bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatocyte Retinoid X Receptor-α-Deficient Mice Have Reduced Food Intake, Increased Body Weight, and Improved Glucose Tolerance
Authors: Wan Yu-Jui Yvonne , Han Guang , Cai Yan , Dai Tiane , Konishi Tamiko , Leng AI-She
Published: January 2003

Hepatocyte retinoid X receptor (RXR)α-deficient mice and wild-type mice were fed either a regular or a high-saturated-fat diet for 12 wk to study the functional role of hepatocyte RXRα in fatty acid and carbohydrate metabolism. Food intake was significantly reduced in hepatocyte RXRα-deficient mice when either diet was used. The amount of food intake was negatively associated with serum leptin level. Although mutant mice ate less, body weight and fat content were significantly higher in mutant than wild-type mice. Examination of the expression of peroxisome proliferator-activated receptor-α target genes indicated that the peroxisome proliferator-activated receptor-α-mediated pathway was compromised in the mutant mice, which, in turn, might affect fatty-acid metabolism and result in increased body weight and fat content. Although mutant mice were obese, they demonstrated the same degree of insulin sensitivity and the same level of serum insulin as the wild-type mice. However, these mutant mice have improved glucose tolerance. To explore a mechanism that may be responsible for the improved glucose tolerance, serum IGF-I level was examined. Serum IGF-1 level was significantly increased in mutant mice compared with wild-type mice. Taken together, hepatocyte RXRα deficiency increases leptin level and reduces food intake. Those mice also develop obesity, with an unexpected improvement of glucose tolerance. The result also suggests that an increase in serum IGF-I level might be one of the mechanisms leading to improved glucose tolerance in hepatocyte RXRα-deficient mice.
Analysis of the mutational effects of theCOP/DET/FUSloci on genome expression profiles reveals their overlapping yet not identical roles in regulatingArabidopsisseedling development
Authors: Ma Ligeng , Zhao Hongyu , Deng Xing Wang
Published: January 2003

Microarray gene expression profiling was used to examine the role of pleiotropic COP/DET/FUS loci as well as other partially photomorphogenic loci during Arabidopsis seedling development and genome expression regulation. Four types of lethal, pleiotropiccop/det/fus mutants exhibit qualitatively similar gene expression profiles, yet each has specific differences. Mutations in COP1 andDET1 show the most similar genome expression profiles, while the mutations in the COP9 signalosome (CSN) and COP10 exhibit increasingly diverged genome expression profiles in both darkness and light. The genome expression profiles of the viable mutants of COP1 andDET1 in darkness mimic those of the physiological light-regulated genome expression profiles, whereas the genome expression profiles of representative lethal mutants belong to another clade and significantly diverge from the normal light control of genome expression. Instead, these lethal pleiotropic mutants show genome expression profiles similar to those from seedlings growth under high light intensity stress. Distinct lethal pleiotropic cop/det/fus mutants also result in distinct expression profiles in the small portion of genes examined and exhibit similar relatedness in both light and darkness. The partial cop/det/fusmutants affected expression of both light regulated and non-light regulated genes. Our results suggest that pleiotropic COP/DET/FUS loci control is largely overlapping but also has separable roles in plant development. The partially photomorphogenic loci regulate a subset of photomorphogenic responses as well as other non-light regulated processes.
Composition and dynamics of theCaenorhabditis elegansearly embryonic transcriptome
Authors: Baugh L. Ryan , Hill Andrew A. , Slonim Donna K. , Brown Eugene L. , Hunter Craig P.
Published: January 2003

Temporal profiles of transcript abundance during embryonic development were obtained by whole-genome expression analysis from precisely staged C. elegans embryos. The result is a highly resolved time course that commences with the zygote and extends into mid-gastrulation, spanning the transition from maternal to embryonic control of development and including the presumptive specification of most major cell fates. Transcripts for nearly half (8890) of the predicted open reading frames are detected and expression levels for the majority of them (>70%) change over time. The transcriptome is stable up to the four-cell stage where it begins rapidly changing until the rate of change plateaus before gastrulation. At gastrulation temporal patterns of maternal degradation and embryonic expression intersect indicating a mid-blastula transition from maternal to embryonic control of development. In addition, we find that embryonic genes tend to be expressed transiently on a time scale consistent with developmental decisions being made with each cell cycle. Furthermore, overall rates of synthesis and degradation are matched such that the transcriptome maintains a steady-state frequency distribution. Finally, a versatile analytical platform based on cluster analysis and developmental classification of genes is provided.
Temporal and cellular requirements for Fms signaling during zebrafish adult pigment pattern development
Authors: Parichy David M. , Turner Jessica M.
Published: January 2003

