bluuebunny/crossref_metadata_2025 huggingface dataset:
A photocleavable fluorescent nucleotide for DNA sequencing and analysis
Authors: Li Zengmin , Bai Xiaopeng , Ruparel Hameer , Kim Sobin , Turro Nicholas J. , Ju Jingyue
Published: January 2003

DNA sequencing by synthesis during a polymerase reaction using laser-induced fluorescence detection is an approach that has a great potential to increase the throughput and data quality of DNA sequencing. We report the design and synthesis of a photocleavable fluorescent nucleoside triphosphate, one of the essential molecules required for the sequencing-by-synthesis approach. We synthesized this nucleoside triphosphate by attaching a fluorophore, 4,4-difluoro-5,7-dimethyl-4-bora-3α,4α-diaza-
s
-indacene propionic acid (BODIPY), to the 5 position of 2′-deoxyuridine triphosphate via a photocleavable 2-nitrobenzyl linker. We demonstrate that the nucleotide analogue can be faithfully incorporated by a DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA-sequencing reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. These results indicate that the nucleotide analogue is an excellent substrate for Thermo Sequenase. We also systematically studied the photocleavage of the fluorescent dye from a DNA molecule that contained the nucleotide analogue. UV irradiation at 340 nm of the DNA molecule led to the efficient release of the fluorescent dye, ensuring that a previous fluorescence signal did not leave any residue that could interfere with the detection of the next nucleotide. Thus, our results indicate that it should be feasible to use four different fluorescent dyes with distinct fluorescence emissions as unique tags to label the four nucleotides (A, C, G, and T) through the photocleavable 2-nitrobenzyl linker. These fluorescent tags can be removed easily by photocleavage after the identification of each nucleotide in the DNA sequencing-by-synthesis approach.
Molecular pathogenesis of inherited hypertension with hyperkalemia: The Na–Cl cotransporter is inhibited by wild-type but not mutant WNK4
Authors: Wilson Frederick H. , Kahle Kristopher T. , Sabath Ernesto , Lalioti Maria D. , Rapson Alicia K. , Hoover Robert S. , Hebert Steven C. , Gamba Gerardo , Lifton Richard P.
Published: January 2003

Mutations in the serine-threonine kinases
WNK1
and
WNK4
[
w
ith
n
o lysine (
K
) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl
−
dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a “mirror image” of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na–Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated Na
+
influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in
Xenopus
oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter (
22
Na-influx reduced 50%,
P
< 1 × 10
−9
, surface expression reduced 75%,
P
< 1 × 10
−14
in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.
Estrogen receptor (ER)β knockout mice reveal a role for ERβ in migration of cortical neurons in the developing brain
Authors: Wang Ling , Andersson Sandra , Warner Margaret , Gustafsson Jan-Åke
Published: January 2003

The present study stems from our previous observations that the brains of adult estrogen receptor β knockout (ERβ−/−) mice show regional neuronal hypocellularity especially in the cerebral cortex. We now show that ERβ is necessary for late embryonic development of the brain and is involved in both neuronal migration and apoptosis. At embryonic day (E)18.5, ERβ−/− mouse brains were smaller than those of the wild-type (WT) littermates, and there were fewer neurons in the cortex. There were no differences in size or cellularity at E14.5. When proliferating cells were labeled with 5′-bromodeoxyuridine (BrdUrd) on E12.5, a time when cortical neurogenesis in mice begins, and examined on E14.5, there was no difference between WT and ERβ−/− mice in the number of labeled cells in the cortex. However, when BrdUrd was administered between E14.5 and E16.5, a time when postmitotic neurons migrate to layers of the cortex, there were fewer BrdUrd-labeled cells in the superficial cortical layers by E18.5 and postnatal day 14 in mice lacking ERβ. At E18.5, there were more apoptotic cells in the ventricular zone of mice lacking ERβ. In addition, the processes of the cortical radial glia, which are essential for guiding the migrating neurons, were fragmented. These findings suggest that by influencing migration and neuronal survival, ERβ has an important role in brain development.
Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry
Authors: Salomon Arthur R. , Ficarro Scott B. , Brill Laurence M. , Brinker Achim , Phung Qui T. , Ericson Christer , Sauer Karsten , Brock Ansgar , Horn David M. , Schultz Peter G. , Peters Eric C.
Published: January 2003

