bluuebunny/crossref_metadata_2025 huggingface dataset:
UVA inactivates protein tyrosine phosphatases by calpain‐mediated degradation
Authors: Gulati Pawan , Markova Boyka , Göttlicher Martin , Böhmer Frank‐D , Herrlich Peter A
Published: July 2004

UV irradiation causes inflammatory and proliferative cellular responses. We have proposed previously that these effects are, to a large extent, caused by the ligand‐independent activation of several receptor tyrosine kinases due to the inactivation of their negative control elements, the protein tyrosine phosphatases (PTPs). We examined the mechanism of this inactivation and found that, in addition to reversible oxidation of PTPs, UV triggers a novel mechanism: induced degradation of PTPs by calpain, which requires both calpain activation and substrate PTP oxidative modification. This as yet unrecognized effect of UV is irreversible, occurs predominantly with UVA and UVB, the range of wavelengths in sunlight that reach the skin surface, and at physiologically relevant doses.
Helicase‐dependent isothermal DNA amplification
Authors: Vincent Myriam , Xu Yan , Kong Huimin
Published: July 2004

Polymerase chain reaction is the most widely used method for in vitro DNA amplification. However, it requires thermocycling to separate two DNA strands. In vivo, DNA is replicated by DNA polymerases with various accessory proteins, including a DNA helicase that acts to separate duplex DNA. We have devised a new in vitro isothermal DNA amplification method by mimicking this in vivo mechanism. Helicase‐dependent amplification (HDA) utilizes a DNA helicase to generate single‐stranded templates for primer hybridization and subsequent primer extension by a DNA polymerase. HDA does not require thermocycling. In addition, it offers several advantages over other isothermal DNA amplification methods by having a simple reaction scheme and being a true isothermal reaction that can be performed at one temperature for the entire process. These properties offer a great potential for the development of simple portable DNA diagnostic devices to be used in the field and at the point‐of‐care.
Terminal differentiation of Sol 8 myoblasts is retarded by a transforming growth factor-β autocrine regulatory loop
Authors: ALLEGRA Séverine , LI Jacques Yuan , SAEZ José Maria , LANGLOIS Dominique
Published: July 2004

In DM (differentiation medium), Sol 8 myoblasts spontaneously form myotubes and express the βMHC (β-myosin heavy chain), their main marker of terminal differentiation. This marker is detectable at 24 h, and increases up to 72 h. Our aim was to define temporal effects of TGFβ (transforming growth factor β) on βMHC expression in Sol 8 cells. TGFβ1 (1 ng/ml) added at time zero to DM decreased MyoD expression and completely inhibited βMHC expression in Sol 8 cells. This inhibition of βMHC expression was progressively lost when TGFβ1 was added from 8 to 34 h. After 34 h, the cells were irreversibly differentiated, and TGFβ1 did not inhibit βMHC accumulation any longer. Two independent approaches showed that a TGFβ autocrine regulatory loop retarded and partially impaired Sol 8 cell terminal differentiation. First, permanent immunoneutralization of the active TGFβs released by the cells into DM increased βMHC levels at 72 h compared with controls. Secondly, a dominant-negative mutant of the TGFβ type II receptor was overexpressed in Sol 8 cells under the control of the βMHC promoter. Both the dominant-negative receptor and the βMHC gene were expressed after 24 h in DM. The delayed blocking of the TGFβ signalling pathway by the dominant-negative receptor was as effective as permanent immunoneutralization to promote βMHC expression. To conclude, TGFβ inhibits Sol 8 cell terminal differentiation within a narrow time interval (24–34 h) that coincides with the onset of βMHC expression.
Dynamic interactions between 14-3-3 proteins and phosphoproteins regulate diverse cellular processes
Authors: MACKINTOSH Carol
Published: July 2004

14-3-3 proteins exert an extraordinarily widespread influence on cellular processes in all eukaryotes. They operate by binding to specific phosphorylated sites on diverse target proteins, thereby forcing conformational changes or influencing interactions between their targets and other molecules. In these ways, 14-3-3s ‘finish the job’ when phosphorylation alone lacks the power to drive changes in the activities of intracellular proteins. By interacting dynamically with phosphorylated proteins, 14-3-3s often trigger events that promote cell survival – in situations from preventing metabolic imbalances caused by sudden darkness in leaves to mammalian cell-survival responses to growth factors. Recent work linking specific 14-3-3 isoforms to genetic disorders and cancers, and the cellular effects of 14-3-3 agonists and antagonists, indicate that the cellular complement of 14-3-3 proteins may integrate the specificity and strength of signalling through to different cellular responses.
Mammalian Hsp22 is a heat-inducible small heat-shock protein with chaperone-like activity
Authors: CHOWDARY Tirumala Kumar , RAMAN Bakthisaran , RAMAKRISHNA Tangirala , RAO Chintalagiri Mohan
Published: July 2004

