bluuebunny/crossref_metadata_2025 huggingface dataset:
The low-spin heme of cytochrome
c
oxidase as the driving element of the proton-pumping process
Authors: Tsukihara Tomitake , Shimokata Kunitoshi , Katayama Yukie , Shimada Hideo , Muramoto Kazumasa , Aoyama Hiroshi , Mochizuki Masao , Shinzawa-Itoh Kyoko , Yamashita Eiki , Yao Min , Ishimura Yuzuru , Yoshikawa Shinya
Published: December 2003

Mitochondrial cytochrome
c
oxidase plays an essential role in aerobic cellular respiration, reducing dioxygen to water in a process coupled with the pumping of protons across the mitochondrial inner membrane. An aspartate residue, Asp-51, located near the enzyme surface, undergoes a redox-coupled x-ray structural change, which is suggestive of a role for this residue in redox-driven proton pumping. However, functional or mechanistic evidence for the involvement of this residue in proton pumping has not yet been obtained. We report that the Asp-51 → Asn mutation of the bovine enzyme abolishes its proton-pumping function without impairment of the dioxygen reduction activity. Improved x-ray structures (at 1.8/1.9-Å resolution in the fully oxidized/reduced states) show that the net positive charge created upon oxidation of the low-spin heme of the enzyme drives the active proton transport from the interior of the mitochondria to Asp-51 across the enzyme via a water channel and a hydrogen-bond network, located in tandem, and that the enzyme reduction induces proton ejection from the aspartate to the mitochondrial exterior. A peptide bond in the hydrogen-bond network critically inhibits reverse proton transfer through the network. A redox-coupled change in the capacity of the water channel, induced by the hydroxyfarnesylethyl group of the low-spin heme, suggests that the channel functions as an effective proton-collecting region. Infrared results indicate that the conformation of Asp-51 is controlled only by the oxidation state of the low-spin heme. These results indicate that the low-spin heme drives the proton-pumping process.
Dissociation of spikes, synaptic activity, and activity-dependent increments in rat cerebellar blood flow by tonic synaptic inhibition
Authors: Caesar Kirsten , Thomsen Kirsten , Lauritzen Martin
Published: December 2003

Functional neuroimaging relies on the robust coupling between neuronal activity, metabolism and cerebral blood flow (CBF) to map the brain, but the physiological basis of the neuroimaging signals is still not well understood. Here we applied a pharmacological approach to separate spiking activity, synaptic activity, and the accompanying changes in CBF in rat cerebellar cortex. We report that tonic synaptic inhibition achieved by topical application of γ-aminobutyric acid type A (GABA
A
) (muscimol) or GABA
B
(baclofen) receptor agonists abolished or reduced spontaneous Purkinje cell spiking activity without affecting basal CBF. The magnitude of CBF responses evoked by climbing fiber stimulation decreased gradually over time after exposure to muscimol, being more pronounced in the superficial than in the deep cortical layers. We provide direct evidence in favor of a laminar-specific regulation of CBF in deep cortical layers, independent of dilatation of surface vessels. With prolonged exposure to muscimol, activity-dependent CBF increments disappeared, despite preserved cerebrovascular reactivity to adenosine and preserved local field potentials (LFP). This dissociation of CBF and LFPs suggests that CBF responses are independent of extracellular synaptic currents that generate LFPs. Our work implies that neuronal and vascular signals evoked by glutamatergic pathways are sensitive to synaptic inhibition, and that local mechanisms independent of transmembrane synaptic currents adjust flow to synaptic activity in distinct cortical layers. Our results provide fundamental insights into the functional regulation of blood flow, showing important interference of GABA
A
receptors in translating excitatory input into blood flow responses.
Unstable mutants in the peripheral endosomal membrane component ALS2 cause early-onset motor neuron disease
Authors: Yamanaka Koji , Vande Velde Christine , Eymard-Pierre Eleonore , Bertini Enrico , Boespflug-Tanguy Odile , Cleveland Don W.
Published: December 2003

Mutations in ALS2, carrying three putative guanine exchange factor (GEF) domains, are causative for a juvenile, autosomal recessive form of amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, and infantile-ascending hereditary spastic paralysis. Endogenous ALS2 is shown here to be enriched in nervous tissue and to be peripherally bound to the cytoplasmic face of endosomal membranes, an association that requires the amino-terminal “RCC1 (regulator of chromatin condensation)-like” GEF domain. Disease-causing mutants and a naturally truncated isoform of ALS2 are shown to be rapidly degraded when expressed in cultured human cells, including lymphocytes derived from patients with ALS2 mutations. Thus, mutations in the ALS2 gene linked to early-onset motor neuron disease uniformly produce loss of activity through decreased protein stability of this endosomal GEF.
Murine and human zona pellucida 3 derived from mouse eggs express identical O-glycans
Authors: Dell Anne , Chalabi Sara , Easton Richard L. , Haslam Stuart M. , Sutton-Smith Mark , Patankar Manish S. , Lattanzio Frank , Panico Maria , Morris Howard R. , Clark Gary F.
Published: December 2003

