bluuebunny/crossref_metadata_2025 huggingface dataset:
An extracellular site on tetraspanin CD151 determines α3 and α6 integrin–dependent cellular morphology
Authors: Kazarov Alexander R. , Yang Xiuwei , Stipp Christopher S. , Sehgal Bantoo , Hemler Martin E.
Published: September 2002

The α3β1 integrin shows strong, stoichiometric, direct lateral association with the tetraspanin CD151. As shown here, an extracellular CD151 site (QRD194–196) is required for strong (i.e., Triton X-100–resistant) α3β1 association and for maintenance of a key CD151 epitope (defined by monoclonal antibody TS151r) that is blocked upon α3 integrin association. Strong CD151 association with integrin α6β1 also required the QRD194–196 site and masked the TS151r epitope. For both α3 and α6 integrins, strong QRD/TS151r-dependent CD151 association occurred early in biosynthesis and involved α subunit precursor forms. In contrast, weaker associations of CD151 with itself, integrins, or other tetraspanins (Triton X-100–sensitive but Brij 96–resistant) were independent of the QRD/TS151r site, occurred late in biosynthesis, and involved mature integrin subunits. Presence of the CD151–QRD194–196→INF mutant disrupted α3 and α6 integrin–dependent formation of a network of cellular cables by Cos7 or NIH3T3 cells on basement membrane Matrigel and markedly altered cell spreading. These results provide definitive evidence that strong lateral CD151–integrin association is functionally important, identify CD151 as a key player during α3 and α6 integrin–dependent matrix remodeling and cell spreading, and support a model of CD151 as a transmembrane linker between extracellular integrin domains and intracellular cytoskeleton/signaling molecules.
TGFβ receptor internalization into EEA1-enriched early endosomes
Authors: Hayes Susan , Chawla Anil , Corvera Silvia
Published: September 2002

Transforming growth factor (TGF)β is an important physiological regulator of cellular growth and differentiation. It activates a receptor threonine/serine kinase that phosphorylates the transcription factor Smad2, which then translocates into the nucleus to trigger specific transcriptional events. Here we show that activated type I and II TGFβ receptors internalize into endosomes containing the early endosomal protein EEA1. The extent of TGFβ-stimulated Smad2 phosphorylation, Smad2 nuclear translocation, and TGFβ-stimulated transcription correlated closely with the extent of internalization of the receptor. TGFβ signaling also requires SARA (Smad anchor for receptor activation), a 135-kD polypeptide that contains a FYVE Zn++ finger motif. Here we show that SARA localizes to endosomes containing EEA1, and that disruption of this localization inhibits TGFβ-induced Smad2 nuclear translocation. These results indicate that traffic of the TGFβ receptor into the endosome enables TGFβ signaling, revealing a novel function for the endosome as a compartment specialized for the amplification of certain extracellular signals.
Tryptophan-derived Catabolites Are Responsible for Inhibition of T and Natural Killer Cell Proliferation Induced by Indoleamine 2,3-Dioxygenase
Authors: Frumento Guido , Rotondo Rita , Tonetti Michela , Damonte Gianluca , Benatti Umberto , Ferrara Giovanni Battista
Published: September 2002

Macrophages exposed to macrophage colony-stimulating factor acquire the capacity to suppress T cell proliferation; this effect is associated with de novo expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). We have purified IDO and tested its activity in in vitro models of T cell activation. IDO was able to inhibit proliferation of CD4+ T lymphocytes, CD8+ T lymphocytes, and natural killer (NK) cells; proliferation of B lymphocytes was not affected. The inhibitory role of tryptophan and of its catabolites was then tested. In the presence of tryptophan, only l-kynurenine and picolinic acid inhibit cell proliferation. In a tryptophan-free medium cell proliferation was not affected. In the absence of tryptophan inhibition induced by l-kynurenine and picolinic acid was observed at concentrations below the lowest concentration that was effective in the presence of tryptophan, and quinolinic acid acquired some inhibitory capacity. Inhibition of cell proliferation induced by the tryptophan catabolites resulting from IDO activity was selective, applying only to cells undergoing activation. Resting cells were not affected and could subsequently activate normally. We suggest that IDO exerts its effect on cell proliferation by (i) starting the cascade of biochemical reactions that produce the three catabolites and by (ii) enhancing their inhibitory potential by depriving the extracellular microenvironment of tryptophan.
Donor-type CD4+CD25+ Regulatory T Cells Suppress Lethal Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation
Authors: Hoffmann Petra , Ermann Joerg , Edinger Matthias , Fathman C. Garrison , Strober Samuel
Published: September 2002