A newly identified 22 kDa protein that interacts with Hsp27 (heat-shock protein 27) was shown to possess the characteristic α-crystallin domain, hence named Hsp22, and categorized as a member of the sHsp (small Hsp) family. Independent studies from different laboratories reported the protein with different names such as Hsp22, H11 kinase, E2IG1 and HspB8. We have identified, on the basis of the nucleotide sequence analysis, putative heat-shock factor 1 binding sites upstream of the Hsp22 translation start site. We demonstrate that indeed Hsp22 is heat-inducible. We show, in vitro, chaperone-like activity of Hsp22 in preventing dithiothreitol-induced aggregation of insulin and thermal aggregation of citrate synthase. We have cloned rat Hsp22, overexpressed and purified the protein to homogeneity and studied its structural and functional aspects. We find that Hsp22 fragments on storage. MS analysis of fragments suggests that the fragmentation might be due to the presence of labile peptide bonds. We have established conditions to improve its stability. Far-UV CD indicates a randomly coiled structure for Hsp22. Quaternary structure analyses by glycerol density-gradient centrifugation and gel filtration chromatography show that Hsp22 exists as a monomer in vitro, unlike other members of the sHsp family. Hsp22 exhibits significantly exposed hydrophobic surfaces as reported by bis-8-anilinonaphthalene-l-sulphonic acid fluorescence. We find that the chaperone-like activity is temperature dependent. Thus Hsp22 appears to be a true member of the sHsp family, which exists as a monomer in vitro and exhibits chaperone-like activity.
Regulation of the cerebellar inositol 1,4,5-trisphosphate receptor by univalent cations
Authors: COQUIL Jean-François , BLAZQUEZ Samantha , SOAVE Sabrina , MAUGER Jean-Pierre
Published: July 2004

In the present study we investigated the effects of K+ and other univalent cations on [3H]InsP3 {[3H]Ins(1,4,5)P3} binding to sheep cerebellar microsomes. In equilibrium binding experiments performed over 4 s at pH 7.1 and 20 °C, the addition of K+ to the binding medium decreased the affinity and increased the total number of binding sites for InsP3 in a dose-dependent manner. At low InsP3 concentration (0.5 nM) these effects resulted in a biphasic dose–response curve, with maximal binding at about 75 mM K+. In contrast, the dose–response curve calculated for InsP3 at the physiological concentration of 5 μM, was linear up to 200 mM K+. Univalent inorganic cations stimulated [3H]InsP3 binding to various extents, with the following descending order of efficiency at 75 mM: Cs+≈Rb+≈K+>Na+>Li+. The effect of K+ on InsP3R affinity was rapidly reversed upon cation removal. We were therefore also able to demonstrate that K+ increased Bmax (maximal specific binding) by pre-treating microsomes with K+ before measuring [3H]InsP3 binding in the absence of that cation. The increase in Bmax was reversible, but this reversal occurred less rapidly than the change in affinity. These results are consistent with a process by which K+ reversibly converted very low-affinity sites into sites with higher affinity, making them detectable in competitive binding experiments. They suggest that interconversion between these two affinity states constitutes the basis of a K+-controlled regulatory mechanism for cerebellar InsP3R.
Regulation of the MST1 kinase by autophosphorylation, by the growth inhibitory proteins, RASSF1 and NORE1, and by Ras
Authors: PRASKOVA Maria , KHOKLATCHEV Andrei , ORTIZ-VEGA Sara , AVRUCH Joseph
Published: July 2004

MST1 (mammalian Sterile20-like 1) and MST2 are closely related Class II GC (protein Ser/Thr) kinases that initiate apoptosis when transiently overexpressed in mammalian cells. In the present study, we show that recombinant MST1/2 undergo a robust autoactivation in vitro, mediated by an intramolecular autophosphorylation of a single site [MST1(Thr183)/MST2(Thr180)] on the activation loop of an MST dimer. Endogenous full-length MST1 is activated by a variety of stressful stimuli, accompanied by the secondary appearance of a 36 kDa Thr183-phosphorylated, caspase-cleaved catalytic fragment. Recombinant MST1 exhibits only 2–5% activation during transient expression; endogenous MST1 in the cycling HeLa or KB cells has a similar low fractional activation, but 2 h incubation with okadaic acid (1 μM) results in 100% activation. Endogenous MST1 immunoprecipitated from KB cells is specifically associated with substoichiometric amounts of the growth inhibitory polypeptides RASSF1A and NORE1A (novel Ras effector 1A; a Ras-GTP-binding protein). Co-expression of RASSF1A, RASSF1C, NORE1A and NORE1B with MST1 markedly suppresses MST1(Thr183) phosphorylation in vivo and abolishes the ability of MST1 to undergo Mg-ATP-mediated autoactivation in vitro; direct addition of purified NORE1A in vitro also inhibits MST1 activation. In contrast, co-transfection of MST1 with NORE1A modified by the addition of a C-terminal CAAX motif results in a substantial increase in MST1(Thr183) phosphorylation, as does fusion of a myristoylation motif directly on to the MST1 N-terminus. Moreover, MST1 polypeptides, bound via wild-type NORE1A to Ras(G12V) (where G12V stands for Gly12→Val), exhibit higher Thr183 phosphorylation compared with MST1 bound to NORE1A alone. Nevertheless, serum stimulation of KB cells does not detectably increase the activation state of endogenous MST1 or MST2 despite promoting the recruitment of the endogenous NORE1–MST1 complex to endogenous Ras. We propose that the NORE1/RASSF1 polypeptides, in addition to their role in maintaining the low activity of MST1 in vivo, direct MST1 to sites of activation and perhaps co-localization with endogenous substrates.
Combinational clustering of receptors following stimulation by bacterial products determines lipopolysaccharide responses
Authors: TRIANTAFILOU Martha , BRANDENBURG Klaus , KUSUMOTO Shoichi , FUKASE Koichi , MACKIE Alan , SEYDEL Ulrich , TRIANTAFILOU Kathy
Published: July 2004