Ectothermic vertebrates exhibit a diverse array of adult pigment patterns. A common element of these patterns is alternating dark and light stripes each comprising different classes of neural crest-derived pigment cells. In the zebrafish, Danio rerio, alternating horizontal stripes of black melanophores and yellow xanthophores are a prominent feature of the adult pigment pattern. In fms mutant zebrafish, however, xanthophores fail to develop and melanophore stripes are severely disrupted. fmsencodes a type III receptor tyrosine kinase expressed by xanthophores and their precursors and is the closest known homologue of kit, which has long been studied for roles in pigment pattern development in amniotes. In this study we assess the cellular and temporal requirements for Fms activity in promoting adult pigment pattern development. By transplanting cells betweenfms mutants and either wild-type or nacre mutant zebrafish,we show that fms acts autonomously to the xanthophore lineage in promoting the striped arrangement of adult melanophores. To identify critical periods for fms activity, we isolated temperature sensitive alleles of fms and performed reciprocal temperature shift experiments at a range of stages from embryo to adult. These analyses demonstrate that Fms is essential for maintaining cells of the xanthophore lineage as well as maintaining the organization of melanophore stripes throughout development. Finally, we show that restoring Fms activity even at late larval stages allows essentially complete recovery of xanthophores and the development of a normal melanophore stripe pattern. Our findings suggest that fms is not required for establishing a population of precursor cells during embryogenesis but is required for recruiting pigment cell precursors to xanthophore fates,with concomitant effects on melanophore organization.
Maelstrom, aDrosophila spindle-class gene, encodes a protein that colocalizes with Vasa and RDE1/AGO1 homolog, Aubergine, in nuage
Authors: Findley Seth D. , Tamanaha Mio , Clegg Nigel J. , Ruohola-Baker Hannele
Published: January 2003

A hallmark of germline cells across the animal kingdom is the presence of perinuclear, electron-dense granules called nuage. In many species examined,Vasa, a DEAD-box RNA helicase, is found in these morphologically distinct particles. Despite its evolutionary conservation, the function of nuage remains obscure. We have characterized a null allele of maelstrom(mael) and shown that Maelstrom protein is localized to nuage in a Vasa-dependent manner. By phenotypic characterization, we have definedmaelstrom as a spindle-class gene that affects Vasa modification. In a nuclear transport assay, we have determined that Maelstrom shuttles between the nucleus and cytoplasm, which may indicate a nuclear origin for nuage components. Interestingly, Maelstrom, but not Vasa, depends on two genes involved in RNAi phenomena, aubergine andspindle-E (spn-E), for its nuage localization. Furthermore,maelstrom mutant ovaries show mislocalization of two proteins involved in the microRNA and/or RNAi pathways, Dicer and Argonaute2,suggesting a potential connection between nuage and the microRNA-pathway.
CRM1-mediated nuclear export and regulated activity of the Receptor Tyrosine Kinase antagonist YAN require specific interactions with MAE
Authors: Tootle Tina L. , Lee Philina S. , Rebay Ilaria
Published: January 2003

ETS family transcription factors serve as downstream effectors of signal transduction pathways, mediating cellular proliferation, differentiation and,when misregulated, tumorigenesis. The transcriptional repressor YAN prevents inappropriate responses to Receptor Tyrosine Kinase signaling by outcompeting POINTED for access to target gene promoters. We demonstrate that the molecular mechanism underlying downregulation of YAN involves CRM1-mediated nuclear export and define a novel role in this context for MAE, a co-factor previously implicated in facilitating MAPK phosphorylation of YAN. In addition to promoting YAN downregulation, MAE also participates in an inhibitory feedback loop that attenuates POINTED-P2 activation. Thus, we propose that MAE plays multiple independent roles in fine-tuning the levels of POINTED and YAN activity in accordance with changing RTK signaling conditions.
Apoptosis-mediated cell death within the ovarian polar cell lineage ofDrosophila melanogaster
Authors: Besse Florence , Pret Anne-Marie
Published: January 2003

Polar cells have been described as pairs of specific follicular cells present at each pole of Drosophila egg chambers. They are required at different stages of oogenesis for egg chamber formation and establishment of both the anteroposterior and planar polarities of the follicular epithelium. We show that definition of polar cell pairs is a progressive process since early stage egg chambers contain a cluster of several polar cell marker-expressing cells at each pole, while as of stage 5, they contain invariantly two pairs of such cells. Using cell lineage analysis, we demonstrate that these pre-polar cell clusters have a polyclonal origin and derive specifically from the polar cell lineage, rather than from that giving rise to follicular cells. In addition, selection of two polar cells from groups of pre-polar cells occurs via an apoptosis-dependent mechanism and is required for correct patterning of the anterior follicular epithelium of vitellogenic egg chambers.
Orbit/Mast, the CLASP orthologue ofDrosophila, is required for asymmetric stem cell and cystocyte divisions and development of the polarised microtubule network that interconnects oocyte and nurse cells during oogenesis
Authors: Máthé Endre , Inoue Yoshihiro H. , Palframan William , Brown Gemma , Glover David M.
Published: January 2003

Drosophila oocyte differentiation is preceded by the formation of a polarised 16-cell cyst from a single progenitor stem cell as a result of four rounds of asymmetric mitosis followed by incomplete cytokinesis. We show that the Orbit/Mast microtubule-associated protein is required at several stages in the formation of such polarised 16-cell cysts. In wild-type cysts,the Orbit/Mast protein not only associates with the mitotic spindle and its poles, but also with the central spindle (spindle remnant), ring canal and fusome, suggesting it participates in interactions between these structures. In orbit mutants, the stem cells and their associated fusomes are eventually lost as Orbit/Mast protein is depleted. The mitotic spindles of those cystocytes that do divide are either diminutive or monopolar, and do not make contact with the fusome. Moreover, the spindle remnants and ring canals fail to differentiate correctly in such cells and the structure of fusome is compromised. The Orbit/Mast protein thus appears to facilitate multiple interactions of the fusome with mitotic spindles and ring canals. This ensures correct growth of the fusome into a branched asymmetrically distributed organelle that is pre-determinative of 16-cell cyst formation and oocyte fate specification. Finally the Orbit/Mast protein is required during mid-oogenesis for the organisation of the polarised microtubule network inside the 16-cell cyst that ensures oocyte differentiation. The localisation of CLIP-190 to such microtubules and to the fusome is dependent upon Orbit/Mast to which it is complexed.
Dax1 regulates testis cord organization during gonadal differentiation
Authors: Meeks Joshua J. , Crawford Susan E. , Russell Theron A. , Morohashi Ken-ichiro , Weiss Jeffrey , Jameson J. Larry
Published: January 2003

