hydrogen protons in the colloidal solutions of nanoparticles have been measured in order to gain information on the relaxation behavior compared to other MRI contrast agents. The in vitro cytotoxicity of the obtained magnetic nanoparticles of iron oxide coated with gummic acid was investigated by two separate methods (MTT and FACS analysis) and by using three different normal and transformed cell lines. Our results showed that the synthesized nanoparticles had no toxic effect on any of the cell lines used.
Synthesis and Preliminary Pharmacological Evaluation of 4′-Arylalkyl Analogues of Clozapine. III. Replacement of the Tricyclic Nucleus with a Bicyclic Template
Authors: Capuano Ben , Crosby Ian T. , Lloyd Edward J. , Taylor David A.
Published: December 2007

As a continuing part of our research program in search of novel compounds for the treatment of schizophrenia, we report the synthesis and preliminary receptor binding affinity for a series of bicyclic analogues of clozapine derived from a selection of promising tricyclic candidates published previously. These bicyclic compounds investigate some substituent effects and the length and nature of the linker between an ionizable nitrogen atom at physiological pH and the introduced aryl moiety. The chemistry, structural characterization, and in vitro evaluation are described. Preliminary findings on the effects on activity of the nature and length of the linker, degree of unsaturation, and selected substituents coupled to the bicyclic nucleus are discussed in relation to affinity for dopamine D4 and serotonin 5-HT2A receptors.
Fluorescent Ethenyl- and Ethynyl-dimesitylboranes Derived from 5-(Dimethylamino)-N-(prop-2-ynyl)naphthalene-1-sulfonamide
Authors: Wong Wai-Yeung , Poon Suk-Yue , Lin Mei-Fang , Wong Wai-Kwok
Published: December 2007

Two new fluorescent ethenyl- and ethynyl-dimesitylboranes functionalized with a dansyl group 1 and 2 have been synthesized in good yields. Compound 1 was prepared by the hydroboration of 5-(dimethylamino)-N-(prop-2-ynyl)naphthalene-1-sulfonamide I with dimesitylborane (HB(Mes)2) in dry tetrahydrofuran at room temperature, and compound 2 was synthesized by Pd-catalyzed cross-coupling of I with 4-I-C6H4B(Mes)2. Both organoborane compounds 1 and 2 have been characterized by infrared spectroscopy, NMR spectroscopy, and mass spectrometry. The molecular structures of I and 1 were confirmed by X-ray crystallography. The electronic absorption and redox properties of 1 and 2 were investigated. They both exhibit large positive solvatochromism and their emission spectra have been recorded in a range of organic solvents with the fluorescence maxima exhibiting large bathochromic shifts in highly polar solvents, indicative of charge transfer which leads to large dipole moments in the excited state. The application of 1 as a blue fluorescent dopant in doped guest–host organic light-emitting diodes is also described.
Chlorination of Aromatics with Trichloroisocyanuric Acid (TCICA) in Brønsted-Acidic Imidazolium Ionic Liquid [BMIM(SO3H)][OTf]: an Economical, Green Protocol for the Synthesis of Chloroarenes
Authors: Hubbard Abigail , Okazaki Takao , Laali Kenneth K.
Published: December 2007

A survey study on electrophilic chlorination of aromatics with trichloroisocyanuric acid (TCICA) in Brønsted-acidic imidazolium ionic liquid [BMIM(SO3H)][OTf] is reported. The reactions are performed under very mild conditions (at ~50°C) and give good to excellent yields, depending on the substrates. Chemoselectivity for mono- v. dichlorination can be tuned by changing the arene-to-TCICA ratio and the reaction time. The survey study and competitive experiments suggest that triprotonated/protosolvated TCICA is a selective/moderately reactive transfer-chlorination electrophile. Density functional theory was used as guide to obtain further insight into the nature of the chlorination electrophile and the transfer-chlorination step.
Salinity tolerance of riverine microinvertebrates from the southern Murray - Darling Basin
Authors: Kefford Ben J. , Fields Elizabeth J. , Clay Colin , Nugegoda Dayanthi
Published: December 2007