The reversible phosphorylation of tyrosine residues is an important mechanism for modulating biological processes such as cellular signaling, differentiation, and growth, and if deregulated, can result in various types of cancer. Therefore, an understanding of these dynamic cellular processes at the molecular level requires the ability to assess changes in the sites of tyrosine phosphorylation across numerous proteins simultaneously as well as over time. Here we describe a sensitive approach based on multidimensional liquid chromatography/mass spectrometry that enables the rapid identification of numerous sites of tyrosine phosphorylation on a number of different proteins from human whole cell lysates. We used this methodology to follow changes in tyrosine phosphorylation patterns that occur over time during either the activation of human T cells or the inhibition of the oncogenic BCR-ABL fusion product in chronic myelogenous leukemia cells in response to treatment with STI571 (Gleevec). Together, these experiments rapidly identified 64 unique sites of tyrosine phosphorylation on 32 different proteins. Half of these sites have been documented in the literature, validating the merits of our approach, whereas motif analysis suggests that a number of the undocumented sites are also potentially involved in biological pathways. This methodology should enable the rapid generation of new insights into signaling pathways as they occur in states of health and disease.
Rational design of low-molecular weight heparins with improved
in
vivo
activity
Authors: Sundaram Mallik , Qi Yiwei , Shriver Zachary , Liu Dongfang , Zhao Ganlin , Venkataraman Ganesh , Langer Robert , Sasisekharan Ram
Published: January 2003

Heparin and low-molecular weight heparins (LMWHs), complex, sulfated polysaccharides isolated from endogenous sources, are potent modulators of hemostasis. Heparin and LMWHs interact with multiple components of the coagulation cascade to inhibit the clotting process. Pharmaceutical preparations of these complex polysaccharides, typically isolated from porcine intestinal mucosa, are heterogeneous in length and composition and, hence, highly polydisperse. Because of the structural heterogeneity of heparin and LMWHs, correlating their activity with a particular structure or structural motif has been a challenging task. Herein, we demonstrate a practical analytical method that enables the measurement of a structural correlate to
in vivo
anticoagulant function. With this understanding we have developed LMWHs with increased anticoagulant activity and decreased polydispersity. In addition to the pronounced anti-Xa and anti-IIa activity of these LMWHs, we also demonstrate that they possess desirable
in vivo
pharmacokinetic properties, the ability to cause the release of tissue factor pathway inhibitor (TFPI) from the endothelium, complete bioavailability through s.c. delivery, and the ability to inhibit both venous and arterial thromboses. Importantly, from a clinical safety point of view, unlike LMWHs presently used in the clinic, we show that these LMWHs are rapidly and completely neutralized by protamine. Together, the findings presented herein demonstrate a facile approach for the creation of designer LMWHs with optimal activity profiles.
An Exploratory Comparison of Electronic Commerce Adoption in Large and Small Enterprises
Authors: Daniel Elizabeth M. , Grimshaw David J.
Published: January 2003

This study compares the reasons why small and large companies are adopting electronic commerce (e-commerce) and compares the benefits realized from the adoption of e-commerce by these two groups of companies. Two surveys that were undertaken contemporaneously on the use of e-commerce by small and large companies in the UK were used as the empirical basis for this study. The study found that the use of e-commerce for responding to competitors, providing enhanced customer services and improving relations with suppliers was driving the uptake by smaller businesses to a greater extent than by their larger counterparts. Only in the area of improved operational efficiency did larger businesses express greater interest in adopting e-commerce. The study also found that smaller businesses believed that they had achieved greater benefits from their e-commerce services than had the larger firms in all areas explored.
The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation
Authors: Diao Aipo , Rahman Dinah , Pappin Darryl J.C. , Lucocq John , Lowe Martin
Published: January 2003

Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only ∼25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.
RhoA is required for cortical retraction and rigidity during mitotic cell rounding
Authors: Maddox Amy Shaub , Burridge Keith
Published: January 2003

Mitotic cell rounding is the process of cell shape change in which a flat interphase cell becomes spherical at the onset of mitosis. Rearrangement of the actin cytoskeleton, de-adhesion, and an increase in cortical rigidity accompany mitotic cell rounding. The molecular mechanisms that contribute to this process have not been defined. We show that RhoA is required for cortical retraction but not de-adhesion during mitotic cell rounding. The mitotic increase in cortical rigidity also requires RhoA, suggesting that increases in cortical rigidity and cortical retraction are linked processes. Rho-kinase is also required for mitotic cortical retraction and rigidity, indicating that the effects of RhoA on cell rounding are mediated through this effector. Consistent with a role for RhoA during mitotic entry, RhoA activity is elevated in rounded, preanaphase mitotic cells. The activity of the RhoA inhibitor p190RhoGAP is decreased due to its serine/threonine phosphorylation at this time. Cumulatively, these results suggest that the mitotic increase in RhoA activity leads to rearrangements of the cortical actin cytoskeleton that promote cortical rigidity, resulting in mitotic cell rounding.
Compensation mechanism in tumor cell migration
Authors: Wolf Katarina , Mazo Irina , Leung Harry , Engelke Katharina , von Andrian Ulrich H. , Deryugina Elena I. , Strongin Alex Y. , Bröcker Eva-B. , Friedl Peter
Published: January 2003