The innate immune system has the capacity to recognize a wide range of pathogens based on conserved PAMPs (pathogen-associated molecular patterns). In the case of bacterial LPS (lipopolysaccharide) recognition, the best studied PAMP, it has been shown that the innate immune system employs at least three cell-surface receptors: CD14, TLR4 (Toll-like receptor 4) and MD-2 protein. CD14 binds LPS from Enterobacteriaceae and then transfers it to MD-2, leading to TLR4 aggregation and signal transduction. LPS analogues such as lipid IVa seem to act as LPS antagonists in human cells, but exhibit LPS mimetic activity in mouse cells. Although TLR4 has been shown to be involved in this species-specific discrimination, the mechanism by which this is achieved has not been elucidated. The questions that remain are how the innate immune system can discriminate between LPS from different bacteria as well as different LPS analogues, and whether or not the structure of LPS affects its interaction with the CD14–TLR4–MD-2 cluster. Is it possible that the ‘shape’ of LPS induces the formation of different receptor clusters, and thus a different immune response? In the present study, we demonstrate using biochemical as well as fluorescence-imaging techniques that different LPS analogues trigger the recruitment of different receptors within microdomains. The composition of each receptor cluster as well as the number of TLR4 molecules that are recruited within the cluster seem to determine whether an immune response will be induced or inhibited.
Reactive oxygen species play no role in the candidacidal activity of the salivary antimicrobial peptide histatin 5
Authors: VEERMAN Enno C. I. , NAZMI Kamran , van 't HOF Wim , BOLSCHER Jan G. M. , den HERTOG Alice L. , NIEUW AMERONGEN Arie V.
Published: July 2004

The mechanism of action of antimicrobial peptides is still a matter of debate. The formation of ROS (reactive oxygen species) has been suggested to be the crucial step in the fungicidal mechanism of a number of antimicrobial peptides, including histatin 5 and lactoferrin-derived peptides. In the present study we have investigated the effects of histatin 5 and of a more amphipathic synthetic derivative, dhvar4, on the generation of ROS in the yeast Candida albicans, using dihydroethidium as an indicator for ROS. With both peptides, a substantial enhancement of fluorescence was observed. However, TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a cell-permeant ROS scavenger, did not have an inhibitory effect on killing or on the enhancement of fluorescence. Furthermore, antimycin and azide, which have been reported to induce ROS in vitro, were not able to enhance the dihydroethidium fluorescence, while chlorhexidine, a non-specific antiseptic agent, enhanced dihydroethidium fluorescence to the same extent as did the peptides. Fluorescence microscopy showed the fluorescence enhancement to be a consequence of the release of unbound preformed ethidium from the mitochondrial matrix within the cell. It is concluded that ROS do not play a role in the histatin 5-mediated killing of C. albicans.
Molecular analysis of muskelin identifies a conserved discoidin-like domain that contributes to protein self-association
Authors: PRAG Soren , COLLETT Georgina D. M. , ADAMS Josephine C.
Published: July 2004

Muskelin is an intracellular protein with a C-terminal kelch-repeat domain that was initially characterized as having functional involvement in cell spreading on the extracellular matrix glycoprotein thrombospondin-1. As one approach to understanding the functional properties of muskelin, we have combined bioinformatic and biochemical studies. Through analysis of a new dataset of eight animal muskelins, we showed that the N-terminal region of the polypeptide corresponds to a predicted discoidin-like domain. This domain architecture is conserved in fungal muskelins and reveals a structural parallel between the muskelins and certain extracellular fungal galactose oxidases, although the phylogeny of the two groups appears distinct. In view of the fact that a number of kelch-repeat proteins have been shown to self-associate, co-immunoprecipitation, protein pull-down assays and studies of cellular localization were carried out with wild-type, deletion mutant and point mutant muskelins to investigate the roles of the discoidin-like and kelch-repeat domains. We obtained evidence for cis- and trans-interactions between the two domains. These studies provide evidence that muskelin self-associates through a head-to-tail mechanism involving the discoidin-like domain.
Reduction of anti-leishmanial pentavalent antimonial drugs by a parasite-specific thiol-dependent reductase, TDR1
Authors: DENTON Helen , McGREGOR Joanne C. , COOMBS Graham H.
Published: July 2004

The reason why Leishmania parasites are susceptible to organic antimonial drugs, the standard chemotherapeutic agents for over 50 years, apparently lies in the fact that the mammalian stage of the parasite reduces the pentavalent form of the administered drug to a trivalent form that causes parasite death. We have identified and characterized a parasite-specific enzyme that can catalyse the reduction of pentavalent antimonials and may therefore be central to the anti-parasite activity of the drug. The unusual protein, a trimer of two-domain monomers in which each domain has some similarity to the Omega class glutathione S-transferases, is a thiol-dependent reductase (designated TDR1) that converts pentavalent antimonials into trivalent antimonials using glutathione as the reductant. The higher abundance of the enzyme in the mammalian stage of the parasite could explain why this parasite form is more susceptible to the drug.
Multisite phosphorylation of doublecortin by cyclin-dependent kinase 5
Authors: GRAHAM Mark E. , RUMA-HAYNES Patricia , CAPES-DAVIS Amanda G. , DUNN Joanne M. , TAN Timothy C. , VALOVA Valentina A. , ROBINSON Phillip J. , JEFFREY Peter L.
Published: July 2004