Acute graft-versus-host disease (aGVHD) is still a major obstacle in clinical allogeneic bone marrow (BM) transplantation. CD4+CD25+ regulatory T (Treg) cells have recently been shown to suppress proliferative responses of CD4+CD25− T cells to alloantigenic stimulation in vitro and are required for ex vivo tolerization of donor T cells, which results in their reduced potential to induce aGVHD. Here we show that CD4+CD25+ T cells isolated from the spleen or BM of donor C57BL/6 (H-2b) mice that have not been tolerized are still potent inhibitors of the alloresponse in vitro and of lethal aGVHD induced by C57BL/6 CD4+CD25− T cells in irradiated BALB/c (H-2d) hosts in vivo. The addition of the CD4+CD25+ Treg cells at a 1:1 ratio with responder/inducer CD4+CD25− T cells resulted in a >90% inhibition of the mixed leukocyte reaction and marked protection from lethal GVHD. This protective effect depended in part on the ability of the transferred CD4+CD25+ T cells to secrete interleukin 10 and occurred if the Treg cells were of donor, but not host, origin. Our results demonstrate that the balance of donor-type CD4+CD25+ Treg and conventional CD4+CD25− T cells can determine the outcome of aGVHD.
Dectin-1 Is A Major β-Glucan Receptor On Macrophages
Authors: Brown Gordon D. , Taylor Philip R. , Reid Delyth M. , Willment Janet A. , Williams David L. , Martinez-Pomares Luisa , Wong Simon Y.C. , Gordon Siamon
Published: September 2002

Zymosan is a β-glucan– and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a β-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the β-glucan receptor mediating this activity. To address the role of the recently described β-glucan receptor, Dectin-1, we generated a novel anti–Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the β-glucan–dependent, nonopsonic recognition of zymosan by primary macro-phages. These findings define Dectin-1 as the leukocyte β-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of β-glucans for therapeutic drug design.
Bacteria-triggered CD4+ T Regulatory Cells Suppress Helicobacter hepaticus–induced Colitis
Authors: Kullberg Marika C. , Jankovic Dragana , Gorelick Peter L. , Caspar Patricia , Letterio John J. , Cheever Allen W. , Sher Alan
Published: September 2002

We have previously demonstrated that interleukin (IL)-10–deficient (IL-10 knockout [KO]) but not wild-type (WT) mice develop colitis after infection with Helicobacter hepaticus. Here, we show that infected recombination activating gene (RAG) KO mice develop intestinal inflammation after reconstitution with CD4+ T cells from IL-10 KO animals and that the cotransfer of CD4+ T cells from H. hepaticus–infected but not uninfected WT mice prevents this colitis. The disease-protective WT CD4+ cells are contained within the CD45RBlow fraction and unexpectedly were found in both the CD25+ and the CD25− subpopulations of these cells, their frequency being higher in the latter. The mechanism by which CD25+ and CD25− CD45RBlow CD4+ cells block colitis involves IL-10 and not transforming growth factor (TGF)-β, as treatment with anti–IL-10R but not anti–TGF-β monoclonal antibody abrogated their protective effect. In vitro, CD45RBlow CD4+ cells from infected WT mice were shown to produce IL-10 and suppress interferon-γ production by IL-10 KO CD4+ cells in an H. hepaticus antigen–specific manner. Together, our data support the concept that H. hepaticus infection results in the induction in WT mice of regulatory T cells that prevent bacteria-induced colitis. The induction of such cells in response to gut flora may be a mechanism protecting normal individuals against inflammatory bowel disease.
Evidence for Replicative Repair of DNA Double-Strand Breaks Leading to Oncogenic Translocation and Gene Amplification
Authors: Difilippantonio Michael J. , Petersen Simone , Chen Hua Tang , Johnson Roger , Jasin Maria , Kanaar Roland , Ried Thomas , Nussenzweig André
Published: September 2002

Nonreciprocal translocations and gene amplifications are commonly found in human tumors. Although little is known about the mechanisms leading to such aberrations, tissue culture models predict that they can arise from DNA breakage, followed by cycles of chromatid fusion, asymmetric mitotic breakage, and replication. Mice deficient in both a nonhomologous end joining (NHEJ) DNA repair protein and the p53 tumor suppressor develop lymphomas at an early age harboring amplification of an IgH/c-myc fusion. Here we report that these chromosomal rearrangements are initiated by a recombination activating gene (RAG)-induced DNA cleavage. Subsequent DNA repair events juxtaposing IgH and c-myc are mediated by a break-induced replication pathway. Cycles of breakage-fusion-bridge result in amplification of IgH/c-myc while chromosome stabilization occurs through telomere capture. Thus, mice deficient in NHEJ provide excellent models to study the etiology of unbalanced translocations and amplification events during tumorigenesis.
Extracellular Sodium Interacts with the HERG Channel at an Outer Pore Site
Authors: Mullins Franklin M. , Stepanovic Svetlana Z. , Desai Reshma R. , George Alfred L. , Balser Jeffrey R.
Published: September 2002