Murine sperm initiate fertilization by binding to the outer covering of the egg known as the murine zona pellucida (mZP). This binding is thought to require the interaction of O-glycans linked to a specific mZP glycoprotein (mZP3) with egg-binding proteins coating the sperm plasma membrane. The precise molecular basis of this interaction remains to be resolved. In this study, we analyzed the O-glycosylation of the individual mZP glycoproteins by using ultrasensitive MS methods. We found that the majority of the O-glycans that are linked to mZP3 are core type 2 sequences terminated with sialic acid, lacNAc (Galβ1-4GlcNAc), lacdiNAc (Gal-NAcβ1-4GlcNAc), Galα1-3Gal, and NeuAcα2-3[GalNAcβ1-4]Galβ1-4 (Sd
a
antigen). Many of these terminal sequences have been implicated previously in murine sperm–egg binding. Core type 1 O-glycans are also present and are generally unmodified, although some are terminated with sialic acid, β-linked
N
-acetylhexosamine, or NeuAcα2-3[GalNAcβ1-4]Galβ1-4. Eggs expressing human ZP (huZP) glycoprotein huZP3, derived from transgenic mice, bind murine but not human sperm, implying that huZP3 acquires the same O-glycans as native mZP3. Sequencing of huZP3-associated O-glycans confirms that this implication is correct. The data obtained in this investigation may prove to be very useful for studies to determine the precise molecular basis of initial murine sperm–egg binding.
p57
Kip2
cooperates with Nurr1 in developing dopamine cells
Authors: Joseph Bertrand , Wallén-Mackenzie Åsa , Benoit Gérard , Murata Takashi , Joodmardi Eliza , Okret Sam , Perlmann Thomas
Published: December 2003

Cyclin-dependent kinase inhibitors of the Cip/Kip family play critical roles in regulating cell proliferation during embryogenesis. However, these proteins also influence cell differentiation by mechanisms that have remained unknown. Here we show that p57
Kip2
is expressed in postmitotic differentiating midbrain dopamine cells. Induction of p57
Kip2
expression depends on Nurr1, an orphan nuclear receptor that is essential for dopamine neuron development. Moreover, analyses of p57
Kip2
gene-targeted mice revealed that p57
Kip2
is required for the maturation of midbrain dopamine neuronal cells. Additional experiments in a dopaminergic cell line demonstrated that p57
Kip2
can promote maturation by a mechanism that does not require p57
Kip2
-mediated inhibition of cyclin-dependent kinases. Instead, evidence indicates that p57
Kip2
functions by a direct protein–protein interaction with Nurr1. Thus, in addition to its established function in control of proliferation, these results reveal a mechanism whereby p57
Kip2
influences postmitotic differentiation of dopamine neurons.
Endonuclease G is required for early embryogenesis and normal apoptosis in mice
Authors: Zhang Jianhua , Dong Mei , Li Lily , Fan Yunxia , Pathre Purnima , Dong Jin , Lou Danwen , Wells James M. , Olivares-Villagómez Danyvid , Van Kaer Luc , Wang Xiaodong , Xu Ming
Published: December 2003

Endonuclease G (EndoG) is a nuclear-encoded mitochondrial protein reported to be important for both nuclear DNA fragmentation during apoptosis and mitochondrial DNA replication. To evaluate thein vivofunction of EndoG, we have investigated the effects of EndoG deficiency in cells and mice. We found that EndoG homozygous mutant embryos die between embryonic days 2.5 and 3.5. Mitochondrial DNA copy numbers in ovulated oocytes from EndoG heterozygous mutant and wild-type mice are similar, suggesting that EndoG is involved in a cellular function unrelated to mitochondrial DNA replication. Interestingly, we found that cells from EndoG heterozygous mutant mice exhibit increased resistance to both tumor necrosis factor α- and staurosporine-induced cell death. Moreover, spontaneous cell death of spermatogonia in EndoG heterozygous mutant mice is significantly reduced compared with wild-type mice. DNA fragmentation is also reduced in EndoG+/-thymocytes and splenocytes compared with wild-type cells, as well as in EndoG+/-thymusin vivocompared with that of the wild-type mice, on activation of apoptosis. These findings indicate that EndoG is essential during early embryogenesis and plays a critical role in normal apoptosis and nuclear DNA fragmentation.
Mullerian Inhibiting Substance inhibits cervical cancer cell growth via a pathway involving p130 and p107
Authors: Barbie Thanh U. , Barbie David A. , MacLaughlin David T. , Maheswaran Shyamala , Donahoe Patricia K.
Published: December 2003

In addition to causing regression of the Mullerian duct in the male embryo, Mullerian Inhibiting Substance (MIS) inhibits the growth of epithelial ovarian cancer cells, which are known to be of Mullerian origin. Because the uterine cervix is derived from the same Mullerian duct precursor as the epithelium of the ovary, we tested the hypothesis that cervical cancer cells might also respond to MIS. A number of cervical cancer cell lines express the MIS type II receptor, and MIS inhibits the growth of both human papilloma virus-transformed and non-human papilloma virus-transformed cervical cell lines, with a more dramatic effect seen in the latter. As in the ovarian cancer cell line OVCAR8, suppression of growth of the C33A cervical cancer cell line by MIS is associated with induction of the p16 tumor suppressor protein. However, in contrast to OVCAR8 cells, induction of p130 and p107 appears to play an important role in the inhibition of growth of C33A cells by MIS. Finally, normal cervical tissue expresses the MIS type II receptor
in vivo
, supporting the idea that MIS could be a targeted therapy for cervical cancer.
Antigen-driven effector CD8 T cell function regulated by T-bet
Authors: Sullivan Brandon M. , Juedes Amy , Szabo Susanne J. , von Herrath Matthias , Glimcher Laurie H.
Published: December 2003

