bluuebunny/crossref_metadata_2025 huggingface dataset:
Drinking and university students: from celebration to inebriation
Authors: ROCHE ANN M. , WATT KERRIANNE
Published: July 2002

There is extensive anecdotal information and considerable interest in the levels and patterns of drinking among university students in Australia. However, few empirical data exist. As a result, a survey was undertaken of students attending the three major universities in the southeast corner of Queensland. Two hundred and nine male and 191 female students, aged 17–25 years, completed a 30‐item survey designed to assess prevalence and patterns of alcohol consumption, associated harms and students' alcohol‐related attitudes. Nearly all (94%) students drank and many drank very heavily. Approximately half the male students and one‐fifth of female students reported drinking to intoxication once or more per week. Consumption of five or more standard drinks on a typical drinking occasion was reported by 54% of students. Overall, more than two‐thirds (69%) of the sample reported drinking at hazardous or harmful levels. About one‐third of the sample reported experiencing an alcohol‐related accident or injury within the last 12 months. Despite this, 62% of students did not believe that a reduction in student drinking was necessary. In addition to examining information on alcohol‐related attitudes and behaviour, this survey identified several groups particularly at risk. [Roche A, Watt K. Drinking and university students: from celebration to inebriation. Drug Alcohol Rev 1999; 18: 389–399]
Anglophilia in crisis: Italian liberals, the ‘English model’ and democracy in the Giolittian era
Authors: Cerasi Laura
Published: July 2002

The image of England was very important to the political culture of Italian liberals during the Risorgimento and the post-unification period. Even if it was viewed as unreachable, it still constituted an example of progress that Italy could strive towards. This article reconstructs the stages of the process that saw a section of Italian political culture become alienated from this image, just at a time when a more complete form of industrial democracy was becoming eswtablished in Britain. This alienation is interpreted here as having its roots in attitudes to the issues raised by the workers’ movement: Italian liberals were reluctant to give it full legitimacy and engage it on the open terrain of social and political competition. The first two sections of the article go over the traits of Italian Anglophilia up to the end of the nineteenth century and single out the earliest reasons for alienation. The third section identifies the Boer War, the figure of Chamberlain and the cluster of issues linking democracy, imperialism and protectionism as key factors in the change of direction. The fourth section shows that the constitutional conflict in Britain and the Parliament Act were a further, decisive, step in Italian liberals’ disaffection with the ‘English model'.
Actin Filament Cables in Drosophila Nurse Cells Are Composed of Modules That Slide Passively Past One Another during Dumping
Authors: Guild Gregory M. , Connelly Patricia S. , Shaw Michael K. , Tilney Lewis G.
Published: July 2002

At a late stage in Drosophila oogenesis, nurse cells rapidly expel their cytoplasm into the oocyte via intracellular bridges by a process called nurse cell dumping. Before dumping, numerous cables composed of actin filaments appear in the cytoplasm and extend inward from the plasma membrane toward the nucleus. This actin cage prevents the nucleus, which becomes highly lobed, from physically blocking the intracellular bridges during dumping. Each cable is composed of a linear series of modules composed of ∼25 cross-linked actin filaments. Adjacent modules overlap in the cable like the units of an extension ladder. During cable formation, individual modules are nucleated from the cell surface as microvilli, released, and then cross-linked to an adjacent forming module. The filaments in all the modules in a cable are unidirectionally polarized. During dumping as the volume of the cytoplasm decreases, the nucleus to plasma membrane distance decreases, compressing the actin cables that shorten as adjacent modules slide passively past one another just as the elements of an extension ladder slide past one another for storage. In Drosophila, the modular construction of actin cytoskeletons seems to be a generalized strategy. The behavior of modular actin cytoskeletons has implications for other actin-based cytoskeletal systems, e.g., those involved in Listeria movement, in cell spreading, and in retrograde flow in growth cones and fibroblasts.
Expression of TRPC3 in Chinese Hamster Ovary Cells Results in Calcium-activated Cation Currents Not Related to Store Depletion
Authors: Zitt Christof , Obukhov Alexander G. , Strübing Carsten , Zobel Andrea , Kalkbrenner Frank , Lückhoff Andreas , Schultz Günter
Published: July 2002

TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.
The Distribution of Polycomb-Group Proteins During Cell Division and Development in Drosophila Embryos: Impact on Models for Silencing
Authors: Buchenau Peter , Hodgson Jacob , Strutt Helen , Arndt-Jovin Donna J.
Published: July 2002

