bluuebunny/crossref_metadata_2025 huggingface dataset:
Difficult Temperament and Behaviour Problems: A Longitudinal Study from 1.5 to 12 Years
Authors: Guerin Diana Wright , Gottfried Allen W. , Thomas Craig W.
Published: July 2002

Results of a 10-year longitudinal study on the developmental-behavioural significance of infant temperamental difficultness are presented. A cross-time, cross-context methodology was employed using data from over 100 children participating in the Fullerton Longitudinal Study. Difficult temperament was assessed at 1.5 years by mothers; behaviour problems were assessed by parents during the third year and annually from 4-12 and by teachers from 6-11 years. Temperamental difficultness correlated significantly, pervasively, and to moderate magnitudes with parent reports of behaviour problems from 3.25-12 years. Additionally, analyses using clinical cutpoints showed that 1.5-year-olds deemed temperamentally difficult evidenced a greater frequency of elevated scores, specifically, attention problems, aggressive behaviour, and thought problems (parents) and attention and thought problems (teachers). These data provide evidence for the ecological validity of parent reports of temperament and for a temperamental component in the development of childhood behaviour problems.
A Longitudinal Study of Phonological Awareness, Visual Skills, and Chinese Reading Acquisition among First-graders in Taiwan
Authors: Huang H. S. , Hanley J. Richard
Published: July 2002

The main purpose of this study was to investigate whether a child’s phonological awareness and visual skills before instruction in school had any predictive power for later Chinese reading ability among 1st-graders in Taiwan. The study also examined the extent to which phonological awareness and visual skills varied in three separate testing sessions during the 1st grade. These testing sessions took place just before the children had learned the alphabetic system Zhu-Yin-Fu-Hao, immediately after the children had learnt Zhu-Yin-Fu-Hao, and, finally, at the end of the first year of schooling. Forty 6-year-old Chinese children from Taiwan took part in the study. The test materials included a Chinese Characters Reading Test, a set of Phonological Awareness tests, a Visual Paired Associates learning test, and a vocabulary and IQ test. Phonological awareness at the first testing session was found to be significantly related to the ability to read Chinese characters at the end of the first year. However, the predictive power of early phonological awareness decreased markedly when the effects of preschool reading scores were partialled out. Therefore, the study provided evidence that phonological processes are significantly related to success in the first year of Chinese reading, but was unable to establish whether or not differences in phonological skills are a cause of differences in the reading ability of Chinese children. In addition 10 weeks of instruction in Zhu-Yin-Fu-Hao led to an increase in performance on all tests of phonological awareness. This is consistent with the view that learning an alphabetic script improves phonological awareness ability.
Functional Parenting in Pakistan
Authors: Stewart Sunita Mahtani , Bond Michael H. , Zaman Riffat Moazam , McBride-Chang Catherine , Rao Nirmala , Ho L. M. , Fielding Richard
Published: July 2002

We obtained information regarding parenting perceptions and psychosocial adjustment from 102 Pakistani young women. Parenting behaviours assessed pertained to warmth and dominating control, as well as to “training” (proposed by Chao as having relevance in other Asian cultures), and indigenously relevant items related to religion and shame/honour. Adjustment measures included self-esteem, relationship harmony, and self-perceived health, shown to have relevance in other Asian cultures. In two-factor analyses, perceptions of parents’ warmth and training behaviours combined into a factor that correlated positively with adjustment measures, whereas the dominating control factor correlated negatively. In three-factor analyses, perceptions of mothers’ training behaviours predicted variance in relationship harmony, independent of perceptions of warmth and dominating control behaviours. These findings support Chao’s views that the dimension of training may play a particularly important role in functional parenting in some non-Western cultures
Overview of Structural and Functional Lesions in Endocrine Organs of Animals
Authors: Capen Charles C.
Published: July 2002

The objective of this review is to summarize the pathogenic mechanisms responsible for perturbations of endocrine function and development of structural lesions that result in important diseases in domestic and laboratory animals. For each major category, several specifi c disease problems have been selected to illustrate the functional and morphologic lesions that are characteristic for either a naturally occurring endocrinopathy or endocrine disturbances induced by the administration of large doses of xenobiotic chemicals. The major pathogenic mechanisms responsible for disruption of endocrine function include primary hyperfunction, secondary hyperfunction, primary hypofunction, secondary hypofunction, endocrine hyperactivity secondary to other conditions, hypersecretion of hormones by nonendocrine tumors, failure of target cells to respond to a hormone, failure of fetal endocrine function, abnormal degradation (increased or decreased rate) of hormone, and iatrogenic syndromes of hormone excess (direct and indirect). Disorders of the endocrine system are encountered in a wide variety of domestic and laboratory animal species and often present challenging diagnostic problems. The development of proliferative lesions, usually hyperplasia and benign tumors, in endocrine organs and hormone-responsive tissues are common findings in chronic studies with high doses of many nongenotoxic xenobiotic chemicals administered to sensitive rodent species and may have limited significance for human safety assessment.
Adrenal Gland: Structure, Function, and Mechanisms of Toxicity
Authors: Rosol Thomas J. , Yarrington John T. , Latendresse John , Capen Charles C.
Published: July 2002

