bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuropoietin, a new IL-6-related cytokine signaling through the ciliary neurotrophic factor receptor
Authors: Derouet Damien , Rousseau François , Alfonsi Fabienne , Froger Josy , Hermann Jacques , Barbier Fabien , Perret David , Diveu Caroline , Guillet Catherine , Preisser Laurence , Dumont Agnès , Barbado Maud , Morel Alain , deLapeyrière Odile , Gascan Hugues , Chevalier Sylvie
Published: March 2004

A structural profile-based computational screen was used to identify neuropoietin (NP), a new cytokine. The
np
gene is localized in tandem with the
cardiotrophin
-
1
gene on mouse chromosome 7. NP shares structural and functional features with ciliary neurotrophic factor (CNTF), cardiotrophin-1, and cardiotrophin-like cytokine. It acts through a membrane receptor complex comprising CNTF receptor-α component (CNTFRα), gp130, and leukemia inhibitory factor receptor to activate signal transducer and activator of transcription 3 signaling pathway. NP is highly expressed in embryonic neuroepithelia. Strikingly, CNTFRα, but not its alternate ligands, CNTF and cardiotrophin-like cytokine, is expressed at the same developmental stages. NP is also observed in retina and to a lesser extent in skeletal muscle. Moreover, NP could sustain the
in vitro
survival of embryonic motor neurons and could increase the proliferation of neural precursors when associated to epidermal growth factor and fibroblast growth factor 2. Thus, NP is a new ligand for CNTFRα, with important implications for murine nervous system development.
Distinguishing human ethnic groups by means of sequences from
Helicobacter pylori
: Lessons from Ladakh
Authors: Wirth Thierry , Wang Xiaoyan , Linz Bodo , Novick Richard P. , Lum J. Koji , Blaser Martin , Morelli Giovanna , Falush Daniel , Achtman Mark
Published: March 2004

The history of mankind remains one of the most challenging fields of study. However, the emergence of anatomically modern humans has been so recent that only a few genetically informative polymorphisms have accumulated. Here, we show that DNA sequences from
Helicobacter pylori
, a bacterium that colonizes the stomachs of most humans and is usually transmitted within families, can distinguish between closely related human populations and are superior in this respect to classical human genetic markers.
H. pylori
from Buddhists and Muslims, the two major ethnic communities in Ladakh (India), differ in their population-genetic structure. Moreover, the prokaryotic diversity is consistent with the Buddhists having arisen from an introgression of Tibetan speakers into an ancient Ladakhi population.
H. pylori
from Muslims contain a much stronger ancestral Ladakhi component, except for several isolates with an Indo-European signature, probably reflecting genetic flux from the Near East. These signatures in
H. pylori
sequences are congruent with the recent history of population movements in Ladakh, whereas similar signatures in human microsatellites or mtDNA were only marginally significant.
H. pylori
sequence analysis has the potential to become an important tool for unraveling short-term genetic changes in human populations.
Exploring amyloid formation by a
de novo
design
Authors: Kammerer Richard A. , Kostrewa Dirk , Zurdo Jesús , Detken Andreas , García-Echeverría Carlos , Green Janelle D. , Müller Shirley A. , Meier Beat H. , Winkler Fritz K. , Dobson Christopher M. , Steinmetz Michel O.
Published: March 2004

Protein deposition as amyloid fibrils underlies many debilitating human disorders. The complexity and size of disease-related polypeptides, however, often hinders a detailed rational approach to study effects that contribute to the process of amyloid formation. We report here a simplified peptide sequence successfully designed
de novo
to fold into a coiled-coil conformation under ambient conditions but to transform into amyloid fibrils at elevated temperatures. We have determined the crystal structure of the coiled-coil form and propose a detailed molecular model for the peptide in its fibrillar state. The relative stabilities of the two structural forms and the kinetics of their interconversion were found to be highly sensitive to small sequence changes. The results reveal the importance of specific packing interactions on the kinetics of amyloid formation and show the potential of this exceptionally favorable system for probing details of the molecular origins of amyloid disease.
Mice lacking pro-opiomelanocortin are sensitive to high-fat feeding but respond normally to the acute anorectic effects of peptide-YY
3-36
Authors: Challis B. G. , Coll A. P. , Yeo G. S. H. , Pinnock S. B. , Dickson S. L. , Thresher R. R. , Dixon J. , Zahn D. , Rochford J. J. , White A. , Oliver R. L. , Millington G. , Aparicio S. A. , Colledge W. H. , Russ A. P. , Carlton M. B. , O'Rahilly S.
Published: March 2004

Inactivating mutations of the pro-opiomelanocortin (POMC) gene in both mice and humans leads to hyperphagia and obesity. To further examine the mechanisms whereby POMC-deficiency leads to disordered energy homeostasis, we have generated mice lacking all POMC-derived peptides. Consistent with a previously reported model,
Pomc
-/-
mice were obese and hyperphagic. They also showed reduced resting oxygen consumption associated with lowered serum levels of thyroxine. Hypothalami from
Pomc
-/-
mice showed markedly increased expression of melanin-concentrating hormone mRNA in the lateral hypothalamus, but expression of neuropeptide Y mRNA in the arcuate nucleus was not altered. Provision of a 45% fat diet increased energy intake and body weight in both
Pomc
-/-
and
Pomc
+/-
mice. The effects of leptin on food intake and body weight were blunted in obese
Pomc
-/-
mice whereas nonobese
Pomc
-/-
mice were sensitive to leptin. Surprisingly, we found that
Pomc
-/-
mice maintained their acute anorectic response to peptide-YY
3-36
(PYY
3-36
). However, 7 days of PYY
3-36
administration had no effect on cumulative food intake or body weight in wild-type or
Pomc
-/-
mice. Thus, POMC peptides seem to be necessary for the normal response of energy balance to high-fat feeding, but not for the acute anorectic effect of PYY
3-36
or full effects of leptin on feeding. The finding that the loss of only one copy of the
Pomc
gene is sufficient to render mice susceptible to the effects of high fat feeding emphasizes the potential importance of this locus as a site for gene-environment interactions predisposing to obesity.
A zebrafish model for pyruvate dehydrogenase deficiency: Rescue of neurological dysfunction and embryonic lethality using a ketogenic diet
Authors: Taylor Michael R. , Hurley James B. , Van Epps Heather A. , Brockerhoff Susan E.
Published: March 2004

