bluuebunny/crossref_metadata_2025 huggingface dataset:
Blarina toxin, a mammalian lethal venom from the short-tailed shrew
Blarina brevicauda
: Isolation and characterization
Authors: Kita Masaki , Nakamura Yasuo , Okumura Yuushi , Ohdachi Satoshi D. , Oba Yuichi , Yoshikuni Michiyasu , Kido Hiroshi , Uemura Daisuke
Published: May 2004

Venomous mammals are rare, and their venoms have not been characterized. We have purified and characterized the blarina toxin (BLTX), a lethal mammalian venom with a tissue kallikrein-like activity from the submaxillary and sublingual glands of the short-tailed shrew
Blarina brevicauda
. Mice administered BLTX i.p. developed irregular respiration, paralysis, and convulsions before dying. Based on the amino acid sequence of purified protein, we cloned the BLTX cDNA. It consists of a prosequence and an active form of 253 aa with a typical catalytic triad of serine proteases, with a high identity with tissue kallikreins. BLTX is an N-linked microheterogeneous glycoprotein with a unique insertion of 10 residues, L
106
TFFYKTFLG
115
. BLTX converted kininogens to kinins, which may be one of the toxic pathogens, and had dilatory effects on the blood vessel walls. The acute toxicity and proteolytic activity of BLTX were strongly inhibited by aprotinin, a kallikrein inhibitor, suggesting that its toxicity is due to a kallikrein-like activity of the venom.
Standardised coding of diet records: experiences from INTERMAP UK
Authors: Conway Rana , Robertson Claire , Dennis Barbara , Stamler Jeremiah , Elliott Paul ,   INTERMAP Research Group
Published: May 2004

Coding diet records is a basic element of most dietary surveys, yet it often receives little attention even though errors in coding can lead to flawed study results. In the INTERnational study of MAcro- and micronutrients and blood Pressure (INTERMAP study), efforts were made to minimise errors in coding the 18 720 diet records. Staff were centrally trained and certified before being able to process study data and ongoing quality control checks were performed. This involved the senior (site) nutritionist re-coding randomly selected diet records. To facilitate standardisation of coding in the UK, a code book was designed; it included information about coding brand items, density and portion size information, and default codes to be assigned when limited information was available for food items. It was found that trainees, despite previous experience in coding elsewhere, made coding errors that resulted in errors in estimates of daily energy and nutrient intakes. As training proceeded, the number of errors decreased. Compilation of the code book was labour-intensive, as information from food manufacturers and retailers had to be collected. Strategies are required to avoid repetition of this effort by other research groups. While the methods used in INTERMAP to reduce coding errors were time consuming, the experiences suggest that such errors are important and that they can be reduced.
Effect of exercise performed immediately before a meal of moderate fat content on postprandial lipaemia
Authors: Petridou Anatoli , Gerkos Nikos , Kolifa Myrsini , Nikolaidis Michalis G. , Simos Dimitris , Mougios Vassilis
Published: May 2004

The majority of the studies that have found a lowering effect of exercise on postprandial lipaemia have employed exercise 12–18 h before a test meal of exaggerated fat content (over 60 % total energy). The aim of the present study was to investigate whether this effect is manifest when exercise is performed immediately before a test meal of moderate fat content. Eleven healthy young men cycled for 45 min at 62 % maximal heart rate or rested, and, immediately afterwards, consumed a meal of moderate fat content (35 % total energy, 0·65 g/kg body mass) in a random counterbalanced design. Blood samples were drawn before exercise, before the meal, and for 8 h postprandially. No significant differences were observed in plasma triacylglycerol concentrations and areas under the triacylglycerol concentration v. time curves between exercise and rest, although exercise reduced the postprandial lipaemic response by 17 %. Non-esterified fatty acids, glucose, and insulin did not differ significantly between the trials. In conclusion, moderate exercise performed immediately before a meal of a fat content typical to the Western diet had only a modest effect on postprandial lipaemia.
Dietary repletion can replenish reduced T cell subset numbers and lymphoid organ weight in zinc-deficient and energy-restricted rats
Authors: Hosea Heather J. , Rector Edward S. , Taylor Carla G.
Published: May 2004

The objective of the present study was to investigate the time course for recovery of lymphoid tissue and T cell subset numbers when Zn-deficient (ZD) or energy-restricted (ER) rats were repleted with control diet; in a second experiment, the link between the stress axis and lymphoid organs was explored. During the deficiency phase, rats were fed a ZD (<1 mg Zn/kg) or control diet (30 mg Zn/kg, nutritionally complete) either as pair-fed controls (ER) or ad libitum-fed controls (CTL) for 3 weeks. During the repletion phase, all rats were fed control diet ad libitum for 3, 7 or 23 d. After the deficiency phase, ZD and ER had lower T cell subset numbers in the thymus compared with CTL, and ZD had reduced T cell subset numbers in the spleen compared with both ER and CTL. T cell subset numbers and lymphoid organ weights recovered from dietary Zn deficiency and energy restriction by 7 d of repletion (except 23 d for thymus weight in ZD), while body weight required more than 23 d for recovery. At the end of the deficiency phase, ZD and ER had higher circulating corticosterone concentrations compared with CTL; plasma TNFα was not detectable and there were no differences in plasma haptoglobin, an acute-phase protein. In conclusion, Zn deficiency and energy restriction elevated circulating corticosterone and reduced T cell subset numbers in the thymus and spleen of growing rats. Repletion with a nutritionally complete diet allowed recovery of T cell subset numbers and lymphoid organ weight.
Vinculin modulation of paxillin–FAK interactions regulates ERK to control survival and motility
Authors: Subauste M. Cecilia , Pertz Olivier , Adamson Eileen D. , Turner Christopher E. , Junger Sachiko , Hahn Klaus M.
Published: May 2004