Most voltage-gated K+ currents are relatively insensitive to extracellular Na+ (Na+o), but Na+o potently inhibits outward human ether-a-go-go–related gene (HERG)–encoded K+ channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429–30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na+ (Na+i) and extracellular Ba2+ (Ba2+o), to define a site where Na+o interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na+o was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K+ selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na+o. Quantitatively similar effects of Na+i to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na+o sensitivity, this result suggested that Na+o and Na+i act at different sites. Extracellular Ba2+ (Ba2+o) blocks K+ channel pores, and thereby serves as a useful probe of K+ channel structure. HERG channel inactivation promotes relief of Ba2+ block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265–278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na+o action. A remote allosteric model predicts that Na+o will speed relief of Ba2+o block by promoting inactivation. Instead, Na+o slowed Ba2+ egress and Ba2+ relieved Na+o inhibition, consistent with Na+o binding to an outer pore site. The apparent affinities of the outer pore for Na+o and K+o as measured by slowing of Ba2+ egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na+o inhibition. Na+o inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A.
The Assay of Mixtures of Sodium-24 and Potassium-42 in Clinical Tracer Studies; with Particular Reference to the Measurement of Exchangeable Sodium and Potassium
Authors: D S Munro  , H Renschler  , G M Wilson
Published: September 2002

1. Combinations of different counting systems have been investigated for their efficiency in assaying mixtures of24Na and42K. The advantages of a combination of thin and thick walled liquid G.M. counter tubes of the Veall pattern are described. Physical methods are unreliable when only a small proportion of the counts is derived from one isotope.

2. The separation of potassium by precipitation with sodium tetraphenylboron is described. Methods dependent on chemical separation are reliable even when42K makes only a small contribution to the counting rate of a mixture.

3. The application of these methods to the simultaneous measurement of exchangeable sodium and potassium is discussed and a rapid method for carrying out these measurements is described.
Localization of electrons in condensed matter
Authors: N H March  , J C Stoddart
Published: September 2002

Recent developments in the electron theory of solids and fluids are leading us more and more to the picture of condensed matter as built up out of localized electron distributions, often only weakly interacting with their environment, and significantly different objects from isolated atoms.

The merits of such an approach seem to be twofold:

In theories where coulombic correlations are not dominant we might hope to obtain a unified description of one-electron states in crystals, amorphous and/or disordered solids, and liquids, in terms of such localized electron distributions. This description will, of course, have to be equivalent to the Bloch-Brillouin theory of energy bands in perfectly periodic structures.

Correlation effects seem to be most usefully described in terms of electron localization, and therefore a description as in (i) promises to be particularly favourable for incorporating many-body effects at a second stage.

In this article we give particular attention to the nature of electron states in the presence of a local potential built up as a superposition of screened one-centre potentials. If these screened potentials are centred on the sites of a regular lattice, it is shown that, once the problem of scattering of electron waves off the one-centre potential is solved, the exact construction of the energy bands is possible, at least in principle. In practice, only for muffin tins, for which potential the density of states can be derived from the phase shifts, has it proved possible to carry through this programme exactly. But we also describe a density matrix method based on the idea of the partition function of a crystal being built up from products of one-centre partition functions, by means of which we can solve approximately for the band structure generated by overlapping screened ion potentials, once the single-centre scattering has been calculated. In this method, the one-centre scattering is defined by an effective potentialUwhich is asymptotically related to the one-centre input potentialVL. The size of the overlaps of the effective potentialUon neighbouring sites determines the accuracy of the approximate theory.

The way in which electron correlations can be incorporated into such a model is then considered. In the Sommerfeld model, where the positive charge is uniformly distributed, a localized orbital method is described. Further, in a lattice, the one-centre method has its counterpart in the many-body problem, though no applications have so far proved possible. Then the relation of the local potential theory to the many-body problem is described. Also included in the discussion are Wigner and Mott transitions, excitonic phases, localized moments in metallic alloys and the recent progress in the theory of strong correlations in narrow energy bands. The role that x-ray scattering measurements can play in helping to define localized electron distributions is also discussed.
Role of the Rab GTP-Binding Protein Ypt3 in the Fission Yeast Exocytic Pathway and Its Connection to Calcineurin Function
Authors: Cheng Hong , Sugiura Reiko , Wu Wenlian , Fujita Masaaki , Lu Yabin , Sio Susie O. , Kawai Rena , Takegawa Kaoru , Shuntoh Hisato , Kuno Takayoshi
Published: September 2002

A genetic screen for mutations synthetically lethal with fission yeast calcineurin deletion led to the identification of Ypt3, a homolog of mammalian Rab11 GTP-binding protein. A mutant with the temperature-sensitive ypt3-i5 allele showed pleiotropic phenotypes such as defects in cytokinesis, cell wall integrity, and vacuole fusion, and these were exacerbated by FK506-treatment, a specific inhibitor of calcineurin. Green fluorescent protein (GFP)-tagged Ypt3 showed cytoplasmic staining that was concentrated at growth sites, and this polarized localization required the actin cytoskeleton. It was also detected as a punctate staining in an actin-independent manner. Electron microscopy revealed thatypt3-i5 mutants accumulated aberrant Golgi-like structures and putative post-Golgi vesicles, which increased remarkably at the restrictive temperature. Consistently, the secretion of GFP fused with the pho1+leader peptide (SPL-GFP) was abolished at the restrictive temperature in ypt3-i5 mutants. FK506-treatment accentuated the accumulation of aberrant Golgi-like structures and caused a significant decrease of SPL-GFP secretion at a permissive temperature. These results suggest that Ypt3 is required at multiple steps of the exocytic pathway and its mutation affects diverse cellular processes and that calcineurin is functionally connected to these cellular processes.
MEC3,MEC1,andDDC2Are Essential Components of a Telomere Checkpoint Pathway Required for Cell Cycle Arrest during Senescence inSaccharomyces cerevisiae
Authors: Enomoto Shinichiro , Glowczewski Lynn , Berman Judith
Published: September 2002