The subcellular three-dimensional distribution of three polycomb-group (PcG) proteins—polycomb, polyhomeotic and posterior sex combs—in fixed whole-mount Drosophila embryos was analyzed by multicolor confocal fluorescence microscopy. All three proteins are localized in complex patterns of 100 or more loci throughout most of the interphase nuclear volume. The rather narrow distribution of the protein intensities in the vast majority of loci argues against a PcG-mediated sequestration of repressed target genes by aggregation into subnuclear domains. In contrast to the case for PEV repression (Csink, A.K., and S. Henikoff. 1996. Nature. 381:529–531), there is a lack of correlation between the occurrence of PcG proteins and high concentrations of DNA, demonstrating that the silenced genes are not targeted to heterochromatic regions within the nucleus. There is a clear distinction between sites of transcription in the nucleus and sites of PcG binding, supporting the assumption that most PcG binding loci are sites of repressive complexes. Although the PcG proteins maintain tissue-specific repression for up to 14 cell generations, the proteins studied here visibly dissociate from the chromatin during mitosis, and disperse into the cytoplasm in a differential manner. Quantitation of the fluorescence intensities in the whole mount embryos demonstrate that the dissociated proteins are present in the cytoplasm. We determined that <2% of PH remains attached to late metaphase and anaphase chromosomes. Each of the three proteins that were studied has a different rate and extent of dissociation at prophase and reassociation at telophase. These observations have important implications for models of the mechanism and maintenance of PcG- mediated gene repression.
Regulation of Angiotensin II–induced Neuromodulation by MARCKS in Brain Neurons
Authors: Lu Di , Yang Hong , Lenox Robert H. , Raizada Mohan K.
Published: July 2002

Angiotensin II (Ang II) exerts chronic stimulatory actions on tyrosine hydroxylase (TH), dopamine β-hydroxylase (DβH), and the norepinephrine transporter (NET), in part, by influencing the transcription of their genes. These neuromodulatory actions of Ang II involve Ras-Raf-MAP kinase signal transduction pathways (Lu, D., H. Yang, and M.K. Raizada. 1997. J. Cell Biol. 135:1609–1617). In this study, we present evidence to demonstrate participation of another signaling pathway in these neuronal actions of Ang II. It involves activation of protein kinase C (PKC)β subtype and phosphorylation and redistribution of myristoylated alanine-rich C kinase substrate (MARCKS) in neurites. Ang II caused a dramatic redistribution of MARCKS from neuronal varicosities to neurites. This was accompanied by a time-dependent stimulation of its phosphorylation, that was mediated by the angiotensin type 1 receptor subtype (AT1). Incubation of neurons with PKCβ subtype specific antisense oligonucleotide (AON) significantly attenuated both redistribution and phosphorylation of MARCKS. Furthermore, depletion of MARCKS by MARCKS-AON treatment of neurons resulted in a significant decrease in Ang II–stimulated accumulation of TH and DβH immunoreactivities and [3H]NE uptake activity in synaptosomes. In contrast, mRNA levels of TH, DβH, and NET were not influenced by MARKS-AON treatment. MARCKS pep148–165, which contains PKC phosphorylation sites, inhibited Ang II stimulation of MARCKS phosphorylation and reduced the amount of TH, DβH, and [3H]NE uptake in neuronal synaptosomes. These observations demonstrate that phosphorylation of MARCKS by PKCβ and its redistribution from varicosities to neurites is important in Ang II–induced synaptic accumulation of TH, DβH, and NE. They suggest that a coordinated stimulation of transcription of TH, DβH, and NET, mediated by Ras-Raf-MAP kinase followed by their transport mediated by PKCβ-MARCKS pathway are key in persistent stimulation of Ang II's neuromodulatory actions.
The Synaptonemal Complex Protein SCP3 Can Form Multistranded, Cross-striated Fibers In Vivo
Authors: Yuan Li , Pelttari Jeanette , Brundell Eva , Björkroth Birgitta , Zhao Jian , Liu Jian-Guo , Brismar Hjalmar , Daneholt Bertil , Höög Christer
Published: July 2002

The synaptonemal complex protein SCP3 is part of the lateral element of the synaptonemal complex, a meiosis-specific protein structure essential for synapsis of homologous chromosomes. We have investigated the fiber-forming properties of SCP3 to elucidate its role in the synaptonemal complex. By synthesis of SCP3 in cultured somatic cells, it has been shown that SCP3 can self-assemble into thick fibers and that this process requires the COOH-terminal coiled coil domain of SCP3, as well as the NH2-terminal nonhelical domain. We have further analyzed the thick SCP3 fibers by transmission electron microscopy and immunoelectron microscopy. We found that the fibers display a transversal striation with a periodicity of ∼20 nm and consist of a large number of closely associated, thin fibers, 5–10 nm in diameter. These features suggest that the SCP3 fibers are structurally related to intermediate filaments. It is known that in some species the lateral elements of the synaptonemal complex show a highly ordered striated structure resembling that of the SCP3 fibers. We propose that SCP3 fibers constitute the core of the lateral elements of the synaptonemal complex and function as a molecular framework to which other proteins attach, regulating DNA binding to the chromatid axis, sister chromatid cohesion, synapsis, and recombination.
ER/Golgi Intermediates Acquire Golgi Enzymes by Brefeldin a–Sensitive Retrograde Transport in Vitro
Authors: Lin Chung-Chih , Love Harold D. , Gushue Jennifer N. , Bergeron John J.M. , Ostermann Joachim
Published: July 2002

