bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet-derived growth factor: mechanism of action and relation to oncogenes
Authors: Heldin C.-H. , Betsholtz C. , Johnsson A. , Nistér M. , Ek B. , Rönnstrand L. , Wasteson Å. , Westermark B.
Published: May 2013

Recent studies of platelet-derived growth factor (PDGF) have revealed several structural and functional similarities between this growth factor or components linked to its mechanism of action and certain oncogene products: PDGF itself has a structural homology with the transforming protein of simian sarcoma virus, the PDGF receptor has a functional homology (tyrosine kinase activity) with a family of oncogene products, and PDGF induces the expression of the cellular counterparts of myc and fos. In addition, several tumour cell lines have been found to produce PDGF-like growth factors, which may cause autocrine stimulation of growth. We interpret these findings as indicating that regulatory components along the PDGF-dependent mitogenic pathway may have oncogenic properties if they are inappropriately expressed or activated.
Dynein Structure And Function
Authors: A. Johnson Kenneth , P. Marchese-Ragona Silvio , B. Clutter Daniel , F. Holzbaur Erika L. , J. Chilcote Tamie
Published: May 2013

The structure of dynein isolated from several sources follows the pattern first observed with Tetrahymena 22 S dynein, which has three globular heads attached by three flexible strands to a root-like base. Recent biochemical data indicate that there is one ATPase site on each dynein head and that all three heads interact with microtubules in an ATP-sensitive manner. Accordingly, images of dynein in situ can be interpreted in terms of a model for crossbridge action where the roots of the bouquet anchor the dynein to the A-tubule and all three heads reach out to interact with the B-tubule in an ATP-dependent reaction to produce a force for sliding.
Mutations Affecting Microtubule Structure In Caenorhabditis Elegans
Authors: Chalfie Martin , Dean Ellen , Reilly Evelyn , Buck Kay , Nichol Thomson J.
Published: May 2013

Three types of microtubules are seen in the neuronal processes of the nematode Caenorhabditis elegans. Single cytoplasmic microtubules of most neurones have 11 protofilaments whereas those of six touch receptor cells have 15 protofilaments. The axonemes of sensory cilia have nine outer doublets with a variable number (up to seven) of singlet microtubules. Mutations in 11 genes affect the appearance of these microtubules.
Cytoskeletal Changes During Oogenesis And Early Development Of Xenopus Laevis
Authors: Wylie C. C. , Heasman Janet , Parke Judy M. , Anderton Brian , Tang Peter
Published: May 2013

The frog oocyte is well known for studies on the control of gene expression, but has been used much less in studies on the cytoskeleton. However, frog oocytes are very large single cells, whose cytoplasmic movements and asymmetries are fundamental to the correct development of the subsequent embryo. One particular example of asymmetrically distributed cytoplasm is germ plasm, thought to be important in the formation of the germ line. Data are presented that show that germ plasm is a highly concentrated mass of cytoskeletal elements, which include tubulin, and an intermediate filament protein of molecular weight 55 × 103. The distribution of these molecules has been studied during oogenesis and during early post-fertilization development. The implications of these findings are discussed.
Enzyme systems initiating replication at the origin of the ESCHERICHIA COLI chromosome
Authors: Kornberg Arthur
Published: May 2013

More than ten proteins are known to participate in replication of plasmids bearing the unique origin of the Escherichia coli chromosome (onC). Initiation of replication of oriC plasmids has been resolved into five separable stages. An initial complex formation (Stage I) requires an oriC plasmid, dnaA protein and HU protein. In the presence of ATP at a temperature of >28°C, a dnaB-C protein complex interacts to form a prepriming complex (Stage II). This is followed by extensive unwinding of the template that depends on the further addition of gyrase and singlestrand binding protein (SSB) (Stage III). Hydrolysis of an rNTP by dnaB protein (a helicase action) and of ATP by gyrase (a swivelling action) drives the extreme unwinding of the template. This unwound template-protein complex is the substrate for priming by primase (Stage IV) and elongation by DNA polymerase III holoenzyme (Stage V). Priming of all DNA chains is done by primase; RNA polymerase functions in template activation rather than priming. DNA polymerase III holoenzyme, composed of at least seven subunits, synthesizes the DNA chains. The α subunit is the polymerase, the ε subunit is the 3′ →5′exonuclease; α+ε is the proofreading activity. Following the synthesis of new DNA chains, DNA polymerase I and ribonuclease H remove the RNA primers, polymerase I fills the gaps, and ligase seals the daughter strands (Stage VI). Replication produces plasmids identical in structure and sequence to the initial template.
Mechanisms of Tumour Cell Metastasis
Authors: Parish Roger W. , Schmidhauser Christian , Schmidt Thomas , Dudler Robert K.
Published: May 2013