Concern about the effect of rising salinity on freshwater biodiversity has led to studies investigating the salt tolerance of macroinvertebrates and fish, with less attention given to microinvertebrates. We investigated the acute lethal effects of salinity on 12 microinvertebrate species from rivers in the southern Murray–Darling Basin in central Victoria, Australia. For a subset of these species, sub-lethal salinity effects and the effect of water temperature on salinity tolerance were also investigated. The most sensitive microinvertebrates had broadly similar 72-h LC50 values to the most sensitive macroinvertebrates, reported in other studies. However, the most tolerant microinvertebrates tested were much more sensitive than the most tolerant macroinvertebrates and the microinvertebrates studied were more sensitive than most freshwater fish. Temperature affected the acute lethal toxicity of salinity but only to a small degree. In three of four species (the exception being Hydra viridissima), the effects of salinity on growth, development and/or reproduction at concentrations below their 72-h LC50 values were observed. However, different endpoints responded differently to salinity. The demonstrated effect of salinity on microinvertebrates has the potential to indirectly affect fish and salt-tolerant macroinvertebrates via changes to their prey species or ecological functions performed by microinvertebrates.
Relating microalgal spatial patterns to flow, mouth and nutrient status in the temporarily open/closed Mngazi estuary, South Africa
Authors: Snow G. C. , Adams J. B.
Published: December 2007

The Mngazi estuary, a near pristine and wave-dominated estuary located on the subtropical east coast of South Africa, requires careful management to ensure that land use does not alter its ecological function. The present study investigated the quality and quantity of water in the estuary and related these to the microalgae. There was no evidence of a persistent elevated phytoplankton biomass in the region of the estuary where river water mixed with brackish estuarine water, even during periods when the estuary mouth was open. Nutrients that determined the microalgal distribution were likely to have come from the mineralisation of organic material, which had been deposited in the estuary following pulses in river water. Results indicate that microalgae were P-limited when the mouth was semi-closed and N-limited during the closed mouth phase. Average benthic chlorophyll a, which ranged from 0.3 to 56.8 µg g–1, was highest in the intertidal middle reaches of the estuary. Organic matter and the proportion of fine sediments (<125 µm) was highest in these sediments. A conceptual model was developed to predict the response of the estuary if further changes in the quality and quantity of river water entering the estuary were to occur.
Faunal relationships with seagrass habitat structure: a case study using shrimp from the Indo-Pacific
Authors: Unsworth Richard K. F. , Grave Sammy De , Jompa Jamaluddin , Smith David J. , Bell James J.
Published: December 2007

Caridean shrimp were used as a model group to investigate the effects of seagrass floral habitat complexity on Indo-Pacific fauna. Relationships between shrimp and seagrass habitat characteristics were explored using both multivariate and multiple linear regression modelling approaches. Epifaunal shrimp assemblages were sampled in the Wakatobi Marine National Park, Indonesia. Seagrass habitat complexity had a significant positive impact on shrimp abundance (F3,59 = 17.51, P < 0.001) and species richness (F3,59 = 10.88, P < 0.001), while significantly altering shrimp assemblage structure (ANOSIM global R = 0.397, P < 0.001). In contrast to studies from other bioregions and faunal groups, species diversity and evenness were inversely related to habitat complexity. Changes in shrimp abundance, diversity and assemblage structure with habitat complexity are considered to reflect changes in individual species habitat specialisation. High complexity habitats were dominated by habitat specialists, whereas low complexity seagrass had higher numbers of habitat generalists. Generalist species may be more adapted to the reduced food availability and increased predatory pressures associated with reduced habitat complexity. Although similar patterns were observed at all sites, inter-site differences in shrimp abundance were observed. This indicates that although the present study demonstrates the importance of small-scale changes in seagrass habitat complexity to faunal assemblages, other factors related to larger spatial-scales are also important.
Influence of area, altitude and hydroperiod on macroinvertebrate communities in southern Brazil wetlands
Authors: Stenert Cristina , Maltchik Leonardo
Published: December 2007