When telomerase is absent and/or telomeres become critically short, cells undergo a progressive decline in viability termed senescence. The telomere checkpoint model predicts that cells will respond to a damaged or critically short telomere by transiently arresting and activating repair of the telomere. We examined the senescence of telomerase-deficient Saccharomyces cerevisiae at the cellular level to ask if the loss of telomerase activity triggers a checkpoint response. As telomerase-deficient mutants were serially subcultured, cells exhibited a progressive decline in average growth rate and an increase in the number of cells delayed in the G2/M stage of the cell cycle. MEC3, MEC1, andDDC2, genes important for the DNA damage checkpoint response, were required for the cell cycle delay in telomerase-deficient cells. In contrast, TEL1,RAD9, and RAD53, genes also required for the DNA damage checkpoint response, were not required for the G2/M delay in telomerase-deficient cells. We propose that the telomere checkpoint is distinct from the DNA damage checkpoint and requires a specific set of gene products to delay the cell cycle and presumably to activate telomerase and/or other telomere repair activities.
Overexpression of Yeast Hsp110 Homolog Sse1p Suppressesydj1-151Thermosensitivity and Restores Hsp90-dependent Activity
Authors: Goeckeler Jennifer L. , Stephens Andi , Lee Paul , Caplan Avrom J. , Brodsky Jeffrey L.
Published: September 2002

The Saccharomyces cerevisiae heat-shock protein (Hsp)40, Ydj1p, is involved in a variety of cellular activities that control polypeptide fate, such as folding and translocation across intracellular membranes. To elucidate the mechanism of Ydj1p action, and to identify functional partners, we screened for multicopy suppressors of the temperature-sensitive ydj1-151 mutant and identified a yeast Hsp110, SSE1. Overexpression of Sse1p also suppressed the folding defect of v-Src kinase in theydj1-151 mutant and partially reversed the α-factor translocation defect. SSE1-dependent suppression ofydj1-151 thermosensitivity required the wild-type ATP-binding domain of Sse1p. However, the Sse1p mutants maintained heat-denatured firefly luciferase in a folding-competent state in vitro and restored human androgen receptor folding in sse1mutant cells. Because the folding of both v-Src kinase and human androgen receptor in yeast requires the Hsp90 complex, these data suggest that Ydj1p and Sse1p are interacting cochaperones in the Hsp90 complex and facilitate Hsp90-dependent activity.
Identification of the Functional Domains of Yeast Sorting Nexins Vps5p and Vps17p
Authors: Seaman Matthew N.J. , Williams Hazel P.
Published: September 2002

Sorting nexins (Snxs) are a recently discovered family of conserved hydrophilic cytoplasmic proteins that have been found associated with membranes of the endocytic system and that are implicated in the trafficking of many endosomal membrane proteins, including the epidermal growth factor receptor and transferrin receptor. Snx proteins are partly defined by the presence of a p40 phox homology domain that has recently been shown to bind phosphatidylinositol 3-phosphate. Most Snx proteins also contain a predicted coiled-coils domain in the carboxyl-terminal half of the protein and have been shown to form dimers with other members of the Snx family. The yeast sorting nexins Vps5p and Vps17p form a dimer and are also components of the retromer complex that mediates endosome-to-Golgi transport of the carboxypeptidase Y receptor Vps10p. To functionally define the different domains of the yeast sorting nexins Vps5p and Vps17p, we have generated various truncations to examine the role that the different domains of Vps5p/Vps17p play in their respective functions. Herein, we show that the C-terminal halves of Vps5p and Vps17p, which contain the coiled-coils domains, are necessary and sufficient for their interaction. We have also mapped the retromer assembly domain to the N-terminal half of Vps5p and found that binding of Vps5p by Vps17p synergizes the interaction between Vps5p and other retromer components. Additionally, we have examined which domain(s) of Vps5p is necessary for membrane association.
CytochromecRelease and Mitochondria Involvement in Programmed Cell Death Induced by Acetic Acid inSaccharomyces cerevisiae
Authors: Ludovico Paula , Rodrigues Fernando , Almeida Agostinho , Silva Manuel T. , Barrientos Antoni , Côrte-Real Manuela
Published: September 2002