Abercrombie and his colleagues have accumulated evidence that changes in the heterotypic contact-inhibition response are largely responsible for the invasiveness of cells, at least in culture. We have identified a 37 000Mr protein on the surface of mouse fibrosarcoma cells that is involved in their in vitro invasion. Blocking this protein with specific antibodies inhibits the invasion of chicken heart fibroblasts by the tumour cells and normal heterotypic contact inhibition is restored. These results are presented in the general framework of metastatic mechanisms and we review a selection of more recent studies aimed at describing the metastatic phenotype in molecular terms.
Integrin, A Transmembrane Glycoprotein Complex Mediating Cell-Substratum Adhesion
Authors: Buck Clayton A. , Horwitz A. F.
Published: May 2013

A monoclonal antibody, CSAT, which inhibits the adhesion of chick cells to substrata coated with fibronectin, laminin and vitronectin, has been used to identify a cell surface receptor required for cell—substratum adhesion. This receptor, termed integrin, is found on the ventral surface of cells in close contact adhesion sites, at the periphery of adhesion plaques and beneath stress fibres. It is a heterodimer consisting of non-covalently linked alpha and beta subunits. Integrin binds directly to laminin, fibronectin and vitronectin with dissociation constants in the micromolar range. The binding of integrin to matrix molecules is sensitive to peptides carrying the cell-binding sequence Arg-Gly-Asp and requires heteromeric integrity. Integrin also binds directly to the cytoskeleton-associated protein talin. Thus, integrin has the properties of a transmembrane molecule capable of bringing extracellular matrix and cytoskeleton-associated molecules in proper juxtaposition to form adhesion structures. The integrin beta subunit is phosphorylated following Rous sarcoma virus transformation. Phosphorylation alters the ability of the receptor to bind extracellular matrix molecules as well as talin, suggesting a mechanism for the alteration of cellular adhesive and morphological properties following malignant transformation. A major phosphorylation site is on the cytoplasmic domain of the beta subunit. Synthetic peptides homologous with this region of integrin inhibit integrin-talin binding. The gene for the beta subunit of integrin has been sequenced. Its structure is consistent with the membrane-spanning properties of the receptor. Integrin is structurally and serologically related to adhesion receptors from mammalian tumour cells, fibroblasts, platelets and lymphocytes. It appears to be a member of a supergene family of receptors involved in cellular adhesive interactions. Antibody and peptide inhibition experiments have suggested a role for integrin and integrin-like molecules in cell migration, neurite extension, •neural differentiation, histogenesis and embryonic development in Drosophila. Thus, integrin appears representative of a set of evolutionarily conserved, biologically important adhesive molecules.
The Influence of Extracellular Matrix on Gene Expression: Is Structure The Message?
Authors: Bissell Mina J. , Barcellos-Hoff Mary Helen
Published: May 2013

The study of the regulation of gene expression in cultured cells, particularly in epithelial cells, has been both hampered and facilitated by the loss of function that accompanies culture on traditional plastic substrata. Initially, investigations of differentiated function were thwarted by the inadequacy of tissue culture methods developed to support growth of mesenchvmal cells. However, with the recognition that the unit of function in higher organisms is larger than the cell itself, and that gene expression is dependent upon cell interactions with hormones, substrata and other cells, came the understanding that the epithelial cell phenotype is profoundly influenced by the extra-cellular environment. In the last decade research on epithelial cells has centred on culture conditions that recreate the appropriate environment for function with very promising and important results. The investigations into the modulation of phenotype in culture produced not only a better model, J>ut also contributed to a better understanding of the regulation of normal function. Using cultured mammary gland epithelial cells as a primary model of these interactions, our studies of gene expression are based on three premises.

That the extracellular matrix (ECM) on which the cells sit is an extension of the cells and an active participant in the regulation of cellular function; i.e. the ECM is an ‘informational’ entity in the sense that it receives, imparts and integrates structural and functional signals. That ECM-induced functional differentiation in the mammary gland is mediated through changes in cell shape, i.e. that the structure is in large part ‘the message’ required to maintain differentiated gene expression. That the unit of function includes the cell plus its extracellular matrix; in a larger context, the unit is the organ itself.

These tenets and the data presented below are consistent with a model of ‘Dvnamic Reciprocity’, where the ECM is postulated to exert an influence on gene expression via transmembrane proteins and cytoskeletal components. In turn, cytoskeletal association with polvribosomes affects mRNA stability and rates of protein synthesis, while its interaction with the nuclear matrix could affect mRNA processing and, possibly, rates of transcription.
Epithelial Cell Polarization In Culture: Orientation of Cell Polarity and Expression of Specific Functions, Studied With Cultured Thyroid Cells
Authors: Mauchamp Jean , Chambard Marianne , Verrier Bernard , Gabrion Jacqueline , Chabaud Odile , Gerard Corinne , Penel Claude , Pialat Bernard , Anfosso Francine
Published: May 2013

Isolated porcine thyroid cells reorganize in culture into various types of multicellular structure, which differ in the orientation of cell polarity and in the surface of the cell layer accessible to molecules present in the culture medium. The types of structure are: (1) follicles-, the basal pole is oriented toward the medium; (2) inside-out follicles or monolayers: the apical pole is facing the culture medium; (3) monolayers on a permeable substratum: both sides of the cell layer are accessible to the medium. Follicles can be transformed into inside-out follicles or monolayers and vice versa by manipulation of the external cell environment and without dissociating the cells. Cells concentrate iodide and respond to acute stimulation by thyroid-stimulating hormone (TSH) when the basal pole is accessible, and organification occurs only when cells form a closed follicular lumen.