Doublecortin (DCX) is a 40 kDa microtubule-associated protein required for normal neural migration and cortical layering during development. Mutations in the human DCX gene cause a disruption of cortical neuronal migration. Defects in cdk5 (cyclin-dependent kinase 5) also cause defects in neural migration and cortical layering. DCX is a substrate for cdk5 in vitro and in vivo and the major site of in vitro phosphorylation is Ser-297. We used a highly developed MS strategy to identify the cdk5 phosphorylation sites and determine the major and minor sites. Several phosphopeptides were identified from a tryptic digest of 32P-labelled, cdk5-phosphorylated DCX using a combination of off-line HPLC and matrix-assisted laser-desorption ionization-MS with alkaline phosphatase treatment. Tandem MS/MS enabled the identification of seven phosphorylation sites for cdk5. Monitoring of 32P label indicated that there was one major site, Ser-28, at the N-terminus, and a major site, Ser-339, in the serine/proline-rich domain at the C-terminus. Five other sites, Ser-287, Thr-289, Ser-297, Thr-326 and Ser-332, were also found in the tail. Site-directed mutagenesis largely supported these findings. Single mutation of Ser-28 reduced but did not abolish phosphorylation. Double, rather than single, mutation for Ser-332 and Ser-339 was required to reduce overall phosphorylation, suggesting an interaction between these sites. Truncations of the tail produced a significant reduction in cdk5 phosphorylation of DCX. These results do not support Ser-297 as the major cdk5 phosphorylation site in DCX, but indicate that DCX is subject to complex multisite phosphorylation. This illustrates the importance of a well-developed MS strategy to identify phosphorylation sites.
Actin filaments play a permissive role in the inhibition of store-operated Ca2+ entry by extracellular ATP in rat brown adipocytes
Authors: OMATSU-KANBE Mariko , SHIBATA Masaru , YAMAMOTO Takefumi , ISONO Takahiro , MATSUURA Hiroshi
Published: July 2004

Stimulation of P2 receptors with micromolar concentration of ATP evokes a transient increase in [Ca2+]i (intracellular free Ca2+ concentration), primarily due to release of Ca2+ from intracellular stores; such stimulation also triggers almost complete suppression of thapsigargin-evoked sustained [Ca2+]i increase mediated through a store-operated Ca2+ entry pathway in rat brown adipocytes. We investigated the role of cytoskeletal actin in the inhibitory effect of the extracellular ATP on store-operated Ca2+ entry, using fura 2 fluorescence for continuous measurement of [Ca2+]i, and using Alexa fluor 488-phalloidin staining of actin. Disassembly of actin networks by cytochalasin D (1 μM) or latrunculin A (3 μM) prevented the inhibitory effect of ATP (10 μM) on the thapsigargin (100 nM)-evoked store-operated Ca2+ entry, without changing the effect of ATP in increasing [Ca2+]i. In normal cells, bath application of ATP induced a transient [Ca2+]i increase, consisting of a rapid increase (the rising phase) and the subsequent decrease (the declining phase) to a lower steady level despite the continued presence of the agonist. Disruption of actin assemblies did not significantly affect the rising phase, but prevented the declining phase. Cells incubated with 10 μM ATP for 4 min demonstrated marked accumulations of actin filaments at the cell periphery, showing protrusions at the cell surface; this actin-assembly process is mediated through P2 receptors. In cells treated with cytochalasin D or latrunculin A, extracellular ATP did not induce actin redistribution. These results suggest that the actin reorganization plays a role in ATP-induced inhibition of store-operated Ca2+ entry in rat brown adipocytes.
Segregation of two glutaminase isoforms in islets of Langerhans
Authors: BAGLIETTO-VARGAS David , LÓPEZ-TÉLLEZ Juan F. , MORENO-GONZÁLEZ Inés , GUTIÉRREZ Antonia , ALEDO J. Carlos
Published: July 2004

Despite the importance of glutamatergic signalling in the co-ordination of hormone secretion, the identity of the enzyme for the production of glutamate in β-cells is still unresolved. We have found that the endocrine pancreas co-expresses two isoforms of GA (glutaminase), denoted as kidney-type (KGA) and liver-type (LGA), with a complementary cellular pattern of expression. Whereas KGA was mainly present in α-cells, LGA was very abundant in β-cells. This spatial segregation may have important functional implications, facilitating a differential regulation of glutamate production in insulin- and glucagon-secreting cells.
Inositol trisphosphate analogues selective for types I and II inositol trisphosphate receptors exert differential effects on vasopressin-stimulated Ca2+ inflow and Ca2+ release from intracellular stores in rat hepatocytes
Authors: GREGORY Roland B. , HUGHES Rachael , RILEY Andrew M. , POTTER Barry V. L. , WILCOX Robert A. , BARRITT Greg J.
Published: July 2004