Type 1 immunity relies on the differentiation of two major subsets of T lymphocytes, the CD4+T helper (Th) cell and the CD8+cytotoxic T cell, that direct inflammatory and cytotoxic responses essential for the destruction of intracellular and extracellular pathogens. In contrast to CD4 cells, little is known about transcription factors that control the transition from the CD8 naïve to effector cell stage. Here, we report that the transcription factor T-bet, known to regulate Th cell differentiation, also controls the generation of the CD8+cytotoxic effector cell. Antigen-driven generation of effector CD8+cells was impaired in OT-I T cell receptor transgenic mice lacking T-bet, resulting in diminished cytotoxicity and a marked shift in cytokine secretion profiles. Furthermore, mice lacking T-bet responded poorly to infection with lymphocytic choriomeningitis virus. T-bet is a key player in the generation of type 1 immunity, in both Th and T cytotoxic cells.
Epstein–Barr virus activates β-catenin in type III latently infected B lymphocyte lines: Association with deubiquitinating enzymes
Authors: Shackelford Julia , Maier Christopher , Pagano Joseph S.
Published: December 2003

β-Catenin is a multifunctional protein, stabilization of which is a critical step in its functional activity. Stabilization can be conferred through several means, including mutations in β-catenin, common in many carcinomas. In this article, we explore the effect of viral infection on the β-catenin signaling pathway in B lymphocytes, including cell lines derived from lymphomas. Infection by the human tumor virus Epstein–Barr virus (EBV) generates several types of latency with different spectra of latent gene expression that are associated with different malignancies. In type III latency, exemplified by EBV lymphoproliferative diseases, the full range of these viral proteins is expressed, whereas in type I latency, only the EBNA1 protein is expressed and functional. We show that β-catenin is rapidly degraded in type I B lymphocytic lines but it is stabilized in type III B cell lines, and that β-catenin/T cell factor transcriptional activity is significantly higher in type III cells. Also we show the association of free cytoplasmic β-catenin with deubiquitinating enzymes, which may play a role in β-catenin stabilization. Activation of the β-catenin/T cell factor pathway by EBV may contribute to the lymphoproliferation characteristic of type III latency.
Motility of dendritic spines in visual cortex
in vivo
: Changes during the critical period and effects of visual deprivation
Authors: Majewska Ania , Sur Mriganka
Published: December 2003

Cortical dendritic spines are highly motile postsynaptic structures onto which most excitatory synapses are formed. It has been postulated that spine dynamics might reflect synaptic plasticity of cortical neurons. To test this hypothesis, we have investigated spine dynamics during the critical period in mouse visual cortex
in vivo
with and without sensory deprivation. The motility of spines on apical dendrites of layer 5 neurons was assayed by time-lapse two-photon microscopy. Spines were motile at the ages examined, postnatal days (P)21–P42, although motility decreased between P21 and P28 and then remained stable through P42. Binocular deprivation from before the time of eye-opening up-regulated spine motility during the peak of the critical period (P28), without affecting average spine length, class distribution, or density. Deprivation at the start of the critical period had no effect on spine motility, whereas continued deprivation through the end of the critical period appeared to reduce spine motility slightly. We conclude that spine motility might be involved in critical-period plasticity and that reduction of activity during the critical period enhances spine dynamics.
Diversity of regulatory CD4+Tcells controlling distinct organ-specific autoimmune diseases
Authors: Alyanakian Marie-Alexandra , You Sylvaine , Damotte Diane , Gouarin Christine , Esling Anne , Garcia Corinne , Havouis Séverine , Chatenoud Lucienne , Bach Jean-François
Published: December 2003

Depletion of selected regulatory CD4+T cell subsets induces the spontaneous onset of various immune or autoimmune disorders. It is not clear, however, whether a given subset, notably CD4+CD25+regulatory T cells, protects from a wide spectrum of immune disorders, or whether specialized subsets of regulatory T cells control each given disease or group of diseases. We report here, using diabetes prone nonobese diabetic (NOD) mice, that depending on the regulatory T cells that are depleted, i.e., CD25+, CD62L+, or CD45RBlow, distinct immune diseases appear after transfer into NOD severe combined immunodeficiency (SCID) recipients. Thus, reconstitution of NOD SCID mice with CD25-T cells induces major gastritis and late-onset diabetes, but no or mild colitis. Reconstitution with CD62L-T cells induces fulminant diabetes with no colitis or gastritis. Reconstitution with CD45RBhighT cells induces major colitis with wasting disease and no or very moderate gastritis and diabetes. Major differences among the three regulatory T cell subsets are also seenin vitro. The bulk of suppressor cells inhibiting the proliferation of CD4+CD25-T cells in coculture is concentrated within the CD25+but not the CD62L+or CD45RBlowT cell subsets. Similarly, cytokine production patterns are significantly different for each regulatory T cell subset. Collectively, these data point to the diversity and organ selectivity of regulatory T cells controlling distinct autoimmune diseases whatever the underlying mechanisms.
Autoubiquitylation of the V(D)J recombinase protein RAG1
Authors: Jones Jessica M. , Gellert Martin
Published: December 2003