Secretory proteins exit the ER in transport vesicles that fuse to form vesicular tubular clusters (VTCs) which move along microtubule tracks to the Golgi apparatus. Using the well-characterized in vitro approach to study the properties of Golgi membranes, we determined whether the Golgi enzyme NAGT I is transported to ER/Golgi intermediates. Secretory cargo was arrested at distinct steps of the secretory pathway of a glycosylation mutant cell line, and in vitro complementation of the glycosylation defect was determined. Complementation yield increased after ER exit of secretory cargo and was optimal when transport was blocked at an ER/Golgi intermediate step. The rapid drop of the complementation yield as secretory cargo progresses into the stack suggests that Golgi enzymes are preferentially targeted to ER/Golgi intermediates and not to membranes of the Golgi stack. Two mechanisms for in vitro complementation could be distinguished due to their different sensitivities to brefeldin A (BFA). Transport occurred either by direct fusion of preexisting transport intermediates with ER/Golgi intermediates, or it occurred as a BFA-sensitive and most likely COP I–mediated step. Direct fusion of ER/Golgi intermediates with cisternal membranes of the Golgi stack was not observed under these conditions.
Cytoplasmic Localization and the Choice of Ligand Determine Aggregate Formation by Androgen Receptor with Amplified Polyglutamine Stretch
Authors: Becker Matthias , Martin Elke , Schneikert Jean , Krug Harald F. , Cato Andrew C.B.
Published: July 2002

Polyglutamine tract expansion in androgen receptor is a recognized cause of spinal and bulbar muscular atrophy (SBMA), an X-linked motor neuronopathy. Similar mutations have been identified in proteins associated with other neurodegenerative diseases. Recent studies have shown that amplified polyglutamine repeat stretches form cellular aggregates that may be markers for these neurodegenerative diseases. Here we describe conditions that lead to aggregate formation by androgen receptor with polyglutamine stretch amplification. In transfection experiments, the mutant, compared with the wild-type receptor, was delayed in its cytoplasmic–nuclear translocation and formed large cytoplasmic aggregates in the presence of androgen. The cytoplasmic environment appears crucial for this aggregation, since retention of both the wild-type and mutant receptors in this cellular compartment by the deletion of their nuclear localization signals resulted in massive aggregation. Conversely, rapid nuclear transport of both receptors brought about by deletion of their ligand binding domains did not result in aggregate formation. However, androgen antagonists that altered the conformation of the ligand binding domain and promoted varying rates of cytoplasmic–nuclear translocation all inhibited aggregate formation. This demonstrates that in addition to the cytoplasmic localization, a distinct contribution of the ligand binding domain of the receptor is necessary for the aggregation. The finding that antiandrogens inhibit aggregate formation may provide the basis for in vivo determination of the role of these structures in SBMA.
Suppression of Pyk2 Kinase and Cellular Activities by Fip200
Authors: Ueda Hiroki , Abbi Smita , Zheng Chuanhai , Guan Jun-Lin
Published: July 2002

Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic tyrosine kinase implicated to play a role in several intracellular signaling pathways. We report the identification of a novel Pyk2-interacting protein designated FIP200 (FAK family kinase–interacting protein of 200 kD) by using a yeast two-hybrid screen. In vitro binding assays and coimmunoprecipitation confirmed association of FIP200 with Pyk2, and similar assays also showed FIP200 binding to FAK. However, immunofluorescent staining indicated that FIP200 was predominantly localized in the cytoplasm. FIP200 bound to the kinase domain of Pyk2 and inhibited its kinase activity in in vitro kinase assays. FIP200 also inhibited the kinase activity of the Pyk2 isolated from SYF cells (deficient in Src, Yes, and Fyn expression) and the Pyk2 mutant lacking binding site for Src, suggesting that it regulated Pyk2 kinase directly rather than affecting the associated Src family kinases. Consistent with its inhibitory effect in vitro, FIP200 inhibited activation of Pyk2 and Pyk2-induced apoptosis in intact cells, which correlated with its binding to Pyk2. Finally, activation of Pyk2 by several biological stimuli correlated with the dissociation of endogenous FIP200–Pyk2 complex, which provided further support for inhibition of Pyk2 by FIP200 in intact cells. Together, these results suggest that FIP200 functions as an inhibitor of Pyk2 via binding to its kinase domain.
Polyglycylation of Tubulin Is Essential and Affects Cell Motility and Division in Tetrahymena thermophila
Authors: Xia Lu , Hai Bing , Gao Yan , Burnette Dylan , Thazhath Rupal , Duan Jianming , Bré Marie-Helene , Levilliers Nicolette , Gorovsky Martin A. , Gaertig Jacek
Published: July 2002

We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies. Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of α- and β-tubulin, respectively. Mutants lacking all polyglycylation sites on α-tubulin have normal phenotype, whereas similar sites on β-tubulin are essential. A viable mutant with three mutated sites in β-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on β-tubulin were mutated were viable if they were cotransformed with an α-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of β-tubulin. In this double mutant, β-tubulin lacked detectable polyglycylation, while the α-β tubulin chimera was hyperglycylated compared with α-tubulin in wild-type cells. Thus, the essential function of polyglycylation of the COOH terminus of β-tubulin can be transferred to α-tubulin, indicating it is the total amount of polyglycylation on both α- and β-tubulin that is essential for survival.
Budding Yeast Chromosome Structure and Dynamics during Mitosis
Authors: Pearson Chad G. , Maddox Paul S. , Salmon E.D. , Bloom Kerry
Published: July 2002

Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation. The rates of movement during these oscillations are similar to those of microtubule plus end dynamics. The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase. Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase. Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B). Chromatin proximal to the centromere is stretched poleward before and during anaphase onset. The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere. These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.
Human Histocompatibility Leukocyte Antigen (HLA)-G Molecules Inhibit NKAT3 Expressing Natural Killer Cells
Authors: Münz Christian , Holmes Nicholas , King Ashley , Loke Yung Wai , Colonna Marco , Schild Hansjörg , Rammensee Hans-Georg
Published: July 2002

The crucial immunological function of the classical human major histocompatibility complex (MHC) class I molecules, human histocompatibility leukocyte antigen (HLA)-A, -B, and -C, is the presentation of peptides to T cells. A secondary function is the inhibition of natural killer (NK) cells, mediated by binding of class I molecules to NK receptors. In contrast, the function of the nonclassical human MHC class I molecules, HLA-E, -F, and -G, is still a mystery. The specific expression of HLA-G in placental trophoblast suggests an important role for this molecule in the immunological interaction between mother and child. The fetus, semiallograft by its genotype, escapes maternal allorecognition by downregulation of HLA-A and HLA-B molecules at this interface. It has been suggested that the maternal NK recognition of this downregulation is balanced by the expression of HLA-G, thus preventing damage to the placenta. Here, we describe the partial inhibition of NK lysis of the MHC class I negative cell line LCL721.221 upon HLA-G transfection. We present three NK lines that are inhibited via the interaction of their NKAT3 receptor with HLA-G and with HLA-Bw4 molecules. Inhibition can be blocked by the anti-NKAT3 antibody 5.133. In conclusion, NK inhibition by HLA-G via NKAT3 may contribute to the survival of the fetal semiallograft in the mother during pregnancy.
Targeted Expression of Major Histocompatibility Complex (MHC) Class II Molecules Demonstrates that Dendritic Cells Can Induce Negative but Not Positive Selection of Thymocytes In Vivo
Authors: Brocker Thomas , Riedinger Mireille , Karjalainen Klaus
Published: July 2002

It is well established that lymphoid dendritic cells (DC) play an important role in the immune system. Beside their role as potent inducers of primary T cell responses, DC seem to play a crucial part as major histocompatibility complex (MHC) class II+ “interdigitating cells” in the thymus during thymocyte development. Thymic DC have been implicated in tolerance induction and also by some authors in inducing major histocompatibility complex restriction of thymocytes. Most of our knowledge about thymic DC was obtained using highly invasive and manipulatory experimental protocols such as thymus reaggregation cultures, suspension cultures, thymus grafting, and bone marrow reconstitution experiments. The DC used in those studies had to go through extensive isolation procedures or were cultured with recombinant growth factors. Since the functions of DC after these in vitro manipulations have been reported to be not identical to those of DC in vivo, we intended to establish a system that would allow us to investigate DC function avoiding artificial interferences due to handling. Here we present a transgenic mouse model in which we targeted gene expression specifically to DC. Using the CD11c promoter we expressed MHC class II I-E molecules specifically on DC of all tissues, but not on other cell types. We report that I-E expression on thymic DC is sufficient to negatively select I-E reactive CD4+ T cells, and to a less complete extent, CD8+ T cells. In contrast, if only DC expressed I-E in a class II–deficient background, positive selection of CD4+ T cells could not be observed. Thus negative, but not positive, selection events can be induced by DC in vivo.
The Role of the Thymus and Recent Thymic Migrants in the Maintenance of the Adult Peripheral Lymphocyte Pool
Authors: Berzins S.P. , Boyd R.L. , Miller J.F.A.P.
Published: July 2002

The thymus is essential for the initial seeding of T cells to the periphery, but its role in maintaining the adult T cell pool remains poorly defined. We investigated whether changes to the rate of T cell export could form part of the mechanism(s) controlling the homeostatic regulation of the size and composition of the peripheral T cell pool. Using neonatal thymi grafted under the kidney capsule, we found that irrespective of whether the pool was oversupplied (by thymic grafts) or undersupplied (due to neonatal thymectomy), the thymic export rate was constant from both the host and graft thymus, and the periphery remained constant in size. Recent thymic emigrants (RTE) were also tracked to determine the extent of their acceptance into the T cell pool of a normal mouse. As a population, RTE are phenotypically mature, but were distinct from resident T cells in the periphery, being released in a CD4/CD8 ratio approximately twice that of established peripheral T cells. This export ratio is similar to that of T cells in the mature thymic compartment, but soon after entry into the periphery, the ratio falls, indicating separate thymic and peripheral regulation of the CD4/CD8 ratio. RTE may also be preferentially incorporated into the periphery, causing displacement of resident T cells, thus maintaining the size of the peripheral pool. Although not vital for the maintenance of a functional T cell pool, the acceptance of RTE in a “full” peripheral pool would ensure that the T cell receptor repertoire is kept diverse and that the T cell population encompasses a broad range of naive as well as memory T cells.
Human Interferon-γ–inducible Protein 10 (IP-10) Inhibits Constitutive Signaling of Kaposi's Sarcoma–associated Herpesvirus G Protein–coupled Receptor
Authors: Geras-Raaka Elizabeth , Varma Anjali , Ho Hao , Clark-Lewis Ian , Gershengorn Marvin C.
Published: July 2002