Previous studies have shown that adenophostin A is a potent initiator of the activation of SOCs (store-operated Ca2+ channels) in rat hepatocytes, and have suggested that, of the two subtypes of Ins(1,4,5)P3 receptor predominantly present in rat hepatocytes [Ins(1,4,5)P3R1 (type I receptor) and Ins(1,4,5)P3R2 (type II receptor)], Ins(1,4,5)P3R1s are required for SOC activation. We compared the abilities of Ins(1,4,6)P3 [with higher apparent affinity for Ins(1,4,5)P3R1] and Ins(1,3,6)P3 and Ins(1,2,4,5)P4 [with higher apparent affinities for Ins(1,4,5)P3R2] to activate SOCs. The Ins(1,4,5)P3 analogues were microinjected into single cells together with fura 2, and dose–response curves for the activation of Ca2+ inflow and Ca2+ release from intracellular stores obtained for each analogue. The concentration of Ins(1,4,6)P3 which gave half-maximal stimulation of Ca2+ inflow was substantially lower than that which gave half-maximal stimulation of Ca2+ release. By contrast, for Ins(1,3,6)P3 and Ins(1,2,4,5)P3, the concentration which gave half-maximal stimulation of Ca2+ inflow was substantially higher than that which gave half-maximal stimulation of Ca2+ release. The distribution of Ins(1,4,5)P3R1 and Ins(1,4,5)P3R2 in rat hepatocytes cultured under the same conditions as those employed for the measurement of Ca2+ inflow and release was determined by immunofluorescence. Ins(1,4,5)-P3R1s were found predominantly at the cell periphery, whereas Ins(1,4,5)P3R2s were found at the cell periphery, the cell interior and nucleus. It is concluded that the idea that a small region of the endoplasmic reticulum enriched in Ins(1,4,5)P3R1 is required for the activation of SOCs is consistent with the present results for hepatocytes.
On the theory of plasma-wall transition layers
Authors: Tskhakaya D. , Eliasson B. , Shukla P. K. , Kuhn S.
Published: July 2004

The problem of the plasma-wall transition (PWT) layer in an unmagnetized plasma is considered, without the usual splitting into a sheath and presheath region. The electrons are assumed to be Boltzmann distributed, while the ion dynamics is described by a kinetic theory which includes ionization, recombination, and charge exchange. A condition is derived, which must be fulfilled by the ion velocity throughout the whole PWT in order to exclude oscillatory behavior of the electrostatic potential. The resulting system of equations is solved numerically for various physical situations. The Harrison–Thomson solution for the Tonks–Langmuir model is extended to the case where the quasineutrality is violated, and it is shown that the parabolic shape of the potential profile is retrieved also in this case. For a complete PWT layer, good agreement with recent experimental observations is demonstrated, whereas marked disagreement is found with results based on theories where no recombination is included and/or the ratio of Debye length to characteristic presheath length is assumed to be infinitesimally small. A comparison between recent experimental results with a numerical solution of the model presented here reveals that the model accurately describes the transition from the bulk plasma to the electron-free plasma sheath.
Origins of recently gained introns in
Caenorhabditis
Authors: Coghlan Avril , Wolfe Kenneth H.
Published: July 2004

The genomes of the nematodes
Caenorhabditis elegans
and
Caenorhabditis briggsae
both contain ≈100,000 introns, of which >6,000 are unique to one or the other species. To study the origins of new introns, we used a conservative method involving phylogenetic comparisons to animal orthologs and nematode paralogs to identify cases where an intron content difference between
C. elegans
and
C. briggsae
was caused by intron insertion rather than deletion. We identified 81 recently gained introns in
C. elegans
and 41 in
C. briggsae
. Novel introns have a stronger exon splice site consensus sequence than the general population of introns and show the same preference for phase 0 sites in codons over phases 1 and 2. More of the novel introns are inserted in genes that are expressed in the
C. elegans
germ line than expected by chance. Thirteen of the 122 gained introns are in genes whose protein products function in premRNA processing, including three gains in the gene for spliceosomal protein SF3B1 and two in the nonsense-mediated decay gene
smg-2
. Twenty-eight novel introns have significant DNA sequence identity to other introns, including three that are similar to other introns in the same gene. All of these similarities involve minisatellites or palindromes in the intron sequences. Our results suggest that at least some of the intron gains were caused by reverse splicing of a preexisting intron.
GTF2IRD2 is located in the Williams–Beuren syndrome critical region 7q11.23 and encodes a protein with two TFII-I-like helix–loop–helix repeats
Authors: Makeyev Aleksandr V. , Erdenechimeg Lkhamsuren , Mungunsukh Ognoon , Roth Jutta J. , Enkhmandakh Badam , Ruddle Frank H. , Bayarsaihan Dashzeveg
Published: July 2004

Williams–Beuren syndrome (also known as Williams syndrome) is caused by a deletion of a 1.55- to 1.84-megabase region from chromosome band 7q11.23.
GTF2IRD1
and
GTF2I
, located within this critical region, encode proteins of the TFII-I family with multiple helix–loop–helix domains known as I repeats. In the present work, we characterize a third member,
GTF2IRD2
, which has sequence and structural similarity to the
GTF2I
and
GTF2IRD1
paralogs. The ORF encodes a protein with several features characteristic of regulatory factors, including two I repeats, two leucine zippers, and a single Cys-2/His-2 zinc finger. The genomic organization of human, baboon, rat, and mouse genes is well conserved. Our exon-by-exon comparison has revealed that
GTF2IRD2
is more closely related to
GTF2I
than to
GTF2IRD1
and apparently is derived from the
GTF2I
sequence. The comparison of
GTF2I
and
GTF2IRD2
genes revealed two distinct regions of homology, indicating that the helix–loop–helix domain structure of the
GTF2IRD2
gene has been generated by two independent genomic duplications. We speculate that
GTF2I
is derived from
GTF2IRD1
as a result of local duplication and the further evolution of its structure was associated with its functional specialization. Comparison of genomic sequences surrounding
GTF2IRD2
genes in mice and humans allows refinement of the centromeric breakpoint position of the primate-specific inversion within the Williams–Beuren syndrome critical region.
Human Haplotype Block Sizes Are Negatively Correlated With Recombination Rates
Authors: Greenwood Tiffany A. , Rana Brinda K. , Schork Nicholas J.
Published: July 2004