Defects in the pyruvate dehydrogenase (PDH) complex result in severe neurological dysfunction, congenital lactic acidosis, growth retardation, and early death. Current treatments for PDH deficiency are administered postnatally and are generally unsuccessful. Because many patients with this disease are born with irreversible defects, a model system for the development of effective pre- and postnatal therapies would be of great value. In a behavioral genetic screen aimed to identify zebrafish with visual function defects, we previously isolated two alleles of the recessive lethal mutant
no optokinetic response a
(
noa
). Here we report that
noa
is deficient for dihydrolipoamide
S
-acetyltransferase (Dlat), the PDH E2 subunit, and exhibits phenotypes similar to human patients with PDH deficiency. To rescue the deficiency, we added ketogenic substrates to the water in which the embryos develop. This treatment successfully restored vision, promoted feeding behavior, reduced lactic acidosis, and increased survival. Our study demonstrates an approach for establishing effective therapies for PDH deficiency and other congenital diseases that affect early embryonic development.
Yeast genome-wide drug-induced haploinsufficiency screen to determine drug mode of action
Authors: Baetz Kristin , McHardy Lianne , Gable Ken , Tarling Tamsin , Rebérioux Delphine , Bryan Jenny , Andersen Raymond J. , Dunn Teresa , Hieter Phil , Roberge Michel
Published: March 2004

Methods to systematically test drugs against all possible proteins in a cell are needed to identify the targets underlying their therapeutic action and unwanted effects. Here, we show that a genome-wide drug-induced haploinsufficiency screen by using yeast can reveal drug mode of action in yeast and can be used to predict drug mode of action in human cells. We demonstrate that dihydromotuporamine C, a compound in preclinical development that inhibits angiogenesis and metastasis by an unknown mechanism, targets sphingolipid metabolism. The systematic, unbiased and genome-wide nature of this technique makes it attractive as a general approach to identify cellular pathways affected by drugs.
Human and avian influenza viruses target different cell types in cultures of human airway epithelium
Authors: Matrosovich Mikhail N. , Matrosovich Tatyana Y. , Gray Thomas , Roberts Noel A. , Klenk Hans-Dieter
Published: March 2004

The recent human infections caused by H5N1, H9N2, and H7N7 avian influenza viruses highlighted the continuous threat of new pathogenic influenza viruses emerging from a natural reservoir in birds. It is generally believed that replication of avian influenza viruses in humans is restricted by a poor fit of these viruses to cellular receptors and extracellular inhibitors in the human respiratory tract. However, detailed mechanisms of this restriction remain obscure. Here, using cultures of differentiated human airway epithelial cells, we demonstrated that influenza viruses enter the airway epithelium through specific target cells and that there were striking differences in this respect between human and avian viruses. During the course of a single-cycle infection, human viruses preferentially infected nonciliated cells, whereas avian viruses as well as the egg-adapted human virus variant with an avian virus-like receptor specificity mainly infected ciliated cells. This pattern correlated with the predominant localization of receptors for human viruses (2-6-linked sialic acids) on nonciliated cells and of receptors for avian viruses (2-3-linked sialic acids) on ciliated cells. These findings suggest that although avian influenza viruses can infect human airway epithelium, their replication may be limited by a nonoptimal cellular tropism. Our data throw light on the mechanisms of generation of pandemic viruses from their avian progenitors and open avenues for cell level-oriented studies on the replication and pathogenicity of influenza virus in humans.
Successful herbivore attack due to metabolic diversion of a plant chemical defense
Authors: Wittstock Ute , Agerbirk Niels , Stauber Einar J. , Olsen Carl Erik , Hippler Michael , Mitchell-Olds Thomas , Gershenzon Jonathan , Vogel Heiko
Published: March 2004

Plants protect themselves against herbivory with a diverse array of repellent or toxic secondary metabolites. However, many herbivorous insects have developed counteradaptations that enable them to feed on chemically defended plants without apparent negative effects. Here, we present evidence that larvae of the specialist insect,
Pieris rapae
(cabbage white butterfly, Lepidoptera: Pieridae), are biochemically adapted to the glucosinolate–myrosinase system, the major chemical defense of their host plants. The defensive function of the glucosinolate–myrosinase system results from the toxic isothiocyanates that are released when glucosinolates are hydrolyzed by myrosinases on tissue disruption. We show that the hydrolysis reaction is redirected toward the formation of nitriles instead of isothiocyanates if plant material is ingested by
P. rapae
larvae, and that the nitriles are excreted with the feces. The ability to form nitriles is due to a larval gut protein, designated nitrile-specifier protein, that by itself has no hydrolytic activity on glucosinolates and that is unrelated to any functionally characterized protein. Nitrile-specifier protein appears to be the key biochemical counteradaptation that allows
P. rapae
to feed with impunity on plants containing glucosinolates and myrosinases. This finding sheds light on the ecology and evolution of plant–insect interactions and suggests novel highly selective pest management strategies.
Biomolecular cryocrystallography: Structural changes during flash-cooling
Authors: Halle Bertil
Published: March 2004