In porous-bottomed culture chambers monolayers are formed with the basal surface accessible to the medium and the apical compartment separated from the medium. Under these conditions 85–95% of the thyroglobulin produced is secreted apically and 5–15% basally. Thyrotropin stimulates (×3) apical accumulation without modifying secretion in the basal compartment.

Sodium transport across the cell layer has been characterized. An amiloride-sensitive influx occurs at the apical pole whereas the Na+/K+-ATPase, localized in the basolateral membrane, mediates ouabain-sensitive efflux at the basal pole. The thyroid epithelium in culture appears therefore as a Na+-absorbing epithelium. The role of this transport in the stabilization of cell polarity is discussed.
The genetics of protein targeting in Escherichia coli K12
Authors: Trun Nancy J. , Silhavy Thomas J.
Published: May 2013

Genetic analysis of protein targeting in Escherichia coli has been facilitated by the use of lacZ gene fusions. Strains which contain a fusion between a gene specifying a noncytoplasmic protein and lacZ exhibit novel phenotypes that can be exploited to obtain export-defective mutations. Analysis of these mutations has demonstrated the importance of the signal sequence and provided some insight into the functions performed by this complex intragenic export signal. In addition, genes that specify components of the cellular protein export machinery have been identified. Three of these genes, secA, secB, and prlA/secY have been studied in some detail and results indicate that the protein products function in the translocation of exported proteins across the cytoplasmic membrane.
Regulation of transforming growth factor-β subtypes by members of the steroid hormone superfamily
Authors: Wakefield Lalage , Kim Seong-Jin , Glick Adam , Winokur Thomas , Colletta Anthony , Sporn Michael
Published: May 2013

Transforming growth factor-/fe (TGF-βs) are potent regulators of cell growth and differentiation. Expression of the closely related TGF-β subtypes in vivo is differentially regulated both temporally and spatially. Members of the steroid hormone superfamily may play an important role in this gene- and tissue-specific regulation. We have shown that anti-estrogens induce the production of TGF-β1 in mammary carcinoma cells and fetal fibroblasts, whereas retinoic acid specifically induces TGF-β2 in primary epidermal kératinocytes. The induction of TGF-β2 by retinoids is accompanied by an increase in TGF-β2 mRNAs, but little change in transcription rates, suggesting an effect of retinoids on message stability or processing. In contrast, TGF-β1 mRNA levels are unchanged by anti-estrogen treatment, suggesting these compounds may regulate the translatability of the TGF-β1 message or some post-translational processing event. We have identified a stable stem-loop structure in the 5′ untranslated region (UTR) of the TGF-β mRNA that inhibits translation of a heterologous reporter gene, and we are investigating the possibility that anti-estrogens may regulate the activity of this element, and hence the translatability of the TGF-β1 message. A significant fraction (25–90%) of the TGF-β induced by retinoids and anti-estrogens is in the biologically active rather than the latent form. We have shown that active TGF-β has a much shorter in vivo half-life than latent TGF-β, suggesting that the TGF-β induced by retinoids and steroids may act locally at the site of production. Since many tumor cells retain sensitivity to the growth inhibitory effects of active TGF-β, the use of members of the steroid hormone superfamily for inducing this potent growth inhibitor locally at the tumor site may have therapeutic potential.
Kinetic analysis of regulated myosin ATPase activity using single and limited turnover assays
Authors: Ankrett R. J. , Walmsley A. R. , Bagshaw C. R.
Published: May 2013

Myosin from molluscan adductor muscle is regulated directly by Ca2+ binding. In the absence of Ca2+ the ATPase activity is greatly inhibited. We review the application of transient kinetic methods to this system and show how they can be simple to perform and less ambiguous than steady-state methods.
Molecular basis of myosin assembly: coiled-coil interactions and the role of charge periodicities
Authors: Atkinson Simon J. , Stewart Murray
Published: May 2013

Complementation of alternating zones of positive and negative charge in the myosin rod enables molecules to interact in a number of ways. This accounts for the complexity of the molecular organisation of thick filaments. However, directed mutagenesis of expressed LMM cDNA indicated that charge zone complementation is not a major driving force in myosin polymerisation. Instead, it probably serves to prevent unfavourable interaction geometries.
A model for kinesin movement from nanometer-level movements of kinesin and cytoplasmic dynein and force measurements
Authors: Kuo Scot C. , Gelles Jeff , Steuer Eric , Sheetz Michael P.
Published: May 2013