Invasive tumor dissemination in vitro and in vivo involves the proteolytic degradation of ECM barriers. This process, however, is only incompletely attenuated by protease inhibitor–based treatment, suggesting the existence of migratory compensation strategies. In three-dimensional collagen matrices, spindle-shaped proteolytically potent HT-1080 fibrosarcoma and MDA-MB-231 carcinoma cells exhibited a constitutive mesenchymal-type movement including the coclustering of β1 integrins and MT1–matrix metalloproteinase (MMP) at fiber bindings sites and the generation of tube-like proteolytic degradation tracks. Near-total inhibition of MMPs, serine proteases, cathepsins, and other proteases, however, induced a conversion toward spherical morphology at near undiminished migration rates. Sustained protease-independent migration resulted from a flexible amoeba-like shape change, i.e., propulsive squeezing through preexisting matrix gaps and formation of constriction rings in the absence of matrix degradation, concomitant loss of clustered β1 integrins and MT1-MMP from fiber binding sites, and a diffuse cortical distribution of the actin cytoskeleton. Acquisition of protease-independent amoeboid dissemination was confirmed for HT-1080 cells injected into the mouse dermis monitored by intravital multiphoton microscopy. In conclusion, the transition from proteolytic mesenchymal toward nonproteolytic amoeboid movement highlights a supramolecular plasticity mechanism in cell migration and further represents a putative escape mechanism in tumor cell dissemination after abrogation of pericellular proteolysis.
Direct visualization of Ras proteins in spatially distinct cell surface microdomains
Authors: Prior Ian A. , Muncke Cornelia , Parton Robert G. , Hancock John F.
Published: January 2003

Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.
Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator
Authors: Fu Lianwu , Sztul Elizabeth
Published: January 2003

Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored. We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation. We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13ts strain (mutant in the Sec13p component of COPII), and sec23ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature. The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic. We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway. These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.
Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development
Authors: Chen Hsiuchen , Detmer Scott A. , Ewald Andrew J. , Griffin Erik E. , Fraser Scott E. , Chan David C.
Published: January 2003

Mitochondrial morphology is determined by a dynamic equilibrium between organelle fusion and fission, but the significance of these processes in vertebrates is unknown. The mitofusins, Mfn1 and Mfn2, have been shown to affect mitochondrial morphology when overexpressed. We find that mice deficient in either Mfn1 or Mfn2 die in midgestation. However, whereas Mfn2 mutant embryos have a specific and severe disruption of the placental trophoblast giant cell layer, Mfn1-deficient giant cells are normal. Embryonic fibroblasts lacking Mfn1 or Mfn2 display distinct types of fragmented mitochondria, a phenotype we determine to be due to a severe reduction in mitochondrial fusion. Moreover, we find that Mfn1 and Mfn2 form homotypic and heterotypic complexes and show, by rescue of mutant cells, that the homotypic complexes are functional for fusion. We conclude that Mfn1 and Mfn2 have both redundant and distinct functions and act in three separate molecular complexes to promote mitochondrial fusion. Strikingly, a subset of mitochondria in mutant cells lose membrane potential. Therefore, mitochondrial fusion is essential for embryonic development, and by enabling cooperation between mitochondria, has protective effects on the mitochondrial population.
Endocytosing the death sentence
Authors: Griffiths Gillian M.
Published: January 2003

A series of recent studies have suggested that endocytosis of the mannose-6-phosphate receptor (MPR)**Abbreviations used in this paper: CTL, cytotoxic T lymphocytes; MPR, mannose-6-phosphate receptor; NK, natural killer. might play a critical role in delivering the death signal to cells targeted for destruction by the immune system (for review see Barry and Bleackley, 2002). These studies have raised a number of controversial issues regarding the trafficking of proteins from the plasma membrane of the target cell to their substrates in the cytosol. In this issue, Trapani and colleagues examine the death of cells in which endocytosis of the MPR is blocked and show that the death signal is delivered effectively in the absence of MPR endocytosis (Trapani et al., 2002, this issue). How then is the death sentence delivered?
Differential Regulation of Cathepsin S and Cathepsin L in Interferon γ–treated Macrophages
Authors: Beers Courtney , Honey Karen , Fink Susan , Forbush Katherine , Rudensky Alexander
Published: January 2003