V(D)J recombination, the rearrangement of gene segments to assemble Ig and T cell receptor coding regions, is vital to B and T lymphocyte development. Here, we demonstrate that the V(D)J recombinase protein RAG1 undergoes ubiquitylation in cells.
In vitro
, the RING finger domain of RAG1 acts as a ubiquitin ligase that mediates its own ubiquitylation at a highly conserved K residue in the RAG1 amino-terminal region. Ubiquitylation is best supported by a specific ubiquitin-conjugating enzyme, UbcH3/CDC34, and requires an intact RAG1 RING finger motif. Disruption of the RING finger and certain RAG1 N-terminal truncations are associated with immunodeficiency in human patients, suggesting that RAG1's ubiquitin ligase is required for its biological role in lymphocyte development.
Myo4p and She3p are required for cortical ER inheritance in Saccharomyces cerevisiae
Authors: Estrada Paula , Kim Jiwon , Coleman Jeff , Walker Lee , Dunn Brian , Takizawa Peter , Novick Peter , Ferro-Novick Susan
Published: December 2003

Myo4p is a nonessential type V myosin required for the bud tip localization of ASH1 and IST2 mRNA. These mRNAs associate with Myo4p via the She2p and She3p proteins. She3p is an adaptor protein that links Myo4p to its cargo. She2p binds to ASH1 and IST2 mRNA, while She3p binds to both She2p and Myo4p. Here we show that Myo4p and She3p, but not She2p, are required for the inheritance of cortical ER in the budding yeast Saccharomyces cerevisiae. Consistent with this observation, we find that cortical ER inheritance is independent of mRNA transport. Cortical ER is a dynamic network that forms cytoplasmic tubular connections to the nuclear envelope. ER tubules failed to grow when actin polymerization was blocked with the drug latrunculin A (Lat-A). Additionally, a reduction in the number of cytoplasmic ER tubules was observed in Lat-A–treated and myo4Δ cells. Our results suggest that Myo4p and She3p facilitate the growth and orientation of ER tubules.
TGFβ3 signaling activates transcription of the LEF1 gene to induce epithelial mesenchymal transformation during mouse palate development
Authors: Nawshad Ali , Hay Elizabeth D.
Published: December 2003

Epithelial mesenchymal transformation (EMT) of the medial edge epithelial (MEE) seam creates palatal confluence. This work aims to elucidate the molecular mechanisms by which TGFβ3 brings about palatal seam EMT. We collected mRNA for PCR analysis from individual transforming MEE cells by laser microdissection techniques and demonstrated that TGFβ3 stimulates lymphoid-enhancing factor 1 (LEF1) mRNA synthesis in MEE cells. We show with antisense β-catenin oligonucleotides that up-regulated LEF1 is not activated by β-catenin in palate EMT. We ruled out other TGFβ3 targets, such as RhoA and MEK1/2 pathways, and we present evidence using dominant-negative Smad4 and dominant-negative LEF1 showing that TGFβ3 uses Smads both to up-regulate synthesis of LEF1 and to activate LEF1 transcription during induction of palatal EMT. When phospho-Smad2 and Smad4 are present in the nucleus, LEF1 is activated without β-catenin. Our paper is the first to show that the Smad2,4/LEF1 complex replaces β-catenin/LEF1 during activation of EMT in vivo by TGFβ3.
Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus
Authors: Henkemeyer Mark , Itkis Olga S. , Ngo Michelle , Hickmott Peter W. , Ethell Iryna M.
Published: December 2003

Here, using a genetic approach, we dissect the roles of EphB receptor tyrosine kinases in dendritic spine development. Analysis of EphB1, EphB2, and EphB3 double and triple mutant mice lacking these receptors in different combinations indicates that all three, although to varying degrees, are involved in dendritic spine morphogenesis and synapse formation in the hippocampus. Hippocampal neurons lacking EphB expression fail to form dendritic spines in vitro and they develop abnormal spines in vivo. Defective spine formation in the mutants is associated with a drastic reduction in excitatory glutamatergic synapses and the clustering of NMDA and AMPA receptors. We show further that a kinase-defective, truncating mutation in EphB2 also results in abnormal spine development and that ephrin-B2–mediated activation of the EphB receptors accelerates dendritic spine development. These results indicate EphB receptor cell autonomous forward signaling is responsible for dendritic spine formation and synaptic maturation in hippocampal neurons.
α3β1 integrin–CD151, a component of the cadherin–catenin complex, regulates PTPμ expression and cell–cell adhesion
Authors: Chattopadhyay Nibedita , Wang Zemin , Ashman Leonie K. , Brady-Kalnay Susann M. , Kreidberg Jordan A.
Published: December 2003