Our detailed measurements of the movements of kinesin- and dynein-coated latex beads have revealed several important features of the motors which underlie basic mechanical aspects of the mechanisms of motor movements. Kinesin-coated beads will move along the paths of individual microtubule protofilaments with high fidelity and will pause at 4nm intervals along the microtubule axis under low ATP conditions. In contrast, cytoplasmic dynein-coated beads move laterally across many protofilaments as they travel along the microtubule, without any regular pauses, suggesting that the movements of kinesin-coated beads are not an artefact of the method. These kinesin bead movements suggest a model for kinesin movement in which the two heads walk along an individual protofilament in a hand-over-hand fashion. A free head would only be able to bind to the next forward tubulin subunit on the protofilament and its binding would pull off the trailing head to start the cycle again. This model is consistent with the observed cooperativity between the heads and with the movement by single dimeric molecules. Several testable predictions of the model are that kinesin should be able to bind to both alpha and beta tubulin and that the length of the neck region of the molecule should control the off-axis motility. In this article, we describe the technology for measuring nanometerlevel movements and the force generated by the kinesin molecule.
Characteristics of the motor responsible for the gliding of native microtubules from squid axoplasm
Authors: Weiss Dieter G. , Seitz-Tutter Dieter , Langford George M.
Published: May 2013

Nucleotide-dependent movement of native microtubules (nMTs) in squid axoplasm has biochemical and biophysical characteristics of kinesin-driven motility. However, the high vanadate and N-ethylmaleimide sensitivity and the velocity demonstrate that the properties of the native motile system differ considerably from those of purified kinesin preparations.
Antibodies against vertebrate microfilament proteins in the analysis of cellular motility and adhesion
Authors: Jockusch Brigitte M. , Zurek Barbara , Zahn Ralph , Westmeyer Annette , Füchtbauer Annette
Published: May 2013

Microinjection of specific antibodies can be an alternative and a supplement to genetic engineering in dissecting the function of individual cytoskeletal components. In this report, we describe some of the requirements for using this technique, its potential application in conjunction with morphological and biochemical analyses, and its limitations. Examples are given for the injection of antibodies to "-actinin, vinculin and myosin, and the effects of such treatment on adhesion, motility and cytokinesis of the recipient cells.
Tau protein and the establishment of an axonal morphology
Authors: Kosik Kenneth S. , Caceres Alfredo
Published: May 2013

Dissociated neuronal cultures from several regions of the nervous system elaborate two populations of neurites which have features of axons and dendrites. The microtubule-associated protein tau appears to segregate to the axon in some of these culture systems, however it does not do so until after the development of morphological polarity. Despite this observation, tau very likely has some role in the development of polarity because in cultured cerebellar macroneurons taken from the rat embryonic day 15 primordial cerebellum, the inhibition of tau expression by antisense techniques resulted in the failure of a single minor neurite to elongate and form an axon-like neurite. Tau antisense given continuously for up to 72 h kept neurons locked in a stage with minor neurites only; however when released from the effects of the antisense they fully recovered. The administration of tau antisense after the development of polarity resulted in the loss of the axonlike neurite, while dendrite-like neurites continued to grow. Together these results suggest that dendritic differentiation in cerebellar macroneurons requires the prior elaboration of an axon-like structure.
Intracellular organization of hippocampal neurons during the development of neuronal polarity
Authors: Dotti Carlos G. , Banker Gary
Published: May 2013

In culture, hippocampal neurons initially establish several short, apparently identical processes; of these, only one acquires axonal characteristics, the remainder becoming dendrites. We examined the organization of cytoplasmic constituents that might influence which of the initial processes becomes the axon. The Golgi complex was visualized using either fluorescent wheat germ agglutinin or a specific antibody. Presumptive microtubule-organizing centers were identified by depolymerizing microtubules with nocodazole, then allowing them to repolymerize for brief periods. As judged by light microscopy, hippocampal neurons contained a single Golgi region and a single microtubule-organizing center, which were frequently localized together adjacent to a shallow indentation in the nucleus. In cells fixed shortly after the axons had emerged, there was no correlation between the position of the Golgi complex or the microtubule organizing center and the site of origin of the axon. Based on nuclear shape, the position of the Golgi complex and microtubuleorganizing center could also be inferred in living cells. When axonal outgrowth was followed in individual cells by time-lapse microscopy, so that the location of the Golgi complex and microtubuleorganizing center could be determined at the exact moment when the axon emerged, no correlation was apparent. Antibodies that recognize specific post-translational modifications of a-tubulin-acetylation and de-tyrosination-were used to assess the distribution of arrays of stable microtubules. Stable microtubules were present in all processes, both before and after the emergence of the axon. They were not confined to the axon. Thus the localization of these cellular constituents does not play a major role in determining which of the processes initially extended by hippocampal neurons becomes the definitive axon.
Regulation of expression of glial filament acidic protein
Authors: Sarkar Srijata , Cowan Nicholas J.
Published: May 2013