Mutations of the DAX1 nuclear receptor gene cause adrenal hypoplasia congenita, an X-linked disorder characterized by adrenal insufficiency and hypogonadotropic hypogonadism. Targeted deletion ofDax1 in mice also reveals primary testicular dysgenesis, which is manifest by obstruction of the rete testis by Sertoli cells and hyperplastic Leydig cells, leading to seminiferous tubule dilation and degeneration of germ cells. Because Dax1 is expressed early in gonadal development, and because Sertoli and Leydig cells are located ectopically in the adult, we hypothesized that these testis abnormalities are the result of an early defect in testis development. In Dax1-/Y males, the gonad develops normally until 12.5 dpc. However, by 13.5 dpc, the testis cords are disorganized and incompletely formed in Dax1-deficient mice. The number of germ and Sertoli cells is unchanged, and the expression of Sertoli-specific markers appears to be normal. However, the number of peritubular myoid cells, which normally surround the testis cords, is reduced. BrdU labeling of peritubular myoid cells is low, consistent with decreased proliferation. The basal lamina produced by peritubular myoid and Sertoli cells is disrupted, leading to open and incompletely formed testis cords. Leydig cells, which normally reside in the peritubular space and extend from the coelomic surface to the dorsal surface of the gonad, are restricted to the coelomic surface ofDax1-deficient testis. We conclude that Dax1 plays a crucial role in testis differentiation by regulating the development of peritubular myoid cells and the formation of intact testis cords. The developmental abnormalities in the Dax1-deficient testis lay the foundation for gonadal dysgenesis and infertility in adult mice and, potentially in humans with DAX1 mutations.
Differential function of RNCAM isoforms in precise target selection of olfactory sensory neurons
Authors: Alenius Mattias , Bohm Staffan
Published: January 2003

Olfactory sensory neurons (OSNs) are individually specified to express one odorant receptor (OR) gene among ∼1000 different and project with precision to topographically defined convergence sites, the glomeruli, in the olfactory bulb. Although ORs partially determine the location of convergence sites, the mechanism ensuring that axons with different OR identities do not co-converge is unknown. RNCAM (OCAM, NCAM2) is assumed to regulate a broad zonal segregation of projections by virtue of being a homophilic cell adhesion molecule that is selectively expressed on axons terminating in a defined olfactory bulb region. We have identified NADPH diaphorase activity as being an independent marker for RNCAM-negative axons. Analyses of transgenic mice that ectopically express RNCAM in NADPH diaphorase-positive OSNs show that the postulated function of RNCAM in mediating zone-specific segregation of axons is unlikely. Instead, analyses of one OR-specific OSN subpopulation (P2)reveal that elevated RNCAM levels result in an increased number of P2 axons that incorrectly co-converge with axons of other OR identities. Both Gpi-anchored and transmembrane-bound RNCAM isoforms are localized on axons in the nerve layer, while the transmembrane-bound RNCAM is the predominant isoform on axon terminals within glomeruli. Overexpressing transmembrane-bound RNCAM results in co-convergence events close to the correct target glomeruli. By contrast, overexpression of Gpi-anchored RNCAM results in axons that can bypass the correct target before co-converging on glomeruli located at a distance. The phenotype specific for Gpi-anchored RNCAM is suppressed in mice overexpressing both isoforms, which suggests that two distinct RNCAM isoform-dependent activities influence segregation of OR-defined axon subclasses.
Functional specificity of the nematode Hox genemab-5
Authors: Gutierrez Arturo , Knoch Lars , Witte Hanh , Sommer Ralf J.
Published: January 2003

Hox genes encode evolutionarily conserved transcription factors involved in morphological specification along the anteroposterior body axis of animals. The two most striking features of Hox genes are colinearity and the strong sequence conservation. Among all animals studied so far, the nematodeCaenorhabditis elegans contains one of the most divergent Hox clusters. The core cluster contains only four members, which in part deviate from the colinearity rule. In addition, orthologous and paralogous nematode Hox sequences diverged substantially. Given these nematode-specific features,we asked how these Hox proteins evolved and how they provide functional specificity. We investigated the role of MAB-5 during ray formation and established an in vivo assay using Cel-mab-5 regulatory elements to express orthologous, paralogous and chimeric cDNAs in a Cel-mab-5mutant background. We show that the MAB-5 ortholog from Pristionchus pacificus, but not the C. elegans paralogous Hox proteins can rescue Cel-mab-5. Experiments with chimeric, truncated and mutagenized Hox proteins suggest the specificity to be conferred by the N-terminal arm and helix I, but not helix II of the homeodomain.
Wnt5aandWnt5bexhibit distinct activities in coordinating chondrocyte proliferation and differentiation
Authors: Yang Yingzi , Topol Lilia , Lee Heuijung , Wu Jinling
Published: January 2003