Evidence is presented that mitochondria are implicated in the previously described programmed cell death (PCD) process induced by acetic acid in Saccharomyces cerevisiae. In yeast cells undergoing a PCD process induced by acetic acid, translocation of cytochrome c (CytC) to the cytosol and reactive oxygen species production, two events known to be proapoptotic in mammals, were observed. Associated with these events, reduction in oxygen consumption and in mitochondrial membrane potential was found. Enzymatic assays showed that the activity of complexbc1was normal, whereas that of cytochrome c oxidase (COX) was strongly decreased. This decrease is in accordance with the observed reduction in the amounts of COX II subunit and of cytochromesa+a3. The acetic acid-induced PCD process was found to be independent of oxidative phosphorylation because it was not inhibited by oligomycin treatment. The inability ofS. cerevisiae mutant strains (lacking mitochondrial DNA, heme lyase, or ATPase) to undergo acetic acid-induced PCD and in the ATPase mutant (knockout in ATP10) the absence of CytC release provides further evidence that the process is mediated by a mitochondria-dependent apoptotic pathway. The understanding of the involvement of a mitochondria-dependent apoptotic pathway inS. cerevisiae PCD process will be most useful in the further elucidation of an ancestral pathway common to PCD in metazoans.
β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast
Authors: Gupta Mohan L. , Bode Claudia J. , Thrower Douglas A. , Pearson Chad G. , Suprenant Kathy A. , Bloom Kerry S. , Himes Richard H.
Published: September 2002

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast β-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.
Septin Function inCandida albicansMorphogenesis
Authors: Warenda Amy J. , Konopka James B.
Published: September 2002

The septin proteins function in the formation of septa, mating projections, and spores in Saccharomyces cerevisiae, as well as in cell division and other processes in animal cells. Candida albicans septins were examined in this study for their roles in morphogenesis of this multimorphic, opportunistically pathogenic fungus, which can range from round budding yeast to elongated hyphae. C. albicans green fluorescent protein labeled septin proteins localized to a tight ring at the bud and pseudohyphae necks and as a more diffuse array in emerging germ tubes of hyphae. Deletion analysis demonstrated that the C. albicans homologs of the S. cerevisiae CDC3 andCDC12 septins are essential for viability. In contrast, the C. albicans cdc10Δ and cdc11Δ mutants were viable but displayed conditional defects in cytokinesis, localization of cell wall chitin, and bud morphology. The mutant phenotypes were not identical, however, indicating that these septins carry out distinct functions. The viable septin mutants could be stimulated to undergo hyphal morphogenesis but formed hyphae with abnormal curvature, and they differed from wild type in the selection of sites for subsequent rounds of hyphal formation. Thecdc11Δ mutants were also defective for invasive growth when embedded in agar. These results further extend the known roles of the septins by demonstrating that they are essential for the proper morphogenesis of C. albicans during both budding and filamentous growth.
Fluorescence Resonance Energy Transfer Detection of Synaptophysin I and Vesicle-associated Membrane Protein 2 Interactions during Exocytosis from Single Live Synapses
Authors: Pennuto Maria , Dunlap David , Contestabile Andrea , Benfenati Fabio , Valtorta Flavia
Published: September 2002

To investigate the molecular interactions of synaptophysin I and vesicle-associated membrane protein 2 (VAMP2)/synaptobrevin II during exocytosis, we have used time-lapse videomicroscopy to measure fluorescence resonance energy transfer in live neurons. For this purpose, fluorescent protein variants fused to synaptophysin I or VAMP2 were expressed in rat hippocampal neurons. We show that synaptophysin I and VAMP2 form both homo- and hetero-oligomers on the synaptic vesicle membrane. When exocytosis is stimulated with α-latrotoxin, VAMP2 dissociates from synaptophysin I even in the absence of appreciable exocytosis, whereas synaptophysin I oligomers disassemble only upon incorporation of the vesicle with the plasma membrane. We propose that synaptophysin I has multiple roles in neurotransmitter release, regulating VAMP2 availability for the solubleN-ethylmaleimide-sensitive factor attachment protein receptor complex and possibly participating in the late steps of exocytosis.
The Shb Adaptor Protein Binds to Tyrosine 766 in the FGFR-1 and Regulates the Ras/MEK/MAPK Pathway via FRS2 Phosphorylation in Endothelial Cells
Authors: Cross Michael J. , Lu Lingge , Magnusson Peetra , Nyqvist Daniel , Holmqvist Kristina , Welsh Michael , Claesson-Welsh Lena
Published: September 2002