Cathepsin S (catS) and cathepsin L (catL) mediate late stages of invariant chain (Ii) degradation in discrete antigen-presenting cell types. Macrophages (Mϕs) are unique in that they express both proteases and here we sought to determine the relative contribution of each enzyme. We observe that catL plays no significant role in Ii cleavage in interferon (IFN)-γ–stimulated Mϕs. In addition, our studies show that the level of catL activity is significantly decreased in Mϕs cultured in the presence of IFN-γ whereas catS activity increases. The decrease in catL activity upon cytokine treatment occurs despite the persistence of high levels of mature catL protein, suggesting that a specific inhibitor of the enzyme is up-regulated in IFN-γ–stimulated peritoneal Mϕs. Similar inhibition of activity is observed in dendritic cells engineered to overexpress catL. Such enzymatic inhibition in Mϕs exhibits only partial dependence upon Ii and therefore, other mechanisms of catL inhibition are regulated by IFN-γ. Thus, during a T helper cell type 1 immune response catL inhibition in Mϕs results in preferential usage of catS, such that major histocompatibility complex class II presentation by all bone marrow–derived antigen-presenting cell is regulated by catS.
Vaginal Submucosal Dendritic Cells, but Not Langerhans Cells, Induce Protective Th1 Responses to Herpes Simplex Virus-2
Authors: Zhao Xinyan , Deak Eszter , Soderberg Kelly , Linehan Melissa , Spezzano David , Zhu Jia , Knipe David M. , Iwasaki Akiko
Published: January 2003

Herpes simplex virus (HSV) type 2 infection occurs primarily at the genital mucosal surfaces and is a leading cause of ulcerative lesions. Despite the availability of animal models for HSV-2 infection, little is known regarding the mechanism of immune induction within the vaginal mucosa. Here, we examined the cell types responsible for the initiation of protective Th1 immunity to HSV-2. Intravaginal inoculation of HSV-2 led to a rapid recruitment of submucosal dendritic cells (DCs) to the infected epithelium. Subsequently, CD11c+ DCs harboring viral peptides in the context of MHC class II molecules emerged in the draining lymph nodes and were found to be responsible for the stimulation of IFNγ secretion from HSV-specific CD4+ T cells. Other antigen-presenting cells including B cells and macrophages did not present viral peptides to T cells in the draining lymph nodes. Next, we assessed the relative contribution to immune generation by the Langerhans cells in the vaginal epithelium, the submucosal CD11b+ DCs, and the CD8α+ lymph node DCs. Analysis of these DC populations from the draining lymph nodes revealed that only the CD11b+ submucosal DCs, but not Langerhans cell–derived or CD8α+ DCs, presented viral antigens to CD4+ T cells and induced IFNγ secretion. These results demonstrate a previously unanticipated role for submucosal DCs in the generation of protective Th1 immune responses to HSV-2 in the vaginal mucosa, and suggest their importance in immunity to other sexually transmitted diseases.
A Single Amino Acid Alteration in Cytoplasmic Domain Determines IL-2 Promoter Activation by Ligation of CD28 but Not Inducible Costimulator (ICOS)
Authors: Harada Yohsuke , Ohgai Daisuke , Watanabe Ryosuke , Okano Kazuhiro , Koiwai Osamu , Tanabe Kazunari , Toma Hiroshi , Altman Amnon , Abe Ryo
Published: January 2003

The CD28 family molecules, CD28, and inducible costimulator (ICOS) all provide positive costimulatory signals. However, unlike CD28, ICOS does not costimulate IL-2 secretion. The YMNM motif that exists in the CD28 cytoplasmic domain is a known binding site for phosphatidylinositol 3-kinase (PI3-K) and Grb2. ICOS possesses the YMFM motif in the corresponding region of CD28 that binds PI3-K but not Grb2. We postulated that the reason that ICOS does not have the ability to induce IL-2 production is because it fails to recruit Grb2. To verify this hypothesis, we generated a mutant ICOS gene that contains the CD28 YMNM motif and measured IL-2 promoter activation after ICOS ligation. The results indicated that ICOS became competent to activate the IL-2 promoter by this single alteration. Further analysis demonstrated that Grb2 binding to ICOS was sufficient to activate the NFAT/AP-1 site in the IL-2 promoter and that the cytoplasmic domain of CD28 outside of the YMNM motif is required for activation of the CD28RE/AP-1 and NF-κB sites. Together, these observations lead us to believe that the difference of a single amino acid, which affects Grb2 binding ability, may define a functional difference between the CD28- and ICOS-mediated costimulatory signals.
Host Prostaglandin E2-EP3 Signaling Regulates Tumor-Associated Angiogenesis and Tumor Growth
Authors: Amano Hideki , Hayashi Izumi , Endo Hirahito , Kitasato Hidero , Yamashina Shohei , Maruyama Takayuki , Kobayashi Michiyoshi , Satoh Kazutoyo , Narita Masami , Sugimoto Yukihiko , Murata Takahiko , Yoshimura Hirokuni , Narumiya Shuh , Majima Masataka
Published: January 2003