Proper longitudinal growth of long bones relies on the regulation of specific spatial patterns of chondrocyte proliferation and differentiation. We have studied the roles of two members of the Wnt family, Wnt5a andWnt5b in long bone development. We show that Wnt5a is required for longitudinal skeletal outgrowth and that both Wnt5a andWnt5b regulate the transition between different chondrocyte zones independently of the Indian hedgehog (Ihh)/parathyroid hormone-related peptide(PTHrP) negative feedback loop. We find that important cell cycle regulators such as cyclin D1 and p130, a member of the retinoblastoma family, exhibit complimentary expression patterns that correlate with the distinct proliferation and differentiation states of chondrocyte zones. Furthermore, we show that Wnt5a and Wnt5b appear to coordinate chondrocyte proliferation and differentiation by differentially regulating cyclin D1 and p130 expression, as well as chondrocyte-specific Col2a1 expression. Our data indicate that Wnt5a and Wnt5b control the pace of transitions between different chondrocyte zones.
Macrophage stimulating protein is a target-derived neurotrophic factor for developing sensory and sympathetic neurons
Authors: Forgie Alison , Wyatt Sean , Correll Pamela H. , Davies Alun M.
Published: January 2003

Macrophage stimulating protein (MSP) is a pleiotropic growth factor that signals via the Ron receptor tyrosine kinase. We report that Ron mRNA is expressed by NGF-dependent sensory and sympathetic neurons and that these neurons survive and grow with MSP at different stages of development. Whereas NGF-dependent sensory neurons become increasingly responsive to MSP with age,sympathetic neurons exhibit an early response to MSP that is lost by birth. MSP mRNA expression increases with age in sensory neuron targets and decreases in sympathetic targets. After the phase of naturally occurring neuronal death,significant numbers of NGF-dependent sensory neurons, but not sensory neurons,dependent on other neurotrophins, are lost in mice lacking a functional Ron receptor. These results show that MSP is a target-derived neurotrophic factor for subsets of sensory and sympathetic neurons at different times during their development.
Analysis of Bub3 spindle checkpoint function inXenopusegg extracts
Authors: Campbell Leigh , Hardwick Kevin G.
Published: January 2003

The spindle checkpoint delays the onset of anaphase if there are any defects in the interactions between spindle microtubules and kinetochores. This checkpoint has been reconstituted in vitro in Xenopus egg extracts, and here we use antibodies to Xenopus Bub3 (XBub3) to show that this protein is required for both the activation and the maintenance of a spindle checkpoint arrest in egg extracts. We detect two forms of XBub3 in egg extracts and find both to be complexed with the XBub1 and XBubR1 kinases. Only one form of XBub3 is apparent in Xenopus tissue culture (XTC) cells,and localisation studies reveal that, unlike the Mad proteins, which are concentrated at the nuclear periphery, XBub3 is diffusely localised throughout the nucleus during interphase. During early prophase it is recruited to kinetochores, where it remains until chromosomes align at the metaphase plate. We discuss the mechanism by which our α-XBub3 antibodies interfere with the checkpoint and possible roles for XBub3 in the spindle checkpoint pathway.
Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells
Authors: Adair-Kirk Tracy L. , Dorsey Frank C. , Cox John V.
Published: January 2003

AE1/Fc receptor chimeras have been used to define the sequences that direct the basolateral sorting, recycling and cytoskeletal association of the chicken AE1-4 anion exchanger in MDCK cells. These analyses revealed that amino acids 1-63 of AE1-4 were sufficient to redirect a cytoplasmic tailless murine IgG FcRII B2 receptor from the apical to the basolateral membrane of MDCK cells, where Fc1-63 associated with elements of the actin cytoskeleton. In contrast to Fc1-63, chimeras containing amino acids 1-37 (Fc1-37) or 38-63 (Fc38-63) of AE1-4 accumulated in intracellular membrane compartments that overlapped late endosomes and the trans-Golgi network (TGN), respectively. Internalization assays indicated that the patterns of localization observed for Fc1-37 and Fc38-63 resulted from the recycling of these chimeras from the cell surface. These assays further indicated that Fc1-37 and Fc38-63 each possess a basolateral sorting activity. Mutagenesis studies revealed that the endocytic and basolateral sorting activities in Fc1-37 are dependent upon serine 25, which is located in a sequence similar to a sorting signal in the polymeric immunoglobulin receptor. In addition, the sorting activities associated with Fc38-63 were dependent upon tyrosine 47 and leucine 50. These residues resided within the sequence, YVEL, which matches the YXXΦ motif(where X is any amino acid and Φ is a hydrophobic residue) that functions as an endocytic and TGN recycling signal for other membrane proteins. Our data indicate that amino acids 1-63 of AE1-4 contain sorting and cytoskeletal binding activities that account for most of the properties previously associated with AE1-4 in MDCK cells. Furthermore, the alternative localization patterns exhibited by chimeras containing various combinations of these activities suggest that interplay between these cytoplasmic activities is critical for specifying AE1-4 localization in epithelial cells.
hRUL138, a novel human RNA-binding RING-H2 ubiquitin-protein ligase
Authors: Kreft Stefan G. , Nassal Michael
Published: January 2003