A G protein–coupled receptor (GPCR) is encoded within the genome of Kaposi's sarcoma– associated herpesvirus (KSHV)/human herpesvirus 8, a virus that may be involved in the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas. KSHV-GPCR exhibits constitutive signaling activity that causes oncogenic transformation. We report that human interferon (IFN)-γ–inducible protein 10 (HuIP-10), a C-X-C chemokine, specifically inhibits signaling of KSHV-GPCR. In contrast, monokine induced by IFN-γ (HuMig), which like HuIP-10 is an agonist of C-X-C chemokine receptor 3, does not inhibit KSHV-GPCR signaling. Moreover, HuIP-10, but not HuMig, inhibits KSHV-GPCR–induced proliferation of NIH 3T3 cells. These results show that HuIP-10 is an inverse agonist that converts KSHV-GPCR from an active to an inactive state. Thus, a human chemokine inhibits constitutive signaling and cellular proliferation that is mediated by a receptor encoded by a human disease-associated herpesvirus.
A Nongenomic Mechanism for Progesterone-mediated Immunosuppression: Inhibition of K+ Channels, Ca2+ Signaling, and Gene Expression in T Lymphocytes
Authors: Ehring George R. , Kerschbaum Hubert H. , Eder Claudia , Neben Amber L. , Fanger Christopher M. , Khoury Rosana M. , Negulescu Paul A. , Cahalan Michael D.
Published: July 2002

The mechanism by which progesterone causes localized suppression of the immune response during pregnancy has remained elusive. Using human T lymphocytes and T cell lines, we show that progesterone, at concentrations found in the placenta, rapidly and reversibly blocks voltage-gated and calcium-activated K+ channels (KV and KCa, respectively), resulting in depolarization of the membrane potential. As a result, Ca2+ signaling and nuclear factor of activated T cells (NF-AT)-driven gene expression are inhibited. Progesterone acts distally to the initial steps of T cell receptor (TCR)-mediated signal transduction, since it blocks sustained Ca2+ signals after thapsigargin stimulation, as well as oscillatory Ca2+ signals, but not the Ca2+ transient after TCR stimulation. K+ channel blockade by progesterone is specific; other steroid hormones had little or no effect, although the progesterone antagonist RU 486 also blocked KV and KCa channels. Progesterone effectively blocked a broad spectrum of K+ channels, reducing both Kv1.3 and charybdotoxin–resistant components of KV current and KCa current in T cells, as well as blocking several cloned KV channels expressed in cell lines. Progesterone had little or no effect on a cloned voltage-gated Na+ channel, an inward rectifier K+ channel, or on lymphocyte Ca2+ and Cl− channels. We propose that direct inhibition of K+ channels in T cells by progesterone contributes to progesterone-induced immunosuppression.
Maturation, Activation, and Protection of Dendritic Cells Induced by Double-stranded RNA
Authors: Cella Marina , Salio Mariolina , Sakakibara Yoichi , Langen Hanno , Julkunen Ilkka , Lanzavecchia Antonio
Published: July 2002

The initiation of an immune response is critically dependent on the activation of dendritic cells (DCs). This process is triggered by surface receptors specific for inflammatory cytokines or for conserved patterns characteristic of infectious agents. Here we show that human DCs are activated by influenza virus infection and by double-stranded (ds)RNA. This activation results not only in increased antigen presentation and T cell stimulatory capacity, but also in resistance to the cytopathic effect of the virus, mediated by the production of type I interferon, and upregulation of MxA. Because dsRNA stimulates both maturation and resistance, DCs can serve as altruistic antigen-presenting cells capable of sustaining viral antigen production while acquiring the capacity to trigger naive T cells and drive polarized T helper cell type 1 responses.
Recombinant Galectin-1 and Its Genetic Delivery Suppress Collagen-Induced Arthritis via T Cell Apoptosis
Authors: Rabinovich Gabriel A. , Daly Gordon , Dreja Hanna , Tailor Hitakshi , Riera Clelia M. , Hirabayashi Jun , Chernajovsky Yuti
Published: July 2002

Galectin-1 (GAL-1), a member of a family of conserved β-galactoside–binding proteins, has been shown to induce in vitro apoptosis of activated T cells and immature thymocytes. We assessed the therapeutic effects and mechanisms of action of delivery of GAL-1 in a collagen-induced arthritis model. A single injection of syngeneic DBA/1 fibroblasts engineered to secrete GAL-1 at the day of disease onset was able to abrogate clinical and histopathological manifestations of arthritis. This effect was reproduced by daily administration of recombinant GAL-1. GAL-1 treatment resulted in reduction in anticollagen immunoglobulin (Ig)G levels. The cytokine profile in draining lymph node cells and the anticollagen IgG isotypes in mice sera at the end of the treatment clearly showed inhibition of the proinflammatory response and skewing towards a type 2–polarized immune reaction. Lymph node cells from mice engaged in the gene therapy protocol increased their susceptibility to antigen-induced apoptosis. Moreover, GAL-1–expressing fibroblasts and recombinant GAL-1 revealed a specific dose-dependent inhibitory effect in vitro in antigen-dependent interleukin 2 production to an Aq-restricted, collagen type 2–specific T cell hybridoma clone. Thus, a correlation between the apoptotic properties of GAL-1 in vitro and its immunomodulatory properties in vivo supports its therapeutic potential in the treatment of T helper cell type 1–mediated autoimmune disorders.
Protective Immunity to Bordetella pertussis Requires Both B Cells and Cd4+ T Cells for Key Functions Other than Specific Antibody Production
Authors: Leef Mary , Elkins Karen L. , Barbic Jerko , Shahin Roberta D.
Published: July 2002