Nonsteroidal antiinflammatories are known to suppress incidence and progression of malignancies including colorectal cancers. However, the precise mechanism of this action remains unknown. Using prostaglandin (PG) receptor knockout mice, we have evaluated a role of PGs in tumor-associated angiogenesis and tumor growth, and identified PG receptors involved. Sarcoma-180 cells implanted in wild-type (WT) mice formed a tumor with extensive angiogenesis, which was greatly suppressed by specific inhibitors for cyclooxygenase (COX)-2 but not for COX-1. Angiogenesis in sponge implantation model, which can mimic tumor-stromal angiogenesis, was markedly suppressed in mice lacking EP3 (EP3−/−) with reduced expression of vascular endothelial growth factor (VEGF) around the sponge implants. Further, implanted tumor growth (sarcoma-180, Lewis lung carcinoma) was markedly suppressed in EP3−/−, in which tumor-associated angiogenesis was also reduced. Immunohistochemical analysis revealed that major VEGF-expressing cells in the stroma were CD3/Mac-1 double-negative fibroblasts, and that VEGF-expression in the stroma was markedly reduced in EP3−/−, compared with WT. Application of an EP3 receptor antagonist inhibited tumor growth and angiogenesis in WT, but not in EP3−/−. These results demonstrate significance of host stromal PGE2-EP3 receptor signaling in tumor development and angiogenesis. An EP3 receptor antagonist may be a candidate of chemopreventive agents effective for malignant tumors.
Unique Resistance of I/LnJ Mice to a Retrovirus Is Due to Sustained Interferon γ–dependent Production of Virus-neutralizing Antibodies
Authors: Purdy Alexandra , Case Laure , Duvall Melody , Overstrom-Coleman Max , Monnier Nilah , Chervonsky Alexander , Golovkina Tatyana
Published: January 2003

Selection of immune escape variants impairs the ability of the immune system to sustain an efficient antiviral response and to control retroviral infections. Like other retroviruses, mouse mammary tumor virus (MMTV) is not efficiently eliminated by the immune system of susceptible mice. In contrast, MMTV-infected I/LnJ mice are capable of producing IgG2a virus-neutralizing antibodies, sustain this response throughout their life, and secrete antibody-coated virions into the milk, thereby preventing infection of their progeny. Antibodies were produced in response to several MMTV variants and were cross-reactive to them. Resistance to MMTV infection was recessive and was dependent on interferon (IFN)-γ production, because I/LnJ mice with targeted deletion of the INF-γ gene failed to produce any virus-neutralizing antibodies. These findings reveal a novel mechanism of resistance to retroviral infection that is based on a robust and sustained IFN-γ–dependent humoral immune response.
Human T Cell Receptor γδ Cells Recognize Endogenous Mevalonate Metabolites in Tumor Cells
Authors: Gober Hans-Jürgen , Kistowska Magdalena , Angman Lena , Jenö Paul , Mori Lucia , De Libero Gennaro
Published: January 2003

T lymphocytes expressing the T cell receptor (TCR)-γδ recognize unknown antigens on tumor cells. Here we identify metabolites of the mevalonate pathway as the tumor ligands that activate TCR-γδ cells. In tumor cells, blockade of hydroxy-methylglutaryl-CoA reductase (HMGR), the rate limiting enzyme of the mevalonate pathway, prevents both accumulation of mevalonate metabolites and recognition by TCR-γδ cells. When metabolite accumulation is induced by overexpressing HMGR or by treatment with nitrogen-containing bisphosphonate drugs, tumor cells derived from many tissues acquire the capacity to stimulate the same TCR-γδ population. Accumulation of mevalonate metabolites in tumor cells is a powerful danger signal that activates the immune response and may represent a novel target of tumor immunotherapy.
Computational and experimental analysis reveals a novel Src family kinase in the C. elegans genome
Authors: Pandey Akhilesh , Peri Suraj , Thacker Colin , Whipple Chery A. , Collins John J. , Mann Matthias
Published: January 2003

Motivation: The complete genomes of a number of organisms have already been sequenced. However, the vast majority of annotated genes are derived by gene prediction methods. It is important to not only validate the predicted coding regions but also to identify genes that may have been missed by these programs.

Methods: We searched the entire C.elegans genomic sequence database maintained by the Sanger Center using human c-Src sequence in a TBLASN search. We have confirmed one of the predicted regions by isolation of a cDNA and carried out a phylogenetic analysis of Src kinase family members in the worm, fly and several vertebrate species.

Results: Our analysis identified a novel tyrosine kinase in the C.elegans genome that contains functional features typical of the Src family kinases that we have designated as Src-1. The open reading frame contains a conserved N-terminal myristoylation site and a tyrosine residue within the C-terminus that is crucial for regulating the activity of Src kinases. Our phylogenetic analysis of Src family members from C.elegans, Drosophila and other higher organisms revealed a relationship among Src kinases from C. elegans and Drosophila.