Cellular as well as viral RNAs are usually found complexed with proteins. In an attempt to identify proteins that interact with transcripts of hepatitis B virus (HBV), a DNA virus that replicates through reverse transcription, a partial cDNA was isolated from a human cDNA expression library whose gene product bound to an HBV-derived RNA. Using an overlapping clone from a molecular hybridization screen a full-length cDNA was assembled. It contained a large open reading frame for a 1208 amino-acid protein of 138 kDa identical to the hypothetical product of the KIAA0675 clone. Closely related sequences are present in mouse cDNA libraries but not in the genomes of lower organisms. The protein sequence contained no known RNA-binding domain and, apart from a probable coiled-coil domain, the only significant homology involved a complete RING-H2 motif. This suggested that the protein might be a novel RNA-binding RING-dependent ubiquitin-protein ligase or E3 enzyme. A motif critical for RNA binding was experimentally mapped to a central Lys-rich region. Binding specificity is either broad or the protein has as yet unknown physiological targets; hence, at present, a potential importance for HBV biology remains open. The RING-H2 domain was functional in and essential for self- and trans-ubiquitylation in vitro and for proteasome-mediated turnover of the protein in vivo. We therefore termed it hRUL138 for human RNA-binding ubiquitin ligase of 138 kDa. hRUL138 mRNAs are expressed at low levels in most tissues. GFP-tagged hRUL138 derivatives were found associated with cytoplasmic structures, possibly the ER, but excluded from the nucleus. The combined presence of RNA binding and E3 activity in hRUL138 raises the possibility that both are mechanistically linked.
Insulin-like growth factor binding protein 5 and apoptosis in mammary epithelial cells
Authors: Marshman Emma , Green Kirsty A. , Flint David J. , White Anne , Streuli Charles H. , Westwood Melissa
Published: January 2003

Insulin-like growth factors (IGFs) are important survival signals that can protect a range of cell types from apoptosis. Although IGF bioavailability is modulated by high affinity interactions with IGF-binding proteins (IGFBPs),there is currently no experimental evidence that IGFBPs regulate the survival function of IGFs in the mammary gland. We have examined IGFBP expression during mammary gland development and studied the effects of IGFBPs on IGF-mediated survival and signalling in mammary epithelial cells in culture. IGFBP-5 protein was greatly increased during days 1-3 of mammary gland involution, when levels of apoptosis are dramatically elevated to remodel the gland after lactation. Primary cultures of mammary epithelial cells (MECs)expressed IGFBP-5 from their basal surface suggesting that IGFBP-5 is suitably located to inhibit IGF signalling. Addition of exogenous IGFBP-5 and IGFBP-3 to MECs suppressed IGF-I-mediated survival, resulting in threefold greater apoptosis in cells incubated with IGF-I and IGFBP-5 compared with IGF-I alone. Examination of signalling pathways involved in apoptosis revealed that phosphorylation of PKB and the forkhead transcription factor, FKHRL1, was induced by IGFs, but that phosphorylation was blocked by IGFBP-5 and IGFBP-3. This study provides evidence that IGFBP-5 plays an important role in the regulation of apoptosis in the mammary gland.
ForC, a novel type of formin family protein lacking an FH1 domain, is involved in multicellular development inDictyostelium discoideum
Authors: Kitayama Chikako , Uyeda Taro Q. P.
Published: January 2003

Formins are highly conserved regulators of cytoskeletal organization and share three regions of homology: the FH1, FH2 and FH3 domains. Of the nine known formin genes or pseudogenes carried by Dictyostelium, forC is novel in that it lacks an FH1 domain. Mutant Dictyostelium lacking forC (ΔforC) grew normally during the vegetative phase and, when starved, migrated normally and formed tight aggregates. Subsequently, however, ΔforC cells made aberrant fruiting bodies with short stalks and sori that remained unlifted. ΔforCaggregates were also unable to migrate as slugs, suggesting forC is involved in mediating cell movement during multicellular stages of Dictyostelium development. Consistent with this idea, expression of forC was increased significantly in aggregates of wild-type cells. GFP-ForC expressed in ΔforC cells was localized at the crowns,which are macropinocytotic structures rich in F-actin, suggesting that, like other formin isoforms, ForC functions in close relation with the actin cytoskeleton. Truncation analysis of GFP-ForC revealed that the FH3 domain is required for ForC localization; moreover, localization of a truncated GFP-ForC mutant at the site of contacts between cells on substrates and along the cortex of cells within a multicellular culminant suggests that ForC is involved in the local actin cytoskeletal reorganization mediating cell-cell adhesion.
Deletion of exon 4 from human surfactant protein C results in aggresome formation and generation of a dominant negative
Authors: Wang Wen-Jing , Mulugeta Surafel , Russo Scott J. , Beers Michael F.
Published: January 2003

