bluuebunny/crossref_metadata_2025 huggingface dataset:
Conceptual and Lexical Knowledge of Shame in Italian Children and Adolescents
Authors: Levorato M. Chiara , Donati Valeria
Published: July 2002

In the present study, the development of knowledge of nonmoral shame in Italian children and adolescents was investigated. The issues addressed were: (a) how the development of the ability to identify appropriate antecedents of shame is achieved (Experiment 1); and (b) the acquisition of the ability to use linguistic labels to refer to this emotion (Experiment 2). Two cross-sectional experiments were conducted with participants aged from 5 years 8 months to 18 years 5 months. In the first experiment, participants were asked for an antecedent of shame; in the second, they were asked to describe an emotion for each of the six antecedents proposed in the experimental setting. Results showed that the conceptual structure of shame is developed during primary school. Among adolescents, however, the need for privacy emerged, and “embarrassment” was given more frequently than shame. In both experiments participants were asked for an explanation of the emotional experience of shame. Correspondence analysis on the explanations given in Experiment 1 showed that two dimensions underlie the conception of shame: The first, which develops in primary school, is intersubjectivity and consideration of the group. The second, which develops during adolescence, is intrasubjectivity and consideration of personal motives. The analysis of the explanations given in Experiment 2 showed that, when the word “shame” was produced, the explanation focused mainly on the fear of being exposed to an audience.
Rethinking Silicon Valley: New Perspectives on Regional Development
Authors: Cook Ian , Joseph Richard
Published: July 2002

Silicon Valley in Southern California has, over the past 30 years, become a model for high technology development in many parts of the world. Associated with Silicon Valley is a common rhetoric and mythology that explains the origins of this area of high technology agglomeration and indeed the business and entrepreneurial attributes needed for success. Governments in many parts of the world (including Southeast Asia and Australia) have tried to emulate this growth through various industry and regional development mechanisms, in particular, the science or technology park. More recently, promoting developments in information technology has come to be seen as an integral feature of these parks' activities. In this paper, we argue that the modeling process used by governments to promote Silicon Valley-like regional development has tended to model the wrong things about Silicon Valley. The models have tended to be mechanical and have failed to reflect the nature of information and information industries. While we have not sought to develop a model for Silicon Valley in this paper,we address a number of issues that require attention on the part of anyone serious about this project. After discussing problems with previous attempts to model Silicon Valley and problems associated with the activity of modeling itself, we move to consider four issues that must be addressed in any real attempt to model Silicon Valley in Southeast Asia. The first is the role of the state and the problems that state involvement may create. The second concerns the contribution that universities can make to the project. The third is the role of firms, particularly Chinese firms. The fourth is the cultural context within which the 'model' will sit. Since technology parks are seen as a popular way of promoting high technology development by governments, the revised history suggested in this paper provides fresh thinking about modeling Silicon Valley in the Southeast Asian region.
Time Dependent Production of Cytokines and Eicosanoids by Human Monocytic Leukaemia U937 Cells; Effects of Glucocorticosteroids
Authors: Garrelds Ingrid M. , Van Hal Peter Th. W. , Haakmat Raquel C. , Hoogsteden Henk C. , Saxena Pramod R. , Zijlstra Frederik J.
Published: July 2002

In the present study the human monoblast cell line U937 has been used as a model to study the function of human mononuclear phagocytes in asthma. The kinetics of the production of eicosanoids and cytokines, which are thought to play a role in the pathogenesis of asthma, were studied. In addition, the effects of glucocorticosteroids were investigated, as these drugs are of great importance for the treatment of asthmatic patients. After stimulation with phorbol‐12 myristate acetate (PMA) for 24h, U937 cells were cultured in the absence or presence of lipopolysaccharide (LPS: 1 and 5 μg ml-1) and glucocorticosteroids (budesonide, fluticasone propionate and prednisolone: 10-11, 10-9 and 10-7 M) for 96h. The production of interleukin‐ 1β (IL‐1β), interleukin‐6 (IL‐6), prostaglandin E2 (PGE2) and thromboxane B2 (TxB2) gradually increased in time after stimulation with LPS, whereas the transient production of tumor necrosis factor alpha (TNF‐α) reached its maximum between 6 and 12 h. Interferon‐gamma (IFN‐γ), interleukin‐10 (IL‐10) and leukotriene B4 (LTB4) were not detectable. All three glucocorticosteroids (budesonide, fluticasone propionate and prednisolone) completely inhibited the production of both eicosanoids and cytokines. The production of eicosanoids was more sensitive to these glucocorticoids than the production of cytokines. The observed differences in the kinetics of the production of eicosanoids and cytokines stress the importance of time course experiments in studies on the effect of drugs on mononuclear cells.
Gender-Based Differences in the Toxicity of Pharmaceuticals—The Food and Drug Administration's Perspective
Authors: Miller Margaret Ann
Published: July 2002