Contact: pandey@cebi.sdu.dk

* To whom correspondence should be addressed.
Chemometric Modelling based on 2D-Fluorescence Spectra without
a Calibration Measurement
Authors: Solle D. , Geissler D. , Stärk E. , Scheper T. , Hitzmann B.
Published: January 2003

Motivation: 2D fluorescence spectra provide information from intracellular compounds. Fluorophores like trytophan, tyrosine and phenylalanin as well as NADH and flavins make the corresponding measurement systems very important for bioprocess supervision and control. The evaluation is usually based on chemometric modelling using for their calibration procedure off-line measurements of the desired process variables. Due to the data driven approach lots of off-line measurements are required. Here a methodology is presented, which enables to perform a calibration procedure of chemometric models without any further measurement.

Results: The necessary information for the calibration procedure is provided by means of the a priori knowledge about the process, i.e. a mathematical model, whose model parameters are estimated during the calibration procedure, as well as the fact that the substrate should be consumed at the end of the process run. The new methodology for chemometric calibration is applied for a batch cultivation of aerobically grown S. cerevisiae on the glucose Schatzmann medium. As will be presented the chemometric models, which are determined by this method, can be used for prediction during new process runs.

Availability:The MATHLAB routine is free available on request from the authors.

Contact: Hitzmann@IFTC.Uni-Hannover.de

* To whom correspondence should be addressed.
A study of accuracy and precision in oligonucleotide
arrays: extracting more signal at large concentrations
Authors: Naef Felix , Socci Nicholas D. , Magnasco Marcelo
Published: January 2003

Motivation: Despite the success and popularity of oligonucleotide arrays as a high-throughput technique for measuring mRNA expression levels, quantitative calibration studies have until now been limited. The main reason is that suitable data was not available. However, calibration data recently produced by Affymetrix now permits detailed studies of the intensity dependent sensitivity. Given a certain transcript concentration, it is of particular interest to know whether current analysis methods are capable of detecting differential expression ratios of 2 or higher.

Results: Using the calibration data, we demonstrate that while current techniques are capable of detecting changes in the low to mid concentration range, the situation is noticeably worse for high concentrations. In this regime, expression changes as large as 4 fold are severely biased, and changes of 2 are often undetectable. Such effects are mainly the consequence of the sequence specific binding properties of probes, and not the result of optical saturation in the fluorescence measurements. GeneChips are manufactured such that each transcript is probed by a set of sequences with a wide affinity range. We show that this property can be used to design a method capable of reducing the high intensity bias. The idea behind our methods is to transfer the weight of a measurement to a subset of probes with optimal linear response at a given concentration, which can be achieved using local embedding techniques.

Availability: Program source code will be sent electronically upon request.

Contact: felix@funes.rockefeller.edu

* To whom correspondence should be addressed.
Combinatorial image analysis of DNA microarray features
Authors: Glasbey C.A. , Ghazal P.
Published: January 2003

Motivation: DNA and protein microarrays have become an established leading-edge technology for large-scale analysis of gene and protein content and activity. Contact-printed microarrays has emerged as a relatively simple and cost effective method of choice but its reliability is especially susceptible to quality of pixel information obtained from digital scans of spotted features in the microarray image.

Results: We address the statistical computation requirements for optimizing data acquisition and processing of digital scans. We consider the use of median filters to reduce noise levels in images and top-hat filters to correct for trends in background values. We also consider, as alternative estimators of spot intensity, discs of fixed radius, proportions of histograms and k-means clustering, either with or without a square-root intensity transformation and background subtraction. We identify, using combinatoric procedures, optimal filter and estimator parameters, in achieving consistency among the replicates of a gene on each microarray. Our results, using test data from microarrays of HCMV, indicate that a highly effective approach for improving reliability and quality of microarray data is to apply a 21 by 21 top-hat filter, then estimate spot intensity as the mean of the largest 20% of pixel values in the target region, after a square-root transformation, and corrected for background, by subtracting the mean of the smallest 70% of pixel values.

Availability: Fortran90 subroutines implementing these methods are available from the authors, or at http://www.bioss.ac.uk/~chris.

Contact: chris@bioss.ac.uk

* To whom correspondence should be addressed.
An associative analysis of gene expression array data
Authors: Dozmorov Igor , Centola Michael
Published: January 2003

Motivation: We face the absence of optimized standards to guide normalization, comparative analysis, and interpretation of data sets. One aspect of this is that current methods of statistical analysis do not adequately utilize the information inherent in the large data sets generated in a microarray experiment and require a tradeoff between detection sensitivity and specificity.