Cells lacking vinculin are highly metastatic and motile. The reasons for this finding have remained unclear. Both enhanced survival and motility are critical to metastasis. Here, we show that vinculin null (vin−/−) cells and cells expressing a vinculin Y822F mutant have increased survival due to up-regulated activity of extracellular signal–regulated kinase (ERK). This increase is shown to result from vinculin's modulation of paxillin–FAK interactions. A vinculin fragment (amino acids 811–1066) containing the paxillin binding site restored apoptosis and suppressed ERK activity in vin−/− cells. Both vinY822F and vin−/− cells exhibit increased interaction between paxillin and focal adhesion kinase (FAK) and increased paxillin and FAK phosphorylation. Transfection with paxillin Y31FY118F dominant-negative mutant in these cells inhibits ERK activation and restores apoptosis. The enhanced motility of vin−/− and vinY822F cells is also shown to be due to a similar mechanism. Thus, vinculin regulates survival and motility via ERK by controlling the accessibility of paxillin for FAK interaction.
The Abl-related gene (Arg) requires its F-actin–microtubule cross-linking activity to regulate lamellipodial dynamics during fibroblast adhesion
Authors: Miller Ann L. , Wang Yinxiang , Mooseker Mark S. , Koleske Anthony J.
Published: May 2004

Microtubules (MTs) help establish and maintain cell polarity by promoting actin-dependent membrane protrusion at the leading edge of the cell, but the molecular mechanisms that mediate cross-talk between actin and MTs during this process are unclear. We demonstrate that the Abl-related gene (Arg) nonreceptor tyrosine kinase is required for dynamic lamellipodial protrusions after adhesion to fibronectin. arg−/− fibroblasts exhibit reduced lamellipodial dynamics as compared with wild-type fibroblasts, and this defect can be rescued by reexpression of an Arg-yellow fluorescent protein fusion. We show that Arg can bind MTs with high affinity and cross-link filamentous actin (F-actin) bundles and MTs in vitro. MTs concentrate and insert into Arg-induced F-actin–rich cell protrusions. Arg requires both its F-actin–binding domains and its MT-binding domain to rescue the defects in lamellipodial dynamics of arg−/− fibroblasts. These findings demonstrate that Arg can mediate physical contact between F-actin and MTs at the cell periphery and that this cross-linking activity is required for Arg to regulate lamellipodial dynamics in fibroblasts.
Regulation of cell migration and survival by focal adhesion targeting of Lasp-1
Authors: Lin Yi Hsing , Park Zee-Yong , Lin Dayin , Brahmbhatt Anar A. , Rio Marie-Christine , Yates John R. , Klemke Richard L.
Published: May 2004

Large-scale proteomic and functional analysis of isolated pseudopodia revealed the Lim, actin, and SH3 domain protein (Lasp-1) as a novel protein necessary for cell migration, but not adhesion to, the extracellular matrix (ECM). Lasp-1 is a ubiquitously expressed actin-binding protein with a unique domain configuration containing SH3 and LIM domains, and is overexpressed in 8–12% of human breast cancers. We find that stimulation of nonmotile and quiescent cells with growth factors or ECM proteins facilitates Lasp-1 relocalization from the cell periphery to the leading edge of the pseudopodium, where it associates with nascent focal complexes and areas of actin polymerization. Interestingly, although Lasp-1 dynamics in migratory cells occur independently of c-Abl kinase activity and tyrosine phosphorylation, c-Abl activation by apoptotic agents specifically promotes phosphorylation of Lasp-1 at tyrosine 171, which is associated with the loss of Lasp-1 localization to focal adhesions and induction of cell death. Thus, Lasp-1 is a dynamic focal adhesion protein necessary for cell migration and survival in response to growth factors and ECM proteins.
atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins
Authors: Smith Matthew D. , Rounds Caleb M. , Wang Fei , Chen Kunhua , Afitlhile Meshack , Schnell Danny J.
Published: May 2004

