bluuebunny/crossref_metadata_2025 huggingface dataset:
A mAb to the β2-leukocyte integrin Mac-1 (CD11b/CD18) reduces intimal thickening after angioplasty or stent implantation in rabbits
Authors: Rogers Campbell , Edelman Elazer R. , Simon Daniel I.
Published: July 2002

Leukocytes are recruited early and abundantly to experimentally injured vessels, in direct proportion to cell proliferation and intimal growth. Activated circulating leukocytes and Mac-1 (CD11b/CD18, αMβ2) expression are markers of restenosis risk in patients undergoing angioplasty. As angioplastied vessels lack endothelium but have extensive fibrin(ogen) and platelet deposition, we hypothesized that Mac-1-dependent adhesion to fibrin(ogen) is an important determinant of leukocyte recruitment and function, which may in turn promote intimal growth. To study this hypothesis we administered M1/70, an anti-CD11b blocking mAb, to rabbits (1 mg/kg i.v.) immediately before, and every 48 hr for 3, 6, or 14 days after, iliac artery balloon denudation or deeper stent-induced injury. M1/70, which bound to isolated rabbit monocytes and dose-dependently inhibited Mac-1-mediated fibrinogen bindingin vitro, reduced leukocyte recruitment more than 2-fold 3, 6, and 14 days after injury. Neointimal growth 14 days after injury was markedly attenuated by treatment with M1/70 (intimal area after balloon injury, 0.12 ± 0.09 mm2, compared with 0.32 ± 0.08 mm2in vehicle-treated controls,P< 0.01, and 0.38 ± 0.08 mm2in IgG-treated controls,P< 0.005; intimal area after stent injury, 0.56 ± 0.16 mm2, compared with 0.84 ± 0.13 mm2in vehicle-treated controls,P< 0.05, and 0.90 ± 0.15 mm2in IgG-treated controls,P< 0.02). Mac-1 blockade reduces experimental neointimal thickening, suggesting that leukocyte recruitment to and infiltration of injured arteries may be a valid target for preventing intimal hyperplasia.
ETO, fusion partner in t(8;21) acute myeloid leukemia, represses transcription by interaction with the human N-CoR/mSin3/HDAC1 complex
Authors: Wang Jianxiang , Hoshino Taizo , Redner Robert L. , Kajigaya Sachiko , Liu Johnson M.
Published: July 2002

The t(8;21) translocation between two genes known asAML1andETOis seen in approximately 12–15% of all acute myeloid leukemia (AML) and is the second-most-frequently observed nonrandom genetic alteration associated with AML. AML1 up-regulates a number of target genes critical to normal hematopoiesis, whereas the AML1/ETO fusion interferes with this trans-activation. We discovered that the fusion partner ETO binds to the human homolog of the murine nuclear receptor corepressor (N-CoR). The interaction is mediated by two unusual zinc finger motifs present at the carboxyl terminus of ETO. Human N-CoR (HuN-CoR), which we cloned and sequenced in its entirety, encodes a 2,440-amino acid polypeptide and has a central domain that binds ETO. N-CoR, mammalian Sin3 (mSin3A and B), and histone deacetylase 1 (HDAC1) form a complex that alters chromatin structure and mediates transcriptional repression by nuclear receptors and by a number of oncoregulatory proteins. We found that ETO, through its interaction with the N-CoR/mSin3/HDAC1 complex, is also a potent repressor of transcription. This observation provides a mechanism for how the AML1/ETO fusion may inhibit expression of AML1-responsive target genes and disturb normal hematopoiesis.
λ Rap protein is a structure-specific endonuclease involved in phage recombination
Authors: Sharples Gary J. , Corbett Lisa M. , Graham Ian R.
Published: July 2002

Bacteriophage λ encodes a number of genes involved in the recombinational repair of DNA double-strand breaks. The product of one of these genes,
rap
, has been purified. Truncated Rap proteins that copurify with the full-length form are derived, at least in part, from a ρ-dependent transcription terminator located within its coding sequence. Full-length and certain truncated Rap polypeptides bind preferentially to branched DNA substrates, including synthetic Holliday junctions and D-loops. In the presence of manganese ions, Rap acts as an endonuclease that cleaves at the branch point of Holliday and D-loop substrates. It shows no obvious sequence preference or symmetry of cleavage on a Holliday junction. The biochemical analysis of Rap gives an insight into how recombinants could be generated by the nicking of a D-loop without the formation of a classical Holliday junction.
Transient cardiac expression of constitutively active Gαqleads to hypertrophy and dilated cardiomyopathy by calcineurin-dependent and independent pathways
Authors: Mende U. , Kagen A. , Cohen A. , Aramburu J. , Schoen F. J. , Neer E. J.
Published: July 2002