To investigate the fundamental nature of protective immunity to Bordetella pertussis, we studied intranasal immunization of adult mice with formalin-fixed B. pertussis (FFBP), followed by aerosol B. pertussis challenge. Mice given two doses of FFBP intranasally completely cleared a subsequent pertussis aerosol challenge from tracheae and lungs (defined as protection), but there was no correlation between levels of specific antibody and clearance of bacteria. Further, transfer of immune serum before aerosol challenge had minimal effects on bacterial burdens. However, pertussis-specific T cells producing interferon γ but not interleukin 4 or interleukin 10 were detected in draining lymph nodes of FFBP-immunized mice. Significantly, repeated immunization of B cell knockout (BKO) mice resulted in partial protection, and complete protection was reconstituted by transfer of pertussis-immune B cells; reconstituted BKO mice had little if any detectable antipertussis antibodies. Immunization of mice lacking all T cells or lacking CD4+ T cells did not lead to protection; in contrast, CD8− mice were protected. Mice depleted of CD4+ T cells after immunization but before aerosol challenge, which thus had normal amounts of specific antibodies, were not optimally protected. Taken together, these data indicate that protective immunity to pertussis is dependent on both CD4+ T cells and B cells, and both cell types provide significant functions other than specific antibody production.
Visualization of Plasmodium falciparum–Endothelium Interactions in Human Microvasculature
Authors: Ho May , Hickey Michael J. , Murray Allan G. , Andonegui Graciela , Kubes Paul
Published: July 2002

Plasmodium falciparum–infected erythrocytes roll on and/or adhere to CD36, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and P-selectin under shear conditions in vitro. However, the lack of an adequate animal model has made it difficult to determine whether infected erythrocytes do indeed interact in vivo in microvessels. Therefore, we made use of an established model of human skin grafted onto severe combined immunodeficient (SCID) mice to directly visualize the human microvasculature by epifluorescence intravital microscopy. In all grafts examined, infected erythrocytes were observed to roll and/or adhere in not just postcapillary venules but also in arterioles. In contrast, occlusion of capillaries by infected erythrocytes was noted only in approximately half of the experiments. Administration of an anti-CD36 antibody resulted in a rapid reduction of rolling and adhesion. More importantly, already adherent cells quickly detached. The residual rolling after anti-CD36 treatment was largely inhibited by an anti–ICAM-1 antibody. Anti–ICAM-1 alone reduced the ability of infected erythrocytes to sustain rolling and subsequent adhesion. These findings provide conclusive evidence that infected erythrocytes interact within the human microvasculature in vivo by a multistep adhesive cascade that mimics the process of leukocyte recruitment.
Caspase-11 Mediates Oligodendrocyte Cell Death and Pathogenesis of Autoimmune-Mediated Demyelination
Authors: Hisahara Shin , Yuan Junying , Momoi Takashi , Okano Hideyuki , Miura Masayuki
Published: July 2002

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory diseases of the central nervous system (CNS) characterized by localized areas of demyelination. The mechanisms underlying oligodendrocyte (OLG) injury in MS and EAE remain unknown. Here we show that caspase-11 plays crucial roles in OLG death and pathogenesis in EAE. Caspase-11 and activated caspase-3 were both expressed in OLGs in spinal cord EAE lesions. OLGs from caspase-11–deficient mice were highly resistant to the cell death induced by cytotoxic cytokines. EAE susceptibility and cytokine concentrations in the CNS were significantly reduced in caspase-11–deficient mice. Our findings suggest that OLG death is mediated by a pathway that involves caspases-11 and -3 and leads to the demyelination observed in EAE.
Enhancement of Osteoclastic Bone Resorption and Suppression of Osteoblastic Bone Formation in Response to Reduced Mechanical Stress Do Not Occur in the Absence of Osteopontin
Authors: Ishijima Muneaki , Rittling Susan R. , Yamashita Teruhito , Tsuji Kunikazu , Kurosawa Hisashi , Nifuji Akira , Denhardt David T. , Noda Masaki
Published: July 2002