Women experience more adverse reactions to treatment with therapeutic drugs than men. Theories proposed to explain this include overdosing, different pharmacokinetics and pharmacodynamics, women are more likely to report adverse events than men, or women take more medications than men. Food and Drug Administration (FDA) Office of Women's Health (OWH) funds research to promote including women in clinical trials and understanding the biology of sex-related differences in the safety of FDA-regulated products. Including women in clinical trials advances the understanding of drug efficacy and safety in women by providing information on drug dosing, pharmacokinetics, and pharmacodynamics. A Baysian statistical analysis of sex differences in adverse events showed that although about the same number of adverse events were reported for men and women, those reported for women were more serious. One example of a sex difference in the toxicity of pharmaceuticals is the drug-induced cardiac arrhythmia, torsades de point. OWH funded studies in animals and humans to investigate the mechanism behind this sex difference. These studies demonstrated that shortening the QT interval increases the risk of developing torsades and that androgens protect against torsades by slowing cardiac repolarization and prolonging the QT interval. Understanding the mechanisms behind other reported sex-related differences in adverse drug effects requires additional research. The preliminary studies conducted to date suggest that this sex-related difference is likely to be a multifactorial problem requiring information from several fields of study. Ideally, individuals at risk for developing an adverse event should be identified prior to therapeutic intervention. The OWH plans to fund more studies to investigate the role of hormonal variations on drug metabolism and drug-drug interactions. Animal and in vitro model systems are needed to fully understand the mechanism of how gender influences drug toxicity.
A Subchronic Toxicity Study of Fyrol PCF in Sprague-Dawley Rats
Authors: Freudenthal Ralph I. , Henrich Richard T.
Published: July 2002

Fyrol PCF is a flame retardant chemical widely used to control the flam mability of rigid polyurethane foams and polyester foam systems. It has moderate acute toxicity and was shown to lack mu-tagenic activity. The present study was conducted to determine the toxicity of Fyrol PCF after repeated dietary exposure. Five groups, each consisting of 20 male and 20 female Sprague-Dawley rats, a diet containing either 0, 800, 2500, 7500, or 20,000 ppm Fyrol PCF for 90 days. Data collected include clinical observations, food consumption, body weights, organ weights, urinalysis, hematology, clinical chemistry, brain cholinesterase activity, and gross and microscopic pathology. The mean body weight of animals receiving 20,000 ppm was significantly lower than control values for most study weeks. No clinical signs were noted during cage-side observations. Liver weights were increased in male rats in all treatment groups, and in female rats in both the mid-and high-dose groups. No treatment-related histopathologic changes were observed in the livers of animals that 800, 2500, or 7500 ppm Fyrol PCF; however very mild periportal hepato-cellular swelling was seen in certain of the 20,000 ppm animals. Mean kidney weights were significantly greater in the male animals given 2500, 7500, and 20,000 ppm. Very mild cortical tubular degenerative changes were found in kidneys of male rats that 7500 ppm or 20,000 ppm and in female animals that ingested 20,000 ppm Fyrol PCF. An increase in the incidence of very mild thyroid follicular changes was found in the two highest dose groups. Fyrol PCF did not affect hematology or clinical chemistry parameters, nor did it significantly inhibit brain cholinesterase activity. None of the observed minimal treatment-related changes appear to have human relevance, because they occurred only at the highest doses used in the study. The no observable effect level in this study is 2500 ppm. Fyrol PCF demonstrated low subchronic toxicity in this study.
Evidentiary Requirements for Risk-Based News
Authors: Rollin Sara Thurin
Published: July 2002

Understanding the criteria journalists use when deciding whether something is newsworthy provides insights into what toxico-logical data and risk-related information gets reported. Although the criteria are common to all media, the level of depth in reporting varies, based on the type of media outlet involved—ranging from daily, general circulation newspapers to the specialized media, including on-line publications. This paper addresses five aspects of the media's perspective on toxicology, risk, and the law. The following questions are addressed. What makes something newsworthy? What is the role of the media in toxicology, risk, and the law? What are common barriers to getting toxicology news reported? What are some potential solutions for overcoming these barriers? What are some take-home tips that toxicologists could use to strengthen their relationships with journalists?
The acute influence of tobacco smoking on adhesion molecule expression on monocytes and neutrophils and on circulating adhesion molecule levels in vivo
Authors: Scott D. A. , Todd D. H. , Coward P. Y. , Wilson R. F. , Odell E. W. , Poston R. N. , Matthews J. P. , Palmer R. M.
Published: July 2002

Soluble adhesion molecules have been reported as risk markers of a wide range of human diseases and specific adhesion molecules may play a direct role in pathological processes. Serum soluble intercellular adhesion molecule‐1 (sICAM‐1) is known to be significantly elevated in smokers compared to non‐smokers. We examined the acute effects of smoking a standard 2R1 research cigarette on the serum concentration of sICAM‐1 and other circulating adhesion molecules (sP‐selectin, sE‐selectin, sL‐selectin, sVCAM‐1 and sPECAM‐1) in heavy smokers (serum cotinine ≥ 100 ng/ml), light smokers (serum cotinine ≤ 60 ng/ml) and non‐smokers (serum cotinine ≤ 10 ng/ml) by ELISA. Adhesion molecule expression on the cell surface of monocytes and neutrophils in peripheral blood was examined by flow cytometry. The sICAM‐1 concentration directly correlated to serum cotinine concentration (p= 0.047) and nicotine load (p= 0.033) in smokers and was significantly elevated compared to non‐smokers (p= 0.037). Other than a decrease in the concentration of sP‐selectin over 1 hour regardless of smoking, no significant temporal alterations of any adhesion molecule were observed following the smoking experience or in the non‐smoking control group. No significant difference in surface expression of ICAM‐1, CD18, PECAM‐1 or L‐selectin on peripheral monocytes or neutrophils was observed over a 1‐hour period following smoking. These data suggest that the elevated concentration of sICAM‐1 in smokers is not due to an immediate effect of smoking.
Characterization of α1(IV) Collagen Mutations in Caenorhabditis elegans and the Effects of α1 and α2(IV) Mutations on Type IV Collagen Distribution
Authors: Gupta Malini C. , Graham Patricia L. , Kramer James M.
Published: July 2002