Wetlands are important ecosystems in southern Brazil because they show high productivity and biological diversity. However, conservative data indicate that ~90% of the wetlands have disappeared as a result of agricultural expansion. In this sense, the understanding of species composition and richness patterns in fragmented and natural wetlands is a priority for biodiversity conservation strategies. The main goal of the present study was to determine how much variation in macroinvertebrate richness and composition is explained by wetland area, hydroperiod and altitude. This survey was carried out in an extensive area of the Neotropical region (~280000 km2 – southern Brazil) with a large number of wetland systems (72) and covering a wide gradient of altitude and wetland surface area. The macroinvertebrate richness was higher in permanent wetlands than intermittent ones, and intermittent wetlands supported a macroinvertebrate composition that clearly differed from those of permanent wetlands. Macroinvertebrate richness was positively affected by wetland area; however, this relationship was significant only in permanent wetlands. There is a need to promote conservation of all wetland systems in southern Brazil, regardless of their hydroperiod and area. These arguments are essential to develop conservation and management programs of wetlands in this region.
Lack of TRF2 in ALT cells causes PML-dependent p53 activation and loss of telomeric DNA
Authors: Stagno D'Alcontres Martina , Mendez-Bermudez Aaron , Foxon Jennifer L. , Royle Nicola J. , Salomoni Paolo
Published: December 2007

Alternative lengthening of telomere (ALT) tumors maintain telomeres by a telomerase-independent mechanism and are characterized by a nuclear structure called the ALT-associated PML body (APB). TRF2 is a component of a telomeric DNA/protein complex called shelterin. However, TRF2 function in ALT cells remains elusive. In telomerase-positive tumor cells, TRF2 inactivation results in telomere de-protection, activation of ATM, and consequent induction of p53-dependent apoptosis. We show that in ALT cells this sequence of events is different. First, TRF2 inactivation/silencing does not induce cell death in p53-proficient ALT cells, but rather triggers cellular senescence. Second, ATM is constitutively activated in ALT cells and colocalizes with TRF2 into APBs. However, it is only following TRF2 silencing that the ATM target p53 is activated. In this context, PML is indispensable for p53-dependent p21 induction. Finally, we find a substantial loss of telomeric DNA upon stable TRF2 knockdown in ALT cells. Overall, we provide insight into the functional consequences of shelterin alterations in ALT cells.
Endoplasmic reticulum remains continuous and undergoes sheet-to-tubule transformation during cell division in mammalian cells
Authors: Puhka Maija , Vihinen Helena , Joensuu Merja , Jokitalo Eija
Published: December 2007

The endoplasmic reticulum (ER) is a multifaceted cellular organelle both structurally and functionally, and its cell cycle–dependent morphological changes are poorly understood. Our quantitative confocal and EM analyses show that the ER undergoes dramatic reorganization during cell division in cultured mammalian cells as mitotic ER profiles become shorter and more branched. 3D modeling by electron tomography reveals that the abundant interphase structures, sheets, are lost and subsequently transform into a branched tubular network that remains continuous. This is confirmed by observing the most prominent ER subdomain, the nuclear envelope (NE). A NE marker protein spreads to the mitotic ER tubules, although it does not show a homogenous distribution within the network. We mimicked the mitotic ER reorganization using puromycin to strip the membrane-bound ribosomes from the interphase ER corresponding to the observed loss of ribosomes normally occurring during mitosis. We propose that the structural changes in mitotic ER are linked to ribosomal action on the ER membranes.
Site-specific ubiquitination exposes a linear motif to promote interferon-α receptor endocytosis
Authors: Kumar K.G. Suresh , Barriere Hervé , Carbone Christopher J. , Liu Jianghuai , Swaminathan Gayathri , Xu Ping , Li Ying , Baker Darren P. , Peng Junmin , Lukacs Gergely L. , Fuchs Serge Y.
Published: December 2007