The β1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for α3β1 integrin in association with the tetraspanin CD151 as a component of a cell–cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin–tetraspanin complex affects epithelial cell–cell adhesion at the level of gene expression both by regulating expression of PTPμ and by organizing a multimolecular complex containing PKCβII, RACK1, PTPμ, β-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.
Isoform-specific insulin receptor signaling involves different plasma membrane domains
Authors: Uhles Sabine , Moede Tilo , Leibiger Barbara , Berggren Per-Olof , Leibiger Ingo B.
Published: December 2003

In pancreatic β-cells, insulin selectively up-regulates the transcription of its own gene and that of the glucokinase gene by signaling through the two isoforms of the insulin receptor, i.e., A-type (Ex11−) and B-type (Ex11+), using different signaling pathways. However, the molecular mechanism(s) that allows the discrete activation of signaling cascades via the two receptor isoforms remains unclear. Here we show that activation of the insulin promoter via A-type and of the glucokinase promoter via B-type insulin receptor is not dependent on receptor isoform–specific differences in internalization but on the different localization of the receptor types in the plasma membrane. Our data demonstrate that localization and function of the two receptor types depend on the 12–amino acid string encoded by exon 11, which acts as a sorting signal rather than as a physical spacer. Moreover, our data suggest that selective activation of the insulin and glucokinase promoters occurs by signaling from noncaveolae lipid rafts that are differently sensitive toward cholesterol depletion.
Mathematical model of the morphogenesis checkpoint in budding yeast
Authors: Ciliberto Andrea , Novak Bela , Tyson John J.
Published: December 2003

The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.
Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 γ–σ1 and AP-3 δ–σ3 hemicomplexes
Authors: Janvier Katy , Kato Yukio , Boehm Markus , Rose Jeremy R. , Martina José A. , Kim Bong-Yoon , Venkatesan Sundararajan , Bonifacino Juan S.
Published: December 2003

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the γ and σ1 subunits of AP-1 and the δ and σ3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the γ/δ and σ subunits of AP-1 and AP-3.
VEGF is a chemoattractant for FGF-2–stimulated neural progenitors
Authors: Zhang Huanxiang , Vutskits Laszlo , Pepper Michael S. , Kiss Jozsef Z.
Published: December 2003

Mmigration of undifferentiated neural progenitors is critical for the development and repair of the nervous system. However, the mechanisms and factors that regulate migration are not well understood. Here, we show that vascular endothelial growth factor (VEGF)-A, a major angiogenic factor, guides the directed migration of neural progenitors that do not display antigenic markers for neuron- or glia-restricted precursor cells. We demonstrate that progenitor cells express both VEGF receptor (VEGFR) 1 and VEGFR2, but signaling through VEGFR2 specifically mediates the chemotactic effect of VEGF. The expression of VEGFRs and the chemotaxis of progenitors in response to VEGF require the presence of fibroblast growth factor 2. These results demonstrate that VEGF is an attractive guidance cue for the migration of undifferentiated neural progenitors and offer a mechanistic link between neurogenesis and angiogenesis in the nervous system.
Latent TGF-β binding protein LTBP-2 decreases fibroblast adhesion to fibronectin
Authors: Hyytiäinen Marko , Keski-Oja Jorma
Published: December 2003

We have analyzed the effects of latent TGF-β binding protein 2 (LTBP-2) and its fragments on lung fibroblast adhesion. Quantitative cell adhesion assays indicated that fibroblasts do not adhere to full-length LTBP-2. Interestingly, LTBP-2 had dominant disrupting effects on the morphology of fibroblasts adhering to fibronectin (FN). Fibroblasts plated on LTBP-2 and FN substratum exhibited less adherent morphology and displayed clearly decreased actin stress fibers than cells plated on FN. These cells formed, instead, extensive membrane ruffles. LTBP-2 had no effects on cells adhering to collagen type I. Fibroblasts adhered weakly to the NH2-terminal fragment of LTBP-2. Unlike FN, this fragment did not augment actin stress fiber formation. Interestingly, the adhesion-mediating and cytoskeleton-disrupting effects were localized to the same NH2-terminal proline-rich region of LTBP-2. LTBP-2 and its antiadhesive fragment bound to FN in vitro, and the antiadhesive fragment associated with the extracellular matrix FN fibrils. These observations reveal a potentially important role for LTBP-2 as an antiadhesive matrix component.
Tyrosine phosphorylation of type Iγ phosphatidylinositol phosphate kinase by Src regulates an integrin–talin switch
Authors: Ling Kun , Doughman Renee L. , Iyer Vidhya V. , Firestone Ari J. , Bairstow Shawn F. , Mosher Deane F. , Schaller Michael D. , Anderson Richard A.
Published: December 2003

Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Iγ phosphatidylinositol phosphate kinase (PIPKIγ661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIγ661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIγ661 and Src. The phosphorylation of Y644 results in an ∼15-fold increase in binding affinity to the talin head domain and blocks β-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a “molecular switch” for talin binding between PIPKIγ661 and β-integrin that may regulate dynamic FA turnover.
A role for cytoplasmic dynein and LIS1 in directed cell movement
Authors: Dujardin Denis L. , Barnhart Lora E. , Stehman Stephanie A. , Gomes Edgar R. , Gundersen Gregg G. , Vallee Richard B.
Published: December 2003