The adrenal gland is one of the most common endocrine organs affected by chemically induced lesions. In the adrenal cortex, lesions are more frequent in the zona fasciculata and reticularis than in the zona glomerulosa. The adrenal cortex produces steroid hormones with a 17-carbon nucleus following a series of hydroxylation reactions that occur in the mitochondria and endoplasmic reticulum. Toxic agents for the adrenal cortex include short-chain aliphatic compounds, lipidosis inducers, amphiphilic compounds, natural and synthetic steroids, and chemicals that affect hydroxylation. Morphologic evaluation of cortical lesions provides insight into the sites of inhibition of steroidogenesis. The adrenal cortex response to injury is varied. Degeneration (vacuolar and granular), necrosis, and hemorrhage are common findings of acute injury. In contrast, chronic reparative processes are typically atrophy, fibrosis, and nodular hyperplasia. Chemically induced proliferative lesions are uncommon in the adrenal cortex. The adrenal medulla contains chromaffin cells (that produce epinephrine, norepinephrine, chromogranin, and neuropeptides) and ganglion cells. Proliferative lesions of the medulla are common in the rat and include diffuse or nodular hyperplasia and benign and malignant pheochromocytoma. Mechanisms of chromaffin cell proliferation in rats include excess growth hormone or prolactin, stimulation of cholinergic nerves, and diet-induced hypercalcemia. There often are species specifi city and age dependence in the development of chemically induced adrenal lesions that should be considered when interpreting toxicity data.
Laser Technologies in Toxicopathology
Authors: Roman Danielle , Greiner Brigitte , Ibrahim Merdol , Pralet Dominique , Germann Paul-Georg
Published: July 2002

One of the main concepts in toxicology and risk assessment is the identification of compound s with the least toxicity, gaining increased understanding of the underlying mechanisms of efficacy and toxicity so as to accelerate the early selection of compound s for development. For this purpose, “cuttingedge” technologies, such as flow cytometry (FC), laser scanning cytometry (LSC) and confocal laser scanning microscopy (CLSM), have proved to be valuable tools. FC, LSC and CLSM have been successfully applied in a wide range of areas within toxicology and research including genetics, reproduction, dermatology, pathology and target organ toxicity. The scope of this paper is to give a short overview of the usefulness of the different laser applications. Specific examples of the impact of these technologies will be presented or can be found in the references. Flow cytometry methods have been successfully applied in immunophenotyping, micronuclei scoring, polyploidy determination, apoptosis and cell cycle evaluation, cell proliferation and quantification. A three-parameter FC method for the analysis of testicular toxicity has also been established as an alternative to traditional histopathological methods. This method allows a large number of cells to be analysed in a short time and provides quantitative values to evaluate testicular damage in the rat. Laser scanning cytometry has been used in our unit for rat blood cell immunophenotyping, tumor proliferation, apoptosis and cell cycle analysis on minipig and rat skin and cardiac cells identification. The wide range of applications that can be applied with the LSC shows the enormous potential of this technology in research and development. Confocal laser scanning microscope was used in our laboratory, in collaboration with the research department, to investigate the mechanisms underlying hepatic lesions found in dogs, to detect fibrinogen influx into rat lung, to explore the mechanism of eye toxicity and to quantify dopaminergic fibers in brain sections. Integrating these technologies within discovery pathology allowed us to understand disease processes with respect to their development and subsequent consequences. It contributes to descriptive pathologic diagnostic and allows a productive interaction with research and development. These technologies offer a range of novel applications and have been shown to be useful tools in terms of specificity, sensitivity, reliability, rapidity and quantification. Expertise in cutting-edge technologies, pathology and cell and molecular biology is essential to a successful and flexible interaction across all therapeutic areas in drug discovery.
Quantitation of Molecular Endpoints for the Dose-Response Component of Cancer Risk Assessment
Authors: Julian Preston R.
Published: July 2002

Cancer risk assessment involves the steps of hazard identification, dose-response assessment, exposure assessment, and risk characterization. The rapid advances in the use of molecular biology approaches has had an impact on all 4 components, but the greatest overall current and future impact will be on the dose-response assessment because this requires an understanding of the mechanisms of carcinogenesis, both background and induced by environmental agents. In this regard, hazard identification is a qualitative assessment and dose-response is a quantitative estimate. Thus, the latter will ultimately require a quantitative assessment of molecular endpoints that are used to describe the dose-response for cancer. It has been possible for many years to quantitate alterations at the level of the single gene. For example, analysis of mutation frequency by phenotypic selection, analysis of transcription (mRNA) by Northern blot, analysis of translation (proteins) by Western blot, and analysis of kinetics of metabolism from metabolite levels. However, it is becoming clear that it is necessary when considering risk for adverse health outcomes to develop quantitative approaches for whole cell phenotypes or organ effects. For example, cancer is a whole tissue phenotype, not a feature of single gene mutations, in spite of the multistep (multimutation) mode of formation of a tumor. Thus, there is the need to quantitate the circuitry of a cell: the metabolic/biochemical pathways, genetic regulation pathways, and signaling pathway s in normal and stressed conditions. The hypothesis presented by Hanahan and Weinberg of the requirement for 6 acquired characteristics for tumor development, independent of tissue type and species or inducer, seems to provide a viable approach. This hypothesi s can be addressed through whole cell molecular assessment using microarray s and quantitative PCR together with the emerging proteomic approaches. This is the world of the new computational cell biology.
Current Methods for Assessing Safety of Genetically Modified Crops as Exemplified by Data on Roundup Ready 1 Soybeans
Authors: Nair Rashmi S. , Fuchs Roy L. , Schuette Sheila A.
Published: July 2002