Ligand-induced endocytosis and lysosomal degradation of cognate receptors regulate the extent of cell signaling. Along with linear endocytic motifs that recruit the adaptin protein complex 2 (AP2)–clathrin molecules, monoubiquitination of receptors has emerged as a major endocytic signal. By investigating ubiquitin-dependent lysosomal degradation of the interferon (IFN)-α/β receptor 1 (IFNAR1) subunit of the type I IFN receptor, we reveal that IFNAR1 is polyubiquitinated via both Lys48- and Lys63-linked chains. The SCFβTrcp (Skp1–Cullin1–F-box complex) E3 ubiquitin ligase that mediates IFNAR1 ubiquitination and degradation in cells can conjugate both types of chains in vitro. Although either polyubiquitin linkage suffices for postinternalization sorting, both types of chains are necessary but not sufficient for robust IFNAR1 turnover and internalization. These processes also depend on the proximity of ubiquitin-acceptor lysines to a linear endocytic motif and on its integrity. Furthermore, ubiquitination of IFNAR1 promotes its interaction with the AP2 adaptin complex that is required for the robust internalization of IFNAR1, implicating cooperation between site-specific ubiquitination and the linear endocytic motif in regulating this process.
Requirements for sulfate transport and the diastrophic dysplasia sulfate transporter in fibronectin matrix assembly
Authors: Galante Leontine L. , Schwarzbauer Jean E.
Published: December 2007

Diastrophic dysplasia sulfate transporter (DTDST) is a sulfate/chloride antiporter whose function is impaired in several human chondrodysplasias. We show that DTDST is upregulated by dexamethasone stimulation of HT1080 fibrosarcoma cells and is required for fibronectin (FN) extracellular matrix deposition by these cells. DTDST imports sulfate for the modification of glycosaminoglycans. We find that N-sulfation of these chains is important for FN matrix assembly and that sulfation of cell surface proteoglycans is reduced in the absence of DTDST. Of the candidate HT1080 cell surface proteoglycans, only loss of syndecan-2 compromises FN assembly, as shown by syndecan-2 small interfering RNA knockdown. DTDST is both necessary and sufficient to induce FN matrix assembly in HT1080 cells. Knockdown of DTDST ablates FN matrix, whereas its overexpression increases assembly without dexamethasone stimulation. These results identify a previously unrecognized regulatory pathway for matrix assembly via modulation of a sulfate transporter and proteoglycan sulfation. These data raise the possibility that FN assembly defects contribute to chondrodysplasias.
Chromatin modification of Apaf-1 restricts the apoptotic pathway in mature neurons
Authors: Wright Kevin M. , Smith Michelle I. , Farrag Lila , Deshmukh Mohanish
Published: December 2007

Although apoptosis has been extensively studied in developing neurons, the dynamic changes in this pathway after neuronal maturation remain largely unexplored. We show that as neurons mature, cytochrome c– mediated apoptosis progresses from inhibitor of apoptosis protein–dependent to –independent regulation because of a complete loss of Apaf-1 expression. However, after DNA damage, mature neurons resynthesize Apaf-1 through the cell cycle–related E2F1 pathway and restore their apoptotic potential. Surprisingly, we find that E2F1 is sufficient to induce Apaf-1 expression in developing but not mature neurons. Rather, Apaf-1 up-regulation in mature neurons requires both chromatin derepression and E2F1 transcriptional activity. This differential capacity of E2F1 to induce Apaf-1 transcription is because of the association of the Apaf-1 promoter with active chromatin in developing neurons and repressed chromatin in mature neurons. These data specifically illustrate how the apoptotic pathway in mature neurons becomes increasingly restricted by a novel mechanism involving the regulation of chromatin structure.
Dual roles of myocardin-related transcription factors in epithelial–mesenchymal transition via slug induction and actin remodeling
Authors: Morita Tsuyoshi , Mayanagi Taira , Sobue Kenji
Published: December 2007