Cytoplasmic dynein has been implicated in numerous aspects of intracellular movement. We recently found dynein inhibitors to interfere with the reorientation of the microtubule cytoskeleton during healing of wounded NIH3T3 cell monolayers. We now find that dynein and its regulators dynactin and LIS1 localize to the leading cell cortex during this process. In the presence of serum, bright diffuse staining was observed in regions of active ruffling. This pattern was abolished by cytochalasin D, and was not observed in cells treated with lysophosphatidic acid, conditions which allow microtubule reorientation but not forward cell movement. Under the same conditions, using total internal reflection fluorescence microscopy, clear punctate dynein/dynactin containing structures were observed along the sides and at the tips of microtubules at the leading edge. Overexpression of dominant negative dynactin and LIS1 cDNAs or injection of antidynein antibody interfered with the rate of cell migration. Together, these results implicate a leading edge cortical pool of dynein in both early and persistent steps in directed cell movement.
Myosin V and the endoplasmic reticulum
Authors: Wagner Wolfgang , Hammer John A.
Published: December 2003

In this issue, Estrada et al. (2003) provide new and important insights into how the endoplasmic reticulum (ER) of budding yeast cells is inherited. Together with other studies in plant and animal cells, the results of Estrada et al. (2003) support the idea that myosin V acts as a universal motor for the transport of ER membranes.
Protein β-turn prediction using nearest-neighbor method
Authors: Kim Saejoon
Published: December 2003

Motivation: With the emerging success of protein secondary structure prediction through the applications of various statistical and machine learning techniques, similar techniques have been applied to protein β-turn prediction. In this study, we perform protein β-turn prediction using a k-nearest neighbor method, which is combined with a filter that uses predicted protein secondary structure information. Traditional β-turn prediction from k-nearest neighbor method is modified to account for the unbalanced ratio of the natural occurrence of β-turns and non-β-turns.

Results: Our prediction scheme is tested on a set of 426 non-homologous protein sequences. The prediction scheme consists of two stages: k-nearest neighbor method stage and filtering stage. Variations of the k-nearest neighbor method were used to take property of β-turns into consideration. Our filtering method uses β-turn/non-β-turn estimates from the k-nearest neighbor method stage and predicted protein secondary structure information from PSI-PRED in order to get new β-turn/non-β-turn estimate. Our result is compared with the previously best known β-turn prediction method on the dataset of 426 non-homologous protein sequences and is shown to give slightly superior performance at significantly lower computational complexity.

Availability: Contact the author for information on the source code of the programs used.
ClusPro: an automated docking and discrimination method for the prediction of protein complexes
Authors: Comeau Stephen R. , Gatchell David W. , Vajda Sandor , Camacho Carlos J.
Published: December 2003

Motivation: Predicting protein interactions is one of the most challenging problems in functional genomics. Given two proteins known to interact, current docking methods evaluate billions of docked conformations by simple scoring functions, and in addition to near-native structures yield many false positives, i.e. structures with good surface complementarity but far from the native.

Results: We have developed a fast algorithm for filtering docked conformations with good surface complementarity, and ranking them based on their clustering properties. The free energy filters select complexes with lowest desolvation and electrostatic energies. Clustering is then used to smooth the local minima and to select the ones with the broadest energy wells—a property associated with the free energy at the binding site. The robustness of the method was tested on sets of 2000 docked conformations generated for 48 pairs of interacting proteins. In 31 of these cases, the top 10 predictions include at least one near-native complex, with an average RMSD of 5 Å from the native structure. The docking and discrimination method also provides good results for a number of complexes that were used as targets in the Critical Assessment of PRedictions of Interactions experiment.

Availability: The fully automated docking and discrimination server ClusPro can be found at http://structure.bu.edu
Light-weight integration of molecular biological databases
Authors: Philippi Stephan
Published: December 2003

Motivation: Due to the increasing number of molecular biological databases and the exponential growth of their contents, database integration is an important topic of research in bioinformatics. Existing approaches in this area have in common that considerable efforts are needed to provide integrated access to heterogeneous data sources.

Results: This article describes the LIMBO architecture as a light-weight approach to molecular biological database integration. By building systems upon this architecture, the efforts needed for database integration can be significantly lowered.

Availability: As an illustration of the principle usefulness of the underlying ideas, a prototypical implementation based upon the LIMBO architecture is described. This implementation is exclusively based on freely available open source components like the PostgreSQL database management system and the BioRuby project. Additional files and modified components are available upon request from the author.
An Iterated loop matching approach to the prediction of RNA secondary structures with pseudoknots
Authors: Ruan Jianhua , Stormo Gary D. , Zhang Weixiong
Published: December 2003

Motivation: Pseudoknots have generally been excluded from the prediction of RNA secondary structures due to its difficulty in modeling. Although, several dynamic programming algorithms exist for the prediction of pseudoknots using thermodynamic approaches, they are neither reliable nor efficient. On the other hand, comparative methods are more reliable, but are often done in an ad hoc manner and require expert intervention. Maximum weighted matching, an algorithm for pseudoknot prediction with comparative analysis, suffers from low-prediction accuracy in many cases.