The International Haplotype Map (“HapMap”) Project is motivated, in part, by the belief that the organization of the human genome, the mechanics of recombination, and the population-level behavior of alleles at adjacent loci should allow researchers to parse the genome into small segments, or “blocks,” that show strong linkage disequilibrium (LD) between alleles at loci within those segments. The discovery and evidence for these blocks is to be based solely on the observed LD strength and patterns between alleles at adjacent loci throughout the genome. Although there are many factors that contribute to LD strength, we assessed the correlation between block structure, in terms of length and percentage of the genome assembled into blocks within a region, and recombination rate obtained from two independent sources. We found evidence of a striking negative correlation between the average recombination rate and average block length, suggesting that recombination rate is a strong contributor to haplotype block structure within the genome. We discuss the potential implications of this negative correlation in the context of the organization, properties, and potential ubiquity of a block-like structure in the human genome.
Gene Expression From the Aneuploid Chromosome in a Trisomy Mouse Model of Down Syndrome
Authors: Lyle Robert , Gehrig Corinne , Neergaard-Henrichsen Charlotte , Deutsch Samuel , Antonarakis Stylianos E.
Published: July 2004

Trisomy 21 is the prototype of human aneuploidies. Since its discovery in 1959, the hypothesis has been that overexpression of the ∼230 human chromosome 21 (Hsa21) genes result in the complex phenotype. However, the level of overexpression of Hsa21 genes in trisomic individuals is presently unknown. We have used Taqman real-time quantitative PCR to accurately measure expression of the mouse orthologs of Hsa21 in the partial trisomy mouse model Ts65Dn. The transcript levels of 78 protein-coding genes present in three copies in Ts65Dn and 21 control genes were compared between Ts65Dn and normal mouse littermates. The mean overexpression of the aneuploid genes is very close to the expected 1.5-fold in all six tissues studied. However, only approximately a third of the genes (37%) are expressed at the theoretical value of 1.5-fold. On average, 45% of the genes are expressed at significantly lower than 1.5-fold, and 9% are not significantly different from 1.0. Interestingly, 18% of the aneuploid genes were expressed at levels significantly greater than 1.5-fold. These data provide candidate genes that might be involved in the phenotypes of Down syndrome, and reveal a complex regulation of gene expression that is not only related to gene copy number.
Single Nucleotide Polymorphisms Associated With Rat Expressed Sequences
Authors: Guryev Victor , Berezikov Eugene , Malik Rainer , Plasterk Ronald H.A. , Cuppen Edwin
Published: July 2004

Single nucleotide polymorphisms (SNPs) are the most common source of genetic variation in populations and are thus most likely to account for the majority of phenotypic and behavioral differences between individuals or strains. Although the rat is extensively studied for the latter, data on naturally occurring polymorphisms are mostly lacking. We have used publicly available sequences consisting of whole-genome shotgun (WGS), expressed sequence tag (EST), and mRNA data as a source for the in silico identification of SNPs in gene-coding regions and have identified a large collection of 33,305 high-quality candidate SNPs. Experimental verification of 471 candidate SNPs using a limited set of rat isolates revealed a confirmation rate of ∼50%. Although the majority of SNPs were identified between Sprague-Dawley (EST data) and Brown Norway (WGS data) strains, we found that 66% of the verified variations are common among different rat strains. All SNPs were extensively annotated, including chromosomal and genetic map information, and nonsynonymous SNPs were analyzed by SIFT and PolyPhen prediction programs for their potential deleterious effect on protein function. Interestingly, we retrieved three SNPs from the database that result in the introduction of a premature stop codon and that could be confirmed experimentally. Two of these “in silico-identified knockouts” reside in interesting QTL regions. Data are publicly available via a Web interface (http://cascad.niob.knaw.nl), allowing simple and advanced search queries.
Genetic Variation in Coding Regions Between and Within Commonly Used Inbred Rat Strains
Authors: Smits Bart M.G. , van Zutphen Bert F.M. , Plasterk Ronald H.A. , Cuppen Edwin
Published: July 2004

Single nucleotide polymorphisms (SNPs) are the most common genetic variation in mammalian populations. Their significance is illustrated by their potential contribution to common disease but also by their potential for use in genetic association and mapping experiments. We have examined the genetic variation between commonly used inbred rat strains by using an efficient SNP discovery and typing assay based on enzyme-based (CEL I) heteroduplex cleavage. Screening of a panel of 96 different rat (sub-)strains for 100 genomic loci in 55 genes, whose human homologs are implicated in clinically relevant diseases like neurological disorder, cancer, schizophrenia, and obesity, resulted in the identification of 103 novel polymorphisms. As all strains are simultaneously genotyped in this setup, this allowed us to make an estimate of the genetic variation between and within commonly used rat inbred strains. Interestingly, we observed substantial genetic variation between colonies of the same inbred strain, maintained at different locations. Furthermore, we identified 17 non-synonymous SNPs that may have an effect on protein function and contribute to phenotypic differences between different laboratory strains.
Mauve: Multiple Alignment of Conserved Genomic Sequence With Rearrangements
Authors: Darling Aaron C.E. , Mau Bob , Blattner Frederick R. , Perna Nicole T.
Published: July 2004