Reduced mechanical stress to bone in bedridden patients and astronauts leads to bone loss and increase in fracture risk which is one of the major medical and health issues in modern aging society and space medicine. However, no molecule involved in the mechanisms underlying this phenomenon has been identified to date. Osteopontin (OPN) is one of the major noncollagenous proteins in bone matrix, but its function in mediating physical-force effects on bone in vivo has not been known. To investigate the possible requirement for OPN in the transduction of mechanical signaling in bone metabolism in vivo, we examined the effect of unloading on the bones of OPN−/− mice using a tail suspension model. In contrast to the tail suspension–induced bone loss in wild-type mice, OPN−/− mice did not lose bone. Elevation of urinary deoxypyridinoline levels due to unloading was observed in wild-type but not in OPN−/− mice. Analysis of the mechanisms of OPN deficiency–dependent reduction in bone on the cellular basis resulted in two unexpected findings. First, osteoclasts, which were increased by unloading in wild-type mice, were not increased by tail suspension in OPN−/− mice. Second, measures of osteoblastic bone formation, which were decreased in wild-type mice by unloading, were not altered in OPN−/− mice. These observations indicate that the presence of OPN is a prerequisite for the activation of osteoclastic bone resorption and for the reduction in osteoblastic bone formation in unloaded mice. Thus, OPN is a molecule required for the bone loss induced by mechanical stress that regulates the functions of osteoblasts and osteoclasts.
Openings of the Rat Recombinant α1 Homomeric Glycine Receptor as a Function of the Number of Agonist Molecules Bound
Authors: Beato Marco , Groot-Kormelink Paul J. , Colquhoun David , Sivilotti Lucia G.
Published: July 2002

The functional properties of rat homomeric α1 glycine receptors were investigated using whole-cell and outside-out recording from human embryonic kidney cells transfected with rat α1 subunit cDNA. Whole-cell dose-response curves gave EC50 estimates between 30 and 120 μM and a Hill slope of ∼3.3. Single channel recordings were obtained by steady-state application of glycine (0.3, 1, or 10 μM) to outside-out patches. Single channel conductances were mostly 60–90 pS, but smaller conductances of ∼40 pS were also seen (10% of the events) with a relative frequency that did not depend on agonist concentration. The time constants of the apparent open time distributions did not vary with agonist concentration, but short events were more frequent at low glycine concentrations. There was also evidence of a previously missed short-lived open state that was more common at lower glycine concentrations. The time constants for the different components of the burst length distributions were found to have similar values at different concentrations. Nevertheless, the mean burst length increased with increasing glycine. This was because the relative area of each burst-length component was concentration dependent and short bursts were favored at lower glycine concentrations. Durations of adjacent open and shut times were found to be strongly (negatively) correlated. Additionally, long bursts were made up of longer than average openings separated by short gaps, whereas short bursts usually consisted of single isolated short openings. The most plausible explanation for these findings is that long bursts are generated when a higher proportion of the five potential agonist binding sites on the receptor is occupied by glycine. On the basis of the concentration dependence and the intraburst structure we provide a preliminary kinetic scheme for the activation of the homomeric glycine receptor, in which any number of glycine molecules from one to five can open the channel, although not with equal efficiency.
Willmore two-spheres in the four-sphere
Authors: Montiel Sebastián
Published: July 2002

Genus zero Willmore surfaces immersed in the three-sphereS3\mathbb {S}^3correspond via the stereographic projection to minimal surfaces in Euclidean three-space with finite total curvature and embedded planar ends. The critical values of the Willmore functional are4πk4\pi k, wherek∈N∗k\in \mathbb {N}^*, withk≠2,3,5,7k\ne 2,3,5,7. When the ambient space is the four-sphereS4\mathbb {S}^4, the regular homotopy class of immersions of the two-sphereS2\mathbb {S}^2is determined by the self-intersection numberq∈Zq\in \mathbb {Z}; here we shall prove that the possible critical values are4π(|q|+k+1)4\pi (|q|+k+1), wherek∈Nk\in \mathbb {N}. Moreover, ifk=0k=0, the corresponding immersion, or its antipodal, is obtained, via the twistor Penrose fibrationP3→S4\mathbb {P}^3\rightarrow \mathbb {S}^4, from a rational curve inP3\mathbb {P}^3and, ifk≠0k\ne 0, via stereographic projection, from a minimal surface inR4\mathbb {R}^4with finite total curvature and embedded planar ends. An immersion lies in both families when the rational curve is contained in someP2⊂P3\mathbb {P}^2\subset \mathbb {P}^3or (equivalently) when the minimal surface ofR4\mathbb {R}^4is complex with respect to a suitable complex structure ofR4\mathbb {R}^4.
InterPro—an integrated documentation resource for protein

families, domains and functional sites
Authors: Apweiler R. , Attwood T. K. , Bairoch A. , Bateman A. , Birney E. , Biswas M. , Bucher P. , Cerutti L. , Corpet F. , Croning M. D. R. , Durbin R. , Falquet L. , Fleischmann W. , Gouzy J. , Hermjakob H. , Hulo N. , Jonassen I. , Kahn D. , Kanapin A. , Karavidopoulou Y. , Lopez R. , Marx B. , Mulder N. J. , Oinn T. M. , Pagni M. , Servant F. , Sigrist C. J. A. , Zdobnov E. M.
Published: July 2002

Motivation: InterPro is a new integrated documentation resource for protein families, domains and functional sites, developed initially as a means of rationalising the complementary efforts of the PROSITE, PRINTS, Pfam and ProDom database projects.