Epithelial–mesenchymal transition (EMT) is a critical process occurring during embryonic development and in fibrosis and tumor progression. Dissociation of cell–cell contacts and remodeling of the actin cytoskeleton are major events of the EMT. Here, we show that myocardin-related transcription factors (MRTFs; also known as MAL and MKL) are critical mediators of transforming growth factor β (TGF-β) 1–induced EMT. In all epithelial cell lines examined here, TGF-β1 triggers the nuclear translocation of MRTFs. Ectopic expression of constitutive-active MRTF-A induces EMT, whereas dominant-negative MRTF-A or knockdown of MRTF-A and -B prevents the TGF-β1–induced EMT. MRTFs form complexes with Smad3. Via Smad3, the MRTF–Smad3 complexes bind to a newly identified cis-element GCCG-like motif in the promoter region of Canis familiaris and the human slug gene, which activates slug transcription and thereby dissociation of cell–cell contacts. MRTFs also increase the expression levels of actin cytoskeletal proteins via serum response factor, thereby triggering reorganization of the actin cytoskeleton. Thus, MRTFs are important mediators of TGF-β1–induced EMT.
A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation
Authors: deSouza Nikhil , Cui Jie , Dura Miroslav , McDonald Thomas V. , Marks Andrew R.
Published: December 2007

Sustained elevation of intracellular calcium by Ca2+ release–activated Ca2+ channels is required for lymphocyte activation. Sustained Ca2+ entry requires endoplasmic reticulum (ER) Ca2+ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP3R)/Ca2+ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca2+, and the mechanism that enables the prolonged activation of IP3R1 required for lymphocyte activation is unclear. We show that IP3R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP3R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP3 and renders the channel less sensitive to Ca2+-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca2+ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca2+ levels during lymphocyte activation.
Mechanism of mRNA deadenylation: evidence for a molecular interplay between translation termination factor eRF3 and mRNA deadenylases
Authors: Funakoshi Yuji , Doi Yusuke , Hosoda Nao , Uchida Naoyuki , Osawa Masanori , Shimada Ichio , Tsujimoto Masafumi , Suzuki Tsutomu , Katada Toshiaki , Hoshino Shin-ichi
Published: December 2007

In eukaryotes, shortening of the 3′-poly(A) tail is the rate-limiting step in the degradation of most mRNAs, and two major mRNA deadenylase complexes—Caf1–Ccr4 and Pan2–Pan3—play central roles in this process, referred to as deadenylation. However, the molecular mechanism triggering deadenylation remains elusive. Previously, we demonstrated that eukaryotic releasing factor eRF3 mediates deadenylation and decay of mRNA in a manner coupled to translation termination. Here, we report the mechanism of mRNA deadenylation. The eRF3-mediated deadenylation is catalyzed by both Caf1–Ccr4 and Pan2–Pan3. Interestingly, translation termination complexes eRF1–eRF3, Pan2–Pan3, and Caf1–Ccr4 competitively interact with polyadenylate-binding protein PABPC1. In each complex, eRF3, Pan3, and Tob, respectively, mediate PABPC1 binding, and a combination of a PAM2 motif and a PABC domain is commonly utilized for their contacts. A translation-dependent exchange of eRF1–eRF3 for the deadenylase occurs on PABPC1. Consequently, PABPC1 binding leads to the activation of Pan2–Pan3 and Caf1–Ccr4. From these results, we suggest a mechanism of mRNA deadenylation by Pan2–Pan3 and Caf1–Ccr4 in cooperation with eRF3 and PABPC1.
Human Y5 RNA specializes a Ro ribonucleoprotein for 5S ribosomal RNA quality control
Authors: Hogg J. Robert , Collins Kathleen
Published: December 2007

Humans express four distinct non-protein-coding Y RNAs (ncRNAs). To investigate Y RNA functional diversification, we exploited an RNA-based affinity purification method to isolate ribonucleoproteins (RNPs) assembled on individual human Y RNAs. Silver staining and mass spectrometry revealed that the Ro and La proteins assemble with all Y RNAs, while additional proteins associate with specific Y RNAs. Unexpectedly, Y5 RNA uniquely copurified ribosomal protein L5 and its binding partner 5S RNA. These findings reveal a contribution of Y5 to 5S surveillance and suggest that interactions between Ro-Y5 and L5–5S RNPs establish 5S RNA as a target of quality control.
Dynamics and interplay of nuclear architecture, genome organization, and gene expression
Authors: Schneider Robert , Grosschedl Rudolf
Published: December 2007