Cardiac hypertrophy and dilatation can result from stimulation of signal transduction pathways mediated by heterotrimeric G proteins, especially Gq, whose α subunit activates phospholipase Cβ (PLCβ). We now report that transient, modest expression of a hemagglutinin (HA) epitope-tagged, constitutively active mutant of the Gqα subunit (HAα*q) in hearts of transgenic mice is sufficient to induce cardiac hypertrophy and dilatation that continue to progress after the initiating stimulus becomes undetectable. At 2 weeks, HAα*qprotein is expressed at less than 50% of endogenous αq/11, and the transgenic hearts are essentially normal morphologically. Although HAα*qprotein declines at 4 weeks and is undetectable by 10 weeks, the animals develop cardiac hypertrophy and dilatation and die between 8 and 30 weeks in heart failure. As the pathology develops, endogenous αq/11rises (2.9-fold in atria; 1.8-fold in ventricles). At 2 weeks, basal PLC activity is increased 9- to 10-fold in atria but not ventricles. By 10 weeks, it is elevated in both, presumably because of the rise in endogenous αq/11. We conclude that the pathological changes initiated by early, transient HAα*qexpression are maintained in part by compensatory changes in signal transduction and other pathways. Cyclosporin A (CsA) prevents hypertrophy caused by activation of calcineurin [Molkentin, J. D., Lu, J.-R., Antos, C. L., Markham, B., Richardson, J., Robbins, J., Grant, S. R. & Olson, E. N. (1998)Cell93, 215–228]. Because HAα*qacts upstream of calcineurin, we hypothesized that HAα*qmight initiate additional pathways leading to hypertrophy and dilatation. Treating HAα*qmice with CsA diminished some, but not all, aspects of the hypertrophic phenotype, suggesting that multiple pathways are involved.
CpG DNA: A potent signal for growth, activation, and maturation of human dendritic cells
Authors: Hartmann G. , Weiner G. J. , Krieg A. M.
Published: July 2002

DNA molecules containing unmethylated CpG-dinucleotides in particular base contexts (“CpG motifs”) are excellent adjuvants in rodents, but their effects on human cells have been less clear. Dendritic cells (DCs) form the link between the innate and the acquired immune system and may influence the balance between T helper 1 (Th1) and Th2 immune responses. We evaluated the effects of CpG oligodeoxynucleotides alone or in combination with granulocyte–macrophage colony-stimulating factor (GMCSF) on different classes of purified human DCs. For primary dendritic precursor cells isolated from human blood, CpG oligonucleotides alone were superior to GMCSF in promoting survival and maturation (CD83 expression) as well as expression of class II MHC and the costimulatory molecules CD40, CD54, and CD86 of DCs. Both CD4-positive and CD4-negative peripheral blood dendritic precursor cells responded to CpG DNA which synergized with GMCSF but these DCs showed little response to lipopolysaccharide (LPS). In contrast, monocyte-derived DCs did not respond to CpG, but they were highly sensitive to LPS, suggesting an inverse correlation between CpG and LPS sensitivity in different subsets of DCs. Compared with GMCSF, CpG-treated peripheral blood DCs showed enhanced functional activity in the mixed lymphocyte reaction and induced T cells to secrete increased levels of Th1 cytokines. These findings demonstrate the ability of specific CpG motifs to strongly activate certain subsets of human DCs to promote Th1-like immune responses, and support the use of CpG DNA-based trials for immunotherapy against cancer, allergy, and infectious diseases.
In vivoblockade of CTLA-4 enhances the priming of responsive T cells but fails to prevent the induction of tumor antigen-specific tolerance
Authors: Sotomayor Eduardo M. , Borrello Ivan , Tubb Erev , Allison James P. , Levitsky Hyam I.
Published: July 2002

The efficacy of therapeutic vaccination for the treatment of cancer is limited by peripheral tolerance to tumor antigens.In vivoblockade of CTLA-4, a negative regulator of T cell function, can induce the regression of established tumors and can augment the tumor rejection achieved through therapeutic vaccination. These outcomes may reflect enhanced tumor-specific T cell priming and/or interference with the development of tolerance to tumor antigens. We examined the effect of CTLA-4 blockade on the fate and function of T cells specific for a model tumor antigen in the tumor-bearing host. We found that while CTLA-4 blockade enhanced the priming of responsive T cells, it did not prevent the induction of tolerance to tumor antigens. These results demonstrate that there is a critical window in which the combination of CTLA-4 blockade and vaccination achieves an optimal response, and they point to mechanisms other than CTLA-4 engagement in mediating peripheral T cell tolerance to tumor antigens.
Effectors of a developmental mitogen-activated protein kinase cascade revealed by expression signatures of signaling mutants
Authors: Madhani H. D. , Galitski T. , Lander E. S. , Fink G. R.
Published: July 2002

Despite the importance of mitogen-activated protein kinase (MAPK) signaling in eukaryotic biology, the mechanisms by which signaling yields phenotypic changes are poorly understood. We have combined transcriptional profiling with genetics to determine how the Kss1 MAPK signaling pathway controls dimorphic development in
Saccharomyces cerevisiae
. This analysis identified dozens of transcripts that are regulated by the pathway, whereas previous work had identified only a single downstream target,
FLO11
. One of the MAPK-regulated genes is
PGU1
, which encodes a secreted enzyme that hydrolyzes polygalacturonic acid, a structural barrier to microbial invasion present in the natural plant substrate of
S. cerevisiae
. A third key transcriptional target is the G
1
cyclin gene
CLN1
, a morphogenetic regulator that we show to be essential for pseudohyphal growth. In contrast, the homologous
CLN2
cyclin gene is dispensable for development. Thus, the Kss1 MAPK cascade programs development by coordinately modulating a cell adhesion factor, a secreted host-destroying activity, and a specialized subunit of the Cdc28 cyclin-dependent kinase.
A 500-bp region, ≈40 kb upstream of the human
CYP19
(aromatase) gene, mediates placenta-specific expression in transgenic mice
Authors: Kamat Amrita , Graves Katherine H. , Smith Margaret E. , Richardson James A. , Mendelson Carole R.
Published: July 2002