Results: Here we present an algorithm, iterated loop matching, for reliably and efficiently predicting RNA secondary structures including pseudoknots. The method can utilize either thermodynamic or comparative information or both, thus is able to predict pseudoknots for both aligned and individual sequences. We have tested the algorithm on a number of RNA families. Using 8–12 homologous sequences, the algorithm correctly identifies more than 90% of base-pairs for short sequences and 80% overall. It correctly predicts nearly all pseudoknots and produces very few spurious base-pairs for sequences without pseudoknots. Comparisons show that our algorithm is both more sensitive and more specific than the maximum weighted matching method. In addition, our algorithm has high-prediction accuracy on individual sequences, comparable with the PKNOTS algorithm, while using much less computational resources.

Availability: The program has been implemented in ANSI C and is freely available for academic use at http://www.cse.wustl.edu/~zhang/projects/rna/ilm/

Supplementary information:  http://www.cse.wustl.edu/~zhang/projects/rna/ilm/
Distribution of words with a predefined range of mismatches to a DNA probe in bacterial genomes
Authors: Melko O. Michael , Mushegian Arcady R.
Published: December 2003

Motivation: Hybridization of oligonucleotides with longer nucleotide sequences is an essential step in nucleic acid biosynthesis in vitro and in vivo, in oligonucleotide-based diagnostics, and in therapeutic applications of oligonucleotides. A major factor determining sensitivity and selectivity of hybridization is the number of base pair mismatches that occur in an ungapped alignment of the oligonucleotide (probe) and a longer sequence (target).

Results: The k-distance match count between the probe and the target is defined as the number of ungapped alignments between the two sequences that have exactly k mismatches, and the k-neighbor match count is defined as the sum of the j-distance match counts for j between 0 and k. We derive a novel formula for the probability of a k-distance match. This formula is based on the assumption that the target is strand-symmetric Bernoulli text (i.e. nucleotides are independently, identically distributed in the target and satisfy Chargaff's second parity rule). Our model predicts that the GC-content in both the probe and the target significantly affects the match count expectation. The ratio of k-neighbor match counts in two distinct genomes for a given probe is a measure of its specificity. We calculated such ratios for pairs of bacterial genomes with different combinations of length, GC-content and phylogenetic distance. Examination of the extreme values of these ratios indicates that probes with a high discriminative power exist for each tested pair.

Supplementary information: Stowers Institute Technical Report No. 0002, C++ source code, Mathematica notebooks and other information is available at http://www.stowers-institute.org/labs/bioinformatics/omm/index.htm
MedBlast: searching articles related to a biological sequence
Authors: Tu Qiang , Tang Haixu , Ding Dafu
Published: December 2003

Summary: In the genomic era, researchers often want to know more information about a biological sequence by retrieving its related articles. However, there is no available tool yet to achieve conveniently this goal. Here we developed a new literature-mining tool MedBlast, which uses natural language processing techniques, to retrieve the related articles of a given sequence. An online server of this program is also provided.

Availability: Both online server and the program are available freely at http://medblast.sibsnet.org
VizStruct: exploratory visualization for gene expression profiling
Authors: Zhang Li , Zhang Aidong , Ramanathan Murali
Published: December 2003

Motivation: DNA arrays provide a broad snapshot of the state of the cell by measuring the expression levels of thousands of genes simultaneously. Visualization techniques can enable the exploration and detection of patterns and relationships in a complex data set by presenting the data in a graphical format in which the key characteristics become more apparent. The dimensionality and size of array data sets however present significant challenges to visualization. The purpose of this study is to present an interactive approach for visualizing variations in gene expression profiles and to assess its usefulness for classifying samples.

Results: The first Fourier harmonic projection was used to map multi-dimensional gene expression data to two dimensions in an implementation called VizStruct. The visualization method was tested using the differentially expressed genes identified in eight separate gene expression data sets. The samples were classified using the oblique decision tree (OC1) algorithm to provide a procedure for visualization-driven classification. The classifiers were evaluated by the holdout and the cross-validation techniques. The proposed method was found to achieve high accuracy.

Availability: Detailed mathematical derivation of all mapping properties as well as figures in color can be found as supplementary on the web page http://www.cse.buffalo.edu/DBGROUP/bioinformatics/supplementary/vizstruct. All programs were written in Java and Matlab and software code is available by request from the first author.
Twelve C2H2 zinc-finger genes on human chromosome 19 can be each translated into the same type of protein after frameshifts
Authors: Meng Shao-Wu , Zhang Zhuo , Li Jinyan
Published: December 2003