Results: We present a multistep procedure for analysis of mRNA expression data obtained from cDNA array methods. To identify and classify differentially expressed genes, results from standard paired t-test of normalized data are compared with those from a novel method, denoted an associative analysis. This method associates experimental gene expressions presented as residuals in regression analysis against control averaged expressions to a common standard—the family of similarly computed residuals for low variability genes derived from control experiments. By associating changes in expression of a given gene to a large family of equally expressed genes of the control group, this method utilizes the large data sets inherent in microarray experiments to increase both specificity and sensitivity. The overall procedure is illustrated by tabulation of genes whose expression differs significantly between Snell dwarf mice (dw/dw) and their phenotypically normal littermates (dw/+, +/+). Of the 2352 genes examined only 450–500 were expressed above the background levels observed in nonexpressed genes and of these 120 were established as differentially expressed in dwarf mice at a significance level that excludes appearance of false positive determinations.

Contact: igor-dozmorov@omrf.ouhsc.edu

* To whom correspondence should be addressed.
Rank order metrics for quantifying the association of sequence
features with gene regulation
Authors: Clarke Neil D. , Granek Joshua A.
Published: January 2003

Motivation: Genome sequences and transcriptome analyses allow the correlation between gene regulation and DNA sequence features to be studied at the whole-genome level. To quantify these correlations, metrics are needed that can be applied to any sequence feature, regardless of its statistical distribution. It is also desirable for the metric values to be determined objectively, that is, without the use of subjective threshold values.

Results: We compare two metrics for quantifying the correlation of DNA sequence features with gene regulation. Each of the metrics is calculated from a rank-ordering of genes based on the value of the sequence feature of interest. The first metric is the area under the curve for a receiver operator characteristic plot (ROC AUC), a common way of summarizing the tradeoff between sensitivity and specificity for different values of a prediction criterion. We call the second metric the mean normalized conditional probability (MNCP). The MNCP can be thought of as the predictive value of the sequence feature averaged over all regulated genes. The statistical significance (P-value) of each metric can be estimated from simulations. Importantly, the P-value of the MNCP metric is less dramatically affected by the presence of false positives among the set of co-regulated genes than is the ROC AUC. This is especially useful in analyzing gene sets identified by DNA microarray analysis because such data cannot distinguish direct regulation by transcription factor binding from indirect regulation. We demonstrate that these two metrics, taken together, are useful tools for defining the binding site representation and regulatory control regions that best explain the difference between genes that are regulated by a given transcription factor and those that are not. Applications to other gene features are also described.

Availability: A Python program for calculating the ROC AUC and MNCP metric values given input rank orders is available from ftp://ftp.bs.jhmi.edu/users/nclarke/MNCP/

Contact: nclarke@jhmi.edu

* To whom correspondence should be addressed.
Gene finding with a hidden Markov model of genome structure and
evolution
Authors: Pedersen Jakob Skou , Hein Jotun
Published: January 2003

Motivation: A growing number of genomes are sequenced. The differences in evolutionary pattern between functional regions can thus be observed genome-wide in a whole set of organisms. The diverse evolutionary pattern of different functional regions can be exploited in the process of genomic annotation. The modelling of evolution by the existing comparative gene finders leaves room for improvement.

Results: A probabilistic model of both genome structure and evolution is designed. This type of model is called an Evolutionary Hidden Markov Model (EHMM), being composed of an HMM and a set of region-specific evolutionary models based on a phylogenetic tree. All parameters can be estimated by maximum likelihood, including the phylogenetic tree. It can handle any number of aligned genomes, using their phylogenetic tree to model the evolutionary correlations. The time complexity of all algorithms used for handling the model are linear in alignment length and genome number. The model is applied to the problem of gene finding. The benefit of modelling sequence evolution is demonstrated both in a range of simulations and on a set of orthologous human/mouse gene pairs.

Availability: Free availability over the Internet on www server: http://www.birc.dk/Software/evogene.

Contact: jsp@daimi.au.dk

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A generalized global alignment algorithm
Authors: Huang Xiaoqiu , Chao Kun-Mao
Published: January 2003

Motivation: Homologous sequences are sometimes similar over some regions but different over other regions. Homologous sequences have a much lower global similarity if the different regions are much longer than the similar regions.

Results: We present a generalized global alignment algorithm for comparing sequences with intermittent similarities, an ordered list of similar regions separated by different regions. A generalized global alignment model is defined to handle sequences with intermittent similarities. A dynamic programming algorithm is designed to compute an optimal general alignment in time proportional to the product of sequence lengths and in space proportional to the sum of sequence lengths. The algorithm is implemented as a computer program named GAP3 (Global Alignment Program Version 3). The generalized global alignment model is validated by experimental results produced with GAP3 on both DNA and protein sequences. The GAP3 program extends the ability of standard global alignment programs to recognize homologous sequences of lower similarity.