Several laboratories have used recombinant DNA technology in plant breeding to improve compositional, processing, and agronomic characteristics of plants. These transformed plants have been extensively tested in fi eld trials, have gained full regulatory approvals and are currently being marketed in a number of countries around the world. This paper briefl y summarizes the approach used to assure the safety of foods and feeds derived from these genetically modified crops, as exemplified by data on Roundup Ready soybeans that has been developed by Monsanto Company using biotechnology in order to confer tolerance to glyphosate, the active ingredient in Roundup herbicide, by the production of the CP4 enolpyruvylshikimate-3-phosphate synthase protein. The results of the studies demonstrate that Roundup Ready soybeans are as safe as traditional soybeans with respect to food and feed safety.
Routine Immunohistochemistry to Simplify Ovarian Follicle Counts
Authors: Muskhelishvili Levan , Latendresse John R. , Bucci Thomas J.
Published: July 2002

U.S. and internationally harmonized Health Effects Test Guidelines for Reproduction and Fertility Effects (USEPA OPPTS 870,3800, 1998: OECD No. 416 C(83)44, 1983) include enumeration of primordial and developing ovarian follicles as endpoints of safety tests, and the number of these structures is also of interest for other aspects of reproductive biology. Performing the counts microscopically on representative hematoxyli n & eosin-stained sections of ovary is tedious and error-prone. The ability to mark oocyte nuclei distinctly with an antibody significantly increases speed and accuracy of counting. We have identified a rabbit polyclonal antibody directed against a synthetic 14-amino acid sequence from human cytochrome P450 1B1 (CYP1B1) that unequivocally marks oocyte nuclei in addition to nuclei of some ovarian granulosa and theca cells. Follicles of all degrees of maturity are easily distinguished from ovarian background; ability to detect and identify primordial follicles is particularly enhanced. Excellent labeling was achieved with routine immunohistochemical procedures using an avidin-biotin-peroxidas e method on rat and mouse tissues fi xed in 10% neutral-buffered formalin. The antigenic peptide and the antiserum were produced by Alpha Diagnostics Incorporated, San Antonio, TX 78238 (www.4adi.com).
Censorship, the Internet and schools: a new moral panic?
Authors: Lawson Tony , Comber Chris
Published: July 2002

As the use of the Internet in schools increases, so too do anxieties over inappropriate access, often fuelled by a popular media focus upon the dangers of children's exposure to pornographic or extremist material. Teachers and school managers, attempting to weigh such hazards against the education potential of access to global information sources, are caught in the crossfire between those who call for rigid controls and those who argue for freedom and access. In the context of these contrary positions, this article begins by exploring the extent to which current public concern over Internet access can be seen as the “moral panic” of the 1990s. Following this, we draw upon examples from Education Departments' Superhighways Initiative (EDSI) to examine the responses of teachers faced with these dilemmas and the effectiveness of various strategies in resolving them. While a combination of control over access (particularly for younger children) and sanctions for inappropriate use was in operation in most schools, many teachers' preferred strategy was to alert pupils to the potential dangers, and to educate them to become responsible users.
Nitric oxide function in atherosclerosis
Authors: Matthys K. E. , Bult H.
Published: July 2002

Atherosclerosis is a chronic inflammatory process in the intima of conduit arteries, which disturbs the endothelium‐dependent regulation of the vascular tone by the labile liposoluble radical nitric oxide (NO) formed by the constitutive endothelial nitric oxide synthase (eNOS). This defect predisposes to coronary vasospasm and cardiac ischaemia, with anginal pain as the typical clinical manifestation. It is now appreciated that endothelial dysfunction is an early event in atherogenesis and that it may also involve the microcirculation, in which atherosclerotic lesions do not develop. On the other hand, the inflammatory environment in atherosclerotic plaques may result in the expression of the inducible NO synthase (iNOS) isozyme. Whether the dysfunction in endothelial NO production is causal to, or the result of, atherosclerotic lesion formation is still highly debated. Most evidence supports the hypothesis that constitutive endothelial NO release protects against atherogenesis e.g. by preventing smooth muscle cell proliferation and leukocyte adhesion. Nitric oxide generated by the inducible isozyme may be beneficial by replacing the failing endothelial production but excessive release may damage the vascular wall cells, especially in combination with reactive oxygen intermediates.
Development and disadvantage: implications for the early years and beyond
Authors: Locke Ann , Ginsborg Jane , Peers Ian
Published: July 2002