To minimize radiation damage, crystal structures of biological macromolecules are usually determined after rapid cooling to cryogenic temperatures, some 150–200 K below the normal physiological range. The biological relevance of such structures relies on the assumption that flash-cooling is sufficiently fast to kinetically trap the macromolecule and associated solvent in a room-temperature equilibrium state. To test this assumption, we use a two-state model to calculate the structural changes expected during rapid cooling of a typical protein crystal. The analysis indicates that many degrees of freedom in a flash-cooled protein crystal are quenched at temperatures near 200 K, where local conformational and association equilibria may be strongly shifted toward low-enthalpy states. Such cryoartifacts should be most important for strongly solvent-coupled processes, such as hydration of nonpolar cavities and surface regions, conformational switching of solvent-exposed side chains, and weak ligand binding. The dynamic quenching that emerges from the model considered here can also rationalize the glass transition associated with the atomic fluctuations in the protein.
Default-mode network activity distinguishes Alzheimer's disease from healthy aging: Evidence from functional MRI
Authors: Greicius Michael D. , Srivastava Gaurav , Reiss Allan L. , Menon Vinod
Published: March 2004

Recent functional imaging studies have revealed coactivation in a distributed network of cortical regions that characterizes the resting state, or default mode, of the human brain. Among the brain regions implicated in this network, several, including the posterior cingulate cortex and inferior parietal lobes, have also shown decreased metabolism early in the course of Alzheimer's disease (AD). We reasoned that default-mode network activity might therefore be abnormal in AD. To test this hypothesis, we used independent component analysis to isolate the network in a group of 13 subjects with mild AD and in a group of 13 age-matched elderly controls as they performed a simple sensory-motor processing task. Three important findings are reported. Prominent coactivation of the hippocampus, detected in all groups, suggests that the default-mode network is closely involved with episodic memory processing. The AD group showed decreased resting-state activity in the posterior cingulate and hippocampus, suggesting that disrupted connectivity between these two regions accounts for the posterior cingulate hypometabolism commonly detected in positron emission tomography studies of early AD. Finally, a goodness-of-fit analysis applied at the individual subject level suggests that activity in the default-mode network may ultimately prove a sensitive and specific biomarker for incipient AD.
Transcript profiling of early lateral root initiation
Authors: Himanen Kristiina , Vuylsteke Marnik , Vanneste Steffen , Vercruysse Steven , Boucheron Elodie , Alard Philippe , Chriqui Dominique , Van Montagu Marc , Inzé Dirk , Beeckman Tom
Published: March 2004

At the onset of lateral root initiation inArabidopsis thaliana, the phytohormone auxin activates xylem pole pericycle cells for asymmetric cell division. However, the molecular events leading from auxin to lateral root initiation are poorly understood, in part because the few responsive cells in the process are embedded in the root and are thus difficult to access. A lateral root induction system, in which most xylem pole pericycle cells were synchronously activated by auxin transport inhibition followed by auxin application, was used for microarray transcript profiling. Of 4,600 genes analyzed, 906 significantly differentially regulated genes were identified that could be grouped into six major clusters. Basically, three major patterns were discerned representing induced, repressed, and transiently expressed genes. Analysis of the coregulated genes, which were specific for each time point, provided new insight into the molecular regulation and signal transduction preceding lateral root initiation inArabidopsis. The reproducible expression profiles during a time course allowed us to define four stages that precede the cell division in the pericycle. These early stages were characterized by G1cell cycle block, auxin perception, and signal transduction, followed by progression over G1/S transition and G2/M transition. All these processes took place within 6 h after transfer fromN-1-naphthylphthalamic acid to 1-naphthalene acetic acid. These results indicate that this lateral root induction system represents a unique synchronized system that allows the systematic study of the developmental program upstream of the cell cycle activation during lateral root initiation.
Phenotypic effects of leptin replacement on morbid obesity, diabetes mellitus, hypogonadism, and behavior in leptin-deficient adults
Authors: Licinio Julio , Caglayan Sinan , Ozata Metin , Yildiz Bulent O. , de Miranda Patricia B. , O'Kirwan Fiona , Whitby Robert , Liang Liyin , Cohen Pinchas , Bhasin Shalender , Krauss Ronald M. , Veldhuis Johannes D. , Wagner Anthony J. , DePaoli Alex M. , McCann Samuel M. , Wong Ma-Li
Published: March 2004