Type IV collagen is a major component of basement membranes. We have characterized 11 mutations in emb-9, the α1(IV) collagen gene of Caenorhabditis elegans, that result in a spectrum of phenotypes. Five are substitutions of glycines in the Gly-X-Y domain and cause semidominant, temperature-sensitive lethality at the twofold stage of embryogenesis. One is a glycine substitution that causes recessive, non–temperature-sensitive larval lethality. Three putative null alleles, two nonsense mutations and a deletion, all cause recessive, non–temperature-sensitive lethality at the threefold stage of embryogenesis. The less severe null phenotype indicates that glycine substitution containing mutant chains dominantly interfere with the function of other molecules. The emb-9 null mutants do not stain with anti–EMB-9 antisera and show intracellular accumulation of the α2(IV) chain, LET-2, indicating that LET-2 assembly and/or secretion requires EMB-9. Glycine substitutions in either EMB-9 or LET-2 cause intracellular accumulation of both chains. The degree of intracellular accumulation differs depending on the allele and temperature and correlates with the severity of the phenotype. Temperature sensitivity appears to result from reduced assembly/secretion of type IV collagen, not defective function in the basement membrane. Because the dominant interference of glycine substitution mutations is maximal when type IV collagen secretion is totally blocked, this interference appears to occur intracellularly, rather than in the basement membrane. We suggest that the nature of dominant interference caused by mutations in type IV collagen is different than that caused by mutations in fibrillar collagens.
Differential Association of Syntrophin Pairs with the Dystrophin Complex
Authors: Peters Matthew F. , Adams Marvin E. , Froehner Stanley C.
Published: July 2002

The syntrophins are a multigene family of intracellular dystrophin-associated proteins comprising three isoforms, α1, β1, and β2. Based on their domain organization and association with neuronal nitric oxide synthase, syntrophins are thought to function as modular adapters that recruit signaling proteins to the membrane via association with the dystrophin complex. Using sequences derived from a new mouse β1-syntrophin cDNA, and previously isolated cDNAs for α1- and β2-syntrophins, we prepared isoform-specific antibodies to study the expression, skeletal muscle localization, and dystrophin family association of all three syntrophins. Most tissues express multiple syntrophin isoforms. In mouse gastrocnemius skeletal muscle, α1- and β1-syntrophin are concentrated at the neuromuscular junction but are also present on the extrasynaptic sarcolemma. β1-syntrophin is restricted to fast-twitch muscle fibers, the first fibers to degenerate in Duchenne muscular dystrophy. β2-syntrophin is largely restricted to the neuromuscular junction.

The sarcolemmal distribution of α1- and β1-syntrophins suggests association with dystrophin and dystrobrevin, whereas all three syntrophins could potentially associate with utrophin at the neuromuscular junction. Utrophin complexes immunoisolated from skeletal muscle are highly enriched in β1- and β2-syntrophins, while dystrophin complexes contain mostly α1- and β1-syntrophins. Dystrobrevin complexes contain dystrophin and α1- and β1-syntrophins. From these results, we propose a model in which a dystrophin–dystrobrevin complex is associated with two syntrophins. Since individual syntrophins do not have intrinsic binding specificity for dystrophin, dystrobrevin, or utrophin, the observed preferential pairing of syntrophins must depend on extrinsic regulatory mechanisms.
Regulation and Function of the CD3γ DxxxLL Motif: A Binding Site for Adaptor Protein-1 and Adaptor Protein-2 in Vitro
Authors: Dietrich Jes , Kastrup Jesper , Nielsen Bodil L. , Ødum Niels , Geisler Carsten
Published: July 2002

Several receptors are downregulated by internalization after ligand binding. Regulation of T cell receptor (TCR) expression is an important step in T cell activation, desensitization, and tolerance induction. One way T cells regulate TCR expression is by phosphorylation/dephosphorylation of the TCR subunit clusters of differentiation (CD)3γ. Thus, phosphorylation of CD3γ serine 126 (S126) causes a downregulation of the TCR. In this study, we have analyzed the CD3γ internalization motif in three different systems in parallel: in the context of the complete multimeric TCR; in monomeric CD4/CD3γ chimeras; and in vitro by binding CD3γ peptides to clathrin-coated vesicle adaptor proteins (APs). We find that the CD3γ D127xxxLL131/132 sequence represents one united motif for binding of both AP-1 and AP-2, and that this motif functions as an active sorting motif in monomeric CD4/ CD3γ molecules independently of S126. An acidic amino acid is required at position 127 and a leucine (L) is required at position 131, whereas the requirements for position 132 are more relaxed. The spacing between aspartic acid 127 (D127) and L131 is crucial for the function of the motif in vivo and for AP binding in vitro. Furthermore, we provide evidence indicating that phosphorylation of CD3γ S126 in the context of the complete TCR induces a conformational change that exposes the DxxxLL sequence for AP binding. Exposure of the DxxxLL motif causes an increase in the TCR internalization rate and we demonstrate that this leads to an impairment of TCR signaling. On the basis of the present results, we propose the existence of at least three different types of L-based receptor sorting motifs.
Nerve Growth Factor–specific Regulation of Protein Methylation during Neuronal Differentiation of PC12 Cells
Authors: Cimato Thomas R. , Ettinger Murray J. , Zhou Xianbo , Aletta John M.
Published: July 2002