In humans, aromatase P450 (product of
CYP19
gene), which catalyzes conversion of C
19
steroids to estrogens, is expressed in a number of tissues, including ovary, adipose, and syncytiotrophoblast of the placenta. The 5′ untranslated regions of
CYP19
mRNA transcripts in these tissues are encoded by different tissue-specific first exons, which are spliced onto a common site just upstream of the translation initiation site in exon II. In placenta, the 5′ untranslated region of
CYP19
mRNA transcripts is encoded by exon I.1, which lies ≈40 kb upstream of exon II. To map genomic sequences required for placenta-specific
CYP19
expression, fusion genes containing 2,400 and 501 bp of placenta-specific exon I.1 5′ flanking DNA linked to the human growth hormone gene (hGH), as reporter, were introduced into transgenic mice. Expression of
CYP19
(I.1):hGH fusion genes containing as little as 501 bp of 5′ flanking DNA was placenta-specific and developmentally regulated. Furthermore, transgene expression occurred specifically in the labyrinthine trophoblast of the mouse placenta, which contains syncytial cells that may be analogous to the human syncytiotrophoblast. We show that a relatively small segment of DNA (≈500 bp) >40 kb upstream of the protein coding region of a human gene is able to direct expression in an appropriate tissue- and cell-specific manner in transgenic mice. These findings suggest that 5′ flanking DNA within 501 bp of exon I.1 of the human
CYP19
gene contains cis-acting elements that bind placenta-specific transcription factors that are conserved between humans and mice.
On the Reliability of OFSTED Inspection of Initial Teacher Training: A case study
Authors: Campbell Jim , Husbands Chris
Published: July 2002

ABSTRACT Inspection has been an increasingly important feature of central monitoring of education in the 1990s. This article examines the technical reliability of inspection processes in initial teacher training, drawing extensively on methodology and procedures adopted by the Office for Standards in Education (OFSTED) between 1996 and 1998. A definition of reliability is advanced and tested against the procedures of inspection, especially by examining the security of internal and external judgements at the pass/fail borderline and the moderation and training procedures deployed by OFSTED. The methodology of inspection is found to be insufficiently reliable for the consequences which flow from it. Tensions are explored between the technicist model of inspection underlying OFSTED initial teacher training procedures and the ‘informed conoisseurship’ model hitherto deployed by members of Her Majesty's Inspectorate. In consequence, there are unresolved tensions between the expressed aspiration for ‘improvement through inspection’ and the use of inspection to police compliance.
Wisdom: A culturally inclusive developmental perspective
Authors: Takahashi Masami , Overton Willis F.
Published: July 2002

This study examines cross-cultural age-related patterns of two modes of wisdom: the analytical (i.e., Knowledge Database, Abstract Reasoning), and the synthetic (i.e., Reflective Understanding, Emotional Empathy, and Emotional Regulation). A total of 68 American and 68 Japanese community-dwelling adults with an equal number of middle-aged (mean age = 45.3 years) and older adults (mean age = 70.1 years) in each group participated in the study. The results demonstrate that across cultures older adults function at a higher level than their middle-aged counterparts on these modes. Specific effects of culture, within each dimension, were also found. The general findings are discussed in terms of their contribution to a culturally inclusive developmental perspective on wisdom.
Emerging Bioinformatic Networks: Contesting the Public Meaning of Private and the Private Meaning of Public
Authors: Brown Nik , Rappert Brian
Published: July 2002

This paper explores the complexity of public/private identities in the emerging global economies of gene sequence mapping and analysis. In so doing we seek to offer a less over-determined acccount of what it means to describe institutional actors as either 'public' or 'private'. Instead, these 'codes' can be seen to offer actors a means of mutual positioning that, more usually conceals broader interdependencies within the world's bioinformatics networks.
What future for initial teacher education? New curriculum and new directions
Authors: Burgess Hilary
Published: July 2002

This article analyses aspects of the implementation of the National Curriculum for initial teacher training (ITT). It focuses on the impact of the standards upon course design and structure and particularly the emphasis on the development of subject knowledge. The assessment of students in terms of subject knowledge and the standards for qualified teacher status (QTS) are explored alongside the development of partnership schools and mentoring programmes. The impact of external agencies such as OFSTED and the TTA is included in the discussion. Future implications for primary and secondary ITT programmes are considered.
Novel distribution of the secretory leucocyte proteinase inhibitor in kidney
Authors: Ohlsson Sophie , Ljungkrantz Irena , Ohlsson Kjell , Segelmark Mårten , Wieslander Jörgen
Published: July 2002

The secretory leucocyte proteinase inhibitor (SLPI) is a low molecular weight, tissue‐specific inhibitor of, for example, elastase and cathepsin G, which also have antimicrobial capacity. SLPI has been localised to the respiratory, gastrointestinal and genital tracts, but so far not to the kidney. The presence of SLPI in renal tubuli cells was demonstrated using immunohistochemistry and, by means of in situ hybridisation on human renal biopsies, we were able to demonstrate SLPI production. In various inflammatory conditions in the kidneys, the protease‐antiprotease balance is disturbed. For this reason, as well as the possible role in the defence against ascending urinary tract infections, it is interesting to establish a source of SLPI in renal tubuli cells.
Cytokines in inflammatory bowel disease
Authors: Beck P. L. , Wallace J. L.
Published: July 2002