Genetic mutations in the leptin pathway can be a cause of human obesity. It is still unknown whether leptin can be effective in the treatment of fully established morbid obesity and its endocrine and metabolic consequences in adults. To test the hypothesis that leptin has a key role in metabolic and endocrine regulation in adults, we examined the effects of human leptin replacement in the only three adults identified to date who have genetically based leptin deficiency. We treated these three morbidly obese homozygous leptin-deficient adult patients with recombinant human leptin at low, physiological replacement doses in the range of 0.01–0.04 mg/kg for 18 months. Patients were hypogonadal, and one of them also had type 2 diabetes mellitus. We chose the doses of recombinant methionyl human leptin that would achieve normal leptin concentrations and administered them daily in the evening to model the normal circadian variation in endogenous leptin. The mean body mass index dropped from 51.2 ± 2.5 (mean ± SEM) at baseline to 26.9 ± 2.1 kg/m
2
after 18 months of treatment, mainly because of loss of fat mass. We document here that leptin replacement therapy in leptin-deficient adults with established morbid obesity results in profound weight loss, increased physical activity, changes in endocrine function and metabolism, including resolution of type 2 diabetes mellitus and hypogonadism, and beneficial effects on ingestive and noningestive behavior. These results highlight the role of the leptin pathway in adults with key effects on the regulation of body weight, gonadal function, and behavior.
Understanding nature's design for a nanosyringe
Authors: Lopez Carlos F. , Nielsen Steve O. , Moore Preston B. , Klein Michael L.
Published: March 2004

Synthetic and natural peptide assemblies can possess transport or conductance activity across biomembranes through the formation of nanopores. The fundamental mechanisms of membrane insertion necessary for antimicrobial or synthetic pore formation are poorly understood. We observe a lipid-assisted mechanism for passive insertion into a model membrane from molecular dynamics simulations. The assembly used in the study, a generic nanotube functionalized with hydrophilic termini, is assisted in crossing the membrane core by transleaflet lipid flips. Lipid tails occlude a purely hydrophobic nanotube. The observed insertion mechanism requirements for hydrophobic-hydrophilic matching have implications for the design of synthetic channels and antibiotics.
Regulation of cyclin-dependent kinase activity during mitotic exit and maintenance of genome stability by p21, p27, and p107
Authors: Chibazakura Taku , McGrew Seth G. , Cooper Jonathan A. , Yoshikawa Hirofumi , Roberts James M.
Published: March 2004

To study the regulation of cyclin-dependent kinase (CDK) activity during mitotic exit in mammalian cells, we constructed murine cell lines that constitutively express a stabilized mutant of cyclin A (cyclin A47). Even though cyclin A47 was expressed throughout mitosis and in G
1
cells, its associated CDK activity was inactivated after the transition from metaphase to anaphase. Cyclin A47 associated with both p21 and p27 during mitotic exit, implicating these proteins in CDK inactivation. However, cyclin A47 was fully inhibited during the M-to-G
1
transition in
p21

–
/
–

p27

–
/
–

fibroblasts. Also, the CDKs associated with cyclin A47 were not inactivated by phosphorylation at tyrosines. The protein responsible for CDK inactivation during mitotic exit in p21/p27 null cells was the Rb family member, p107. p107 bound to cyclin A47 when p21 and p27 were absent, and cyclin A47–CDK activity was not inactivated during the M-to-G
1
transition in
p21

–
/
–

p27

–
/
–

p107

–
/
–

null fibroblasts. Enforced expression of cyclin A in cells lacking all three CDK inhibitors induced rapid tetraploidization, indicative of mitotic failure/endoreduplication. We concluded that cyclin proteolysis and CDK inhibitors constitute redundant pathways that control cyclin A–CDK activity during mitotic exit in mammalian cells and that loss of these pathways can cause genetic instability.
The role of RNA polymerase σ subunit in promoter-independent initiation of transcription
Authors: Zenkin Nikolay , Severinov Konstantin
Published: March 2004

In bacteria, initiation of transcription depends on the RNA polymerase σ subunit, which brings catalytically proficient RNA polymerase core to promoters by binding to specific DNA elements located upstream of the transcription start point. Here, we study σ-dependent synthesis of a transcript that is used to prime replication of the single-stranded genome of bacteriophage M13. We show that, in this system, σ plays no role in DNA recognition, which is accomplished solely through RNA polymerase core interaction with DNA downstream of the transcription start point. However, σ is required for full-sized transcript synthesis by allowing RNA polymerase core to escape into productive elongation. RNA polymerase σ may play a similar role during replication primer synthesis in other bacterial mobile elements whose life cycle involves a single-stranded DNA stage.
D2 receptor regulation of synaptic burst firing in prefrontal cortical pyramidal neurons
Authors: Wang Yun , Goldman-Rakic Patricia S.
Published: March 2004

The efficacy of antipsychotics in the treatment of schizophrenia depends on their ability to block dopamine (DA) D2 receptors. D2 receptor excitatory mediation of glutamatergic receptors has been implicated inin vivostudies. However, D2 receptor enhancement of glutamatergic transmission has rarely been reported in slice recordings. Instead, D2 receptor depression of both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) andN-methyl-d-aspartate (NMDA) action was obtained in previous slice studies. To obtain insight into this paradox, we examined DA's actions on synaptic responses of layer V pyramidal cells to minimal extracellular stimulation in layer III of ferret prefrontal cortical slices under NMDA and γ-aminobutyric acid type A blockade. This experimental design models the proposed hypofunction of NMDA receptor and γ-aminobutyric acid type A deficiency in schizophrenia. We found that DA and D2 receptor agonists promoted burst firing in a subset of pyramidal cells, which was reversed by haloperidol, a D2 antagonist and a D3 agonist, compounds having antipsychotic efficacy. In contrast, a D4 antagonist, which has not proven clinically effective, was not effective in blocking DA-promoted bursts. These results revealed excitatory effects of DA mediated mainly via D2 receptors, potentially providing a cellular mechanism for the D2 antagonism in treating schizophrenia.
RBSP3 (HYA22)
is a tumor suppressor gene implicated in major epithelial malignancies
Authors: Kashuba Vladimir I. , Li Jingfeng , Wang Fuli , Senchenko Vera N. , Protopopov Alexey , Malyukova Alena , Kutsenko Alexey S. , Kadyrova Elena , Zabarovska Veronika I. , Muravenko Olga V. , Zelenin Alexander V. , Kisselev Lev L. , Kuzmin Igor , Minna John D. , Winberg Gösta , Ernberg Ingemar , Braga Eleonora , Lerman Michael I. , Klein George , Zabarovsky Eugene R.
Published: March 2004