The regulation of cell type-specific expression of the gene encoding glial filament acidic protein (GFAP) was examined by introducing various deletion mutants of the gene into GFAP-expressing (U251 human astrocytoma) and non-expressing (HeLa) cell lines, and measuring their transcriptional activity in an RNAase protection assay. The expression of GFAP is influenced by a number of cis-acting elements. A domain that resides between nucleotides-1631 and — 1479 can confer cell type-specific expression when coupled to a heterologous gene. We also present evidence for the existence of a negative regulatory element that resides within the first intron of the GFAP gene.
The expression and distribution of laminin in the developing nervous system
Authors: Edgar David
Published: May 2013

The extracellular matrix glycoprotein laminin exerts profound effects on the survival and differentiation of neurons in vitro. Although principally confined to the basement membranes of the adult extracellular matrix, during development laminin immunoreactivity may be found both within the interstitial extracellular matrix and on the membranes of neural cells, in which location it may be expected to affect their development in vivo. To investigate the reasons for the occurrence of laminin outside basement membranes, the expression of genes coding for laminin subunits has been analysed in mouse sciatic nerves at different postnatal ages. The results show that the expression of genes coding for laminin subunits decreases from high levels at birth, very low steady state levels of the individual mRNAs being found in the adult. Thus, the predicted high rate of laminin synthesis during development might exceed the rate at which it may be incorporated into basement membranes. The effective change in distribution of laminin into the basement membrane is therefore likely to be simply a consequence of the down-regulation of gene expression, rather than being caused, for example, by the differential expression of laminin variants.
Microtubule-associated proteins and neuronal morphogenesis
Authors: Matus Andrew
Published: May 2013

The microtubule-associated proteins (MAPs) are a set of structural proteins that bind to microtubules in vitro. Several of them occur at high levels in neurons where their expression is under strong developmental regulation, suggesting that they are involved in neuronal morphogenesis. Recently cDNAs for several of them have been cloned and sequenced revealing their primary structures and opening the way for genetic manipulation experiments aimed at determining their functions. Such experiments have shown that there are at least two classes of MAPs that are distinguished by the amino acid sequence motifs by which they bind to tubulin. One of these classes contains three known genes, two of which have been expressed in non-neuronal cells where the proteins cause bundling of microtubules and their rearrangement within the cytoplasm. The mechanism of this rearrangement is at present controversial. Another significant feature of these proteins is that several of them are differentially distributed within the neuronal cytoplasm; for example, some forms of MAP2 are selectively located in dendrites while in many situations MAP tau is limited to axons. In the case of MAP2, the mRNA that encodes the protein is also located in dendrites. This suggests that synthesis of MAP2 may be regulated locally in the dendritic cytoplasm. The molecular mechanism underlying the sorting of these proteins within neurons is unknown at present.
Developmental regulation of globin gene expression
Authors: Minie Mark , Clark David , Trainor Cecelia , Evans Todd , Reitman Marc , Hannon Robert , Gould Hannah , Felsenfeld Gary
Published: May 2013

We have used the globin family of genes in chicken to study developmental regulation of gene expression, both at the level of individual interaction of trans-acting factors with local promoters and enhancers, and at the level of chromatin structure. Regulation of all members of the a- and [3-globin clusters is affected by the erythroid regulatory factor GATA-1. Separate mechanisms exist for regulation of individual members of the family. As an example, we describe the control mechanisms that play a role in the expression of the p-globin gene, which is expressed only in primitive lineage erythroid cells. In addressing the involvement of chromatin structure in gene activation, we have examined the role of locus control elements, and also considered the way in which RNA polymerase molecules might accommodate to the presence of nucleosomes on transcribed genes.
Retinoid receptors and binding proteins
Authors: Lohnes David , Dierich Andrée , Ghyselinck Norbert , Kastner Phillipe , Lampron Carmen , Lemeur Marianne , Lufkin Thomas , Mendelsohn Cathy , Nakshatri Hari , Chambon Pierre
Published: May 2013

Retinoids, in particular all-trans retinoic acid (T-RA), are essential for normal development and homeostasis of vertebrates. Although many effects of retinoids, particularity with regard to teratogenicity, have been described in the literature, the mechanisms by which these simple signalling molecules work has only recently begun to be elucidated. We now recognize at least two classes of retinoid-binding proteins and two families of retinoid receptors. The ultimate interpretation of the retinoid signal within a given cell is probably the result of a complex series of interactions between these proteins, yet little is understood concerning the role each member of this signalling pathway plays. It is therefore imperative to dissect the molecular mechanisms which transduce the effects of these ligands, both in vivo and in isolated systems. One approach we are employing is gene targeting of retinoic acid receptors (RARs) and cellular retinoid-binding proteins to generate mice in which one or more of these genes has been functionally inactivated.
Synaptic vesicle proteins and regulated exocytosis
Authors: Elferink Lisa A. , Scheller Richard H.
Published: May 2013

The recent identification of novel proteins associated with the membranes of synaptic vesicles has ignited the field of molecular neurobiology to probe the function of these molecules. Evidence is mounting that the vesicle proteins vamp (synaptobrevin), rab3A, synaptophysin, synaptotagmin (p65) and SV2 play an important role in regulated exocytosis, by regulating neurotransmitter uptake, vesicle targeting and fusion with the presynaptic plasma membrane.
Membrane traffic in polarized neurons in culture
Authors: Hoop Meltsje J. de , Dotti Carlos G.
Published: May 2013