Over the past decade, much has been learned regarding the role of various cytokines in the pathogenesis of inflammatory bowel disease. Several cytokine ‘knockout’ models in mice have been shown to develop colitis, while alterations in the production of various cytokines has been documented in human Crohn′s disease and ulcerative colitis. In recent years, attempts have been made to treat these diseases through modulation of cytokine production or action. This review focuses on the cytokines that have been implicated in the pathogenesis of inflammatory bowel disease. The evidence for and against a role for particular cytokines in intestinal inflammation is reviewed, as is the experimental and clinical data suggesting that cytokines are rational targets for the development of new therapies.
The role of cyclooxygenase‐1 and cyclooxygenase‐2 in lipopolysaccharide and interleukin‐1 stimulated enterocyte prostanoid formation
Authors: Longo W. E. , Damore L. J. , Mazuski J. E. , Smith G. S. , Panesar N. , Kaminski D. L.
Published: July 2002

Lipopolysaccharide is an inflammatory agent and interleukin‐1 is a cytokine. Their pro‐inflammatory effects may be mediated by prostanoids produced by inducible cyclooxygenase‐2. The aim of this study was to determine the prostanoids produced by lipopolysaccharide and interleukin‐1 stimulated enterocytes through the cyclooxygenase‐1 and 2 pathways. Cultured enterocytes were stimulated with lipopolysaccharide or interleukin‐1 β with and without cyclooxygenase inhibitors. Low concentrations of indomethacin and valerylsalicylic acid (VSA) were evaluated as cyclooxygenase‐1 inhibitors and their effects compared with the effects of a specific cyclooxygenase‐2 inhibitor, SC‐58125. Prostaglandin E2 , 6‐keto prostaglandin F1α , prostaglandin D2 and leukotriene B4 levels were determined by radio immunoassay. Immunoblot analysis using isoformspecific antibodies showed that the inducible cyclooxygenase enzyme (COX‐2) was expressed by 4 h in LPS and IL‐1β treated cells while the constitutive COX‐1 remained unaltered in its expression. Interleukin‐1β and lipopolysaccharide stimulated the formation of all prostanoids compared with untreated cells, but failed to stimulate leukotriene B4. Indomethacin at 20 μ M concentration, and VSA inhibited lipopolysaccharide and interleukin 1β stimulated prostaglandin E2 , but not 6‐keto prostaglandin F1α formation. SC‐58125 inhibited lipopolysaccharide and interleukin‐1β stimulated 6‐keto prostaglandin F1α but not prostaglandin E2 release. The specific cyclooxygenase‐2 inhibitor also inhibited lipopolysaccharide produced prostaglandin D2 but not interleukin‐1β stimulated prostaglandin D2 While SC‐58125 inhibited basal 6‐keto prostaglandin‐F1α formation it significantly increased basal prostaglandin E2 and prostaglandin D2 formation. As SC‐58125 inhibited lipopolysaccharide and interleukin‐1β induced 6‐keto prostaglandin F1α production but not prostaglandin E2 production, it suggests that these agents stimulate prostacyclin production through a cyclooxygenase‐2 mediated mechanism and prostaglandin E2 production occurs through a cyclooxygenase‐1 mediated mechanism. Prostaglandin D2 production appeared to be variably produced by cyclooxygenase‐1 or cyclooxygenase‐2, depending on the stimulus.
Is Dependence on One Drug Associated with Dependence on Other Drugs? The Cases of Alcohol, Caffeine and Nicotine
Authors: Hughes John R. , Oliveto Alison H. , MacLaughlin Mark
Published: July 2002

Several studies have correlated the use of one drug with that of another drug; however, whether dependence on one drug is associated with dependence on another drug, independent of any use/use association, is unclear. We asked 196 randomly‐selected subjects the DSM‐IV criteria for dependence as applied to alcohol, caffeine, and nicotine. Among ever users, the severity of alcohol vs nicotine dependence and alcohol vs caffeine dependence was related, but this relationship was weak (r = .22 & .31). Nicotine and caffeine dependence were not correlated. These results fail to confirm theories of commonality that hypothesize dependence on one drug predisposes to dependence on another drug.
Utility of Crossover Designs in Clinical Trials: Efficacy of Desipramine vs. Placebo in Opioid‐Dependent Cocaine Abusers
Authors: Feingold Alan , Oliveto Alison , Schottenfeld Richard , Kosten Thomas R.
Published: July 2002

The utility of the crossover design in substance abuse research was examined in a 26‐week, double‐blind clinical trial that evaluated the efficacy of desipramine (0 or 150 mg/day) in 109 male and female cocaine‐ and opiate‐dependent patients maintained on buprenorphine (12 mg/day) or methadone (65 mg/day). After being stabilized on buprenorphine or methadone (weeks 1–2), half of the patients were randomly assigned to receive desipramine for the first half of the trial and placebo for the second, with the order reversed for the second half. Analyses using hierarchical linear models (HLM) indicated that desipramine reduced the use of opiates only when administered at the start (rather than the middle) of the trial, whereas cocaine use was reduced when desipramine was introduced at either time.
Sequential Actions of Rab5 and Rab7 Regulate Endocytosis in the Xenopus Oocyte
Authors: Mukhopadhyay Amitabha , Barbieri Alejandro M. , Funato Kouichi , Roberts Richard , Stahl Philip D.
Published: July 2002