We report a discovery that, of the 226 C2H2 zinc-finger (C2H2-ZNF) genes on human chromosome 19, 12 genes each have two open reading frames (ORFs) that are in different reading frames but that can be translated into the same type of C2H2-ZNF proteins. We came to this observation after using standard tools in an original manner. First, we found that the both ORFs of such a gene contained the same type of significant C2H2-ZNF domain with e-values of e–2 or better. Second, the both ORFs had a promoter, a transcription start site, a start codon, a Kozak pattern and a poly(A) site; hence, each of them can be viewed as a gene in terms of a gene's primary structure. Third, both the ORFs matched not only human C2H2-ZNF expressed sequence tags (ESTs) but also human C2H2-ZNF proteins with e-values of e–50 or better. This indicates that the both ORFs can be transcribed and translated into the same zinc-finger proteins. More importantly, we observed that the phenomenon—a DNA can be translated into the same type of proteins after a frameshift—also occurred in a set of 160 human C2H2-ZNF ESTs and in a set of nine cDNAs of human C2H2-ZNF proteins. This observation based on the two sets of wet-experimental data much strengthened our confidence on the discovery. Our discovery is useful in the deeper understanding of a gene's regulatory mechanism to maintain its function.

Supplementary information:  http://zfgene.i2r.a-star.edu.sg
Adjustment of systematic microarray data biases
Authors: Benito Monica , Parker Joel , Du Quan , Wu Junyuan , Xiang Dong , Perou Charles M. , Marron J. S.
Published: December 2003

Motivation: Systematic differences due to experimental features of microarray experiments are present in most large microarray data sets. Many different experimental features can cause biases including different sources of RNA, different production lots of microarrays or different microarray platforms. These systematic effects present a substantial hurdle to the analysis of microarray data.

Results: We present here a new method for the identification and adjustment of systematic biases that are present within microarray data sets. Our approach is based on modern statistical discrimination methods and is shown to be very effective in removing systematic biases present in a previously published breast tumor cDNA microarray data set. The new method of ‘Distance Weighted Discrimination (DWD)’ is shown to be better than Support Vector Machines and Singular Value Decomposition for the adjustment of systematic microarray effects. In addition, it is shown to be of general use as a tool for the discrimination of systematic problems present in microarray data sets, including the merging of two breast tumor data sets completed on different microarray platforms.

Availability: Matlab software to perform DWD can be retrieved from https://genome.unc.edu/pubsup/dwd/

Supplementary information: The complete figures that represent the cluster diagrams in Figure 6 and other figures are available at https://genome.unc.edu/pubsup/dwd/
The Genomic Threading Database
Authors: McGuffin Liam J. , Street Stefano , Sørensen Søren-Aksel , Jones David T.
Published: December 2003

Summary: The Genomic Threading Database currently contains structural annotations for the genomes of over 100 recently sequenced organisms. Annotations are carried out by using our modified GenTHREADER software and through implementing grid technology.

Availability:  http://bioinf.cs.ucl.ac.uk/GTD

Supplementary information:  http://bioinf.cs.ucl.ac.uk/GTD/demo.avi, http://bioinf.cs.ucl.ac.uk/GTD/summaryView.html, http://bioinf.cs.ucl.ac.uk/GTD/figure1.pdf, http://www.e-protein.org
TM or not TM: transmembrane protein prediction with low false positive rate using DAS-TMfilter
Authors: Cserzo Miklos , Eisenhaber Frank , Eisenhaber Birgit , Simon Istvan
Published: December 2003

Summary: Web-based servers implementing the DAS-TMfilter algorithm have been launched at three mirror sites and their usage is described. The underlying computer program is an upgraded and modified version of the DAS-prediction method. The new server is designed to make distinction between protein sequences with and without transmembrane (TM) helices at a reasonably low rate of false positive prediction (∼1 among 100 unrelated queries) while the high efficiency of the original algorithm locating TM segments in queries is preserved (sensitivity of ∼95% among documented proteins with helical TM regions).

Availability: The server operates at three mirror sites: http://mendel.imp.univie.ac.at/sat/DAS/DAS.html, http://wooster.bip.bham.ac.uk/DAS.html and http://www.enzim.hu/DAS/DAS.html. The program is available on request.
The c-myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer
Authors: Lewis Brian C. , Klimstra David S. , Varmus Harold E.
Published: December 2003

We have generated a mouse model for pancreatic cancer through the somatic delivery of oncogene-bearing avian retroviruses to mice that express TVA, the receptor for avian leukosis sarcoma virus subgroup A (ALSV-A), under the control of the elastase promoter. Delivery of ALSV-A-based RCAS vectors encoding either mouse polyoma virus middle T antigen (PyMT) or c-Myc to elastase-tv-a transgenic, Ink4a/Arf null mice induced the formation of pancreatic tumors. RCAS-PyMT induced pancreatic tumors with the histologic features of acinar or ductal carcinomas. The induced pancreatic lesions express Pdx1, a marker for pancreas progenitor cells, and many tumors express markers for both exocrine and endocrine cell lineages, suggesting that the tumors may be derived from progenitor cells. In contrast, RCAS-c-myc induced endocrine tumors exclusively, as determined by histology and detection of differentiation markers. Thus, specific oncogenes can induce the formation of different pancreatic tumor types in a single transgenic line, most likely from one or more types of multipotential progenitor cells. Our model appears to be useful for elucidating the genetic alterations, target cells, and signaling pathways that are important in the genesis of different types of pancreatic cancer.