Fetal hippocampal neurons develop axons and dendrites in culture. To study how neurons form and maintain different plasma membrane domains, hippocampal neurons were infected with RNA viruses and the distribution of the viral glycoproteins was analyzed by light and electron microscopy. Infection of hippocampal cells with vesicular stomatitis virus (VSV) and fowl plague virus (FPV) resulted in the polarized distribution of the newly synthesized viral glycoproteins. The VSV glycoprotein appeared firstly in the Golgi apparatus and then in the dendrites. In contrast, the hemagglutinin of FPV, after accumulation in the Golgi apparatus, moved to the axons. These results suggest that the mechanism of sorting of viral glycoproteins might be similar in neurons and MDCK cells, a cell line of epithelial origin. In these cells the VSV glycoprotein and the hemagglutinin of FPV distribute to the basolateral and apical membranes, respectively. Transport of viral glycoproteins to both neuronal domains was microtubule dependent. Nocoda- zole treatment of infected neurons inhibited the delivery of axonal and dendritic viral glycoproteins equally.

To investigate if the analogy between epithelial cells and neurons extended to include an endogenous plasma membrane protein, the distribution of Thy-1, a GPI- linked protein, was analyzed. By immunofluorescence and immunoelectron microscopy, Thy-1 was found exclusively along the axonal surface. In epithelial cells GPI-anchored proteins are located apically.

The existence of a barrier on the neuronal plasma membrane that would prevent intermixing of axonal and dendritic proteins was analyzed by a liposomefusion assay. Fluorescently labeled liposomes containing the GDia ganglioside were added to FPV-infected neurons. The liposomes bound specifically to the hemagglutinin protein, expressed on the axonal surface. After fusion, fluorescent labelling was observed along the axon but not diffusing into the cell body and dendrites. The barrier that prevented lipid diffusion appeared to be located in the axonal hillock region.

Our work shows that experimental strategies that have proven useful in the understanding of membrane sorting in epithelial cells, can also be used to unveil the mechanism of neuronal sorting.
Traffic of synaptic vesicle proteins in polarized and nonpolarized cells
Authors: Cameron Patricia , Mundigl Olaf , Camilli Pietro De
Published: May 2013

Neurons have at least two pathways of regulated secretion, which involve two classes of secretory organelles: typical synaptic vesicles (SVs) and large dense-core vesicles. Large dense-core vesicles store and secrete peptide neurotransmitters and amines, and may be seen as the neuronal counterpart of secretory granules of endocrine cells. SVs are highly specialized secretory organelles, which store and secrete non-peptide hormones and play a dominant role in the fast, point-to-point signalling typical of the nervous system. Microvesicles that share a variety of biochemical and functional similarities with SVs (synaptic-like micro vesicles) have recently been described in endocrine cells. SVs and synaptic-like microvesicles are closely related to vesicular carriers of the receptor-mediated recycling pathway. They undergo repeated cycles of exo-endocytosis, which are thought to involve endosomal intermediates.

In mature neurons, SVs are concentrated in axon endings. To gain insight into the mechanisms responsible for SV targeting, we have studied the traffic of SV proteins in both endocrine cells and developing hippocampal neurons in primary culture at different stages of differentiation. Additionally, the distribution of the SV protein synaptophysin, when expressed by transfection in fibroblastic cells or in polarized epithelial cells (MDCK cells), was investigated.

SV proteins are already present in developing neurons at stages preceding the establishment of neuronal polarity. As axons and dendrites form, SV proteins are found in both types of processes, although they become progressively more concentrated in the axon. Throughout these developmental stages SVs undergo active exo-endocytotic recycling. The nonpolarized distribution of SV proteins is observed even at stages when the transferrin receptor, a protein that is present in epithelial cells only at the basolateral surface, is already restricted to dendrites. This indicates that, in immature neurons, SV proteins are not selectively targeted to axons and that the accumulation in axons may at least partially result from a specific retention. In agreement with this finding, synaptophysin, when transfected into MDCK cells, was targeted to both the basolateral and the apical plasma membrane. Brefeldin A, a fungal metabolite that induces a modification of the steady-state localization of endosomal proteins in a variety of cell types, was found to have a different effect on the distribution of SV proteins in dendrites and in axons. Taken together, these observations support the existence of two separate endosomal systems in axons and dendrites, which have differential properties, are enriched in different proteins, and may be related to the basolateral and apical endosomes of epithelial cells.
Biogenesis of structural intercellular junctions during cleavage in the mouse embryo
Authors: Fleming Tom P. , Javed Qamar , Collins Jane , Hay Mark
Published: May 2013