Protein methylation is a posttranslational modification that can potentially regulate signal transduction pathways in a similar manner as protein phosphorylation. The role of protein methylation in NGF signaling was examined by metabolic labeling of PC12 cell proteins with l-[methyl-3H]methionine and by in vitro labeling of cell proteins with l-[methyl-3H]S-adenosylmethionine. Effects of NGF were detected within 15 min. Methyl-labeled proteins were resolved by one and two dimensional SDS-PAGE. NGF affected the methylation of several 68–60-kD proteins (pI 5.8–6.4) and 50-kD proteins (isoelectric point pH 6.7–6.8 and 5.8–6.2). Several NGF-induced changes in methylation increased over several hours and through 4 d. Moreover, methyl labeling of several specific proteins was only detected after NGF treatment, but not in nontreated controls. The effects of NGF on protein methylation were NGF specific since they were not observed with EGF or insulin. A requirement for protein methylation for neurite outgrowth was substantiated with either of two methylation inhibitors: dihydroxycyclopentenyl adenine (DHCA) and homocysteine. DHCA, the more potent of the two, markedly inhibits protein methylation and neurite outgrowth without affecting cell growth, NGF-induced survival, cell flattening, or several protein phosphorylations that are associated with early signaling events. Removal of DHCA leads to rapid protein methylation of several proteins and concurrent neurite outgrowth. The results indicate that NGF regulates the methylation of several specific proteins and that protein methylation is involved in neurite outgrowth from PC12 cells.
Precocious Mammary Gland Development in P-Cadherin–deficient Mice
Authors: Radice Glenn L. , Ferreira-Cornwell M. Celeste , Robinson Stephen D. , Rayburn Helen , Chodosh Lewis A. , Takeichi Masatoshi , Hynes Richard O.
Published: July 2002

To investigate the functions of P-cadherin in vivo, we have mutated the gene encoding this cell adhesion receptor in mice. In contrast to E- and N-cadherin– deficient mice, mice homozygous for the P-cadherin mutation are viable. Although P-cadherin is expressed at high levels in the placenta, P-cadherin–null females are fertile. P-cadherin expression is localized to the myoepithelial cells surrounding the lumenal epithelial cells of the mammary gland. The role of the myoepithelium as a contractile tissue necessary for milk secretion is clear, but its function in the nonpregnant animal is unknown. The ability of the P-cadherin mutant female to nurse and maintain her litter indicates that the contractile function of the myoepithelium is not dependent on the cell adhesion molecule P-cadherin. The virgin P-cadherin–null females display precocious differentiation of the mammary gland. The alveolar-like buds in virgins resemble the glands of an early pregnant animal morphologically and biochemically (i.e., milk protein synthesis). The P-cadherin mutant mice develop hyperplasia and dysplasia of the mammary epithelium with age. In addition, abnormal lymphocyte infiltration was observed in the mammary glands of the mutant animals. These results indicate that P-cadherin–mediated adhesion and/or signals derived from cell–cell interactions are important determinants in negative growth control in the mammary gland. Furthermore, the loss of P-cadherin from the myoepithelium has uncovered a novel function for this tissue in maintaining the undifferentiated state of the underlying secretory epithelium.
The Caspase-3 Precursor Has a Cytosolic and Mitochondrial Distribution: Implications for Apoptotic Signaling
Authors: Mancini Marie , Nicholson Donald W. , Roy Sophie , Thornberry Nancy A. , Peterson Erin P. , Casciola-Rosen Livia A. , Rosen Antony
Published: July 2002

Caspase-3–mediated proteolysis is a critical element of the apoptotic process. Recent studies have demonstrated a central role for mitochondrial proteins (e.g., Bcl-2 and cytochrome c) in the activation of caspase-3, by a process that involves interaction of several protein molecules. Using antibodies that specifically recognize the precursor form of caspase-3, we demonstrate that the caspase-3 proenzyme has a mitochondrial and cytosolic distribution in nonapoptotic cells. The mitochondrial caspase-3 precursor is contained in the intermembrane space. Delivery of a variety of apoptotic stimuli is accompanied by loss of mitochondrial caspase-3 precursor staining and appearance of caspase-3 proteolytic activity. We propose that the mitochondrial subpopulation of caspase-3 precursor molecules is coupled to a distinct subset of apoptotic signaling pathways that are Bcl-2 sensitive and that are transduced through multiple mitochondrion-specific protein interactions.
Altered Cellular Responses by Varying Expression of a Ribosomal Protein Gene: Sequential Coordination of Enhancement and Suppression of Ribosomal Protein S3a Gene Expression Induces Apoptosis
Authors: Naora Honami , Takai Izumi , Adachi Masakazu , Naora Hiroto
Published: July 2002