Stimulation of fibroblast growth factor receptor-1 (FGFR-1) is known to result in phosphorylation of tyrosine 766 and the recruitment and subsequent activation of phospholipase C-γ (PLC-γ). To assess the role of tyrosine 766 in endothelial cell function, we generated endothelial cells expressing a chimeric receptor, composed of the extracellular domain of the PDGF receptor-α and the intracellular domain of FGFR-1. Mutation of tyrosine 766 to phenylalanine prevented PLC-γ activation and resulted in a reduced phosphorylation of FRS2 and reduced activation of the Ras/MEK/MAPK pathway relative to the wild-type chimeric receptor. However, FGFR-1–mediated MAPK activation was not dependent on PKC activation or intracellular calcium, both downstream mediators of PLC-γ activation. We report that the adaptor protein Shb is also able to bind tyrosine 766 in the FGFR-1, via its SH2 domain, resulting in its subsequent phosphorylation. Overexpression of an SH2 domain mutant Shb caused a dramatic reduction in FGFR-1–mediated FRS2 phosphorylation with concomitant perturbment of the Ras/MEK/MAPK pathway. Expression of the chimeric receptor mutant and the Shb SH2 domain mutant resulted in a similar reduction in FGFR-1–mediated mitogenicity. We conclude, that Shb binds to tyrosine 766 in the FGFR-1 and regulates FGF-mediated mitogenicity via FRS2 phosphorylation and the subsequent activation of the Ras/MEK/MAPK pathway.
Casein Kinase I Regulates Membrane Binding by ARF GAP1
Authors: Yu Sidney , Roth Michael G.
Published: September 2002

ARF GAP1, a 415-amino acid GTPase activating protein (GAP) for ADP-ribosylation factor (ARF) contains an amino-terminal 115-amino acid catalytic domain and no other recognizable features. Amino acids 203–334 of ARF GAP1 were sufficient to target a GFP-fusion protein to Golgi membranes in vivo. When overexpressed in COS-1 cells, this protein domain inhibited protein transport between the ER and Golgi and, in vitro, competed with the full-length ARF GAP1 for binding to membranes. Membrane binding by ARF GAP1 in vitro was increased by a factor in cytosol and this increase was inhibited by IC261, an inhibitor selective for casein kinase Iδ (CKIδ), or when cytosol was treated with antibody to CKIδ. The noncatalytic domain of ARF GAP1 was phosphorylated both in vivo and in vitro by CKI. IC261 blocked membrane binding by ARF GAP1 in vivo and inhibited protein transport in the early secretory pathway. Overexpression of a catalytically inactive CKIδ also inhibited the binding of ARF GAP1 to membranes and interfered with protein transport. Thus, a CKI isoform is required for protein traffic through the early secretory pathway and can modulate the amount of ARF GAP1 that can bind to membranes.
Detection of cis -element clusters in higher
eukaryotic DNA
Authors: Frith Martin C. , Hansen Ulla , Weng Zhiping
Published: September 2002

Motivation: Computational prediction and analysis of transcription regulatory regions in DNA sequences has the potential to accelerate greatly our understanding of how cellular processes are controlled. We present a hidden Markov model based method for detecting regulatory regions in DNA sequences, by searching for clusters of cis -elements.

Results: When applied to regulatory targets of the transcription factor LSF, this method achieves a sensitivity of 67%, while making one prediction per 33 kb of non-repetitive human genomic sequence. When applied to muscle specific regulatory regions, we obtain a sensitivity and prediction rate that compare favorably with one of the best alternative approaches. Our method, which we call Cister, can be used to predict different varieties of regulatory region by searching for clusters of cis -elements of any type chosen by the user. Cister is simple to use and is available on the web.

Availability: http://sullivan.bu.edu/~mfrith/cister.shtml

Contact: mfrith@bu.edu; zhiping@bu.edu

* To whom correspondence should be addressed.
Presence of ATG triplets in

5′ untranslated regions of

eukaryotic cDNAs correlates with a ‘weak’ context of the

start codon
Authors: Rogozin Igor B. , Kochetov Alexey V. , Kondrashov Fyodor A. , Koonin Eugene V. , Milanesi Luciano
Published: September 2002

Motivation: The context of the start codon (typically, AUG) and the features of the 5′ Untranslated Regions (5′ UTRs) are important for understanding translation regulation in eukaryotic mRNAs and for accurate prediction of the coding region in genomic and cDNA sequences. The presence of AUG triplets in 5′ UTRs (upstream AUGs) might effect the initiation rate and, in the context of gene prediction, could reduce the accuracy of the identification of the authentic start. To reveal potential connections between the presence of upstream AUGs and other features of 5′ UTRs, such as their length and the start codon context, we undertook a systematic analysis of the available eukaryotic 5′ UTR sequences.

Results: We show that a large fraction of 5′ UTRs in the available cDNA sequences, 15–53% depending on the organism, contain upstream ATGs. A negative correlation was observed between the information content of the translation start signal and the length of the 5′ UTR. Similarly, a negative correlation exists between the ‘strength’ of the start context and the number of upstream ATGs. Typically, cDNAs containing long 5′ UTRs with multiple upstream ATGs have a ‘weak’ start context, and in contrast, cDNAs containing short 5′ UTRs without ATGs have ‘strong’ starts. These counter-intuitive results may be interpreted in terms of upstream AUGs having an important role in the regulation of translation efficiency by ensuring low basal translation level via double negative control and creating the potential for additional regulatory mechanisms. One of such mechanisms, supported by experimental studies of some mRNAs, includes removal of the AUG-containing portion of the 5′ UTR by alternative splicing.