Links have long been made between literacy and economic development, and recent governments in the UK have put great emphasis on the teaching of literacy to raise educational standards. There is substantial evidence to show that spoken and written language share some processes in common and that the development of literacy is supported by the development of spoken language. Anecdotal evidence from early years practitioners suggests that many children coming into early years education, particularly those from disadvantaged socioeconomic backgrounds, may not have the spoken language skills needed to develop reading and writing. This preliminary study aimed to investigate the extent to which the spoken language skills of children reared in poverty are depressed in comparison with the general population, and in comparison with their general cognitive abilities. Two tests were administered to children in their first term at nursery school to measure their spoken language skills and cognitive abilities: CELF‐PUK and BAS II. More than half of the children were found to be language‐delayed, although girls' receptive language abilities were significantly better than those of boys. Participants' language skills were also significantly depressed in comparison with their cognitive abilities. Government initiatives to raise awareness of spoken language in the early years are discussed, and implications for the future role of speech and language therapists working in the pre‐school sector are considered.
GKAP, a Novel Synaptic Protein That Interacts with the Guanylate Kinase-like Domain of the PSD-95/SAP90 Family of Channel Clustering Molecules
Authors: Kim Eunjoon , Naisbitt Scott , Hsueh Yi-Ping , Rao Anuradha , Rothschild Adam , Craig Ann Marie , Sheng Morgan
Published: July 2002

The molecular mechanisms underlying the organization of ion channels and signaling molecules at the synaptic junction are largely unknown. Recently, members of the PSD-95/SAP90 family of synaptic MAGUK (membrane-associated guanylate kinase) proteins have been shown to interact, via their NH2-terminal PDZ domains, with certain ion channels (NMDA receptors and K+ channels), thereby promoting the clustering of these proteins. Although the function of the NH2-terminal PDZ domains is relatively well characterized, the function of the Src homology 3 (SH3) domain and the guanylate kinase-like (GK) domain in the COOH-terminal half of PSD-95 has remained obscure. We now report the isolation of a novel synaptic protein, termed GKAP for guanylate kinase-associated protein, that binds directly to the GK domain of the four known members of the mammalian PSD-95 family. GKAP shows a unique domain structure and appears to be a major constituent of the postsynaptic density. GKAP colocalizes and coimmunoprecipitates with PSD-95 in vivo, and coclusters with PSD-95 and K+ channels/ NMDA receptors in heterologous cells. Given their apparent lack of guanylate kinase enzymatic activity, the fact that the GK domain can act as a site for protein– protein interaction has implications for the function of diverse GK-containing proteins (such as p55, ZO-1, and LIN-2/CASK).
Ezrin/Radixin/Moesin (ERM) Proteins Bind to a Positively Charged Amino Acid Cluster in the Juxta-Membrane Cytoplasmic Domain of CD44, CD43, and ICAM-2
Authors: Yonemura Shigenobu , Hirao Motohiro , Doi Yoshinori , Takahashi Nobuyuki , Kondo Takahisa , Tsukita Sachiko , Tsukita Shoichiro
Published: July 2002

Abstract. CD44 has been identified as a membrane-binding partner for ezrin/radixin/moesin (ERM) proteins, plasma membrane/actin filament cross-linkers. ERM proteins, however, are not necessarily colocalized with CD44 in tissues, but with CD43 and ICAM-2 in some types of cells. We found that glutathione-S-transferase fusion proteins with the cytoplasmic domain of CD43 and ICAM-2, as well as CD44, bound to moesin in vitro. The regions responsible for the in vitro binding of CD43 and CD44 to moesin were narrowed down to their juxta-membrane 20–30–amino acid sequences in the cytoplasmic domain. These sequences and the cytoplasmic domain of ICAM-2 (28 amino acids) were all characterized by the positively charged amino acid clusters. When E-cadherin chimeric molecules bearing these positively charged amino acid clusters of CD44, CD43, or ICAM-2 were expressed in mouse L fibroblasts, they were co-concentrated with ERM proteins at microvilli, whereas those lacking these clusters were diffusely distributed on the cell surface. The specific binding of ERM proteins to the juxta-membrane positively charged amino acid clusters of CD44, CD43, and ICAM-2 was confirmed by immunoprecipitation and site-directed mutagenesis. From these findings, we conclude that ERM proteins bind to integral membrane proteins bearing a positively charged amino acid cluster in their juxta-membrane cytoplasmic domain.
Growing Pollen Tubes Possess a Constitutive Alkaline Band in the Clear Zone and a Growth-dependent Acidic Tip
Authors: Feijó J.A. , Sainhas J. , Hackett G.R. , Kunkel J.G. , Hepler P.K.
Published: July 2002