To explore the role of GTPases in endocytosis, we developed an assay using Xenopus oocytes injected with recombinant proteins to follow the uptake of the fluid phase marker HRP. HRP uptake was inhibited in cells injected with GTPγS or incubated with aluminum fluoride, suggesting a general role for GTPases in endocytosis. Injection of Rab5 into oocytes, as well as Rab5:Q79L, a mutant with decreased GTPase activity, increased HRP uptake. Injection of Rab5:S34N, the dominant-negative mutant, inhibited HRP uptake. Injection of N-ethylmaleimide–sensitive factor (NSF) stimulated HRP uptake, and ATPase-defective NSF mutants inhibited HRP uptake when coinjected with Rab5:Q79L, confirming a requirement for NSF in endocytosis. Surprisingly, injection of Rab7:WT stimulated both uptake and degradation/activation of HRP. The latter appears to be due to enhanced transport to a late endosomal/prelysosomal degradative compartment that is monensin sensitive. Enhancement of uptake by Rab7 appears to function via an Rab5-sensitive pathway in oocytes since the stimulatory effect of Rab7 was blocked by coinjection of Rab5:S34N. Stimulation of uptake by Rab5 was blocked by Rab5:S34N but not by Rab7:T22N. Our results suggest that Rab7, while functioning downstream of Rab5, may be rate limiting for endocytosis in oocytes.
Epithelial V-like Antigen (EVA), a Novel Member of the Immunoglobulin Superfamily, Expressed in Embryonic Epithelia with a Potential Role as Homotypic Adhesion Molecule in Thymus Histogenesis
Authors: Guttinger Maria , Sutti Francesca , Panigada Maddalena , Porcellini Simona , Merati Barbara , Mariani Margherita , Teesalu Tambet , Consalez G. Giacomo , Grassi Fabio
Published: July 2002

Thymus development depends on a complex series of interactions between thymocytes and the stromal component of the organ. To identify regulated genes during this codependent developmental relationship, we have applied an RNA fingerprinting technique to the analysis of thymus expansion and maturation induced in recombinase-deficient mice injected with anti-CD3 antibodies. This approach led us to the identification of a gene encoding a new member of the immunoglobulin superfamily, named epithelial V-like antigen (EVA), which is expressed in thymus epithelium and strongly downregulated by thymocyte developmental progression. This gene is expressed in the thymus and in several epithelial structures early in embryogenesis. EVA is highly homologous to the myelin protein zero and, in thymus-derived epithelial cell lines, is poorly soluble in nonionic detergents, strongly suggesting an association to the cytoskeleton. Its capacity to mediate cell adhesion through a homophilic interaction and its selective regulation by T cell maturation might imply the participation of EVA in the earliest phases of thymus organogenesis.
Exogenous Expression of the Amino-Terminal Half of the Tight Junction Protein Zo-3 Perturbs Junctional Complex Assembly
Authors: Wittchen Erika S. , Haskins Julie , Stevenson Bruce R.
Published: July 2002

The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and β-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100–soluble, signaling-active β-catenin was increased in NZO-3–expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or β-catenin.
Integrin-linked kinase (ILK) and its interactors
Authors: Wu Chuanyue , Dedhar Shoukat
Published: July 2002

How intracellular cytoskeletal and signaling proteins connect and communicate with the extracellular matrix (ECM) is a fundamental question in cell biology. Recent biochemical, cell biological, and genetic studies have revealed important roles of cytoplasmic integrin-linked kinase (ILK) and its interactive proteins in these processes. Cell adhesion to ECM is an important process that controls cell shape change, migration, proliferation, survival, and differentiation. Upon adhesion to ECM, integrins and a selective group of cytoskeletal and signaling proteins are recruited to cell matrix contact sites where they link the actin cytoskeleton to the ECM and mediate signal transduction between the intracellular and extracellular compartments. In this review, we discuss the molecular activities and cellular functions of ILK, a protein that is emerging as a key component of the cell–ECM adhesion structures.
Human Speedy
Authors: Porter Lisa A. , Dellinger Ryan W. , Tynan John A. , Barnes Elizabeth A. , Kong Monica , Lenormand Jean-Luc , Donoghue Daniel J.
Published: July 2002

The decision for a cell to self-replicate requires passage from G1 to S phase of the cell cycle and initiation of another round of DNA replication. This commitment is a critical one that is tightly regulated by many parallel pathways. Significantly, these pathways converge to result in activation of the cyclin-dependent kinase, cdk2. It is, therefore, important to understand all the mechanisms regulating cdk2 to determine the molecular basis of cell progression. Here we report the identification and characterization of a novel cell cycle gene, designated Speedy (Spy1). Spy1 is 40% homologous to the Xenopus cell cycle gene, X-Spy1. Similar to its Xenopus counterpart, human Speedy is able to induce oocyte maturation, suggesting similar biological characteristics. Spy1 mRNA is expressed in several human tissues and immortalized cell lines and is only expressed during the G1/S phase of the cell cycle. Overexpression of Spy1 protein demonstrates that Spy1 is nuclear and results in enhanced cell proliferation. In addition, flow cytometry profiles of these cells demonstrate a reduction in G1 population. Changes in cell cycle regulation can be attributed to the ability of Spy1 to bind to and prematurely activate cdk2 independent of cyclin binding. We demonstrate that Spy1-enhanced cell proliferation is dependent on cdk2 activation. Furthermore, abrogation of Spy1 expression, through the use of siRNA, demonstrates that Spy1 is an essential component of cell proliferation pathways. Hence, human Speedy is a novel cell cycle protein capable of promoting cell proliferation through the premature activation of cdk2 at the G1/S phase transition.
Transfection of the Primate Malaria Parasite Plasmodium knowlesi Using Entirely Heterologous Constructs
Authors: Wel Anna M. van der , Tomás Ana M. , Kocken Clemens H.M. , Malhotra Pawan , Janse Chris J. , Waters Andrew P. , Thomas Alan W.
Published: July 2002