The members of the Toc159 family of GTPases act as the primary receptors for the import of nucleus-encoded preproteins into plastids. Toc159, the most abundant member of this family in chloroplasts, is required for chloroplast biogenesis (Bauer, J., K. Chen, A. Hiltbunner, E. Wehrli, M. Eugster, D. Schnell, and F. Kessler. 2000. Nature. 403:203–207) and has been shown to covalently cross-link to bound preproteins at the chloroplast surface (Ma, Y., A. Kouranov, S. LaSala, and D.J. Schnell. 1996. J. Cell Biol. 134:1–13; Perry, S.E., and K. Keegstra. 1994. Plant Cell. 6:93–105). These reports led to the hypothesis that Toc159 functions as a selective import receptor for preproteins that are required for chloroplast development. In this report, we provide evidence that Toc159 is required for the import of several highly expressed photosynthetic preproteins in vivo. Furthermore, we demonstrate that the cytoplasmic and recombinant forms of soluble Toc159 bind directly and selectively to the transit peptides of these representative photosynthetic preproteins, but not representative constitutively expressed plastid preproteins. These data support the function of Toc159 as a selective import receptor for the targeting of a set of preproteins required for chloroplast biogenesis.
Golgi duplication in Trypanosoma brucei
Authors: He Cynthia Y. , Ho Helen H. , Malsam Joerg , Chalouni Cecile , West Christopher M. , Ullu Elisabetta , Toomre Derek , Warren Graham
Published: May 2004

Duplication of the single Golgi apparatus in the protozoan parasite Trypanosoma brucei has been followed by tagging a putative Golgi enzyme and a matrix protein with variants of GFP. Video microscopy shows that the new Golgi appears de novo, near to the old Golgi, about two hours into the cell cycle and grows over a two-hour period until it is the same size as the old Golgi. Duplication of the endoplasmic reticulum (ER) export site follows exactly the same time course. Photobleaching experiments show that the new Golgi is not the exclusive product of the new ER export site. Rather, it is supplied, at least in part, by material directly from the old Golgi. Pharmacological experiments show that the site of the new Golgi and ER export is determined by the location of the new basal body.
Surface Sialic Acids Taken from the Host Allow Trypanosome Survival in Tsetse Fly Vectors
Authors: Nagamune Kisaburo , Acosta-Serrano Alvaro , Uemura Haruki , Brun Reto , Kunz-Renggli Christina , Maeda Yusuke , Ferguson Michael A.J. , Kinoshita Taroh
Published: May 2004

The African trypanosome Trypanosoma brucei, which causes sleeping sickness in humans and Nagana disease in livestock, is spread via blood-sucking Tsetse flies. In the fly's intestine, the trypanosomes survive digestive and trypanocidal environments, proliferate, and translocate into the salivary gland, where they become infectious to the next mammalian host. Here, we show that for successful survival in Tsetse flies, the trypanosomes use trans-sialidase to transfer sialic acids that they cannot synthesize from host's glycoconjugates to the glycosylphosphatidylinositols (GPIs), which are abundantly expressed on their surface. Trypanosomes lacking sialic acids due to a defective generation of GPI-anchored trans-sialidase could not survive in the intestine, but regained the ability to survive when sialylated by means of soluble trans-sialidase. Thus, surface sialic acids appear to protect the parasites from the digestive and trypanocidal environments in the midgut of Tsetse flies.
KLF2 Is a Novel Transcriptional Regulator of Endothelial Proinflammatory Activation
Authors: SenBanerjee Sucharita , Lin Zhiyong , Atkins G. Brandon , Greif Daniel M. , Rao Ravi M. , Kumar Ajay , Feinberg Mark W. , Chen Zhiping , Simon Daniel I. , Luscinskas F. William , Michel Thomas M. , Gimbrone Michael A. , García-Cardeña Guillermo , Jain Mukesh K.
Published: May 2004

The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1β and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element–binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli.
Increased Expression of Human T Lymphocyte Virus Type I (HTLV-I) Tax11-19 Peptide–Human Histocompatibility Leukocyte Antigen A*201 Complexes on CD4+ CD25+T Cells Detected by Peptide-specific, Major Histocompatibility Complex–restricted Antibodies in Patients with HTLV-I–associated Neurologic Disease
Authors: Yamano Yoshihisa , Cohen Cyril J. , Takenouchi Norihiro , Yao Karen , Tomaru Utano , Li Hong-Chuan , Reiter Yoram , Jacobson Steven
Published: May 2004

Human T lymphocyte virus type I (HTLV-I)–associated chronic inflammatory neurological disease (HTLV-I–associated myelopathy/tropical spastic paraparesis [HAM/TSP]) is suggested to be an immunopathologically mediated disorder characterized by large numbers of HTLV-I Tax–specific CD8+ T cells. The frequency of these cells in the peripheral blood and cerebrospinal fluid is proportional to the amount of HTLV-I proviral load and the levels of HTLV-I tax mRNA expression. As the stimulus for these virus-specific T cells are immunodominant peptide–human histocompatibility leukocyte antigen (HLA) complexes expressed on antigen-presenting cells, it was of interest to determine which cells express these complexes and at what frequency. However, until now, it has not been possible to identify and/or quantify these peptide–HLA complexes. Using a recently developed antibody that specifically recognizes Tax11-19 peptide–HLA-A*201 complexes, the level of Tax11-19–HLA-A*201 expression on T cells was demonstrated to be increased in HAM/TSP and correlated with HTLV-I proviral DNA load, HTLV-I tax mRNA load, and HTLV-I Tax–specific CD8+ T cell frequencies. Furthermore, CD4+ CD25+ T cells were demonstrated to be the major reservoir of HTLV-I provirus as well as Tax11-19 peptide–HLA-A*201 complexes. These results indicate that the increased detection and visualization of peptide–HLA complexes in HAM/TSP CD4+ CD25+ T cell subsets that are shown to stimulate and expand HTLV-I Tax–specific CD8+ T cells may play an important role in the pathogenesis of HTLV-I–associated neurological disease.
DNAM-1 and PVR Regulate Monocyte Migration through Endothelial Junctions
Authors: Reymond Nicolas , Imbert Anne-Marie , Devilard Elisabeth , Fabre Stéphanie , Chabannon Christian , Xerri Luc , Farnarier Catherine , Cantoni Claudia , Bottino Cristina , Moretta Alessandro , Dubreuil Patrice , Lopez Marc
Published: May 2004