Using both the proton selective vibrating electrode to probe the extracellular currents and ratiometric wide-field fluorescence microscopy with the indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-dextran to image the intracellular pH, we have examined the distribution and activity of protons (H+) associated with pollen tube growth. The intracellular images reveal that lily pollen tubes possess a constitutive alkaline band at the base of the clear zone and an acidic domain at the extreme apex. The extracellular observations, in close agreement, show a proton influx at the extreme apex of the pollen tube and an efflux in the region that corresponds to the position of the alkaline band. The ability to detect the intracellular pH gradient is strongly dependent on the concentration of exogenous buffers in the cytoplasm. Thus, even the indicator dye, if introduced at levels estimated to be of 1.0 μM or greater, will dissipate the gradient, possibly through shuttle buffering. The apical acidic domain correlates closely with the process of growth, and thus may play a direct role, possibly in facilitating vesicle movement and exocytosis. The alkaline band correlates with the position of the reverse fountain streaming at the base of the clear zone, and may participate in the regulation of actin filament formation through the modulation of pH-sensitive actin binding proteins. These studies not only demonstrate that proton gradients exist, but that they may be intimately associated with polarized pollen tube growth.
The Kinesin-Related Protein, Hset, Opposes the Activity of Eg5 and Cross-Links Microtubules in the Mammalian Mitotic Spindle
Authors: Mountain Vicki , Simerly Calvin , Howard Louisa , Ando Asako , Schatten Gerald , Compton Duane A.
Published: July 2002

We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors. Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function. Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells. However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis. We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization. In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity. Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation. Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.
Filamin 2 (FLN2): A Muscle-specific Sarcoglycan Interacting Protein
Authors: Thompson Terri G. , Chan Yiu-Mo , Hack Andrew A. , Brosius Melissa , Rajala Michael , Lidov Hart G.W. , McNally Elizabeth M. , Watkins Simon , Kunkel Louis M.
Published: July 2002

Mutations in genes encoding for the sarcoglycans, a subset of proteins within the dystrophin–glycoprotein complex, produce a limb-girdle muscular dystrophy phenotype; however, the precise role of this group of proteins in the skeletal muscle is not known. To understand the role of the sarcoglycan complex, we looked for sarcoglycan interacting proteins with the hope of finding novel members of the dystrophin–glycoprotein complex. Using the yeast two-hybrid method, we have identified a skeletal muscle-specific form of filamin, which we term filamin 2 (FLN2), as a γ- and δ-sarcoglycan interacting protein. In addition, we demonstrate that FLN2 protein localization in limb-girdle muscular dystrophy and Duchenne muscular dystrophy patients and mice is altered when compared with unaffected individuals. Previous studies of filamin family members have determined that these proteins are involved in actin reorganization and signal transduction cascades associated with cell migration, adhesion, differentiation, force transduction, and survival. Specifically, filamin proteins have been found essential in maintaining membrane integrity during force application. The finding that FLN2 interacts with the sarcoglycans introduces new implications for the pathogenesis of muscular dystrophy.
The Mkk3/6-p38–Signaling Cascade Alters the Subcellular Distribution of Hnrnp A1 and Modulates Alternative Splicing Regulation
Authors: van der Houven van Oordt Willemien , Diaz-Meco María T. , Lozano José , Krainer Adrian R. , Moscat Jorge , Cáceres Javier F.
Published: July 2002

Individual members of the serine-arginine (SR) and heterogeneous nuclear ribonucleoprotein (hnRNP) A/B families of proteins have antagonistic effects in regulating alternative splicing. Although hnRNP A1 accumulates predominantly in the nucleus, it shuttles continuously between the nucleus and the cytoplasm. Some but not all SR proteins also undergo nucleo-cytoplasmic shuttling, which is affected by phosphorylation of their serine/arginine (RS)–rich domain. The signaling mechanisms that control the subcellular localization of these proteins are unknown. We show that exposure of NIH-3T3 and SV-40 transformed green monkey kidney (COS) cells to stress stimuli such as osmotic shock or UVC irradiation, but not to mitogenic activators such as PDGF or EGF, results in a marked cytoplasmic accumulation of hnRNP A1, concomitant with an increase in its phosphorylation. These effects are mediated by the MKK3/6-p38 pathway, and moreover, p38 activation is necessary and sufficient for the induction of hnRNP A1 cytoplasmic accumulation. The stress-induced increase in the cytoplasmic levels of hnRNP A/B proteins and the concomitant decrease in their nuclear abundance are paralleled by changes in the alternative splicing pattern of an adenovirus E1A pre-mRNA splicing reporter. These results suggest the intriguing possibility that signaling mechanisms regulate pre-mRNA splicing in vivo by influencing the subcellular distribution of splicing factors.
Actopaxin, a New Focal Adhesion Protein That Binds Paxillin Ld Motifs and Actin and Regulates Cell Adhesion
Authors: Nikolopoulos Sotiris N. , Turner Christopher E.
Published: July 2002