The preimplantation embryo differentiates the trophec-toderm epithelium which, from the 32-cell stage, generates the blastocoel of the blastocyst and, after implantation, gives rise to most extraembryonic lineages of the conceptus. Trophectoderm differentiation begins at compaction (8-cell stage) when cell-cell adhesion, mediated by uvomorulin, and epithelial cell polarisation first occur. Here, we review our work on the biogenesis of tight junctions and desmosomes during epithelial differentiation. Tight junction construction begins at compaction and appears to be a gradual process, both at morphological and molecular levels. This maturation pattern may be due in part to sequential expression of tight junction constituents from the embryonic genome. Tight junction formation is dependent upon uvomorulin adhesion but can be inhibited by different means without apparently disturbing cell adhesion or polarisation. Cell interactions appear to regulate tight junction tissue specificity, in part by controlling the level of synthesis of constituents. Desmosome formation begins at the 32cell stage, particularly as the embryo initiates blastocoel accumulation, and, in contrast with tight junction formation, does not appear to be a gradual process. Thus, nascent desmosomes appear mature in terms of their molecular composition. Desmosomal proteins are synthesised well in advance of desmosome formation but the synthesis of the principal glycoprotein components begins at the blastocyst stage and may regulate the timing of junction assembly. Implications of these differing patterns of biogenesis for the embryo are discussed.
Epithelial cell adhesion and development of cell surface polarity: possible mechanisms for modulation of cadherin function, organization and distribution
Authors: Näthke Inke S. , Hinck Lindsay E. , Nelson W. James
Published: May 2013

Epithelial cell adhesion is principally regulated by calcium-dependent cell adhesion proteins, termed cadherins. Recent studies indicate that cadherin function is modulated by a class of proteins, termed catenins, that bind to the cytoplasmic domain of cadherin. Here we review the evidence that catenins regulate cadherin function in cell-cell adhesion, and discuss their role in inititiating cell surface polarity in epithelial cells.
Protein traffic in polarized epithelial cells: the polymeric immunoglobulin receptor as a model system
Authors: Mostov Keith
Published: May 2013

As a model system to study protein traffic in polarized epithelial cells, we have used the polymeric immunoglobulin receptor. This receptor travels first to the basolateral surface, where it can bind polymeric IgA or IgM. The receptor is then endocytosed and delivered to endosomes. The receptor is sorted into transcytotic vesicles, which are exocytosed at the apical surface. The 103-amino acid cytoplasmic domain of the receptor contains several sorting signals. The 17 residues closest to the membrane are an autonomous signal that is necessary and sufficient for basolateral sorting. For rapid endocytosis there are two independent signals, both of which contain critical tyrosine residues. Finally, transcytosis is signaled by phosphorylation of a particular serine.
Role of heterotrimeric G proteins in polarized membrane transport
Authors: Pimplikar Sanjay W. , Simons Kai
Published: May 2013

MDCK cells maintain the polarized distribution of surface proteins mainly by sorting the newly synthesized proteins in the irans-Golgi network (TGN). In order to identify the components of the putative sorting machinery and to study factors that affect the sorting process, we have developed an in vitro system that reconstitutes the transport of viral glycoproteins from the TGN to the apical or basolateral surface. We have used this system to study effects of membrane impermeable reagents (such as peptides and antibodies) on the polarized transport. We observed that reagents affecting the stimulatory class (Gs) of heterotrimeric GTP binding proteins (G proteins) influenced the apical but not the basolat-eral transport. In contrast, reagents specific for the inhibitory class of G proteins (Gi) affected the basolateral but not the apical transport. These results show that the heterotrimeric G proteins differentially regulate the two pathways of polarized transport. The G proteins may regulate the process of polarized sorting of proteins in a fashion analogous to their role in signal transduction by providing a communication link with the cytosolic side of the membrane.
Endosomal pathways for water channel and proton pump recycling in kidney epithelial cells
Authors: Brown Dennis , Sabolic Ivan
Published: May 2013