DNAX accessory molecule 1 (DNAM-1; CD226) is a transmembrane glycoprotein involved in T cell and natural killer (NK) cell cytotoxicity. We demonstrated recently that DNAM-1 triggers NK cell–mediated killing of tumor cells upon engagement by its two ligands, poliovirus receptor (PVR; CD155) and Nectin-2 (CD112). In the present paper, we show that PVR and Nectin-2 are expressed at cell junctions on primary vascular endothelial cells. Moreover, the specific binding of a soluble DNAM-1–Fc molecule was detected at endothelial junctions. This binding was almost completely abrogated by anti-PVR monoclonal antibodies (mAbs), but not modified by anti–Nectin-2 mAbs, which demonstrates that PVR is the major DNAM-1 ligand on endothelial cells. Because DNAM-1 is highly expressed on leukocytes, we investigated the role of the DNAM-1–PVR interaction during the monocyte transendothelial migration process. In vitro, both anti–DNAM-1 and anti-PVR mAbs strongly blocked the transmigration of monocytes through the endothelium. Moreover, after anti–DNAM-1 or anti-PVR mAb treatment, monocytes were arrested at the apical surface of the endothelium over intercellular junctions, which strongly suggests that the DNAM-1–PVR interaction occurs during the diapedesis step. Altogether, our results demonstrate that DNAM-1 regulates monocyte extravasation via its interaction with PVR expressed at endothelial junctions on normal cells.
Exclusion of Lipid Rafts and Decreased Mobility of CD94/NKG2A Receptors at the Inhibitory NK Cell Synapse
Authors: Sanni Tolib B. , Masilamani Madhan , Kabat Juraj , Coligan John E. , Borrego Francisco
Published: May 2004

CD94/NKG2A is an inhibitory receptor expressed by most human natural killer (NK) cells and a subset of T cells that recognizes human leukocyte antigen E (HLA-E) on potential target cells. To elucidate the cell surface dynamics of CD94/NKG2A receptors, we have expressed CD94/NKG2A-EGFP receptors in the rat basophilic leukemia (RBL) cell line. Photobleaching experiments revealed that CD94/NKG2A-EGFP receptors move freely within the plasma membrane and accumulate at the site of contact with ligand. The enriched CD94/NKG2A-EGFP is markedly less mobile than the nonligated receptor. We observed that not only are lipid rafts not required for receptor polarization, they are excluded from the site of receptor contact with the ligand. Furthermore, the lipid raft patches normally observed at the sites where FcϵR1 activation receptors are cross-linked were not observed when CD94/NKG2A was coengaged along with the activation receptor. These results suggest that immobilization of the CD94/NKG2A receptors at ligation sites not only promote sustenance of the inhibitory signal, but by lipid rafts exclusion prevent formation of activation signaling complexes.
ArabidopsisImmunophilin-like TWD1 Functionally Interacts with Vacuolar ABC Transporters
Authors: Geisler Markus , Girin Marjolaine , Brandt Sabine , Vincenzetti Vincent , Plaza Sonia , Paris Nadine , Kobae Yoshihiro , Maeshima Masayoshi , Billion Karla , Kolukisaoglu Üner H. , Schulz Burkhard , Martinoia Enrico
Published: May 2004

Previously, the immunophilin-like protein TWD1 from Arabidopsis has been demonstrated to interact with the ABC transporters AtPGP1 and its closest homologue, AtPGP19. Physiological and biochemical investigation of pgp1/pgp19 and of twd1 plants suggested a regulatory role of TWD1 on AtPGP1/AtPGP19 transport activities. To further understand the dramatic pleiotropic phenotype that is caused by loss-of-function mutation of the TWD1 gene, we were interested in other TWD1 interacting proteins. AtMRP1, a multidrug resistance-associated (MRP/ABCC)-like ABC transporter, has been isolated in a yeast two-hybrid screen. We demonstrate molecular interaction between TWD1 and ABC transporters AtMRP1 and its closest homologue, AtMRP2. Unlike AtPGP1, AtMRP1 binds to the C-terminal tetratricopeptide repeat domain of TWD1, which is well known to mediate protein-protein interactions. Domain mapping proved that TWD1 binds to a motif of AtMRP1 that resembles calmodulin-binding motifs; and calmodulin binding to the C-terminus of MRP1 was verified. By membrane fractionation and GFP-tagging, we localized AtMRP1 to the central vacuolar membrane and the TWD1-AtMRP1 complex was verified in vivo by coimmunoprecipitation. We were able to demonstrate that TWD1 binds to isolated vacuoles and has a significant impact on the uptake of metolachlor-GS and estradiol-β-glucuronide, well-known substrates of vacuolar transporters AtMRP1 and AtMRP2.
IRI-1, a LIN-15B Homologue, Interacts with Inositol-1,4,5-Triphosphate Receptors and Regulates Gonadogenesis, Defecation, and Pharyngeal Pumping inCaenorhabditis elegans
Authors: Walker Denise S. , Ly Sung , Gower Nicholas J.D. , Baylis Howard A.
Published: May 2004