A growing body of evidence indicates that individual ribosomal proteins and changes in their expression, participate in, and modulate, a variety of cellular activities. Our earlier studies have found that apoptosis could be induced by inhibiting expression of ribosomal protein S3a (RPS3a) in many tumor cells which constitutively express RPS3a at levels much higher than in normal cells. This study aimed to investigate cellular responses to enhancement of RPS3a expression, and whether apoptosis could be induced by sequential alterations in RPS3a expression involving enhancement from an initially low constitutive level, followed by suppression. Stably transfected NIH 3T3– derived cell lines were established in which exogenous RPS3a expression could be readily manipulated. Enhancement of RPS3a expression appeared to induce transformation as assessed by well-established criteria such as foci formation and anchorage-independent growth in vitro, and formation of tumors in nude mice. These properties were compared with those observed in ras-transformed NIH 3T3 cells. Apparent transformation occurred only when enhanced RPS3a-expressing cells were in close cell–cell contact. Suppression of enhanced RPS3a expression was observed to induce apoptosis as assessed by various morphological and biochemical characteristics including cell shrinkage, membrane blebbing, chromatin condensation, nuclear and cell fragmentation, phosphatidylserine externalization, and internucleosomal DNA fragmentation. This induction of apoptosis was not specific to apparently transformed cells, as cells at low confluence, which likewise expressed RPS3a at enhanced levels but exhibited no morphological transformation, underwent apoptosis when RPS3a expression was inhibited. These results support a role for RPS3a in the apoptotic process, but not as an oncoprotein per se.
γ-Sarcoglycan Deficiency Leads to Muscle Membrane Defects and Apoptosis Independent of Dystrophin
Authors: Hack Andrew A. , Ly Chantal T. , Jiang Fang , Clendenin Cynthia J. , Sigrist Kirsten S. , Wollmann Robert L. , McNally Elizabeth M.
Published: July 2002

γ-Sarcoglycan is a transmembrane, dystrophin-associated protein expressed in skeletal and cardiac muscle. The murine γ-sarcoglycan gene was disrupted using homologous recombination. Mice lacking γ-sarcoglycan showed pronounced dystrophic muscle changes in early life. By 20 wk of age, these mice developed cardiomyopathy and died prematurely. The loss of γ-sarcoglycan produced secondary reduction of β- and δ-sarcoglycan with partial retention of α- and ε-sarcoglycan, suggesting that β-, γ-, and δ-sarcoglycan function as a unit. Importantly, mice lacking γ-sarco- glycan showed normal dystrophin content and local- ization, demonstrating that myofiber degeneration occurred independently of dystrophin alteration. Furthermore, β-dystroglycan and laminin were left intact, implying that the dystrophin–dystroglycan–laminin mechanical link was unaffected by sarcoglycan deficiency. Apoptotic myonuclei were abundant in skeletal muscle lacking γ-sarcoglycan, suggesting that programmed cell death contributes to myofiber degeneration. Vital staining with Evans blue dye revealed that muscle lacking γ-sarcoglycan developed membrane disruptions like those seen in dystrophin-deficient muscle. Our data demonstrate that sarcoglycan loss was sufficient, and that dystrophin loss was not necessary to cause membrane defects and apoptosis. As a common molecular feature in a variety of muscular dystrophies, sarcoglycan loss is a likely mediator of pathology.
The Muscle Regulatory Factors MyoD and Myf-5 Undergo Distinct Cell Cycle–specific Expression in Muscle Cells
Authors: Kitzmann Magali , Carnac Gilles , Vandromme Marie , Primig Michael , Lamb Ned J.C. , Fernandez Anne
Published: July 2002

The muscle regulators MyoD and Myf-5 control cell cycle withdrawal and induction of differentiation in skeletal muscle cells. By immunofluorescence analysis, we show that MyoD and Myf-5 expression patterns become mutually exclusive when C2 cells are induced to differentiate with Myf-5 staining present in cells which fail to differentiate. Isolation of these undifferentiated cells reveals that upon serum stimulation they reenter the cell cycle, express MyoD and downregulate Myf-5. Similar regulations of MyoD and Myf-5 were observed using cultured primary myoblasts derived from satellite cells. To further analyze these regulations of MyoD and Myf-5 expression, we synchronized proliferating myoblasts. Analysis of MyoD and Myf-5 expression during cell cycle progression revealed distinct and contrasting profiles of expression. MyoD is absent in G0, peaks in mid-G1, falls to its minimum level at G1/S and reaugments from S to M. In contrast, Myf-5 protein is high in G0, decreases during G1 and reappears at the end of G1 to remain stable until mitosis. These data demonstrate that the two myogenic factors MyoD and Myf-5 undergo specific and distinct cell cycle–dependent regulation, thus establishing a correlation between the cell cycle–specific ratios of MyoD and Myf-5 and the capacity of cells to differentiate: (a) in G1, when cells express high levels of MyoD and enter differentiation; (b) in G0, when cells express high levels of Myf-5 and fail to differentiate.
Progressive Muscular Dystrophy in α-Sarcoglycan–deficient Mice
Authors: Duclos Franck , Straub Volker , Moore Steven A. , Venzke David P. , Hrstka Ron F. , Crosbie Rachelle H. , Durbeej Madeleine , Lebakken Connie S. , Ettinger Audrey J. , van der Meulen Jack , Holt Kathleen H. , Lim Leland E. , Sanes Joshua R. , Davidson Beverly L. , Faulkner John A. , Williamson Roger , Campbell Kevin P.
Published: July 2002