Human surfactant protein C (hSP-C) is synthesized by the alveolar type 2 cell as a 197 amino acid integral membrane proprotein and proteolytically processed to a secreted 3.7 kDa mature form. Although the SP-C null mouse possesses a non-lethal phenotype, a heterozygous substitution of A for G in the first base of intron 4 of the human SP-C gene (c.460+1A>G) has been reported in association with familial interstitial lung disease and absence of mature protein. This mutation produces a splice deletion of exon 4(ΔExon4) resulting in removal of a positionally conserved cysteine in the C-terminal flanking propeptide. Based on a prior study showing that an identical deletion in the rat isoform diverted mutant protein to stable aggregates, we hypothesized that expression of the ΔExon4 mutation would result in disruption of intracellular trafficking of both mutant and wild-type proSP-C. We tested this in vitro using fusion proteins of EGFP conjugated either to wild-type SP-C (EGFP/hSP-C1-197) or to SP-C deleted of Exon4 (EGFP/hSP-CΔExon4). Fluorescence microscopy showed that EGFP/hSP-C1-197 transfected into A549 cells was expressed in a punctuate pattern in CD63 (+) cytoplasmic vesicles, whereas EGFP/hSP-CΔExon4 accumulated in ubiquitinated perinuclear inclusions linked to the microtubule organizing center. A similar juxtanuclear pattern was observed following transfection of SP-C cDNA lacking only cysteine residues in the C-terminal propeptide encoded by Exon 4(EGFP/hSP-CC120/121G). To evaluate whether mutant proSP-C could function as a dominant negative, EGFP/hSP-CΔExon4 was cotransfected with HA-tagged hSP-C1-197 and resulted in the restriction of both forms to perinuclear compartments. Addition of Na+ 4-phenylbutyrate, a facilitator of trafficking of other misfolded proteins, attenuated the aggregation of EGFP/hSP-CΔExon4. We conclude that c.460+1A>G mutation of human SP-C results in disruption of disulfide-mediated folding encoded by Exon 4 leading to diversion of unprocessed proSP-C to aggresomes. The heterotypic oligomerization of hSP-C1-197 and hSP-CΔExon4provides a molecular mechanism for the dominant-negative effect observed in vivo.
Nuclear RNA export
Authors: Cullen Bryan R.
Published: January 2003

Eukaryotic cells export several different classes of RNA molecule from the nucleus, where they are transcribed, to the cytoplasm, where the majority participate in different aspects of protein synthesis. It is now clear that these different classes of RNA, including rRNAs, tRNAs, mRNAs and snRNAs, are specifically directed into distinct but in some cases partially overlapping nuclear export pathways. All non-coding RNAs are now known to depend on members of the karyopherin family of Ran-dependent nucleocytoplasmic transport factors for their nuclear export. In contrast, mRNA export is generally mediated by a distinct, Ran-independent nuclear export pathway that is both complex and, as yet, incompletely understood. However, for all classes of RNA molecules, nuclear export is dependent on the assembly of the RNA into the appropriate ribonucleoprotein complex, and nuclear export therefore also appears to function as an important proofreading mechanism.
Plastic adult stem cells: will they graduate from the school of hard knocks?
Authors: Alison Malcolm R. , Poulsom Richard , Otto William R. , Vig Pamela , Brittan Mairi , Direkze Natalie C. , Preston Sean L. , Wright Nicholas A.
Published: January 2003

Notwithstanding the fact that adult bone marrow cell engraftment to epithelial organs seems a somewhat uncommon event, there is no doubt it does occur, and under appropriate conditions of a strong and positive selection pressure these cells will expand clonally and make a significant contribution to tissue replacement. Likewise, bone-marrow-derived cells can be amplified in vitro and differentiated into a multitude of tissues. These in essence are the goals of regenerative medicine using any source of stem cells, be it embryonic or adult. Despite such irrefutable evidence of what is possible, a veritable chorus of detractors of adult stem cell plasticity has emerged, some doubting its very existence, motivated perhaps by more than a little self-interest. The issues that have led to this state of affairs have included the inability to reproduce certain widely quoted data, one case where the apparent transdifferentiation was due to contamination of the donor tissue with haematopoietic cells and, most notoriously, extrapolating from the behaviour of embryonic stem cells to suggest that adult bone marrow cells simply fuse with other cells and adopt their phenotype. While these issues need resolving,slamming this whole new field because not everything is crystal clear is not good science. The fact that a phenomenon is quite rare in no way mitigates against its very existence: asteroid collisions with the Earth are rare, but try telling the dinosaurs they do not occur! When such events do occur(transdifferentiation or collision), they certainly can make an impact.
ABA- and cADPR-mediated effects on respiration and filtration downstream of the temperature-signaling cascade in sponges
Authors: Zocchi Elena , Basile Giovanna , Cerrano Carlo , Bavestrello Giorgio , Giovine Marco , Bruzzone Santina , Guida Lucrezia , Carpaneto Armando , Magrassi Raffaella , Usai Cesare
Published: January 2003

Recently, the thermosensing pathway in sponges (Porifera) was elucidated. The thermosensor triggering this cascade is a heat-activated cation channel,with the phytohormone abscisic acid (ABA), cyclic ADP-ribose (cADPR) and calcium acting as intracellular messengers, similarly to the drought-stress signaling cascade in higher plants. Here, we investigated the functional effects downstream of the temperature-signaling pathway in Axinella polypoides (Porifera, Demonspongiae).

Short-term stimulation followed by long-term depression of amino acid incorporation, oxygen consumption and water filtration were observed after exposure of the sponge to a brief heat stress or to micromolar ABA. These effects could be prevented by the targeted interruption of the signaling pathway either at the level of the cation channel thermosensor or at the level of the cADPR-induced intracellular calcium increase. Moreover, release of cyclase activity into the sea water and generation of extracellular cADPR were observed following brief heat stress. Intact sponge cells were sensitive to extracellular cADPR and addition of purified cyclase increased sponge respiration similarly to heat stress.