Inositol-1,4,5-triphosphate receptors (IP3Rs) are ligand-gated Ca2+channels that control Ca2+release from intracellular stores. They are central to a wide range of cellular responses. IP3Rs in Caenorhabditis elegans are encoded by a single gene, itr-1, and are widely expressed. Signaling through IP3and IP3Rs is important in ovulation, control of the defecation cycle, modulation of pharyngeal pumping rate, and embryogenesis. To further elucidate the molecular basis of the diversity of IP3R function, we used a yeast two-hybrid screen to search for proteins that interact with ITR-1. We identified an interaction between ITR-1 and IRI-1, a previously uncharacterized protein with homology to LIN-15B. Iri-1 is widely expressed, and its expression overlaps significantly with that of itr-1. In agreement with this observation, iri-1 functions in known itr-1-mediated processes, namely, upregulation of pharyngeal pumping in response to food and control of the defecation cycle. Knockdown of iri-1 in an itr-1 loss-of-function mutant potentiates some of these effects and sheds light on the signaling pathways that control pharyngeal pumping rate. Knockdown of iri-1 expression also results in a sterile, evl phenotype, as a consequence of failures in early Z1/Z4 lineage divisions, such that gonadogenesis is severely disrupted.
Use of Fluorescent Protein Tags to Study Nuclear Organization of the Spliceosomal Machinery in Transiently Transformed Living Plant Cells
Authors: Lorković Zdravko J. , Hilscher Julia , Barta Andrea
Published: May 2004

Although early studies suggested that little compartmentalization exists within the nucleus, more recent studies on metazoan systems have identified a still increasing number of specific subnuclear compartments. Some of these compartments are dynamic structures; indeed, protein and RNA-protein components can cycle between different domains. This is particularly evident for RNA processing components. In plants, lack of tools has hampered studies on nuclear compartmentalization and dynamics of RNA processing components. Here, we show that transient expression of fluorescent protein fusions of U1 and U2 small nuclear ribonucleoprotein particle (snRNP)-specific proteins U1-70K, U2B″, and U2A ′, nucleolar proteins Nop10 and PRH75, and serine-arginine-rich proteins in plant protoplasts results in their correct localization. Furthermore, snRNP-specific proteins also were correctly assembled into mature snRNPs. This system allowed a systematic analysis of the cellular localization of Arabidopsis serine-arginine-rich proteins, which, like their animal counterparts, localize to speckles but not to nucleoli and Cajal bodies. Finally, markers for three different nuclear compartments, namely, nucleoli, Cajal bodies, and speckles, have been established and were shown to be applicable for colocalization studies in living plant protoplasts. Thus, transient expression of proteins tagged with four different fluorescent proteins is a suitable system for studying the nuclear organization of spliceosomal proteins in living plant cells and should therefore allow studies of their dynamics as well.
SGEF, a RhoG Guanine Nucleotide Exchange Factor that Stimulates Macropinocytosis
Authors: Ellerbroek Shawn M. , Wennerberg Krister , Arthur William T. , Dunty Jill M. , Bowman Dan R. , DeMali Kris A. , Der Channing , Burridge Keith
Published: May 2004

SGEF (SH3-containing Guanine Nucleotide Exchange Factor) is a RhoGEF of unknown function. We found the SGEF protein to be expressed in many established cell lines and highly expressed in human liver tissue. SGEF stimulated the formation of large interconnected membrane ruffles across dorsal surfaces when expressed in fibroblasts. SGEF required its proline-rich amino-terminus to generate dorsal, but not lateral, membrane ruffles and a functional SH3 domain to colocalize with filamentous actin at sites of membrane protrusion. Full-length SGEF activated RhoG, but not Rac, when expressed in fibroblasts. Further, recombinant SGEF DH/PH protein exchanged nucleotide on RhoG, but not on Rac1 or Rac3, in vitro. Scanning electron microscopy of fibroblasts demonstrated that SGEF induced dorsal ruffles that were morphologically similar to those generated by constitutively active RhoG, but not constitutively active Rac1. Transient expression of SGEF stimulated fibroblast uptake of 10-kDa dextran, a marker of macropinocytosis. This required the full-length protein and a catalytically active DH domain. Finally, activated RhoG was found to be more effective than activated Rac, and comparable to SGEF, in its ability to trigger dextran uptake. Together, this work establishes SGEF as a RhoG exchange factor and provides evidence that both SGEF and RhoG regulate membrane dynamics in promotion of macropinocytosis.
Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals
Authors: Rottensteiner Hanspeter , Kramer Achim , Lorenzen Stephan , Stein Katharina , Landgraf Christiane , Volkmer-Engert Rudolf , Erdmann Ralf
Published: May 2004