The organization of the genome in the nucleus of a eukaryotic cell is fairly complex and dynamic. Various features of the nuclear architecture, including compartmentalization of molecular machines and the spatial arrangement of genomic sequences, help to carry out and regulate nuclear processes, such as DNA replication, DNA repair, gene transcription, RNA processing, and mRNA transport. Compartmentalized multiprotein complexes undergo extensive modifications or exchange of protein subunits, allowing for an exquisite dynamics of structural components and functional processes of the nucleus. The architecture of the interphase nucleus is linked to the spatial arrangement of genes and gene clusters, the structure of chromatin, and the accessibility of regulatory DNA elements. In this review, we discuss recent studies that have provided exciting insight into the interplay between nuclear architecture, genome organization, and gene expression.
The C. elegans protein CEH-30 protects male-specific neurons from apoptosis independently of the Bcl-2 homolog CED-9
Authors: Schwartz Hillel T. , Horvitz H. Robert
Published: December 2007

The developmental control of apoptosis is fundamental and important. We report that the Caenorhabditis elegans Bar homeodomain transcription factor CEH-30 is required for the sexually dimorphic survival of the male-specific CEM (cephalic male) sensory neurons; the homologous cells of hermaphrodites undergo programmed cell death. We propose that the cell-type-specific anti-apoptotic gene ceh-30 is transcriptionally repressed by the TRA-1 transcription factor, the terminal regulator of sexual identity in C. elegans, to cause hermaphrodite-specific CEM death. The established mechanism for the regulation of specific programmed cell deaths in C. elegans is the transcriptional control of the BH3-only gene egl-1, which inhibits the Bcl-2 homolog ced-9; similarly, most regulation of vertebrate apoptosis involves the Bcl-2 superfamily. In contrast, ceh-30 acts within the CEM neurons to promote their survival independently of both egl-1 and ced-9. Mammalian ceh-30 homologs can substitute for ceh-30 in C. elegans. Mice lacking the ceh-30 homolog Barhl1 show a progressive loss of sensory neurons and increased sensory-neuron cell death. Based on these observations, we suggest that the function of Bar homeodomain proteins as cell-type-specific inhibitors of apoptosis is evolutionarily conserved.
Genome-wide view of cell fate specification: ladybird acts at multiple levels during diversification of muscle and heart precursors
Authors: Junion Guillaume , Bataillé Laetitia , Jagla Teresa , Da Ponte Jean Philippe , Tapin Romain , Jagla Krzysztof
Published: December 2007

Correct diversification of cell types during development ensures the formation of functional organs. The evolutionarily conserved homeobox genes from ladybird/Lbx family were found to act as cell identity genes in a number of embryonic tissues. A prior genetic analysis showed that during Drosophila muscle and heart development ladybird is required for the specification of a subset of muscular and cardiac precursors. To learn how ladybird genes exert their cell identity functions we performed muscle and heart-targeted genome-wide transcriptional profiling and a chromatin immunoprecipitation (ChIP)-on-chip search for direct Ladybird targets. Our data reveal that ladybird not only contributes to the combinatorial code of transcription factors specifying the identity of muscle and cardiac precursors, but also regulates a large number of genes involved in setting cell shape, adhesion, and motility. Among direct ladybird targets, we identified bric-a-brac 2 gene as a new component of identity code and inflated encoding αPS2-integrin playing a pivotal role in cell–cell interactions. Unexpectedly, ladybird also contributes to the regulation of terminal differentiation genes encoding structural muscle proteins or contributing to muscle contractility. Thus, the identity gene-governed diversification of cell types is a multistep process involving the transcriptional control of genes determining both morphological and functional properties of cells.
The influence of genetic variation on gene expression
Authors: Williams Rohan B.H. , Chan Eva K.F. , Cowley Mark J. , Little Peter F.R.
Published: December 2007