Results: Merged annotations from PRINTS, PROSITE and Pfam form the InterPro core. Each combined InterPro entry includes functional descriptions and literature references, and links are made back to the relevant parent database(s), allowing users to see at a glance whether a particular family or domain has associated patterns, profiles, fingerprints, etc. Merged and individual entries (i.e. those that have no counterpart in the companion resources) are assigned unique accession numbers. Release 1.2 of InterPro (June 2000) contains over 3000 entries, representing families, domains, repeats and sites of post-translational modification (PTMs) encoded by 6581 different regular expressions, profiles, fingerprints and Hidden Markov Models (HMMs). Each InterPro entry lists all the matches against SWISS-PROT and TrEMBL (more than 1000000 hits from 264333 different proteins out of 384572 in SWISS-PROT and TrEMBL).

Availability: The database is accessible for text- and sequence-based searches at http://www.ebi.ac.uk/interpro/.

Contact: Interhelp@ebi.ac.uk

* To whom correspondence should be addressed.
A homolog of mammalian antizyme is present in fission yeast
Schizosaccharomyces pombe but not detected in budding yeast
Saccharomyces cerevisiae
Authors: Zhu Chang , Karplus Kevin , Grate Leslie , Coffino Philip
Published: July 2002

* To whom correspondence should be addressed.

Motivation: The antizymes (AZ) are proteins that regulate cellular polyamine pools in metazoa. To search for remote homologs in single-celled eukaryotes, we used computer software based on hidden Markov models. The most divergent homolog detected was that of the fission yeast Schizosaccharomyces pombe. Sequence identities between S.pombe AZ and known AZs are as low as 18–22% in the most conserved C-terminal regions. The authenticity of the S.pombe AZ is validated by the presence of a conserved nucleotide sequence that, in metazoa, promotes a +1 programmed ribosomal frameshift required for AZ expression. However, no homolog was detected in the completed genome of the budding yeast Saccharomyces cerevisiae. Procedural details and supplementary information can be found at http://itsa.ucsf.edu/~czhu/AZ.

Contact: pcoffin@itsa.ucsf.edu
Transcription-associated protein families are primarily

taxon-specific
Authors: Coulson Richard M.R. , Enright Anton J. , Ouzounis Christos A.
Published: July 2002

The mechanisms controlling gene regulation appear to be fundamentally different in eukaryotes and prokaryotes (Struhl (1999) Cell , 98, 1–4). To investigate this diversity further, we have analysed the distribution of all known transcription-associated proteins (TAPs), as reflected by sequence database annotations. Our results for the primary phylogenetic domains (Archaea, Bacteria and Eukaryota) show that TAP families are mostly taxon-specific and very few transcriptional regulators are common across these domains.

Contact: ouzounis@ebi.ac.uk
Concepts to automate the theoretical design of effective antisense oligonucleotides
Authors: Kretschmer-Kazemi Far Rosel , Nedbal Wolfgang , Sczakiel Georg
Published: July 2002

Among the large number of possible antisense species against a given target RNA, only a small number shows effective suppression of the target gene in living cells. In the case of short-chain antisense oligonucleotides (asON) which usually comprise less than approximately 25 nucleotides, local structures of the target RNA seem to be of particular importance for the extent of gene suppression. Experimental approaches to identify promising local target sequences and, hence, complementary asON sequences, have provided tools to define asON that are biologically active at higher than statistical probability. However, experimental protocols are expensive, time consuming, and are associated with intrinsic basic and technical limitations. As insights into the structure–function relationship of asON as well as the role of sequence motifs increase, it becomes feasible to consider computer-based theoretical approaches for the design of effective asON. In the following we describe how individual steps of the theoretical design of asON may be automated by establishing and implementing suitable algorithms.
The Cdc7/Dbf4 protein kinase: target of the S phase checkpoint?
Authors: Jares Pedro , Donaldson Anne , Blow J Julian
Published: July 2002

Cdc7/Dbf4 is a protein kinase that is required for the initiation of DNA replication in eukaryotes. Recent work has provided new clues to the role that Cdc7/Dbf4 plays in this process. A range of other observations suggest that Cdc7/Dbf4 also plays another, less well characterized, role in checkpoint function and in the maintenance of genomic integrity. In this review we attempt to bring together new information to explain how Cdc7/Dbf4 may perform these two distinct functions.
Improved Thermodynamic Parameters and Helix Initiation Factor to Predict Stability of DNA Duplexes
Authors: Sugimoto Naoki , Nakano Shu-ich , Yoneyama Mari , Honda Kei-ich
Published: July 2002

To improve the previous DNA/DNA nearest-neighbor parameters, thermodynamic parameters (Δ H °, Δ S ° and Δ G °) of 50 DNA/DNA duplexes were measured. Enthalpy change of a helix initiation factor is also considered though the parameters reported recently did not contain the factor. A helix initiation factor for DNA/DNA duplex determined here was the same as that of RNA/RNA duplex (ΔG° 37 = 3.4 kcal/mol). The improved nearest-neighbor parameters reproduced not only these 50 experimental values used here but also 15 other experimental values obtained in different studies. Comparing Δ G ° 37 values of DNA/DNA nearest-neighbor parameters obtained here with those of RNA/RNA and RNA/DNA, RNA/RNA duplex was generally the most stable of the three kinds of duplexes with the same nearest-neighbor sequences. Which is more stable between DNA/DNA and RNA/DNA duplexes is sequence dependent.