Availability: An ATG_ EVALUATOR program is available upon request or at www.itba.mi.cnr.it/webgene.

Contact: rogozin@ncbi.nlm.nih.gov, milanesi@itba.mi.cnr.it

4 To whom correspondence should be addressed at NCBI/NLM/NIH, 8600 Rockville Pike, Bldg 38A, Rm 805, Bethesda, MD 20894, USA.
Markovian domain fingerprinting: statistical segmentation of
protein sequences
Authors: Bejerano Gill , Seldin Yevgeny , Margalit Hanah , Tishby Naftali
Published: September 2002

Motivation: Characterization of a protein family by its distinct sequence domains is crucial for functional annotation and correct classification of newly discovered proteins. Conventional Multiple Sequence Alignment (MSA) based methods find difficulties when faced with heterogeneous groups of proteins. However, even many families of proteins that do share a common domain contain instances of several other domains, without any common underlying linear ordering. Ignoring this modularity may lead to poor or even false classification results. An automated method that can analyze a group of proteins into the sequence domains it contains is therefore highly desirable.

Results: We apply a novel method to the problem of protein domain detection. The method takes as input an unaligned group of protein sequences. It segments them and clusters the segments into groups sharing the same underlying statistics. A Variable Memory Markov (VMM) model is built using a Prediction Suffix Tree (PST) data structure for each group of segments. Refinement is achieved by letting the PSTs compete over the segments, and a deterministic annealing framework infers the number of underlying PST models while avoiding many inferior solutions. We show that regions of similar statistics correlate well with protein sequence domains, by matching a unique signature to each domain. This is done in a fully automated manner, and does not require or attempt an MSA. Several representative cases are analyzed. We identify a protein fusion event, refine an HMM superfamily classification into the underlying families the HMM cannot separate, and detect all 12 instances of a short domain in a group of 396 sequences.

Contact: jill@cs.huji.ac.il; tishby@cs.huji.ac.il

* To whom correspondence should be addressed.
Clustering protein sequences—structure prediction by

transitive homology
Authors: Bolten Eva , Schliep Alexander , Schneckener Sebastian , Schomburg Dietmar , Schrader Rainer
Published: September 2002

Motivation: It is widely believed that for two proteins Aand Ba sequence identity above some threshold implies structural similarity due to a common evolutionary ancestor. Since this is only a sufficient, but not a necessary condition for structural similarity, the question remains what other criteria can be used to identify remote homologues. Transitivity refers to the concept of deducing a structural similarity between proteins Aand Cfrom the existence of a third protein B, such that Aand Bas well as Band Care homologues, as ascertained if the sequence identity between Aand Bas well as that between Band Cis above the aforementioned threshold. It is not fully understood if transitivity always holds and whether transitivity can be extended ad infinitum.

Results: We developed a graph-based clustering approach, where transitivity plays a crucial role. We determined all pair-wise similarities for the sequences in the SwissProt database using the Smith–Waterman local alignment algorithm. This data was transformed into a directed graph, where protein sequences constitute vertices. A directed edge was drawn from vertex Ato vertex Bif the sequences Aand Bshowed similarity, scaled with respect to the self-similarity of A, above a fixed threshold. Transitivity was important in the clustering process, as intermediate sequences were used, limited though by the requirement of having directed paths in both directions between proteins linked over such sequences. The length dependency—implied by the self-similarity—of the scaling of the alignment scores appears to be an effective criterion to avoid clustering errors due to multi-domain proteins. To deal with the resulting large graphs we have developedan efficient library. Methods include the novel graph-based clustering algorithm capable of handling multi-domain proteins and cluster comparison algorithms. Structural Classification of Proteins (SCOP) was used as an evaluation data set for our method, yielding a 24% improvement over pair-wise comparisons in terms of detecting remote homologues.

Availability: The software is available to academic users on request from the authors.

Contact: e.bolten@science-factory.com; schliep@zpr.uni-koeln.de; s.schneckener@science-factory.com; d.schomburg@uni-koeln.de; schrader@zpr.uni-koeln.de

Supplementary information: http://www.zaik.uni-koeln.de/~schliep/ProtClust.html

* To whom correspondence should be addressed.
Prediction of disulfide connectivity in proteins
Authors: Fariselli Piero , Casadio Rita
Published: September 2002

Motivation: A major problem in protein structure predictionis the correct location of disulfide bridges in cysteine-rich proteins. In protein-folding prediction, the location of disulfide bridges can strongly reduce the search in the conformational space. Therefore the correct prediction of the disulfide connectivity starting from the protein residue sequencemay also help in predicting its 3D structure.