Targeting of peroxisomal membrane proteins (PMPs) is a multistep process that requires not only recognition of PMPs in the cytosol but also their insertion into the peroxisomal membrane. As a consequence, targeting signals of PMPs (mPTS) are rather complex. A candidate protein for the PMP recognition event is Pex19p, which interacts with most PMPs. However, the respective Pex19p-binding sites are ill-defined and it is currently disputed whether these sites are contained within mPTS. By using synthetic peptide scans and yeast two-hybrid analyses, we determined and characterized Pex19p-binding sites in Pex11p and Pex13p, two PMPs from Saccharomyces cerevisiae. The sites turned out to be composed of a short helical motif with a minimal length of 11 amino acids. With the acquired data, it proved possible to predict and experimentally verify Pex19p-binding sites in several other PMPs by applying a pattern search and a prediction matrix. A peroxisomally targeted Pex13p fragment became mislocalized to the endoplasmic reticulum in the absence of its Pex19p-binding site. By adding the heterologous binding site of Pex11p, peroxisomal targeting of the Pex13p fragment was restored. We conclude that Pex19p-binding sites are well-defined entities that represent an essential part of the mPTS.
Na,K-ATPase β1-Subunit Increases the Translation Efficiency of the α1-Subunit in MSV-MDCK Cells
Authors: Rajasekaran Sigrid A. , Gopal Jegan , Willis Dianna , Espineda Cromwell , Twiss Jeffery L. , Rajasekaran Ayyappan K.
Published: May 2004

The Na,K-ATPase consists of an α- and β-subunit. Moloney sarcoma virus-transformed MDCK cells (MSV-MDCK) express low levels of Na,K-ATPase β1-subunit. Ectopic expression of Na,K-ATPase β1-subunit in these cells increased the protein levels of the α1-subunit of Na,K-ATPase. This increase was not due to altered transcription of the α1-subunit gene or half-life of the α1-subunit protein because both α1-subunit mRNA levels and half-life of the α1-subunit protein were comparable in MSV-MDCK and β1-subunit expressing MSV-MDCK cells. However, short pulse labeling revealed that the initial translation rate of the α1-subunit in β1-subunit expressing MSV-MDCK cells was six- to sevenfold higher compared with MSV-MDCK cells. The increased translation was specific to α1-subunit because translation rates of occludin and β-catenin, membrane and cytosolic proteins, respectively, were not altered. In vitro cotranslation/translocation experiments using rabbit reticulocyte lysate and rough microsomes revealed that the α1-subunit mRNA is more efficiently translated in the presence of β1-subunit. Furthermore, sucrose density gradient analysis revealed significantly more α1-subunit transcript associated with the polysomal fraction in β1-subunit expressing MSV-MDCK cells compared with MSV-MDCK cells, indicating that in mammalian cells the Na,K-ATPase β1-subunit is involved in facilitating the translation of the α1-subunit mRNA in the endoplasmic reticulum.
Clathrin Isoform CHC22, a Component of Neuromuscular and Myotendinous Junctions, Binds Sorting Nexin 5 and Has Increased Expression during Myogenesis and Muscle Regeneration
Authors: Towler Mhairi C. , Gleeson Paul A. , Hoshino Sachiko , Rahkila Paavo , Manalo Venus , Ohkoshi Norio , Ordahl Charles , Parton Robert G. , Brodsky Frances M.
Published: May 2004

The muscle isoform of clathrin heavy chain, CHC22, has 85% sequence identity to the ubiquitously expressed CHC17, yet its expression pattern and function appear to be distinct from those of well-characterized clathrin-coated vesicles. In mature muscle CHC22 is preferentially concentrated at neuromuscular and myotendinous junctions, suggesting a role at sarcolemmal contacts with extracellular matrix. During myoblast differentiation, CHC22 expression is increased, initially localized with desmin and nestin and then preferentially segregated to the poles of fused myoblasts. CHC22 expression is also increased in regenerating muscle fibers with the same time course as embryonic myosin, indicating a role in muscle repair. CHC22 binds to sorting nexin 5 through a coiled-coil domain present in both partners, which is absent in CHC17 and coincides with the region on CHC17 that binds the regulatory light-chain subunit. These differential binding data suggest a mechanism for the distinct functions of CHC22 relative to CHC17 in membrane traffic during muscle development, repair, and at neuromuscular and myotendinous junctions.
Gene co-expression network topology provides a framework for molecular characterization of cellular state
Authors: Carter Scott L. , Brechbühler Christian M. , Griffin Michael , Bond Andrew T.
Published: May 2004

Motivation: Gene expression data have become an instrumental resource in describing the molecular state associated with various cellular phenotypes and responses to environmental perturbations. The utility of expression profiling has been demonstrated in partitioning clinical states, predicting the class of unknown samples and in assigning putative functional roles to previously uncharacterized genes based on profile similarity. However, gene expression profiling has had only limited success in identifying therapeutic targets. This is partly due to the fact that current methods based on fold-change focus only on single genes in isolation, and thus cannot convey causal information. In this paper, we present a technique for analysis of expression data in a graph-theoretic framework that relies on associations between genes. We describe the global organization of these networks and biological correlates of their structure. We go on to present a novel technique for the molecular characterization of disparate cellular states that adds a new dimension to the fold-based methods and conclude with an example application to a human medulloblastoma dataset.