Availability: The GAP3 program is freely available for academic use at http://bioinformatics.iastate.edu/aat/align/align.html.

Contact: xqhuang@cs.iastate.edu; kmchao@csie.ntu.edu.tw

* To whom correspondence should be addressed.
A naive Bayes model to predict coupling between seven
transmembrane domain receptors and G-proteins
Authors: Cao Jack , Panetta Rosemarie , Yue Shiyi , Steyaert Alain , Young-Bellido Michele , Ahmad Sultan
Published: January 2003

Motivation: An understanding of the coupling between a G-protein coupled receptor (GPCR) and a specific class of heterotrimeric GTP-binding proteins (G-proteins) is vital for further comprehending the function of the receptor within a cell. However, predicting G-protein coupling based on the amino acid sequence of a receptor has been a daunting task. While experimental data for G-protein coupling exist, published models that rely on sequence based prediction are few. In this study, we have developed a Naive Bayes model to successfully predict G-protein coupling specificity by training over 80 GPCRs with known coupling. Each intracellular domain of GPCRs was treated as a discrete random variable, conditionally independent of one another. In order to determine the conditional probability distributions of these variables, ClustalW-generated phylogenetic trees were used as an approximation for the clustering of the intracellular domain sequences. The sampling of an intracellular domain sequence was achieved by identifying the cluster containing the homologue with the highest sequence similarity.

Results: Out of 55 GPCRs validated, the model yielded a correct classification rate of 72%. Our model also predicted multiple G-protein coupling for most of the GPCRs in the validation set. The Bayesian approach in this work offers an alternative to the experimental approach in order to answer the biological problem of GPCR/G-protein coupling selectivity.

Availability: Academic users should send their request for the perl program for calculating likelihood probabilities at jack.cao@astrazeneca.com.

Supplementary Information: The materials can be viewed at http://www.astrazeneca-montreal.com/AZRDM_info/supporting_info.pdf.

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Knowledge acquisition, consistency checking and
concurrency control for Gene Ontology (GO)
Authors: Yeh Iwei , Karp Peter D. , Noy Natalya F. , Altman Russ B.
Published: January 2003

Motivation: A critical element of the computational infrastructure required for functional genomics is a shared language for communicating biological data and knowledge. The Gene Ontology (GO; http://www.geneontology.org) provides a taxonomy of concepts and their attributes for annotating gene products. As GO increases in size, its ongoing construction and maintenance becomes more challenging. In this paper, we assess the applicability of a Knowledge Base Management System (KBMS), Protégé-2000, to the maintenance and development of GO.

Results: We transferred GO to Protégé-2000 in order to evaluate its suitability for GO. The graphical user interface supported browsing and editing of GO. Tools for consistency checking identified minor inconsistencies in GO and opportunities to reduce redundancy in its representation. The Protégé Axiom Language proved useful for checking ontological consistency. The PROMPT tool allowed us to track changes to GO. Using Protégé-2000, we tested our ability to make changes and extensions to GO to refine the semantics of attributes and classify more concepts.

Availability: Gene Ontology in Protégé-2000 and the associated code are located at http://smi.stanford.edu/projects/helix/gokbms/. Protégé-2000 is available from http://protege.stanford.edu.

Contact: russ.altman@stanford.edu

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Positional candidate gene selection from livestock EST databases
using Gene Ontology
Authors: Harhay Gregory P. , Keele John W.
Published: January 2003

Motivation: The number of expressed sequence tags (ESTs) in GenBank has now surpassed 200 000 for cattle and 100 000 for swine. The Institute of Genome Research (TIGR) has organized these sequences into approximately 60 000 non-redundant consensus sequences (identified by TIGR Gene Indices) for cattle and 40 000 for swine. Anonymous ESTs are of limited value unless they are connected to function. Functional information is difficult to manage electronically because of heterogeneity of meaning and form among databases. The Gene Ontology (GO) Consortium has produced ontologies for gene function with consistent meaning and form across species. Linking livestock EST to gene function through similarity with sequences from other annotation-rich mammals could accelerate: (1) the discovery of positional candidate genes underlying a livestock quantitative trait locus (QTL) and (2) comparative mapping between livestock and other mammals (e.g. humans, mouse and rat). We initiated this investigation to determine if incorporation of the GO into the annotation process could accelerate livestock positional candidate gene discovery.

Results: We have associated livestock ESTs with GO nodes through sequence similarity to the NCBI Reference Sequences (RefSeq). Positional candidate genes are identified within minutes that otherwise required days. The schema described here accommodates queries that return GO nodes from terms familiar to biologists, such as gene name, alternate/alias symbol, and OMIM phenotype.

Availability: Scripts and schema are available on request from the authors.

Contact: harhay@email.marc.usda.gov

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