Chromosome 3p21.3 region is frequently (>90%) deleted in lung and other major human carcinomas. We subdivided 3p21.3 into LUCA and AP20 subregions and discovered frequent homozygous deletions (10-18%) in both subregions. This finding strongly implies that they harbor multiple tumor suppressor genes involved in the origin and/or development of major epithelial cancers. In this study, we performed an initial analysis of
RBSP3/HYA22
, a candidate tumor suppressor genes located in the AP20 region. Two sequence splice variants of
RBSP3/HYA22
(A and B) were identified, and we provide evidence for their tumor suppressor function. By sequence analysis
RBSP3/HYA22
belongs to a gene family of small C-terminal domain phosphatases that may control the RNA polymerase II transcription machinery. Expression of the gene was drastically (>20-fold) decreased in 11 of 12 analyzed carcinoma cell lines and in three of eight tumor biopsies. We report missense and nonsense mutations in tumors where
RBSP3/HYA22
was expressed, growth suppression with regulated transgenes in culture, suppression of tumor formation in severe combined immunodeficient mice, and dephosphorylation of ppRB by RBSP3/HYA22, presumably leading to a block of the cell cycle at the G
1
/S boundary.
Prevention of Autoimmunity by Targeting a Distinct, Noninvariant CD1d-reactive T Cell Population Reactive to Sulfatide
Authors: Jahng Alex , Maricic Igor , Aguilera Carlos , Cardell Susanna , Halder Ramesh C. , Kumar Vipin
Published: March 2004

Class I and class II MHC-restricted T cells specific for proteins present in myelin have been shown to be involved in autoimmunity in the central nervous system (CNS). It is not yet known whether CD1d-restricted T cells reactive to myelin-derived lipids are present in the CNS and might be targeted to influence the course of autoimmune demyelination. Using specific glycolipid-CD1d tetramers and cloned T cells we have characterized a T cell population reactive to a myelin-derived glycolipid, sulfatide, presented by CD1d. This population is distinct from the invariant Vα14+ NK T cells, and a panel of Vα3/Vα8+ CD1d-restricted NK T cell hybridomas is unable to recognize sulfatide in the presence of CD1d+ antigen-presenting cells. Interestingly, during experimental autoimmune encephalomyelitis a model for human multiple sclerosis, sulfatide-reactive T cells but not invariant NK T cells are increased severalfold in CNS tissue. Moreover, treatment of mice with sulfatide prevents antigen-induced experimental autoimmune encephalomyelitis in wild-type but not in CD1d-deficient mice. Disease prevention correlates with the ability of sulfatide to suppress both interferon-γ and interleukin-4 production by pathogenic myelin oligodendrocyte glycoprotein-reactive T cells. Since recognition of sulfatide by CD1d-restricted T cells has now been shown both in mice and humans, study of murine myelin lipid-reactive T cells may form a basis for the development of intervention strategies in human autoimmune demyelinating diseases.
Immediate Cytotoxicity But Not Degranulation Distinguishes Effector and Memory Subsets of CD8+ T Cells
Authors: Wolint Petra , Betts Michael R. , Koup Richard A. , Oxenius Annette
Published: March 2004

CD8+ T cells play a central role in the resolution and containment of viral infections. A key effector function of CD8+ T cells is their cytolytic activity toward infected cells. Here, we studied the regulation of cytolytic activity in naive, effector, and central versus effector memory CD8+ T cells specific for the same glycoprotein-derived epitope of lymphocytic choriomeningitis virus. Our results show that the kinetics of degranulation, assessed by a novel flow cytometric based assay, were identical in effector and both subsets of memory CD8+ T cells, but absent in naive CD8+ T cells. However, immediate cytolytic activity was most pronounced in effector T cells, low in effector memory T cells, and absent in central memory T cells, correlating with the respective levels of cytolytic effector molecules present in lytic granules. These results indicate that an inherent program of degranulation is a feature of antigen-experienced cells as opposed to naive CD8+ T cells and that the ability of CD8+ T cells to induce target cell apoptosis/death is dependent on granule protein content rather than on the act of degranulation itself. Furthermore, these results provide a potential mechanism by which central memory CD8+ T cell–mediated death of antigen-presenting cells within the lymph node is avoided.
The Sec1/Munc18 Protein, Vps33p, Functions at the Endosome and the Vacuole ofSaccharomyces cerevisiae
Authors: Subramanian Shoba , Woolford Carol A. , Jones Elizabeth W.
Published: March 2004