Results: We have shown that expression networks generated from large model-organism expression datasets are scale-free and that the average clustering coefficient of these networks is several orders of magnitude higher than would be expected for similarly sized scale-free networks, suggesting an inherent hierarchical modularity similar to that previously identified in other biological networks. Furthermore, we have shown that these properties are robust with respect to the parameters of network construction. We have demonstrated an enrichment of genes having lethal knockout phenotypes in the high-degree (i.e. hub) nodes in networks generated from aggregate condition datasets; using process-focused Saccharomyces cerivisiae datasets we have demonstrated additional high-degree enrichments of condition-specific genes encoding proteins known to be involved in or important for the processes interrogated by the microarrays. These results demonstrate the utility of network analysis applied to expression data in identifying genes that are regulated in a state-specific manner. We concluded by showing that a sample application to a human clinical dataset prominently identified a known therapeutic target.

Availability: Software implementing the methods for network generation presented in this paper is available for academic use by request from the authors in the form of compiled linux binary executables.
Analyzing time series gene expression data
Authors: Bar-Joseph Ziv
Published: May 2004

Motivation: Time series expression experiments are an increasingly popular method for studying a wide range of biological systems. However, when analyzing these experiments researchers face many new computational challenges. Algorithms that are specifically designed for time series experiments are required so that we can take advantage of their unique features (such as the ability to infer causality from the temporal response pattern) and address the unique problems they raise (e.g. handling the different non-uniform sampling rates).

Results: We present a comprehensive review of the current research in time series expression data analysis. We divide the computational challenges into four analysis levels: experimental design, data analysis, pattern recognition and networks. For each of these levels, we discuss computational and biological problems at that level and point out some of the methods that have been proposed to deal with these issues. Many open problems in all these levels are discussed. This review is intended to serve as both, a point of reference for experimental biologists looking for practical solutions for analyzing their data, and a starting point for computer scientists interested in working on the computational problems related to time series expression analysis.
Incremental generation of summarized clustering hierarchy for protein family analysis
Authors: Chen Chien-Yu , Oyang Yen-Jen , Juan Hsueh-Fen
Published: May 2004

Motivation: Protein sequence clustering has been widely exploited to facilitate in-depth analysis of protein functions and families. For some applications of protein sequence clustering, it is highly desirable that a hierarchical structure, also referred to as dendrogram, which shows how proteins are clustered at various levels, is generated. However, as the sizes of contemporary protein databases continue to grow at rapid rates, it is of great interest to develop some summarization mechanisms so that the users can browse the dendrogram and/or search for the desired information more effectively.

Results: In this paper, the design of a novel incremental clustering algorithm aimed at generating summarized dendrograms for analysis of protein databases is described. The proposed incremental clustering algorithm employs a statistics-based model to summarize the distributions of the similarity scores among the proteins in the database and to control formation of clusters. Experimental results reveal that, due to the summarization mechanism incorporated, the proposed incremental clustering algorithm offers the users highly concise dendrograms for analysis of protein clusters with biological significance. Another distinction of the proposed algorithm is its incremental nature. As the sizes of the contemporary protein databases continue to grow at fast rates, due to the concern of efficiency, it is desirable that cluster analysis of a protein database can be carried out incrementally, when the protein database is updated. Experimental results with the Swiss-Prot protein database reveal that the time complexity for carrying out incremental clustering with k new proteins added into the database containing n proteins is O(n2βlogn), where β ≅ 0.865, provided that k ≪ n.

Availability: The Linux executable is available on the following supplementary page.

Supplementary information:  http://mars.csie.ntu.edu.tw/~cychen/protein_clustering/psc.htm
Distribution of information in biomedical abstracts and full-text publications
Authors: Schuemie M. J. , Weeber M. , Schijvenaars B. J. A. , van Mulligen E. M. , van der Eijk C. C. , Jelier R. , Mons B. , Kors J. A.
Published: May 2004

Motivation: Full-text documents potentially hold more information than their abstracts, but require more resources for processing. We investigated the added value of full text over abstracts in terms of information content and occurrences of gene symbol—gene name combinations that can resolve gene-symbol ambiguity.

Results: We analyzed a set of 3902 biomedical full-text articles. Different keyword measures indicate that information density is highest in abstracts, but that the information coverage in full texts is much greater than in abstracts. Analysis of five different standard sections of articles shows that the highest information coverage is located in the results section. Still, 30–40% of the information mentioned in each section is unique to that section. Only 30% of the gene symbols in the abstract are accompanied by their corresponding names, and a further 8% of the gene names are found in the full text. In the full text, only 18% of the gene symbols are accompanied by their gene names.
GO-Mapper: functional analysis of gene expression data using the expression level as a score to evaluate Gene Ontology terms
Authors: Smid Marcel , Dorssers Lambert C. J.
Published: May 2004

Motivation: Retrieval of information on biological processes from large-scale expression data is still a time-consuming task. An automated analysis utilizing all expression information would greatly increase our understanding of the samples under study.