This is the first observation of functional effects exerted on Metazoa by the phytohormone ABA: conservation of the ABA/cADPR stress-signaling cascade points to its early evolution in a common precursor of modern Metazoa and Metaphyta. The functional effects induced by extracellular cyclase/cADPR suggest an evolutionary origin of cADPR as an ancient stress hormone in Porifera.
VEGF expression in human macrophages is NF-κB-dependent: studies using adenoviruses expressing the endogenous NF-κB inhibitor IκBα and a kinase-defective form of the IκB kinase 2
Authors: Kiriakidis Serafim , Andreakos Evangelos , Monaco Claudia , Foxwell Brian , Feldmann Marc , Paleolog Ewa
Published: January 2003

Vascular endothelial growth factor (VEGF) is the most endothelial cell-specific angiogenic factor characterised to date, and it is produced by a variety of cell types. In macrophages, VEGF has been shown to be upregulated by the inflammatory mediator lipopolysaccharide (LPS) and by engagement of CD40 by CD40 ligand (CD40L). Because LPS and CD40L activate nuclear factor-κB (NF-κB) in monocytes, we investigated in this study whether VEGF production in macrophages, when stimulated with either LPS or CD40L, is NF-κB-dependent. We used adenoviral constructs over-expressing either IκBα (AdvIκBα), the endogenous inhibitor of NF-κB, or a kinase-defective mutant of IKK-2 (AdvIKK-2dn), an upstream activator of IκBα, to infect normal human monocyte-derived macrophages. We observed that LPS-induced production of VEGF in human macrophages was almost completely inhibited (>90%) following adenoviral transfer of IκBα. In addition, we observed significant inhibition of the CD40L-induced VEGF production in macrophages following infection with AdvIκBα. Expression of IKK-2dn in macrophages decreased VEGF production in response to LPS or CD40L by approximately 50%, suggesting that in addition to IKK-2, other kinases might be involved in NF-κB activation. These results show for the first time that VEGF production in human macrophages is NF-κB dependent. NF-κB regulates many of the genes involved in immune and inflammatory responses, and our study adds the angiogenic cytokine VEGF to the list of NF-κB-dependent cytokines.
Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function
Authors: Hopkins Ann M. , Walsh Shaun V. , Verkade Paul , Boquet Patrice , Nusrat Asma
Published: January 2003

The apical-most epithelial intercellular junction, referred to as the tight junction (TJ), regulates paracellular solute flux in diverse physiological and pathological states. TJ affiliations with the apical filamentous actin(F-actin) cytoskeleton are crucial in regulating TJ function. F-actin organization is influenced by the Rho GTPase family, which also controls TJ function. To explore the role of Rho GTPases in regulating TJ structure and function, we utilized Escherichia coli cytotoxic necrotizing factor-1(CNF-1) as a tool to activate constitutively Rho, Rac and Cdc42 signaling in T84 polarized intestinal epithelial monolayers. The biological effects of the toxin were polarized to the basolateral membrane, and included profound reductions in TJ gate function, accompanied by displacement of the TJ proteins occludin and zonula occludens-1 (ZO-1), and reorganization of junction adhesion molecule-1 (JAM-1) away from the TJ membrane. Immunogold electron microscopy revealed occludin and caveolin-1 internalization in endosomal/caveolar-like structures in CNF-treated cells. Immunofluorescence/confocal microscopy suggested that a pool of internalized occludin went to caveolae, early endosomes and recycling endosomes, but not to late endosomes. This provides a novel mechanism potentially allowing occludin to evade a degradative pathway, perhaps allowing efficient recycling back to the TJ membrane. In contrast to the TJ, the characteristic ring structure of proteins in adherens junctions (AJs) was largely preserved despite CNF-1 treatment. CNF-1 also induced displacement of a TJ-associated pool of phosphorylated myosin light chain (p-MLC), which is normally also linked to the F-actin contractile machinery in epithelial cells. The apical perjunctional F-actin ring itself was maintained even after toxin exposure,but there was a striking effacement of microvillous F-actin and its binding protein, villin, from the same plane. However, basal F-actin stress fibers became prominent and cabled following basolateral CNF-1 treatment, and the focal adhesion protein paxillin was tyrosine phosphorylated. This indicates differences in Rho GTPase-mediated control of distinct F-actin pools in polarized cells. Functionally, CNF-1 profoundly impaired TJ/AJ assembly in calcium switch assays. Re-localization of occludin but not E-cadherin along the lateral membrane during junctional reassembly was severely impaired by the toxin. A balance between activity and quiescence of Rho GTPases appears crucial for both the generation and maintenance of optimal epithelial barrier function. Overactivation of Rho, Rac and Cdc42 with CNF-1 seems to mirror key barrier-function disruptions previously reported for inactivation of RhoA.
Application of dynamic band brake model for enhanced drivetrain simulation
Authors: Fujii Y , Tobler W E , Clausing E M , Megli T W , Haghgooie M
Published: January 2003