Paxillin is a focal adhesion adapter protein involved in the integration of growth factor– and adhesion-mediated signal transduction pathways. Paxillin LD motifs have been demonstrated to bind to several proteins associated with remodeling of the actin cytoskeleton including the focal adhesion kinase, vinculin, and a complex of proteins comprising p95PKL, PIX, and PAK (Turner, C.E., M.C. Brown, J.A. Perrotta, M.C. Riedy, S.N. Nikolopoulos, A.R. McDonald, S. Bagrodia, S. Thomas, and P.S. Leventhal. 1999. J. Cell Biol. 145:851–863). In this study, we report the cloning and initial characterization of a new paxillin LD motif–binding protein, actopaxin. Analysis of the deduced amino acid sequence of actopaxin reveals a 42-kD protein with two calponin homology domains and a paxillin-binding subdomain (PBS). Western blotting identifies actopaxin as a widely expressed protein. Actopaxin binds directly to both F-actin and paxillin LD1 and LD4 motifs. It exhibits robust focal adhesion localization in several cultured cell types but is not found along the length of the associated actin-rich stress fibers. Similar to paxillin, it is absent from actin-rich cell–cell adherens junctions. Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells. An actopaxin PBS mutant incapable of binding paxillin in vitro cannot target to focal adhesions when expressed in fibroblasts. In addition, ectopic expression of the PBS mutant and/or the COOH terminus of actopaxin in HeLa cells resulted in substantial reduction in adhesion to collagen. Together, these results suggest an important role for actopaxin in integrin-dependent remodeling of the actin cytoskeleton during cell motility and cell adhesion.
Rho-Kinase–Mediated Contraction of Isolated Stress Fibers
Authors: Katoh Kazuo , Kano Yumiko , Amano Mutsuki , Onishi Hirofumi , Kaibuchi Kozo , Fujiwara Keigi
Published: July 2002

It is widely accepted that actin filaments and the conventional double-headed myosin interact to generate force for many types of nonmuscle cell motility, and that this interaction occurs when the myosin regulatory light chain (MLC) is phosphorylated by MLC kinase (MLCK) together with calmodulin and Ca2+. However, recent studies indicate that Rho-kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have recently isolated reactivatable stress fibers from cultured cells and established them as a model system for actomyosin-based contraction in nonmuscle cells. Here, using isolated stress fibers, we show that Rho-kinase mediates MLC phosphorylation and their contraction in the absence of Ca2+. More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho-kinase. When the activity of Rho-kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscle contractility is regulated by two kinase systems: the Ca2+-dependent MLCK and the Rho-kinase systems. We propose that Ca2+ is used to generate rapid contraction, whereas Rho-kinase plays a major role in maintaining sustained contraction in cells.
Activation of the Met Receptor by Cell Attachment Induces and Sustains Hepatocellular Carcinomas in Transgenic Mice
Authors: Wang Rong , Ferrell Linda D. , Faouzi Saadia , Maher Jacquelyn J. , Bishop J. Michael
Published: July 2002

Overexpression is the most common abnormality of receptor tyrosine kinases (RTKs) in human tumors. It is presumed that overexpression leads to constitutive activation of RTKs, but the mechanism of that activation has been uncertain. Here we show that overexpression of the Met RTK allows activation of the receptor by cell attachment and that this form of activation can be tumorigenic. Transgenic mice that overexpressed Met in hepatocytes developed hepatocellular carcinoma (HCC), one of the human tumors in which Met has been implicated previously. The tumorigenic Met was activated by cell attachment rather than by ligand. Inactivation of the transgene led to regression of even highly advanced tumors, apparently mediated by apoptosis and cessation of cellular proliferation. These results reveal a previously unappreciated mechanism by which the tumorigenic action of RTKs can be mediated, provide evidence that Met may play a role in both the genesis and maintenance of HCC, and suggest that Met may be a beneficial therapeutic target in tumors that overexpress the receptor.
A Novel Integrin-Linked Kinase–Binding Protein, Affixin, Is Involved in the Early Stage of Cell–Substrate Interaction
Authors: Yamaji Satoshi , Suzuki Atsushi , Sugiyama Yuki , Koide Yu-ichi , Yoshida Michihiko , Kanamori Heiwa , Mohri Hiroshi , Ohno Shigeo , Ishigatsubo Yoshiaki
Published: July 2002

Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of β1 integrin, seems to be a key component of FAs, but its exact role in cell–substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin–ILK signaling required for the establishment of cell–substrate adhesion.
Sodium channel β1 and β3 subunits associate with neurofascin through their extracellular immunoglobulin-like domain
Authors: Ratcliffe Charlotte F. , Westenbroek Ruth E. , Curtis Rory , Catterall William A.
Published: July 2002

Sequence homology predicts that the extracellular domain of the sodium channel β1 subunit forms an immunoglobulin (Ig) fold and functions as a cell adhesion molecule. We show here that β1 subunits associate with neurofascin, a neuronal cell adhesion molecule that plays a key role in the assembly of nodes of Ranvier. The first Ig-like domain and second fibronectin type III–like domain of neurofascin mediate the interaction with the extracellular Ig-like domain of β1, confirming the proposed function of this domain as a cell adhesion molecule. β1 subunits localize to nodes of Ranvier with neurofascin in sciatic nerve axons, and β1 and neurofascin are associated as early as postnatal day 5, during the period that nodes of Ranvier are forming. This association of β1 subunit extracellular domains with neurofascin in developing axons may facilitate recruitment and concentration of sodium channel complexes at nodes of Ranvier.
Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor
Authors: Kato Masaki , Patel Millan S. , Levasseur Regis , Lobov Ivan , Chang Benny H.-J. , Glass Donald A. , Hartmann Christine , Li Lan , Hwang Tae-Ho , Brayton Cory F. , Lang Richard A. , Karsenty Gerard , Chan Lawrence
Published: July 2002