Results: We describe here a novel method to obtain a functional analysis of complex gene expression data. Instead of applying a predefined expression threshold, Gene Ontology (GO) terms are weighted using the actual measured levels of expression of all associated genes. Based on this concept, the application GO-Mapper was developed to quantitatively link gene expression levels to GO-terms for multiple experiments in an automated way. The applicability of GO-Mapper was developed and validated on in house and public human microarray data and mouse SAGE data. We demonstrate that the GO-Mapper allows for interrelating relevant biological functions with the experiments under study.

Availability: The GO-Mapper application is free of charge available from our website.

Supplementary information:  http://www.erasmusmc.nl/gatcplatform
THEA: ontology-driven analysis of microarray data
Authors: Pasquier C. , Girardot F. , Jevardat de Fombelle K. , Christen R.
Published: May 2004

Motivation: Microarray technology makes it possible to measure thousands of variables and to compare their values under hundreds of conditions. Once microarray data are quantified, normalized and classified, the analysis phase is essentially a manual and subjective task based on visual inspection of classes in the light of the vast amount of information available. Currently, data interpretation clearly constitutes the bottleneck of such analyses and there is an obvious need for tools able to fill the gap between data processed with mathematical methods and existing biological knowledge.

Results: THEA (Tools for High-throughput Experiments Analysis) is an integrated information processing system allowing convenient handling of data. It allows to automatically annotate data issued from classification systems with selected biological information coming from a knowledge base and to either manually search and browse through these annotations or automatically generate meaningful generalizations according to statistical criteria (data mining).

Availability: The software is available on the website http://thea.unice.fr/

Supplementary information: Supplementary tables as well as files containing the biological data used in this publication can be downloaded from our website http://bioinfo.unice.fr/publications/thea_article/
Eigenvalue analysis of amino acid substitution matrices reveals a sharp transition of the mode of sequence conservation in proteins
Authors: Kinjo Akira R. , Nishikawa Ken
Published: May 2004

Summary: The pattern of amino acid substitutions and sequence conservation over many structure-based alignments of protein sequences was analyzed as a function of percentage sequence identity. The statistics of the amino acid substitutions were converted into the form of log-odds amino acid substitution matrices to which eigenvalue decomposition was applied. It was found that the most important component of the substitution matrices exhibited a sharp transition at the sequence identity of 30–35%, which coincides with the twilight zone. Above the transition point, the most dominant component is related to the mutability of amino acids and it acts to disfavor any substitutions, whereas below the transition point, the most dominant component is related to the hydrophobicity of amino acids and substitutions between residues of similar hydrophobic character are positively favored. Implications for protein evolution and sequence analysis are discussed.

Supplementary information:  http://maccl01.genes.nig.ac.jp/~akinjo/aasm/
A collaborative database of inbred mouse strain characteristics
Authors: Grubb Stephen C. , Churchill Gary A. , Bogue Molly A.
Published: May 2004

Summary: A database and website (MPD: Mouse Phenome Database) have been developed to serve as a consolidated home for mouse strain characterization data being generated by the scientific community. Physiological, anatomical and behavioral data are being collected and integrated into a common framework for tabulation by strain and sex. Genotypic data are being collected as well. The current focus is on a set of 40 inbred strains. The MPD as of February 2004 contains approximately 500 phenotypic parameters relevant to human health, voluntarily contributed by several dozen investigators and laboratories.

Availability:  http://www.jax.org/phenome

Supplementary information: Guidelines for investigators who may be interested in contributing data are available at http://aretha.jax.org/phenome/dataguide.html
Lucy2: an interactive DNA sequence quality trimming and vector removal tool
Authors: Li Song , Chou Hui-Hsien
Published: May 2004

Summary: Lucy2 is a raw DNA sequence trimming and visualization tool based on the popular command-line Lucy1. Users can change parameters, trim multiple sequences and visualize the results within an integrated, easy-to-use graphical user interface. Lucy2 is designed specifically for non-programmers to use, and is currently available on Windows, Linux and MacOS X. Source code is also available for porting to the other platforms.

Availability: Lucy2 is distributed under the GNU General Public License and can be downloaded from www.complex.iastate.edu
WebSIDD: server for predicting stress-induced duplex destabilized (SIDD) sites in superhelical DNA
Authors: Bi Chengpeng , Benham Craig J.
Published: May 2004

Summary: WebSIDD is a Web-based service designed to predict locations and extents of stress-induced duplex destabilization (SIDD) that occur in a double-stranded DNA molecule of specified base sequence, on which a specified level of superhelical stress is imposed. The algorithm calculates the approximate equilibrium statistical mechanical distribution of a population of identical molecules among its accessible states. The user inputs the DNA sequence, and the program outputs the calculated transition probability and destabilization energy of each base pair in the sequence. As options, the user can specify the temperature and the level of superhelicity. The values of all structural and energy parameters used in the calculation have been experimentally measured. WebSIDD should prove useful for finding SIDD-susceptible sites in genomic sequences, and correlating their occurrence with locations involved in regulatory and pathological processes. This strategy already has illuminated the roles of SIDD in diverse biological regulatory processes, including transcriptional initiation and termination, and the eukaryotic nuclear scaffold attachments that partition chromosomes into domains.

Availability:  http://orange.genomecenter.ucdavis.edu/benham/sidd/index.html