The recently developed transfection systems for Plasmodium berghei and Plasmodium falciparum offer important new tools enabling further insight into the biology of malaria parasites. These systems rely upon artificial parasite–host combinations which do not allow investigation into the complex interactions between parasites and their natural hosts. Here we report on stable transfection of Plasmodium knowlesi (a primate malaria parasite that clusters phylogenetically with P. vivax) for which both natural and artificial experimental hosts are available. Transfection of this parasite offers the opportunity to further analyze the biology of antigens not only in a natural host but also in hosts that are closely related to humans. To facilitate future development of integration-dependent transfection in P. knowlesi, completely heterologous plasmids that would reduce homologous recombination at unwanted sites in the genome were constructed. These plasmids contained the pyrimethamine-resistant form of dihydrofolate reductase-thymidylate synthase (dhfr-ts) from Toxoplasma gondii or P. berghei, under control of either (a) P. berghei or (b) P. falciparum promoters. Plasmids were electroporated into mature P. knowlesi schizonts and these cells were injected into rhesus monkeys (Macaca mulatta). After pyrimethamine treatment of these monkeys, resistant parasites were obtained that contained the plasmids. Promoter regions of both P. berghei and P. falciparum controlling dhfr-ts expression were effective in conferring pyrimethamine resistance in P. knowlesi, indicating that common signals control gene expression in phylogenetically distant Plasmodium species.
Interleukin 4 (IL-4) or IL-7 Prevents the Death of Resting T Cells: Stat6 Is Probably Not Required for the Effect of IL-4
Authors: Vella Anthony , Teague T. Kent , Ihle James , Kappler John , Marrack Philippa
Published: July 2002

Although much is known about the activation, proliferation, and function of CD4+ T cells, little is known about how they survive as resting T cells in animals. Resting T cells have a half-life in animals of more than a week; however, when they are removed from animals and placed in tissue culture their half-life falls to ∼24 h. In this paper, we show that the survival of resting T cells in vitro is promoted by two cytokines, interleukins 4 and 7 (IL-4, IL-7). They may do this in part by maintaining levels of survival-promoting proteins such as Bcl-2 in the cells, because the levels of Bcl-2 and Bcl-Xl in resting T cells fall rapidly after the cells are isolated from animals, and are maintained by culture in IL-4. Because the IL-4 receptor is known to signal through the JAK1 and JAK3/Stat6 pathway, we tested whether Stat6 was required for IL-4– dependent T cell survival. Surprisingly, we found that IL-4 rescued T cells from apoptosis in what appeared to be a Stat6-independent manner. These results demonstrate that the survival of resting T cells is an active process that can be affected by signals delivered by cytokines and also suggest that the IL-4 receptor on resting T cells may use a novel signaling pathway to facilitate T cell viability.
CD4 T Cell Cytokine Differentiation: The B Cell Activation Molecule, OX40 Ligand, Instructs CD4 T Cells to Express Interleukin 4 and Upregulates Expression of the Chemokine Receptor, Blr-1
Authors: Flynn Sarah , Toellner Kai-Michael , Raykundalia Chandra , Goodall Margaret , Lane Peter
Published: July 2002

This report investigates the role of OX40 ligand (OX40L) and its receptor, OX40, expressed on activated B and T cells, respectively, in promoting the differentiation of T helper type 2 (Th2) CD4 T cells. These molecules are expressed in vivo by day 2 after priming with T cell– dependent antigens. Their expression coincides with the appearance of immunoglobulin (Ig)G switch transcripts and mRNA for interleukin (IL)-4 and interferon (IFN)-γ, suggesting that this molecular interaction plays a role in early cognate interactions between B and T cells. In vitro, we report that costimulation of naive, CD62Lhigh CD4 T cells through OX40 promotes IL-4 expression and upregulates mRNA for the chemokine receptor, blr-1, whose ligand is expressed in B follicles and attracts lymphocytes to this location. Furthermore, T cell stimulation through OX40 inhibits IFN-γ expression in both CD8 T cells and IL-12–stimulated CD4 T cells. Although this signal initiates IL-4 expression, IL-4 itself is strongly synergistic. Our data suggest that OX40L on antigen-activated B cells instructs naive T cells to differentiate into Th2 cells and migrate into B follicles, where T cell–dependent germinal centers develop.
Exercise Provides Direct Biphasic Cardioprotection via Manganese Superoxide Dismutase Activation
Authors: Yamashita Nobushige , Hoshida Shiro , Otsu Kinya , Asahi Michio , Kuzuya Tsunehiko , Hori Masatsugu
Published: July 2002