Limb-girdle muscular dystrophy type 2D (LGMD 2D) is an autosomal recessive disorder caused by mutations in the α-sarcoglycan gene. To determine how α-sarcoglycan deficiency leads to muscle fiber degeneration, we generated and analyzed α-sarcoglycan– deficient mice. Sgca-null mice developed progressive muscular dystrophy and, in contrast to other animal models for muscular dystrophy, showed ongoing muscle necrosis with age, a hallmark of the human disease. Sgca-null mice also revealed loss of sarcolemmal integrity, elevated serum levels of muscle enzymes, increased muscle masses, and changes in the generation of absolute force. Molecular analysis of Sgca-null mice demonstrated that the absence of α-sarcoglycan resulted in the complete loss of the sarcoglycan complex, sarcospan, and a disruption of α-dystroglycan association with membranes. In contrast, no change in the expression of ε-sarcoglycan (α-sarcoglycan homologue) was observed. Recombinant α-sarcoglycan adenovirus injection into Sgca-deficient muscles restored the sarcoglycan complex and sarcospan to the membrane. We propose that the sarcoglycan–sarcospan complex is requisite for stable association of α-dystroglycan with the sarcolemma. The Sgca-deficient mice will be a valuable model for elucidating the pathogenesis of sarcoglycan deficient limb-girdle muscular dystrophies and for the development of therapeutic strategies for this disease.
Role of Xklp3, a Subunit of the Xenopus Kinesin II Heterotrimeric Complex, in Membrane Transport between the Endoplasmic Reticulum and the Golgi Apparatus
Authors: Le Bot Nathalie , Antony Claude , White Jamie , Karsenti Eric , Vernos Isabelle
Published: July 2002

The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.
Laminin 5 in the Human Thymus: Control of T Cell Proliferation via α6β4 Integrins
Authors: Vivinus-Nebot Mylène , Ticchioni Michel , Mary Florence , Hofman Paul , Quaranta Vito , Rousselle Patricia , Bernard Alain
Published: July 2002

Laminin 5 (α3β3γ2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-α3, -β3, and -γ2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. We also found that laminin 5 may influence mature human thymocyte expansion; while bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59fyn inhibition, but not overall inhibition of p56lck. Using a mAb specific for α6β4 integrins, we observed that while α3β1 are known to be uniformly present on all thymocytes, α6β4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and α6β4 on mature thymocytes. Moreover, the soluble Ab against α6β4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that α6β4 is involved in laminin 5–induced modulation of T cell activation.
Local Control of Neurofilament Accumulation during Radial Growth of Myelinating Axons in Vivo
Authors: Sánchez Ivelisse , Hassinger Linda , Sihag Ram K. , Cleveland Don W. , Mohan Panaiyur , Nixon Ralph A.
Published: July 2002

The accumulation of neurofilaments required for postnatal radial growth of myelinated axons is controlled regionally along axons by oligodendroglia. Developmentally regulated processes previously suspected of modulating neurofilament number, including heavy neurofilament subunit (NFH) expression, attainment of mature neurofilament subunit stoichiometry, and expansion of interneurofilament spacing cannot be primary determinants of regional accumulation as we show each of these factors precede accumulation by days or weeks. Rather, we find that regional neurofilament accumulation is selectively associated with phosphorylation of a subset of Lys-Ser-Pro (KSP) motifs on heavy neurofilament subunits and medium-size neurofilament subunits (NFMs), rising >50-fold selectively in the expanding portions of optic axons. In mice deleted in NFH, substantial preservation of regional neurofilament accumulation was accompanied by increased levels of the same phosphorylated KSP epitope on NFM. Interruption of oligodendroglial signaling to axons in Shiverer mutant mice, which selectively inhibited this site-specific phosphorylation, reduced regional neurofilament accumulation without affecting other neurofilament properties or aspects of NFH phosphorylation. We conclude that phosphorylation of a specific KSP motif triggered by glia is a key aspect of the regulation of neurofilament number in axons during axonal radial growth.
Endothelial Cell Laminin Isoforms, Laminins 8 and 10, Play Decisive Roles in T Cell Recruitment across the Blood–Brain Barrier in Experimental Autoimmune Encephalomyelitis
Authors: Sixt Michael , Engelhardt Britta , Pausch Friederike , Hallmann Rupert , Wendler Olaf , Sorokin Lydia M.
Published: July 2002

An active involvement of blood–brain barrier endothelial cell basement membranes in development of inflammatory lesions in the central nervous system (CNS) has not been considered to date. Here we investigated the molecular composition and possible function of the extracellular matrix encountered by extravasating T lymphocytes during experimental autoimmune encephalomyelitis (EAE).