The Sec1/Munc18 (SM) family of proteins is thought to impart compartmental specificity to vesicle fusion reactions. Here we report characterization of Vps33p, an SM family member previously thought to act exclusively at the vacuolar membrane with the vacuolar syntaxin Vam3p. Vacuolar morphology of vps33Δ cells resembles that of cells lacking both Vam3p and the endosomal syntaxin Pep12p, suggesting that Vps33p may function with these syntaxins at the vacuole and the endosome. Consistent with this, vps33 mutants secrete the Golgi precursor form of the vacuolar hydrolase CPY into the medium. We also demonstrate that Vps33p acts at other steps, for vps33 mutants show severe defects in endocytosis at the late endosome. At the endosome, Vps33p and other class C members exist as a complex with Vps8p, a protein previously known to act in transport between the late Golgi and the endosome. Vps33p also interacts with Pep12p, a known interactor of the SM protein Vps45p. High copy PEP7/VAC1 suppresses vacuolar morphology defects of vps33 mutants. These findings demonstrate that Vps33p functions at multiple trafficking steps and is not limited to action at the vacuolar membrane. This is the first report demonstrating the involvement of a single syntaxin with two SM proteins at the same organelle.
Gap Junction Channel Protein Innexin 2 Is Essential for Epithelial Morphogenesis in theDrosophilaEmbryo
Authors: Bauer Reinhard , Lehmann Corinna , Martini Julia , Eckardt Franka , Hoch Michael
Published: March 2004

Direct communication of neighboring cells by gap junction channels is essential for the development of tissues and organs in the body. Whereas vertebrate gap junctions are composed of members of the connexin family of transmembrane proteins, in invertebrates gap junctions consist of Innexin channel proteins. Innexins display very low sequence homology to connexins. In addition, very little is known about their cellular role during developmental processes. In this report, we examined the function and the distribution of Drosophila Innexin 2 protein in embryonic epithelia. Both loss-of-function and gain-of-function innexin 2 mutants display severe developmental defects due to cell death and a failure of proper epithelial morphogenesis. Furthermore, immunohistochemical analyses using antibodies against the Innexins 1 and 2 indicate that the distribution of Innexin gap junction proteins to specific membrane domains is regulated by tissue specific factors. Finally, biochemical interaction studies together with genetic loss- and gain-of-function experiments provide evidence that Innexin 2 interacts with core proteins of adherens and septate junctions. This is the first study, to our knowledge, of cellular distribution and protein–protein interactions of an Innexin gap junctional channel protein in the developing epithelia of Drosophila.
Identification of a Novel Microtubule-destabilizing Motif in CPAP That Binds to Tubulin Heterodimers and Inhibits Microtubule Assembly
Authors: Hung Liang-Yi , Chen Hua-Ling , Chang Ching-Wen , Li Bor-Ran , Tang Tang K.
Published: March 2004

We have previously identified a new centrosomal protein, centrosomal protein 4.1-associated protein (CPAP), which is associated with the γ-tubulin complex. Here, we report that CPAP carries a novel microtubule-destabilizing motif that not only inhibits microtubule nucleation from the centrosome but also depolymerizes taxol-stabilized microtubules. Deletion mapping and functional analyses have defined a 112-residue CPAP that is necessary and sufficient for microtubule destabilization. This 112-residue CPAP directly recognizes the plus end of a microtubule and inhibits microtubule nucleation from the centrosome. Biochemical and functional analyses revealed that this 112-residue CPAP also binds to tubulin dimers, resulting in the destabilization of microtubules. Using the tetracycline-controlled system (tet-off), we observed that overexpression of this 112-residue CPAP inhibits cell proliferation and induces apoptosis after G2/M arrest. The possible mechanisms of how this 112-residue motif in CPAP that inhibits microtubule nucleation from the centrosome and disassembles preformed microtubules are discussed.
Role for Actin Filament Turnover and a Myosin II Motor in Cytoskeleton-driven Disassembly of the Epithelial Apical Junctional Complex
Authors: Ivanov Andrei I. , McCall Ingrid C. , Parkos Charles A. , Nusrat Asma
Published: March 2004

Disassembly of the epithelial apical junctional complex (AJC), composed of the tight junction (TJ) and adherens junction (AJ), is important for normal tissue remodeling and pathogen-induced disruption of epithelial barriers. Using a calcium depletion model in T84 epithelial cells, we previously found that disassembly of the AJC results in endocytosis of AJ/TJ proteins. In the present study, we investigated the role of the actin cytoskeleton in disassembly and internalization of the AJC. Calcium depletion induced reorganization of apical F-actin into contractile rings. Internalized AJ/TJ proteins colocalized with these rings. Both depolymerization and stabilization of F-actin inhibited ring formation and disassembly of the AJC, suggesting a role for actin filament turnover. Actin reorganization was accompanied by activation (dephosphorylation) of cofilin-1 and its translocation to the F-actin rings. In addition, Arp3 and cortactin colocalized with these rings. F-actin reorganization and disassembly of the AJC were blocked by blebbistatin, an inhibitor of nonmuscle myosin II. Myosin IIA was expressed in T84 cells and colocalized with F-actin rings. We conclude that disassembly of the AJC in calcium-depleted cells is driven by reorganization of apical F-actin. Mechanisms of such reorganization involve cofilin-1-dependent depolymerization and Arp2/3-assisted repolymerization of actin filaments as well as myosin IIA-mediated contraction.
Simultaneous search for multiple QTL using the global optimization algorithm DIRECT
Authors: Ljungberg K. , Holmgren S. , Carlborg Ö.
Published: March 2004

Motivation: A simultaneous search is necessary for maximizing the power to detect epistatic quantitative trait loci (QTL). The computational complexity demands that the traditional exhaustive search be replaced by a more efficient global optimization algorithm.

Results: We have the previously known algorithm adapted DIRECT, to the problem of simultaneous mapping of multiple QTL. We have compared DIRECT with standard exhaustive search and a genetic algorithm previously used for QTL mapping in two dimensions. In all two- and three-QTL test cases, DIRECT accurately finds the global optimum two to four orders of magnitude faster than when using an exhaustive search, and one order of magnitude faster than when using the genetic algorithm. Thus, randomization testing for determining empirical significance thresholds for at least three QTL is made feasible by the use of DIRECT.