Results: In this paper we equate the problem of predicting the disulfide connectivity in proteins to a problem of finding the graph matching with the maximum weight. The graph vertices are the residues of cysteine-forming disulfide bridges, and the weight edges are contact potentials. In order to solve this problem we develop and test different residue contact potentials. The best performing one, based on the Edmonds–Gabow algorithm and Monte-Carlo simulated annealing reaches an accuracy significantly higher than that obtained with a general mean force contact potential. Significantly, in the case of proteins with four disulfide bonds in the structure, the accuracy is 17 times higher than that of a random predictor. The method presented here can be used to locate putative disulfide bridges in protein-folding.

Availability: The program is available upon request from the authors.

Contact: Casadio@alma.unibo.it; Piero@biocomp.unibo.it
Birth of scale-free molecular networks and the number of

distinct DNA and protein domains per genome
Authors: Rzhetsky Andrey , Gomez Shawn M.
Published: September 2002

Motivation: Current growth in the field of genomics has provided a number of exciting approaches to the modeling of evolutionary mechanisms within the genome. Separately, dynamical and statistical analyses of networks such as the World Wide Web and the social interactions existing between humans have shown that these networks can exhibit common fractal properties—including the property of being scale-free. This work attempts to bridge these two fields and demonstrate that the fractal properties of molecular networks are linked to the fractal properties of their underlying genomes.

Results: We suggest a stochastic model capable of describing the evolutionary growth of metabolic or signal-transduction networks. This model generates networks that share important statistical properties (so-called scale-free behavior) with real molecular networks. In particular, the frequency of vertices connected to exactly k other vertices follows a power-law distribution. The shape of this distribution remains invariant to changes in network scale: a small subgraph has the same distribution as the complete graph from which it is derived. Furthermore, the model correctly predicts that the frequencies of distinct DNA and protein domains also follow a power-law distribution. Finally, the model leads to a simple equation linking the total number of different DNA and protein domains in a genome with both the total number of genes and the overall network topology.

Availability: MatLab (MathWorks, Inc.) programs described in this manuscript are available on request from the authors.

Contact: ar345@columbia.edu

* To whom correspondence should be addressed.
A non-parametric approach to translating gene region
heterogeneity associated with phenotype into location heterogeneity
Authors: Kowalski Jeanne
Published: September 2002

Motivation: The analysis of genetic data poses statistical problems in the form of high dimensionality with small sample sizes. The construction of a composite gene region (sequence pair) heterogeneity measure is one technique for reducing the dimensionality of the problem. This approach however is not without cost, since the contribution of locations to observed gene region differences between groups becomes entangled in this summary measure. This is problematic since it is of scientific interest to identify locations that together depict phenotype.

Results: A method is proposed for relating observed gene region heterogeneity back to the location level. In the spirit of a factor analysis-type setting, the approach focuses on identifying a latent variable structure among locations to explain within and between group genetic differences associated with phenotype. The method is flexible for identifying either the additive contribution from individual locations or the additive contribution from a group of locations, to observed gene region heterogeneity, depending upon the weighting scheme used in constructing a gene region heterogeneity measure. The approach is illustrated with clinical trial data, where the problem of altered HIV drug susceptibility is examined through characterizing location contributions to HIV protease gene region differences associated with a phenotypic treatment response.

Availability: The Splus (MathSoft, Inc. S-Plus 2000, Seattle, WA, 1999) developed menu-driven functions for obtaining results, GENE_ S (J.Kowalski, Harvard School of Public Health, Boston, MA 2001), is available from the author upon request.
Evolutionary HMMs: a Bayesian approach to multiple alignment
Authors: Holmes Ian , Bruno William J.
Published: September 2002

Motivation: We review proposed syntheses of probabilistic sequence alignment, profiling and phylogeny. We develop a multiple alignment algorithm for Bayesian inference in the links model proposed by Thorne et al. (1991, J. Mol. Evol. , 33, 114–124). The algorithm, described in detail in Section 3, samples from and/or maximizes the posterior distribution over multiple alignments for any number of DNA or protein sequences, conditioned on a phylogenetic tree. The individual sampling and maximization steps of the algorithm require no more computational resources than pairwise alignment.

Methods: We present a software implementation (Handel) of our algorithm and report test results on (i) simulated data sets and (ii) the structurally informed protein alignments of BAliBASE (Thompson et al. , 1999, Nucleic Acids Res. , 27, 2682–2690).

Results: We find that the mean sum-of-pairs score (a measure of residue-pair correspondence) for the BAliBASE alignments is only 13% lower for Handelthan for CLUSTALW(Thompson et al. , 1994, Nucleic Acids Res. , 22, 4673–4680), despite the relative simplicity of the links model (CLUSTALW uses affine gap scores and increased penalties for indels in hydrophobic regions). With reference to these benchmarks, we discuss potential improvements to the links model and implications for Bayesian multiple alignment and phylogenetic profiling.

Availability: The source code to Handelis freely distributed on the Internet at http://www.biowiki.org/Handel under the terms of the GNU Public License (GPL, 2000, http://www.fsf.org./copyleft/gpl.html).

Contact: ihh@fruitfly.org