In a modern vehicle design process, analytical tools are widely employed to complement experimental approaches for design evaluation. When effectively utilized, they lead to a reduced development time with improved vehicle performance. The development process of an automatic transmission (AT) system can benefit from an analytical representation which accurately captures AT shifting behaviours. In a typical AT system, friction components such as wet clutches and band brakes are utilized to alter planetary gear configurations for automatic shifting. Thus, an accurate representation of friction component dynamics is critical in predicting AT shifting behaviour. Engagement characteristics of friction components vary widely under different operating conditions. Although the basic engagement physics was identified in the 1970s, it is relatively recently that a predictive, yet computationally efficient model became available. This paper describes the first attempt to utilize a dynamic friction component model in drivetrain simulations. Specifically, a dynamic band brake model is implemented to predict the up-shift behaviour of a four-speed AT system under various operating conditions. Simulation results are qualitatively validated with experimental data obtained from a dynamometer test stand. The dynamic band brake model enhances the shift predictability of a drivetrain model and potentially allows analytical evaluation of shift quality and control strategy.
A thermomechanical theory of impact trauma
Authors: Neal-Sturgess C E
Published: January 2003

A review of injuries in impact trauma reveals a plethora of ‘injury criteria’, many of which are enshrined in legislation. It is assumed here that injuries can be modelled as mechanical dissipative processes, and the formalism of continuum damage mechanics based on irreversible thermodynamics is applied to impact trauma. It is shown that peak virtual power (PVP) predicts the severity of injury, measured on the abbreviated injury scale, in around 90 per cent of cases for all types of injury to all body regions (brain, skull, thorax, spine, upper and lower extremities) for car occupants from the CCIS and NASS-CDC databases. Consideration of injury to body regions shows that PVP predicts the form of acceleration-based criteria, the head injury criterion and the viscous criterion. It is shown that in general the lower bound of severity of injury is proportional to δ V3 or (ETS)3, where ETS is equivalent test speed, for restrained vehicle occupants, and the upper bound proportional to δ V2 or (ETS)2 for unrestrained occupants. It is concluded that PVP is a suitable candidate for an objective universal injury criterion which can be correlated to real-world injury experience.
Self-learning approach to automatic transmission shift control in a commercial construction vehicle during the inertia phase
Authors: Hahn J-O , Hur J-W , Choi G-W , Cho Y M , Lee K I
Published: January 2003

Electrohydraulic shift control of a vehicle automatic transmission has been predominantly carried out via open-loop control based on numerous time-consuming calibrations. Despite remarkable success in practice, the variations of system characteristics inevitably cause the performance of the tuned open-loop controller to deteriorate. As a result, the controller parameters need to be continuously updated in order to maintain satisfactory shift quality. This paper presents a selflearning algorithm for automatic transmission shift control in a commercial construction vehicle during the inertia phase. First, an observer reconstructs the turbine acceleration signal (impossible to measure in a commercial construction vehicle) from the readily accessible turbine speed measurement. Then, a control algorithm based on a quadratic cost function of the turbine acceleration is shown to guarantee the asymptotic convergence (within a specified target bound) of the error between the actual and the desired turbine accelerations. A Lyapunov argument plays a crucial role in deriving adaptive laws for control parameters. The simulation and hardware-in-the-loop simulation studies show that the proposed algorithm actually delivers the promise of satisfactory performance despite the variations and uncertainties of system characteristics.
Dynamic analysis and control of an active engine mount system
Authors: Lee Y-W , Lee C-W
Published: January 2003

Dynamic characteristics of a prototype active engine mount (AEM), designed on the basis of a hydraulic engine mount, have been investigated and an adaptive controller for the AEM has been designed. An equivalent mass-spring-damper AEM model is proposed, and the transfer function that describes the dynamic characteristics of the AEM is deduced from mathematical analysis of the model. The damping coefficient of the model is derived by considering the non-linear flow effect in the inertia track. Experiments confirmed that the model precisely describes the dynamic characteristics of the AEM. An adaptive controller using the filtered-X LMS algorithm is designed to cancel the force transmitted through the AEM. The stability of the LMS algorithm is guaranteed by using the secondary path transfer function derived on the basis of the dynamic model of the AEM. The performance test in the laboratory shows that the AEM system is capable of significantly reducing the force transmitted through the AEM.
Structural integrity of polypropylene prosthetic sockets manufactured using the polymer deposition technique
Authors: Goh J C H , Lee P V S , Ng P
Published: January 2003

Rapid prototyping (RP) technology has been used recently as a means for automated socket fabrication. Although the technology has proven to be promising and has truly automated the socket fabrication process, the structural integrity of RP sockets remains questionable. For the long term, unsupervised use of these ‘unconventional’ sockets, their material properties and structural integrity must be determined. This study investigated the structural integrity of polypropylene sockets manufactured using a polymer deposition technique, in which a socket is formed by a continuous strand of partially melted polypropylene that is spirally deposited according to the socket's cross-sectional contour. To investigate the problem of delamination of the socket, the tensile properties of the socket material were determined according to ASTM D638-99. The ultimate tensile strength was found to be approximately 13–23 per cent lower than that of polypropylene sheets that are at present normally used for socket fabrication. In order to improve the load-bearing capacity of the socket, it was reinforced using a double-wall arrangement at the distal region, where failure normally occurs. The structural integrity of the complete prosthesis was then investigated according to ISO 10328 (loading condition II). The prosthesis passed the static loading test registering only 12 mm permanent deformation, and it successfully completed a preliminary cyclic test of 250000 cycles with no observable failure.