Availability: The code of the prototype implementation is available at http://user.it.uu.se/~kl/qtl_software.html
CEBS object model for systems biology data, SysBio-OM
Authors: Xirasagar Sandhya , Gustafson Scott , Merrick B. Alex , Tomer Kenneth B. , Stasiewicz Stanley , Chan Denny D. , Yost Kenneth J. , Yates John R. , Sumner Susan , Xiao Nianqing , Waters Michael D.
Published: March 2004

Motivation: To promote a systems biology approach to understanding the biological effects of environmental stressors, the Chemical Effects in Biological Systems (CEBS) knowledge base is being developed to house data from multiple complex data streams in a systems friendly manner that will accommodate extensive querying from users. Unified data representation via a single object model will greatly aid in integrating data storage and management, and facilitate reuse of software to analyze and display data resulting from diverse differential expression or differential profile technologies. Data streams include, but are not limited to, gene expression analysis (transcriptomics), protein expression and protein–protein interaction analysis (proteomics) and changes in low molecular weight metabolite levels (metabolomics).

Results: To enable the integration of microarray gene expression, proteomics and metabolomics data in the CEBS system, we designed an object model, Systems Biology Object Model (SysBio-OM). The model is comprehensive and leverages other open source efforts, namely the MicroArray Gene Expression Object Model (MAGE-OM) and the Proteomics Experiment Data Repository (PEDRo) object model. SysBio-OM is designed by extending MAGE-OM to represent protein expression data elements (including those from PEDRo), protein–protein interaction and metabolomics data. SysBio-OM promotes the standardization of data representation and data quality by facilitating the capture of the minimum annotation required for an experiment. Such standardization refines the accuracy of data mining and interpretation. The open source SysBio-OM model, which can be implemented on varied computing platforms is presented here.

Availability: A universal modeling language depiction of the entire SysBio-OM is available at http://cebs.niehs.nih.gov/SysBioOM/. The Rational Rose object model package is distributed under an open source license that permits unrestricted academic and commercial use and is available at http://cebs.niehs.nih.gov/cebsdownloads. The database and interface are being built to implement the model and will be available for public use at http://cebs.niehs.nih.gov.
DNA microarray normalization methods can remove bias from differential protein expression analysis of 2D difference gel electrophoresis results
Authors: Kreil David P. , Karp Natasha A. , Lilley Kathryn S.
Published: March 2004

Motivation: Two-dimensional Difference Gel Electrophoresis (DIGE) measures expression differences for thousands of proteins in parallel. In contrast to DNA microarray analysis, however, there have been few systematic studies on the validity of differential protein expression analysis, and the effects of normalization methods have not yet been investigated. To address this need, we assessed a series of same–same comparisons, evaluating how random experimental variance influenced differential expression analysis.

Results: The strong fluctuations observed were reflected in large discrepancies between the distributions of the spot intensities for different gels. Correct normalization for pooling of multiple gels for analysis is, therefore, essential. We show that both dye-specific background levels and the differences in scale of the spot intensity distributions must be accounted for. A variance stabilizing transform that had been developed for DNA microarray analysis combined with a robust Z-score allowed the determination of gel-independent signal thresholds based on the empirical distributions from same–same comparisons. In contrast, similar thresholds holding up to cross-validation could not be proposed for data normalized using methods established in the field of proteomics.

Availability: Software is available on request from the authors.

Supplementary information: There is supplementary material available online at http://www.flychip.org.uk/kreil/pub/2dgels/
Identifying multiple alignment regions satisfying simple formulas and patterns
Authors: Stojanovic Nikola , Dewar Ken
Published: March 2004

Motivation: When studying multiple alignments of genomic sequences one frequently aims to locate and count regions which satisfy a set of constraints. These regions may be putatively functional, but researchers may also be interested in quantifying the frequency of occurrences of certain patterns.

Results: We have developed a program that applies simple formulas and pattern specifications to multiple alignments, reporting the positions and counts of conforming regions. As an example, we have navigated a 15-species alignment of the CAV2–CAV1 region and outlined some findings regarding PPARγ binding sites.

Availability: Our software and the accompanying documentation can be obtained at no charge by contacting the authors. It can also be accessed at http://ranger.uta.edu/~nick/compgen
Major Histocompatibility Gene Therapy: The Importance of Haplotype and β2‐Microglobulin
Authors: Salamone Frank N. , Gleich Lyon L. , Li Ya‐Qin , Stambrook Peter J.
Published: March 2004

Objectives/Hypothesis Alloantigen gene therapy with the genes for the Class I major histocompatibility complex (MHC) HLA‐B7 and β2‐microglobulin in HLA‐B7–negative patients has potential efficacy in the treatment of head and neck cancer, although the mechanism of response is unclear. Whether tumor regression is due to a response to HLA‐B7 in HLA‐B7–negative patients (i.e., due to “foreign” antigen) or simply to MHC overexpression is unknown. Therefore, a mouse model was used to compare tumor growth following syngeneic MHC transfection to alloantigenic MHC transfection. The importance of the β2‐microglobulin gene was also evaluated.

Study Design Prospective animal study.