The low-density lipoprotein receptor–related protein (Lrp)-5 functions as a Wnt coreceptor. Here we show that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype. In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In addition, Lrp5-deficient mice display persistent embryonic eye vascularization due to a failure of macrophage-induced endothelial cell apoptosis. These results implicate Wnt proteins in the postnatal control of vascular regression and bone formation, two functions affected in many diseases. Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.
Identification of High Potency Microbial and Self Ligands for a Human Autoreactive Class II–restricted T Cell Clone
Authors: Hemmer Bernhard , Fleckenstein Burkhard T. , Vergelli Marco , Jung Günther , McFarland Henry , Martin Roland , Wiesmüller Karl-Heinz
Published: July 2002

CD4+ class II–restricted T cells specific for self antigens are thought to be involved in the pathogenesis of most human autoimmune diseases and molecular mimicry between foreign and self ligands has been implicated as a possible mechanism for their activation. In this report we introduce combinatorial peptide libraries as a powerful tool to identify cross-reactive ligands for these T cells. The antigen recognition of a CD4+ T cell clone (TCC) specific for myelin basic protein peptide (MBP) (86-96) was dissected by the response to a set of 220 11-mer peptide sublibraries. Based on the results obtained with the libraries for each position of the antigen, artificial peptides were found that induced proliferative responses at much lower concentrations than MBP(86-96). In addition stimulatory ligands derived from protein sequences of self and microbial proteins were identified, some of them even more potent agonists than MBP(86-96). These results indicate that: (a) for at least some autoreactive CD4+ T cells antigen recognition is highly degenerate; (b) the autoantigen used to establish the TCC represents only a suboptimal ligand for the TCC; (c) a completely random and unbiased approach such as combinatorial peptide libraries can decrypt the spectrum of stimulatory ligands for a T cell receptor (TCR).
Antigen Receptor Engagement Turns off the V(D)J Recombination Machinery in Human Tonsil B Cells
Authors: Meffre Eric , Papavasiliou Fotini , Cohen Paul , de Bouteiller Odette , Bell Diana , Karasuyama Hajime , Schiff Claudine , Banchereau Jacques , Liu Yong-Jun , Nussenzweig Michel C.
Published: July 2002

The germinal center (GC) is an anatomic compartment found in peripheral lymphoid organs, wherein B cells undergo clonal expansion, somatic mutation, switch recombination, and reactivate immunoglobulin gene V(D)J recombination. As a result of somatic mutation, some GC B cells develop higher affinity antibodies, whereas others suffer mutations that decrease affinity, and still others may become self-reactive. It has been proposed that secondary V(D)J rearrangements in GCs might rescue B cells whose receptors are damaged by somatic mutations. Here we present evidence that mature human tonsil B cells coexpress conventional light chains and recombination associated genes, and that they extinguish recombination activating gene and terminal deoxynucleotidyl transferase expression when their receptors are cross-linked. Thus, the response of the recombinase to receptor engagement in peripheral B cells is the opposite of the response in developing B cells to the same stimulus. These observations suggest that receptor revision is a mechanism for receptor diversification that is turned off when antigen receptors are cross-linked by the cognate antigen.
Molecular Cloning of NKp46: A Novel Member of the Immunoglobulin Superfamily Involved in Triggering of Natural Cytotoxicity
Authors: Pessino Anna , Sivori Simona , Bottino Cristina , Malaspina Angela , Morelli Luigia , Moretta Lorenzo , Biassoni Roberto , Moretta Alessandro
Published: July 2002

NKp46 has been shown to represent a novel, natural killer (NK) cell–specific surface molecule, involved in human NK cell activation. In this study, we further analyzed the role of NKp46 in natural cytotoxicity against different tumor target cells. We provide direct evidence that NKp46 represents a major activating receptor involved in the recognition and lysis of both human and murine tumor cells. Although NKp46 may cooperate with other activating receptors (including the recently identified NKp44 molecule) in the induction of NK-mediated lysis of human tumor cells, it may represent the only human NK receptor involved in recognition of murine target cells. Molecular cloning of the cDNA encoding the NKp46 molecule revealed a novel member of the immunoglobulin (Ig) superfamily, characterized by two C2-type Ig-like domains in the extracellular portion. The transmembrane region contains the positively charged amino acid Arg, which is possibly involved in stabilizing the association with CD3ζ chain. The cytoplasmic portion, spanning 30 amino acids, does not contain immunoreceptor tyrosine-based activating motifs.