As genomes evolve, they undergo large-scale evolutionary processes that present a challenge to sequence comparison not posed by short sequences. Recombination causes frequent genome rearrangements, horizontal transfer introduces new sequences into bacterial chromosomes, and deletions remove segments of the genome. Consequently, each genome is a mosaic of unique lineage-specific segments, regions shared with a subset of other genomes and segments conserved among all the genomes under consideration. Furthermore, the linear order of these segments may be shuffled among genomes. We present methods for identification and alignment of conserved genomic DNA in the presence of rearrangements and horizontal transfer. Our methods have been implemented in a software package called Mauve. Mauve has been applied to align nine enterobacterial genomes and to determine global rearrangement structure in three mammalian genomes. We have evaluated the quality of Mauve alignments and drawn comparison to other methods through extensive simulations of genome evolution.
Evolution and Topology in the Yeast Protein Interaction Network
Authors: Wuchty Stefan
Published: July 2004

The integrity of the yeast protein–protein interaction network is maintained by a few highly connected proteins, or hubs, which hold the numerous less-connected proteins together. The structural importance and the increased essentiality of these proteins suggest that they are likely to be conserved in evolution, implying a strong relationship between the number of interactions and their evolutionary distance to its orthologs in other organisms. The existence of this coherence was recently reported to strongly depend on the quality of the protein interaction and orthologs data. Here, we introduce a novel method, the evolutionary excess retention (ER), allowing us to uncover a robust and strong correlation between the conservation, essentiality, and connectivity of a yeast protein. We conclude that the relevance of the hubs for the network integrity is simultaneously reflected by a considerable probability of simultaneously being evolutionarily conserved and essential, an observation that does not have an equivalent for nonessential proteins. Providing a thorough assessment of the impact noisy and incomplete data have on our findings, we conclude that our results are largely insensitive to the quality of the utilized data.
Two Breakpoint Clusters at Fragile Site FRA3B Form Phased Nucleosomes
Authors: Mulvihill David J. , Wang Yuh-Hwa
Published: July 2004

Fragile sites are gaps and breaks in metaphase chromosomes generated by specific culture conditions. Fragile site FRA3B is the most unstable site and is directly involved in the breakpoints of deletion and translocation in a wide spectrum of cancers. To learn about the general characteristics of common fragile sites, we investigated the chromatin structure of the FRA3B site. Because FRA3B spans several hundred kilobases, we focused our study on two breakpoint clusters found in FRA3B. Using various nucleases, we demonstrated that these two regions contain phased nucleosomes, regardless of treatment with aphidicolin. Because these regions are located in intron 4 of the FHIT gene, it is very interesting to observe phased nucleosomes over these regions, which are several hundred kilobases downstream from the promoter. Further, by using nucleosome assembly assays, we demonstrate that these two regions do not contain strong nucleosome positioning elements. These results suggest that other factors appear to cooperate with the DNA sequence of these regions to impart nucleosome phasing. This study provides the first information on the chromatin structure of breakpoint regions in a common fragile site. The observation of phased nucleosomes over these breakpoint regions could offer a foundation to understand the expression of fragile sites.
SNP Discovery in Pooled Samples With Mismatch Repair Detection
Authors: Fakhrai-Rad Hossein , Zheng Jianbiao , Willis Thomas D. , Wong Kee , Suyenaga Kent , Moorhead Martin , Eberle Jim , Thorstenson Yvonne R. , Jones Ted , Davis Ronald W. , Namsaraev Eugeni , Faham Malek
Published: July 2004

A targeted discovery effort is required to identify low frequency single nucleotide polymorphisms (SNPs) in human coding and regulatory regions. We here describe combining mismatch repair detection (MRD) with dideoxy terminator sequencing to detect SNPs in pooled DNA samples. MRD enriches for variant alleles in the pooled sample, and sequencing determines the nature of the variants. By using a genomic DNA pool as a template, ∼100 fragments were amplified and subsequently combined and subjected en masse to the MRD procedure. The variant-enriched pool from this one MRD reaction is enriched for the population variants of all the tested fragments. Each fragment was amplified from the variant-enriched pool and sequenced, allowing the discovery of alleles with frequencies as low as 1% in the initial population. Our results support that MRD-based SNP discovery can be used for large-scale discovery of SNPs at low frequencies in a population.
A 1463 Gene Cattle–Human Comparative Map With Anchor Points Defined by Human Genome Sequence Coordinates
Authors: Everts-van der Wind Annelie , Kata Srinivas R. , Band Mark R. , Rebeiz Mark , Larkin Denis M. , Everts Robin E. , Green Cheryl A. , Liu Lei , Natarajan Shreedhar , Goldammer Tom , Lee Jun Heon , McKay Stephanie , Womack James E. , Lewin Harris A.
Published: July 2004

A second-generation 5000 rad radiation hybrid (RH) map of the cattle genome was constructed primarily using cattle ESTs that were targeted to gaps in the existing cattle–human comparative map, as well as to sparsely populated map intervals. A total of 870 targeted markers were added, bringing the number of markers mapped on the RH5000 panel to 1913. Of these, 1463 have significant BLASTN hits (E < e–5) against the human genome sequence. A cattle–human comparative map was created using human genome sequence coordinates of the paired orthologs. One-hundred and ninety-five conserved segments (defined by two or more genes) were identified between the cattle and human genomes, of which 31 are newly discovered and 34 were extended singletons on the first-generation map. The new map represents an improvement of 20% genome-wide comparative coverage compared with the first-generation map. Analysis of gene content within human genome regions where there are gaps in the comparative map revealed gaps with both significantly greater and significantly lower gene content. The new, more detailed cattle–human comparative map provides an improved resource for the analysis of mammalian chromosome evolution, the identification of candidate genes for economically important traits, and for proper alignment of sequence contigs on cattle chromosomes.
Need a university adopt a formal environmental management system?
Authors: Spellerberg Ian F. , Buchan Graeme D. , Englefield Russell
Published: July 2004