Endothelial basement membranes contained laminin 8 (α4β1γ1) and/or 10 (α5β1γ1) and their expression was influenced by proinflammatory cytokines or angiostatic agents. T cells emigrating into the CNS during EAE encountered two biochemically distinct basement membranes, the endothelial (containing laminins 8 and 10) and the parenchymal (containing laminins 1 and 2) basement membranes. However, inflammatory cuffs occurred exclusively around endothelial basement membranes containing laminin 8, whereas in the presence of laminin 10 no infiltration was detectable. In vitro assays using encephalitogenic T cell lines revealed adhesion to laminins 8 and 10, whereas binding to laminins 1 and 2 could not be induced. Downregulation of integrin α6 on cerebral endothelium at sites of T cell infiltration, plus a high turnover of laminin 8 at these sites, suggested two possible roles for laminin 8 in the endothelial basement membrane: one at the level of the endothelial cells resulting in reduced adhesion and, thereby, increased penetrability of the monolayer; and secondly at the level of the T cells providing direct signals to the transmigrating cells.
A New Mouse Gene, SRG3, Related to the SWI3 of Saccharomyces cerevisiae, Is Required for Apoptosis Induced by Glucocorticoids in a Thymoma Cell Line
Authors: Jeon Sung H. , Kang Myeong G. , Kim Young H. , Jin Yong H. , Lee Changjin , Chung Hee-Yong , Kwon Hyockman , Park Sang D. , Seong Rho H.
Published: July 2002

We isolated a new mouse gene that is highly expressed in thymocytes, testis, and brain. This gene, SRG3, showed a significant sequence homology to SWI3, a yeast transcriptional activator, and its human homolog BAF155. SRG3 encodes 1,100 amino acids and has 33–47% identity with SWI3 protein over three regions. The SRG3 protein contains an acidic NH2 terminus, a myb-like DNA binding domain, a leucine-zipper motif, and a proline- and glutamine-rich region at its COOH terminus. Rabbit antiserum raised against a COOH-terminal polypeptide of the SRG3 recognized a protein with an apparent molecular mass of 155 kD. The serum also detected a 170-kD protein that seems to be a mouse homologue of human BAF170. Immunoprecipitation of cell extract with the antiserum against the mouse SRG3 also brought down a 195-kD protein that could be recognized by an antiserum raised against human SWI2 protein. The results suggest that the SRG3 protein associates with a mouse SWI2. The SRG3 protein is expressed about three times higher in thymocytes than in peripheral lymphocytes. The expression of anti-sense RNA to SRG3 mRNA in a thymoma cell line, S49.1, reduced the expression level of the SRG3 protein, and decreased the apoptotic cell death induced by glucocorticoids. These results suggest that the SRG3 protein is involved in the glucocorticoid-induced apoptosis in the thymoma cell line. This implicates that the SRG3 may play an important regulatory role during T cell development in thymus.
The Active Site of ICP47, a Herpes Simplex Virus–encoded Inhibitor of the Major Histocompatibility Complex (MHC)-encoded Peptide Transporter Associated with Antigen Processing (TAP), Maps to the NH2-terminal 35 Residues
Authors: Galocha Begoña , Hill Ann , Barnett Barbara C. , Dolan Aidan , Raimondi Alejandra , Cook Richard F. , Brunner Joseph , McGeoch Duncan J. , Ploegh Hidde L.
Published: July 2002

The herpes simplex virus (HSV) immediate early protein ICP47 inhibits the transporter associated with antigen processing (TAP)-dependent peptide translocation. As a consequence, empty major histocompatibility complex (MHC) class I molecules are retained in the endoplasmic reticulum and recognition of HSV-infected cells by cytotoxic T lymphocytes is abolished. We chemically synthesized full-length ICP47 (sICP47) and show that sICP47 inhibits TAP-dependent peptide translocation in human cells. Its biological activity is indistinguishable from that of recombinant ICP47 (rICP47). By using synthetic peptides, we mapped the core sequence of ICP47 minimally required for TAP inhibition to residues 2–35. This segment is located within the region of the molecule conserved between ICP47 from HSV-1 and HSV-2. Through alanine scanning substitution we identified three segments within this region that are critical for the ability to inhibit TAP function. The interaction of ICP47 with TAP is unlikely to mimic precisely that of the transported peptides, as deduced from differential labeling of the TAP1 and TAP2 subunits using sICP47 fragments with chemical cross-linkers.
Identification of CCR8: A Human Monocyte and Thymus Receptor for the CC Chemokine I-309
Authors: Tiffany H. Lee , Lautens Laura L. , Gao Ji-Liang , Pease James , Locati Massimo , Combadiere Christophe , Modi William , Bonner Tom I. , Murphy Philip M.
Published: July 2002

The human CC chemokine I-309 is a potent monocyte chemoattractant and inhibits apoptosis in thymic cell lines. Here, we identify a specific human I-309 receptor, and name it CCR8 according to an accepted nomenclature system. The receptor has seven predicted transmembrane domains, is expressed constitutively in monocytes and thymus, and is encoded by a previously reported gene of previously unknown function named, alternatively, CY6, TER1, and CKR-L1. After transfection with the CY6 open reading frame, a mouse pre–B cell line exhibited calcium flux and chemotaxis in response to I-309 (EC50 = 2 nM for each), whereas 20 other chemokines were inactive. Signaling was sensitive to pertussis toxin, suggesting coupling to a Gi-type G protein. These properties parallel those of endogenous I-309 receptors expressed in an HL-60 clone 15 cell line model. The apparent monogamous relationship between I-309 and CCR8 is unusual among known CC chemokines and known CC chemokine receptors. CCR8 may regulate monocyte chemotaxis and thymic cell line apoptosis.
Identification of a Thymic Epithelial Cell Subset Sharing Expression of the Class Ib HLA-G Molecule with Fetal Trophoblasts
Authors: Crisa Laura , McMaster Michael T. , Ishii Jennifer K. , Fisher Susan J. , Salomon Daniel R.
Published: July 2002