Methods The head and neck cancer cell line SCC‐VII that grows in immunocompetent C3H mice, which are MHC haplotype H2‐Kk, was used. Stable transfections were made with H2‐Kb, H2‐Kk, and β2‐microglobulin in the SCC‐VII cells. To test the importance of MHC “foreignness,” mice were injected with SCC‐VII cells, SCC‐VII plus H2‐Kb plus β2‐microglobulin transfected cells, and SCC‐VII plus H2‐Kk plus β2‐microglobulin transfected cells. To evaluate β2‐microglobulin, mice were injected with SCC‐VII cells, SCC‐VII plus H2‐Kb plus β2‐microglobulin transfected cells, SCC‐VII plusH2‐Kb transfected cells, and SCC‐VII plus β2‐microglobulin transfected cells. Tumor growth in all groups was compared statistically.

Results Major histocompatibility complex foreignness was a part of the antitumor response. Foreign MHC routinely abrogated tumor growth, whereas syngeneic MHC only slowed tumor growth. β2‐microglobulin aided the MHC tumor inhibition but did not inhibit tumor without the MHC.

Conclusion The antitumor response was greater when the MHC gene used was foreign. β2‐microglobulin increased the efficacy of MHC gene therapy. Both of these findings are important when designing clinical trials of immunologically based gene therapies for head and neck cancer.
Cochlear Changes in Chronic Otitis Media
Authors: Cureoglu Sebahattin , Schachern Patricia A. , Paparella Michael M. , Lindgren Bruce R.
Published: March 2004

Objectives/Hypothesis The objective was to describe the morphological changes in the cochlea in chronic otitis media.

Study Design Retrospective human temporal bone analysis.

Methods Fifteen temporal bones with unilateral chronic otitis media were selected and compared with contralateral normal temporal bones. Standard cytocochleograms and spiral ganglion cell reconstructions were performed on all temporal bones. Spiral ligament was divided into four segments according to the locations of different types of fibrocytes. The average loss of fibrocytes in each segment was estimated. Morphometric measurements of areas of stria vascularis and spiral ligament were made in all turns of the cochlea on mid modiolar sections.

Results Loss of outer and inner hair cells was common in the basal turn of the cochlea in temporal bones with chronic otitis media compared with control ears. There was no difference in the number of spiral ganglion cells in the chronic otitis media and contralateral ears. The areas of stria vascularis and spiral ligament in the basal turn decreased significantly in the ears with chronic otitis media compared with control ears. There were no significant differences between the ears with chronic otitis media and the contralateral ears for any of the regions characterized by the presence of types I–IV fibrocytes.

Conclusion The results of the study are consistent with the hypothesis that chronic otitis media causes cochlear disease.
Chemokine RANTES Promoter Polymorphisms in Allergic Rhinitis
Authors: Kim Jeong Joong , Lee Jung Hun , Jang Chul Ho , Kim Yong Shin , Chae Soo Cheon , Chung Hun Taeg , Choi Tae Wook , Lee Jae Hoon
Published: March 2004

Objectives/Hypothesis RANTES is one of the most widely studied of the chemokines linked to allergic diseases. Two polymorphisms of the RANTES promoter region (–403 G/A and –28 C/G) have been found. The authors investigated whether these RANTES promoter polymorphisms were associated with allergic rhinitis.

Study Design Case‐control study.

Methods Blood samples for genetic analysis were obtained from 151 individuals with allergic rhinitis and from 278 healthy individuals without atopic disease. Polymerase chain reaction–based assays for detection of the –403 G/A and –28 C/G polymorphisms of the RANTES gene were used for genotyping.

Results The frequencies of both the RANTES –403A and –28G alleles were significantly higher in patients with allergic rhinitis than in control subjects (P < .05 for both).

Conclusion The study results indicated that the –403 and –28 alleles in the RANTES promoter region belong to the predictor gene set for allergic rhinitis and could be used in genomic analysis.
Inner Ear Changes With Intracochlear Gentamicin Administration in Guinea Pigs
Authors: Okuda Takeshi , Sugahara Kazuma , Shimogori Hiroaki , Yamashita Hiroshi
Published: March 2004

Objectives/Hypothesis Transtympanic administration of gentamicin is reported to be a useful treatment for vertigo in such conditions as Meniere's disease, and determining appropriate clinical dosage of gentamicin is difficult. The authors examined the relation between gentamicin dosages and inner ear function in guinea pigs.

Study Design This study is a basic science project designed to examine cochlear and vestibular function in animal models.

Methods Various concentrations of gentamicin solution were infused into the right inner ear of guinea pigs by osmotic pumps. Caloric nystagmus as a marker of vestibular function and the change in auditory brainstem response (ABR) threshold as a marker of cochlear function were observed.

Results After 14 days of treatment, high gentamicin concentrations of 40 mg/mL caused canal paralysis and a rapid shift in ABR threshold. Animals exposed to low gentamicin concentrations of 4 mg/mL showed no obvious change in either vestibular or cochlear function. Animals exposed to moderate gentamicin concentrations of 12 mg/mL showed a moderate shift in ABR threshold and caloric malfunction. Histopathological examination revealed that after 14 days of treatment with 40 mg/mL gentamicin, severe cytoplasmic damage occurred in both vestibular and cochlear end organs. In animals treated with 12 mg/mL gentamicin, hair cells remained in the cochlear third turn and ampulla of the lateral semicircular canal.

Conclusion The authors established an animal model that showed the moderate damage of inner ear with moderate‐dose gentamicin. The study results indicated that the appropriate administration of gentamicin could establish a stable effect on the inner ear. It may be important to select the protocol that delivers a stable dosage of gentamicin to treat patients with Meniere's disease safely and effectively.