What system does a university need to optimise its progress to sustainability? Discusses the gradation of approaches possible for a university as it strives to improve its environmental performance. Argues that an environmental policy plus mechanisms for its implementation can be adequate, and endorsement of a single formal EMS need not be necessary. Illustrates this with progress at Lincoln University, the smallest public university in New Zealand. Lincoln University introduced its environmental policy in 1992, but its full implementation was delayed due to pressures on the tertiary sector in New Zealand. However, a new policy has now been adopted and an environmental task force established. Also briefly addresses the following topics: why have an environmental policy?; the establishment and content of the Lincoln University environmental policy; putting the policy into practice – an example based on waste reduction; and a discussion of the main obstacles to progress, in the current university climate.
Environmental certification at Mälardalen University
Authors: von Oelreich Kristina
Published: July 2004

Aims to describe how the concept of sustainable development, with a focus on the ecological dimension, can be applied practically in an institution for higher education and research. The institution used as an example is Mälardalen University in Västerås, Sweden. Encompasses literature studies and conclusions from practical experience in implementing and maintaining the environmental management system at Mälardalen University. Begins with a review of the concept of sustainable development based on the report “Our common future”, presented by the Commission on Environment and Development in 1987. Then briefly summarises “Agenda 21”, the document from the Rio conference in 1992. Then describes the sector principle, which has been adopted in Sweden by its Riksdag (the Swedish parliament) and government. Then reviews European, regional, and national strategies for a sustainable development within the bounds of higher education and research, followed by a report of the University's work to translate these strategies into practice. The report describes the University's work to integrate the environmental management system within the bounds of training and research and daily operations.
Implementing the sustainable development policy at the University of British Columbia
Authors: Gudz Nadine A.
Published: July 2004

Rethinking our organisational learning processes is critical to society's transition to sustainability – a transition toward living respectfully with one another on a planet with finite resources. Presents the concept of sustainability as a central organising focus for higher education and draws on theories of organisational learning to help inform the transition. Specifically, the central case study focuses on the University of British Columbia (UBC), Canada's first university to adopt a sustainable development policy, and explores how well the policy is implemented in practice. Explores some of the barriers to effective implementation, and provides insights into why the University operates as a “knowledge institution” rather than a “learning institution”. An embedded case study examines the learning experience of the Faculty of Agricultural Sciences at UBC based on the recent transformation of its core curriculum to incorporate sustainability principles.
Comparative analysis of CD45 proteins in primate context: owl monkeys vs humans
Authors: Montoya G.E. , Vernot J‐P. , Patarroyo M.E.
Published: July 2004

Abstract:  Transmembrane protein tyrosine phosphatase (PTPase) CD45 has been implicated in activating, differentiating and the development of different immune system cells. It regulates T‐or B‐cell activation during receptor‐specific recognition by dephosphorylating tyrosine residues in protein kinase substrates. Aotus nancymaae, Aotus nigriceps, and Aotus vociferans CD45 nucleotide and deduced amino acid sequences are presented here, where we found 90–92% identity with the human counterpart in the nucleotide sequence and 83–86% in the amino acid sequence. Aotus CD45 alternative splicing isoforms include the same exons used in human CD45, producing several identical molecular weight nucleotide fragments. Most of the non‐synonymous substitutions were found in the extracellular domain. The more conserved CD45 cytoplasmic portion has two intracellular phosphatase domains (D1 and D2) separated by a short spacer and some residues and motifs involved in signaling or molecular docking, intra‐ and intermolecular interactions and CD45 activity and activity regulation. All invariant residues and structural/functional motifs found in PTPases were totally conserved, suggesting that Aotus CD45 is a functional enzyme. Phylogenetic analysis has shown that the Aotus CD45 molecules are more related to the human homologs than to other reported vertebrate sequences and that the ancestral group of Aotus clade is A. vociferans. When Aotus species were compared, A. nigriceps and A. vociferans were the two most distant species, while A. nancymaae and A. nigriceps appeared to be a sister group. This could be relevant in deciding which Aotus species is to be used for studying particular immunological processes during lymphocyte activation or development.
Lack of association between the –2518G/A polymorphism of the MCP‐1 gene and ischaemic heart disease: a family‐based investigation
Authors: McGlinchey P.G. , Spence M.S. , Patterson C.C. , Allen A. R. , Murphy G. , Belton C. , McKeown P.P.
Published: July 2004

Abstract:  Using two recently described family‐based tests of association, the possible role of the functional –2518G/A polymorphism in the promoter region of the monocyte chemoattractant protein‐1 (MCP‐1) gene in the susceptibility to ischaemic heart disease (IHD) was investigated in a well‐defined Irish population. One thousand and twelve individuals from 386 families with at least one member prematurely affected with IHD were genotyped for the MCP‐1 –2518G/A polymorphism. Using the combined transmission disequilibrium test and the pedigree disequilibrium test, no association between the MCP‐1 –2518G/A polymorphism and IHD was found. Our data demonstrate that, in an Irish population, the MCP‐1 –2518G/A polymorphism is not strongly associated with IHD.