HLA-G is the only class I determinant of the major histocompatibility complex (MHC) expressed by the trophoblasts, the fetal cells invading the maternal decidua during pregnancy. A unique feature of this nonclassical HLA molecule is its low polymorphism, a property that has been postulated to play an important role in preventing local activation of maternal alloreactive T and natural killer cells against the fetus. Yet, the mechanisms by which fetal HLA-G can be recognized as a self-MHC molecule by the maternal immune system remain unclear. Here we report the novel observation that HLA-G is expressed in the human thymus. Expression is targeted to the cell surface of thymic medullary and subcapsular epithelium. Thymic epithelial cell lines were generated and shown to express three alternatively spliced HLA-G transcripts, previously identified in human trophoblasts. Sequencing of HLA-G1 transcripts revealed a few nucleotide changes resulting in amino acid substitutions, all clustered within exon 3 of HLA-G, encoding for the α2 domain of the molecule. Our findings raise the possibility that maternal unresponsiveness to HLA-G–expressing fetal tissues may be shaped in the thymus by a previously unrecognized central presentation of this MHC molecule on the medullary epithelium.
The Superantigen Streptococcal Pyrogenic Exotoxin C (SPE-C) Exhibits a Novel Mode of Action
Authors: Li Pei-Lin , Tiedemann Rodger E. , Moffat S. Louise , Fraser John D.
Published: July 2002

Recombinant streptococcal pyrogenic exotoxin C (SPE-C) is a potent superantigen that stimulates Vβ2-bearing human T cells, but is inactive in mice. SPE-C binds with high affinity to both human HLA-DR and murine I-E molecules, but not to murine I-A molecules in a zinc-dependent fashion. Competition binding studies with other recombinant toxins revealed that SPE-C lacks the generic low affinity major histocompatibility complex (MHC) class II α-chain binding site common to all other bacterial superantigens. Despite this, SPE-C cross-links MHC class II to induce homotypic aggregation of class II–bearing B cells. Nondenaturing sodium dodecyl sulfate electrophoresis and size exclusion chromatography revealed that both wild-type and recombinant SPE-C exist in a stable dimer at neutral or alkaline pH. These data support a recent crystal structure of SPE-C and reveal yet another mechanism by which bacterial superantigens ligate and cross-link MHC class II.
Influences of Transporter Associated with Antigen Processing (TAP) on the Repertoire of Peptides Associated with the Endoplasmic Reticulum–resident Stress Protein gp96
Authors: Arnold Danièle , Wahl Claudia , Faath Stefan , Rammensee Hans-Georg , Schild Hansjörg
Published: July 2002

The endoplasmic reticulum (ER)-resident stress protein gp96 induces protective immunity and specific cytotoxic T lymphocyte (CTL) responses against antigens expressed in those cells it has been isolated from. This ability is based on peptides associated with gp96. Because gp96 is located inside the ER, our experiments address the question whether or not the repertoire of peptides associated with gp96 is influenced by the transporter associated with antigen processing (TAP). For this purpose, gp96 was isolated from cells with and without a TAP defect and used for immunization of mice. We found that for some antigens the association of peptides with gp96 required functional TAP molecules, whereas the association of peptides from other antigens was TAP independent. In the case of a TAP-dependent association of peptides with gp96, our results prove that peptide binding by gp96 in vivo occurs inside the ER and is not an artifact induced by cell lysis during the gp96 purification. The finding that some antigens can also associate with gp96 in the absence of functional TAP molecules indicates that the repertoire of peptides bound by gp96 truly reflects the entire repertoire of peptides present inside the ER and not only those peptides transported by TAP. These results, together with the earlier finding that the gp96 peptide repertoire is independent of the major histocompatibility complex molecules expressed by the cell gp96 is isolated from, give the theoretical foundation for the ability of gp96 to induce CTL responses against all kinds of intracellular antigens.
Abnormalities in Monocyte Recruitment and Cytokine Expression in Monocyte Chemoattractant Protein 1–deficient Mice
Authors: Lu Bao , Rutledge Barbara J. , Gu Long , Fiorillo Joseph , Lukacs Nicholas W. , Kunkel Steven L. , North Robert , Gerard Craig , Rollins Barrett J.
Published: July 2002

Monocyte chemoattractant protein 1 (MCP-1) is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. Because other chemokines have similar target cell specificities and because CCR2, a cloned MCP-1 receptor, binds other ligands, it has been uncertain whether MCP-1 plays a unique role in recruiting mononuclear cells in vivo. To address this question, we disrupted SCYA2 (the gene encoding MCP-1) and tested MCP-1–deficient mice in models of inflammation. Despite normal numbers of circulating leukocytes and resident macrophages, MCP-1−/− mice were specifically unable to recruit monocytes 72 h after intraperitoneal thioglycollate administration. Similarly, accumulation of F4/80+ monocytes in delayed-type hypersensitivity lesions was impaired, although the swelling response was normal. Development of secondary pulmonary granulomata in response to Schistosoma mansoni eggs was blunted in MCP-1−/− mice, as was expression of IL-4, IL-5, and interferon γ in splenocytes. In contrast, MCP-1−/− mice were indistinguishable from wild-type mice in their ability to clear Mycobacterium tuberculosis. Our data indicate that MCP-1 is uniquely essential for monocyte recruitment in several inflammatory models in vivo and influences expression of cytokines related to T helper responses.