Analysis of a panel of human/hamster somatic cell hybrids revealed segregation of the NKp46 gene on human chromosome 19. Assessment of the NKp46 mRNA expression in different tissues and cell types unambiguously confirmed the strict NK cell specificity of the NKp46 molecule. Remarkably, in line with the ability of NKp46 to recognize ligand(s) on murine target cells, the cDNA encoding NKp46 was found to be homologous to a cDNA expressed in murine spleen. In conclusion, this study reports the first characterization of the molecular structure of a NK-specific receptor involved in the mechanism of NK cell activation during natural cytotoxicity.
Interferon γ Derived from CD4+ T Cells Is Sufficient to Mediate T Helper Cell Type 1 Development
Authors: Wakil Adil E. , Wang Zhi-En , Ryan James C. , Fowell Deborah J. , Locksley Richard M.
Published: July 2002

Interferon γ (IFN-γ) has been implicated in T helper type 1 (Th1) cell development through its ability to optimize interleukin 12 (IL-12) production from macrophages and IL-12 receptor expression on activated T cells. Various systems have suggested a role for IFN-γ derived from the innate immune system, particularly natural killer (NK) cells, in mediating Th1 differentiation in vivo. We tested this requirement by reconstituting T cell and IFN-γ doubly deficient mice with wild-type CD4+ T cells and challenging the mice with pathogens that elicited either minimal or robust IL-12 in vivo (Leishmania major or Listeria monocytogenes, respectively). Th1 cells developed under both conditions, and this was unaffected by the presence or absence of IFN-γ in non-T cells. Reconstitution with IFN-γ–deficient CD4+ T cells could not reestablish control over L. major, even in the presence of IFN-γ from the NK compartment. These data demonstrate that activated T cells can maintain responsiveness to IL-12 through elaboration of endogenous IFN-γ without requirement for an exogenous source of this cytokine.
Inhibition of Ca2+ Signaling by Mycobacterium tuberculosisIs Associated with Reduced Phagosome–Lysosome Fusion and Increased Survival within Human Macrophages
Authors: Malik Zulfiqar A. , Denning Gerene M. , Kusner David J.
Published: July 2002

Complement receptor (CR)-mediated phagocytosis of Mycobacterium tuberculosis by macrophages results in intracellular survival, suggesting that M. tuberculosis interferes with macrophage microbicidal mechanisms. As increases in cytosolic Ca2+ concentration ([Ca2+]c) promote phagocyte antimicrobial responses, we hypothesized that CR phagocytosis of M. tuberculosis is accompanied by altered Ca2+ signaling. Whereas the control complement (C)-opsonized particle zymosan (COZ) induced a 4.6-fold increase in [Ca2+]c in human macrophages, no change in [Ca2+]c occurred upon addition of live, C-opsonized virulent M. tuberculosis. Viability of M. tuberculosis and ingestion via CRs was required for infection of macrophages in the absence of increased [Ca2+]c, as killed M. tuberculosis or antibody (Ab)-opsonized, live M. tuberculosis induced elevations in [Ca2+]c similar to COZ. Increased [Ca2+]c induced by Ab-opsonized bacilli was associated with a 76% reduction in intracellular survival, compared with C-opsonized M. tuberculosis. Similarly, reversible elevation of macrophage [Ca2+]c with the ionophore A23187 reduced intracellular viability by 50%. Ionophore-mediated elevation of [Ca2+]c promoted the maturation of phagosomes containing live C-opsonized bacilli, as evidenced by acidification and accumulation of lysosomal protein markers. These data demonstrate that M. tuberculosis inhibits CR-mediated Ca2+ signaling and indicate that this alteration of macrophage activation contributes to inhibition of phagosome–lysosome fusion and promotion of intracellular mycobacterial survival.
Carbon Monoxide Generated by Heme Oxygenase 1 Suppresses Endothelial Cell Apoptosis
Authors: Brouard Sophie , Otterbein Leo E. , Anrather Josef , Tobiasch Edda , Bach Fritz H. , Choi Augustine M.K. , Soares Miguel P.
Published: July 2002

Heme oxygenase 1 (HO-1) inhibits apoptosis by regulating cellular prooxidant iron. We now show that there is an additional mechanism by which HO-1 inhibits apoptosis, namely by generating the gaseous molecule carbon monoxide (CO). Overexpression of HO-1, or induction of HO-1 expression by heme, protects endothelial cells (ECs) from apoptosis. When HO-1 enzymatic activity is blocked by tin protoporphyrin (SnPPIX) or the action of CO is inhibited by hemoglobin (Hb), HO-1 no longer prevents EC apoptosis while these reagents do not affect the antiapoptotic action of bcl-2. Exposure of ECs to exogenous CO, under inhibition of HO-1 activity by SnPPIX, substitutes HO-1 in preventing EC apoptosis. The mechanism of action of HO-1/CO is dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) signaling transduction pathway. Expression of HO-1 or exposure of ECs to exogenous CO enhanced p38 MAPK activation by TNF-α. Specific inhibition of p38 MAPK activation by the pyridinyl imidazol SB203580 or through overexpression of a p38 MAPK dominant negative mutant abrogated the antiapoptotic effect of HO-1. Taken together, these data demonstrate that the antiapoptotic effect of HO-1 in ECs is mediated by CO and more specifically via the activation of p38 MAPK by CO.