The view that changes to the control of gene expression rather than alterations to protein sequence are central to the evolution of organisms has become something of a truism in molecular biology. In reality, the direct evidence for this is limited, and only recently have we had the ability to look more globally at how genetic variation influences gene expression, focusing upon inter-individual variation in gene expression and using microarrays to test for differences in mRNA levels. Here, we review the scope of these experimental analyses, what they are designed to tell us about genetic variation, and what are their limitations from both a technical and a conceptual viewpoint. We conclude that while we are starting to understand the impact of this class of genetic variation upon steady-state mRNA levels, we are still far from identifying the potential phenotypic and evolutionary outcomes.
Generating Imaginal Disc Clones in Drosophila
Authors: Blair Seth S.
Published: December 2007

INTRODUCTION

Imaginal disc primordia appear in Drosophila embryos as clusters of cells that invaginate from the embryonic epithelium. During metamorphosis, the imaginal discs form much of the outer covering of the developing adult. Generation of genetic mosaics is useful for removing or adding gene function to imaginal cells. Mitotic recombinants can be generated in a small percentage of disc cells at any stage; each cell then divides normally, forming a clone of genetically altered tissue. Mitotic recombination is induced between homologous chromosomes to generate homozygotic cells in heterozygotic flies. This is done either by irradiating heterozygotic larvae, or by using heat-shock-induced expression of flippase recombinase (FLPase) to induce recombination between FLPase recombination targets (FRTs) inserted into selected chromosome arms. In the FLPout technique, heat-shock-induced expression of FLPase joins a ubiquitous promoter to a selected coding sequence by removing blocking DNA flanked by FRTs. This technique can be used with an upstream activation sequence (UAS)-GAL4. A GAL4 FLPout clone expresses GAL4, which in turn drives the expression of any gene coupled to the UAS promoter. This protocol describes methods for generating such mosaics, by which genetic changes can be limited to small groups of imaginal cells.
Dissection of Imaginal Discs in Drosophila: Figure 1.
Authors: Blair Seth S.
Published: December 2007

INTRODUCTION

Imaginal disc primordia appear in embryos as clusters of 20-40 cells that invaginate from the embryonic epithelium. Each disc is a single-layered epithelial sheet or sac that stays connected to the embryonic and larval epithelium by a thin stalk. Eventually, each sac flattens and the two sides of the sac take on different characteristics, forming on one side the thicker, highly folded disc epithelium and on the other side, the thinner, unfolded peripodial membrane. The imaginal discs are set aside during embryonic development and do not participate appreciably in larval life. However, the discs undergo extensive proliferation during the three larval instars, and by late in the third instar, just before metamorphosis, each disc contains tens of thousands of cells. During metamorphosis, the larval epidermis is converted into the pupal case, and the imaginal discs (along with the histoblast nests) form the outer covering of the developing adult. Most of the ectodermal adult structures are derived from the disc epithelium. This protocol describes methods for dissecting imaginal discs from Drosophila larvae.
Fixation of Imaginal Discs in Drosophila
Authors: Blair Seth S.
Published: December 2007

INTRODUCTION

Imaginal disc primordia appear in Drosophila embryos as clusters of cells that invaginate from the embryonic epithelium. During metamorphosis, the imaginal discs form much of the outer covering of the developing adult. Generation of genetic mosaics is useful for removing or adding gene function to imaginal cells. This protocol describes the methods for fixing such tissues in preparation for immunohistological techniques to monitor gene expression.
Triple-Label Fluorescent Antibody Staining of Imaginal Discs in Drosophila
Authors: Blair Seth S.
Published: December 2007

INTRODUCTION

This protocol describes a triple-label, fluorescent-antibody staining procedure that uses three different primary antisera generated in three different species. Biotin and rhodamine-streptavidin are used for the most critical of these antibodies, as they have the best signal-to-noise ratio. Fluorescein is used to visualize the second antibody, while Cy5 is used for the third. Cy5 is only poorly visible using a standard fluorescence microscope, but is used by the third channel on many laser-scanning confocal microscopes. Obviously, other fluorescent tags, as well as double and single fluorescent stains, are possible. Although these methods are useful for visualizing genetic mosaic clones in wandering larval stage imaginal discs and pupal tissues from Drosophila, the immunohistological techniques are generally applicable to the description of protein expression.
Analysis of siRNA Knockdown of Cell-Cycle Control Genes in G1/S and G2/M Cell-Cycle Phase Marker Cell Lines Using Multiplexed High-Content Analysis: Figure 1.
Authors: Roquemore Elizabeth P.
Published: December 2007