Epidemiologic investigations have shown that exercise reduces morbidity and mortality from coronary artery disease. In this study, using a rat model, we attempted to determine whether exercise can reduce ischemic injury to the heart and elucidate a mechanism for the cardioprotective effect of exercise. Results showed that exercise significantly reduced the magnitude of a myocardial infarction in biphasic manner. The time course for cardioprotection resembled that of the change in manganese superoxide dismutase (Mn-SOD) activity. The administration of the antisense oligodeoxyribonucleotide to Mn-SOD abolished the expected decrease in infarct size. We showed that the level of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) increased after exercise. The simultaneous administration of the neutralizing antibodies to the cytokines abolished the exercise-induced cardioprotection and the activation of Mn-SOD. Furthermore, TNF-α can mimic the biphasic pattern of cardioprotection and activation of Mn-SOD. An antioxidant completely abolished cardioprotection and the activation of Mn-SOD by exercise or the injection of TNF-α as well as exercise-induced increase in TNF-α and IL-1β. The production of reactive oxygen species and endogenous TNF-α and IL-1β induced by exercise leads to the activation of Mn-SOD, which plays major roles in the acquisition of biphasic cardioprotection against ischemia/reperfusion injury in rats.
Protection from Lethal Gram-Positive Infection by Macrophage Scavenger Receptor–Dependent Phagocytosis
Authors: Thomas Christian A. , Li Yongmei , Kodama Tatsuhiko , Suzuki Hiroshi , Silverstein Samuel C. , El Khoury Joseph
Published: July 2002

Infections with gram-positive bacteria are a major cause of morbidity and mortality in humans. Opsonin-dependent phagocytosis plays a major role in protection against and recovery from gram-positive infections. Inborn and acquired defects in opsonin generation and/or recognition by phagocytes are associated with an increased susceptibility to bacterial infections. In contrast, the physiological significance of opsonin-independent phagocytosis is unknown. Type I and II class A scavenger receptors (SR-AI/II) recognize a variety of polyanions including bacterial cell wall products such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), suggesting a role for SR-AI/II in innate immunity to bacterial infections. Here, we show that SR-AI/II–deficient mice (MSR-A−/−) are more susceptible to intraperitoneal infection with a prototypic gram-positive pathogen, Staphylococcus aureus, than MSR-A+/+ control mice. MSR-A−/− mice display an impaired ability to clear bacteria from the site of infection despite normal killing of S. aureus by neutrophils and die as a result of disseminated infection. Opsonin-independent phagocytosis of gram-positive bacteria by MSR-A−/− macrophages is significantly decreased although their phagocytic machinery is intact. Peritoneal macrophages from control mice phagocytose a variety of gram-positive bacteria in an SR-AI/II–dependent manner. Our findings demonstrate that SR-AI/II mediate opsonin-independent phagocytosis of gram-positive bacteria, and provide the first evidence that opsonin-independent phagocytosis plays a critical role in host defense against bacterial infections in vivo.
Inhibition of E-Selectin Gene Expression by Transforming Growth Factor β in Endothelial Cells Involves Coactivator Integration of Smad and Nuclear Factor κB–Mediated Signals
Authors: DiChiara Maria R. , Kiely Jeanne Marie , Gimbrone Michael A. , Lee Mu-En , Perrella Mark A. , Topper James N.
Published: July 2002

Transforming growth factor (TGF)-β1 is a pleiotropic cytokine/growth factor that is thought to play a critical role in the modulation of inflammatory events. We demonstrate that exogenous TGF-β1 can inhibit the expression of the proinflammatory adhesion molecule, E-selectin, in vascular endothelium exposed to inflammatory stimuli both in vitro and in vivo. This inhibitory effect occurs at the level of transcription of the E-selectin gene and is dependent on the action of Smad proteins, a class of intracellular signaling proteins involved in mediating the cellular effects of TGF-β1. Furthermore, we demonstrate that these Smad-mediated effects in endothelial cells result from a novel competitive interaction between Smad proteins activated by TGF-β1 and nuclear factor κB (NFκB) proteins activated by inflammatory stimuli (such as cytokines or bacterial lipopolysaccharide) that is mediated by the transcriptional coactivator cyclic AMP response element–binding protein (CREB)-binding protein (CBP). Augmentation of the limited amount of CBP present in endothelial cells (via overexpression) or selective disruption of Smad–CBP interactions (via a dominant negative strategy) effectively antagonizes the ability of TGF-β1 to block proinflammatory E-selectin expression. These data thus demonstrate a novel mechanism of interaction between TGF-β1–regulated Smad proteins and NFκB proteins regulated by inflammatory stimuli in vascular endothelial cells. This type of signaling mechanism may play an important role in the immunomodulatory actions of this cytokine/growth factor in the cardiovascular system.
Fatal Leukemia in Interleukin 15 Transgenic Mice Follows Early Expansions in Natural Killer and Memory Phenotype Cd8+ T Cells
Authors: Fehniger Todd A. , Suzuki Kazuhiro , Ponnappan Anand , VanDeusen Jeffrey B. , Cooper Megan A. , Florea Sorin M. , Freud Aharon G. , Robinson Michael L. , Durbin Joan , Caligiuri Michael A.
Published: July 2002

Inflammation likely has a role in the early genesis of certain malignancies. Interleukin (IL)-15, a proinflammatory cytokine and growth factor, is required for lymphocyte homeostasis. Intriguingly, the expression of IL-15 protein is tightly controlled by multiple posttranscriptional mechanisms. Here, we engineered a transgenic mouse to overexpress IL-15 by eliminating these posttranscriptional checkpoints. IL-15 transgenic mice have early expansions in natural killer (NK) and CD8+ T lymphocytes. Later, these mice develop fatal lymphocytic leukemia with a T-NK phenotype. These data provide novel evidence that leukemia, like certain other cancers, can arise as the result of chronic stimulation by a proinflammatory cytokine.
Inhibition of Natural Killer Cells through Engagement of CD81 by the Major Hepatitis C Virus Envelope Protein
Authors: Crotta Stefania , Stilla Annalisa , Wack Andreas , D'Andrea Annalisa , Nuti Sandra , D'Oro Ugo , Mosca Marta , Filliponi Franco , Brunetto R. Maurizia , Bonino Ferruccio , Abrignani Sergio , Valiante Nicholas M.
Published: July 2002