INTRODUCTION

RNA interference using siRNA libraries is a powerful technology for elucidating gene function by downregulating gene expression at the post-transcriptional level. Phenotypic changes associated with siRNA knockdown can be monitored using cell lines expressing fluorescent reporter proteins. Test siRNAs are transiently transfected into the reporter cell line and behavior of the fluorescent reporter probe is monitored using a high-content imaging system. A range of cell features and additional fluorescent probes can be monitored to assess the effects of knockdown. This article provides a method of screening an siRNA cell-cycle array using cell lines that report cell-cycle phase. The subcellular distribution and intensity of the fluorescent phase reporter changes in a cell-cycle-dependent manner. The phase marker assay is multiplexed with an assay for bromodeoxyuridine (BrdU) incorporation, which identifies cells that have undergone DNA replication. When a G2/M cell-cycle phase reporter cell line is multiplexed with BrdU incorporation, information from both assays enables assignment of each cell to one of five distinct phases of the cell cycle (G1, S, G2, prophase, or mitosis).
Construction of a Single-Axis Molecular Puller for Measuring Polysaccharide and Protein Mechanics by Atomic Force Microscopy
Authors: Rabbi Mahir , Marszalek Piotr E.
Published: December 2007

INTRODUCTION

Polysaccharides and proteins are frequently subjected to mechanical forces in vivo. Because these forces affect a wide range of biological activities, it is important to develop methods that directly investigate the mechanical properties of these molecules. Recent progress in techniques that allow the mechanical manipulation of biopolymers at a single-molecule level has revealed the complex nature of the elasticity of proteins and polysaccharides. The atomic force microscope (AFM) is an excellent force spectrometer for probing the mechanical properties (e.g., length and tension) of individual polysaccharides and proteins. The following protocol describes the basic design and construction of an AFM (a single-axis molecular puller) that has four parts: a head, a base, electronics, and software. Those with a background in mechanical engineering, basic knowledge of electronics and data acquisition techniques, and some computer programming skills (e.g., with LabView, Matlab, or Igor) should be able to construct this instrument. It is advisable to inspect commercial AFMs before constructing one from scratch.
Measuring Protein Mechanics by Atomic Force Microscopy: Figure 1.
Authors: Rabbi Mahir , Marszalek Piotr E.
Published: December 2007

INTRODUCTION

Proteins are frequently subjected to mechanical forces in vivo. Because these forces affect a wide range of biological activities, it is important to develop methods that directly investigate the mechanical properties of these molecules. Force spectroscopy of individual proteins (modular or single-domain) allows one to characterize their entropic elasticity under low stretching forces and to determine their persistence length by fitting the worm-like chain (WLC) model to the force-extension curve. At higher stretching forces, it is possible to mechanically unravel these proteins, study their mechanical strength, and examine their unfolding and refolding properties. This provides data on their rates of mechanical unfolding and refolding, as well as on the position of the transition state along the unfolding reaction coordinate, which is defined as the extension of the protein module along the direction of the applied force. Thus, single-protein force spectroscopy may generate a plethora of interesting data that cannot be obtained using traditional biophysical methods. The atomic force microscope (AFM) is an excellent force spectrometer for probing the mechanical properties (e.g., length and tension) of individual proteins. The following protocol describes the use of AFM for measurements of protein mechanics under the constant extension rate regime.
Dot Matrix Pairwise Sequence Comparison
Authors: Mount David W.
Published: December 2007

INTRODUCTION

A dot matrix analysis is primarily a method for comparing two sequences to look for possible alignment of characters between the sequences. The method is also used for finding direct or inverted repeats in protein and DNA sequences, and for predicting regions in RNA that are self-complementary and that, therefore, have the potential of forming secondary structure through base-pairing.