The immune response against hepatitis C virus (HCV) is rarely effective at clearing the virus, resulting in ∼170 million chronic HCV infections worldwide. Here we report that ligation of an HCV receptor (CD81) inhibits natural killer (NK) cells. Cross-linking of CD81 by the major envelope protein of HCV (HCV-E2) or anti-CD81 antibodies blocks NK cell activation, cytokine production, cytotoxic granule release, and proliferation. This inhibitory effect was observed using both activated and resting NK cells. Conversely, on NK-like T cell clones, including those expressing NK cell inhibitory receptors, CD81 ligation delivered a costimulatory signal. Engagement of CD81 on NK cells blocks tyrosine phosphorylation through a mechanism which is distinct from the negative signaling pathways associated with NK cell inhibitory receptors for major histocompatibility complex class I. These results implicate HCV-E2–mediated inhibition of NK cells as an efficient HCV evasion strategy targeting the early antiviral activities of NK cells and allowing the virus to establish itself as a chronic infection.
Ion Channel Selectivity through Stepwise Changes in Binding Affinity
Authors: Dang Thieu X. , McCleskey Edwin W.
Published: July 2002

Voltage-gated Ca2+ channels select Ca2+ over competing, more abundant ions by means of a high affinity binding site in the pore. The maximum off rate from this site is ∼1,000× slower than observed Ca2+ current. Various theories that explain how high Ca2+ current can pass through such a sticky pore all assume that flux occurs from a condition in which the pore's affinity for Ca2+ transiently decreases because of ion interactions. Here, we use rate theory calculations to demonstrate a different mechanism that requires no transient changes in affinity to quantitatively reproduce observed Ca2+ channel behavior. The model pore has a single high affinity Ca2+ binding site flanked by a low affinity site on either side; ions permeate in single file without repulsive interactions. The low affinity sites provide steps of potential energy that speed the exit of a Ca2+ ion off the selectivity site, just as potential energy steps accelerate other chemical reactions. The steps could be provided by weak binding in the nonselective vestibules that appear to be a general feature of ion channels, by specific protein structures in a long pore, or by stepwise rehydration of a permeating ion. The previous ion-interaction models and this stepwise permeation model demonstrate two general mechanisms, which might well work together, to simultaneously generate high flux and high selectivity in single file pores.
The Barium Site in a Potassium Channel by X-Ray Crystallography
Authors: Jiang Youxing , MacKinnon Roderick
Published: July 2002

X-ray diffraction data were collected from frozen crystals (100°K) of the KcsA K+ channel equilibrated with solutions containing barium chloride. Difference electron density maps (Fbarium − Fnative, 5.0 Å resolution) show that Ba2+ resides at a single location within the selectivity filter. The Ba2+ blocking site corresponds to the internal aspect (adjacent to the central cavity) of the “inner ion” position where an alkali metal cation is found in the absence of the blocking Ba2+ ion. The location of Ba2+ with respect to Rb+ ions in the pore is in good agreement with the findings on the functional interaction of Ba2+ with K+ (and Rb+) in Ca2+-activated K+ channels (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549–567). Taken together, these structural and functional data imply that at physiological ion concentrations a third ion may interact with two ions in the selectivity filter, perhaps by entering from one side and displacing an ion on the opposite side.
Temporal Variations in the Circumstellar Shell IRC+10216
Authors: Sada Pedro Antonio Valdes
Published: July 2002

The dynamical evolution of the inner sections of the circumstellar shell (CS) surrounding the prototypical evolved carbon star IRC+10216 is studied through the analysis and modeling of high resolution (<0.02 cm-1) infrared 12CO and 13CO vibrational-rotational fundamental (4.6 microns) and overtone (2.3 microns) band absorption line profiles. The overtone band lines in particular sample a zone within the circumstellar shell, near the Long-Period Variable (LPV) central star, where the mass ejection and dust formation and acceleration mechanisms dominate (2-10 R*).Temporal changes in the line profiles observed over the past 12 years indicates evolution of this region of the CS. Modeling of the dust and gas components of the circumstellar shell in the observer's frame of reference is performed for each available spectroscopic data set in an effort to reproduce broadband photometric flux measurements and the observed molecular line profiles. The dust density distribution and the gas velocity fields are adjusted in the model, yielding a time sequence of synthetic lines which implies evolution of the CS structure. Short-term (phase dependent) and long-term (years) systematic changes in line shapes are successfully reproduced by this method, yielding further insight on the physical conditions governing this region of the circumstellar shell. The dust condensation point in the model roughly coincides with the location of the largest gas velocity gradient, supporting the notion that the dust formation and gas mass loss mechanisms are associated. Radiation pressure, acting on co-spatial momentum-coupled dust particles, accelerates the ambient gas component by 7.3 ± 1.8 x 10-4 cm s-2 (0.23 ± 0.06 km s-1 yr-1) throughout the probed region of the CS. Evolution of the observed absorption line components suggests that the principal mass loss mechanism in cool evolved stars is not related to the pulsation period of the central star, but may be stochastic in nature. Broadband photometric observational data indicates that an episodic disturbance that modifies the line profiles between 1979 and 1991 may have originated sometime between 1975 and 1978, and that it progressed outward through the CS. This analysis indicates that broadband IR monitoring of thermal dust emission from carbon stars can be used as an indicator of the onset of episodic mass loss events.