The plasma membrane composition of virtually all eukaryotic cells is maintained and continually modified by the recycling of specific protein and lipid components. In the kidney collecting duct, urinary acidification and urinary concentration are physiologically regulated at the cellular level by the shuttling of proton pumps and water channels between intracellular vesicles and the plasma membrane of highly specialized cell types. In the intercalated cell, hydrogen ion secretion into the urine is modulated by the recycling of vesicles carrying a proton pumping ATPase to and from the plasma membrane. In the principal cell, the antidiuretic hormone, vasopressin, induces the insertion of vesicles that contain proteinaceous water channels into the apical cell membrane, thus increasing the permeability to water of the epithelial layer. In both cell types, ‘coated’ carrier vesicles are involved in this process, but whereas clathrin-coated vesicles are involved in the endocytotic phase of water channel recycling, the transporting vesicles in intercalated cells are coated with the cytoplasmic domains of the proton pumping ATPase. By a combination of morphological and functional techniques using FITC-dextran as an endosomal marker, we have shown that recycling endosomes from intercalated cells are acidifying vesicles but that they do not contain water channels. In contrast, principal cell vesicles that recycle water channels do not acidify their lumens in response to ATP. These non-acidic vesicles lack functionally important subunits of the vacuolar proton ATPase, including the 16 kDa proteolipid that forms the transmembrane proton pore. Because these endosomes are directly derived via clathrin-mediated endocytosis, our results indicate that endocytotic clathrin-coated vesicles are non-acidic compartments in principal cells. In contrast, recycling vesicles in intercalated cells contain large numbers of proton pumps, arranged in hexagonally packed arrays on the vesicle membrane. These pumps are inserted into the apical plasma membrane of A-type (acid-secreting) intercalated cells, and the baso-lateral plasma membrane of B-type (bicarbonate-secreting) cells in the collecting duct. Both apical and baso-lateral targeting of H+-ATPase-containing vesicles in these cells may be directed by microtubules, because polarized insertion of the pump into both membrane domains is disrupted by microtubule depolymerizing agents. However, the basolateral localization of other transporting proteins in intercalated cells, including the band 3-like anion exchanger and facilitated glucose transporters, is not affected by microtubule disruption.
The role of PRP8 protein in nuclear pre-mRNA splicing in yeast
Authors: Beggs Jean D. , Teigelkamp Stefan , Newman Andrew J.
Published: May 2013

The removal of introns from precursor messenger RNAs occurs in a large complex, the spliceosome, that contains many proteins and five small nuclear RNAs (snRNAs). The snRNAs interact with the intron-containing substrate RNA and with each other to form a dynamic network of RNA interactions that define the intron and promote splicing. There is evidence that protein splicing factors play important roles in regulating RNA interactions in the spliceosome. PRP8 is a highly conserved protein that is associated in particles with the U5 snRNA and directly binds the substrate RNA in spliceosomes. UV crosslinking has been used to map the binding sites, and shows extensive interaction between PRP8 protein and the 5′exon prior to the first step of splicing and with the 3′splice site region subsequently. It is proposed that PRP8 protein may stabilize fragile interactions between the U5 snRNA and exon sequences at the splice sites, to anchor and align them in the catalytic centre of the spliceosome.
Studies of DNA methylation in animals
Authors: Bird Adrian , Tate Peri , Nan Xinsheng , Campoy Javier , Meehan Richard , Cross Sally , Tweedie Susan , Charlton Jillian , Macleod Donald
Published: May 2013

We have been studying the evolution and function of DNA methylation in vertebrate animals using three related approaches. The first is to further characterise proteins that bind to methylated DNA. Such proteins can be viewed as ‘receptors′ of the methyl-CpG ‘ligand’ that mediate downstream consequences of DNA modification. The second approach involves CpG islands. These patches of non-methylated DNA coincide with most gene promoters, but their origin and functional significance have only recently become the subject of intensive study. The third approach is to trace the evolution of DNA methylation. Genomic methylation patterns of vertebrates are strikingly different from those of invertebrates. By studying methylation in animals that diverged from common ancestors near to the invertebrate/vertebrate boundary, we will assess the possibility that changes in DNA methylation contributed causally to the evolution of the complex vertebrate lineage.
Remarkable Effect of Bases on Core–Shell AgNP@CeO2 Nanocomposite-catalyzed Highly Chemoselective Reduction of Unsaturated Aldehydes
Authors: Mitsudome Takato , Matoba Motoshi , Yamamoto Masaaki , Mizugaki Tomoo , Jitsukawa Koichiro , Kaneda Kiyotomi
Published: May 2013

A highly dispersed core–shell silver nanoparticle–ceria nanocomposite catalyst (AgNP@CeO2-D) was prepared. The addition of bases was found to enhance the catalytic efficiency of AgNP@CeO2-D significantly in the chemoselective reduction of diverse unsaturated aldehydes to the corresponding unsaturated alcohols.
Boundary mechanisms in adverts from Silesian Catholic periodicals from the second half of the 19th and early 20th centuries
Authors: Haładewicz-Grzelak Małgorzata , Lubos-Kozieł Joanna
Published: May 2013

The paper provides an empirical study of semiotic mechanisms of culture. We apply the methodology developed by the Tartu-Moscow School of Semiotics, building also on the criteria of boundary-work dynamics to examine a collected corpus of adverts appearing in Silesian Catholic periodicals (in German and in Polish) from the second half of the 19th and early 20th centuries. We discuss the cultural implications of the differences and similarities in German and Polish ads and propose functional explanations of the results in terms of the notion of boundary configurations in a region as a particular structuring of cultural codes. The two analytical axes are the social boundary implicated in the use of German vs. Polish on the parameter of 'sacred' (sacrum) reference, and the symbolic border in the use of Fraktur (German script) versus Antiqua (Latin script) (boundary objects).
Enchantment of the past and semiocide. Remembering Ivar Puura
Authors: Maran Timo
Published: May 2013

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Zawłaszczenie
Authors: Esposito Roberto
Published: May 2013

Immunizująca funkcja, jaką względem wspólnoty pełni prawo, jest oczywista i ogólnie uznana, także poza obszarem literatury prawniczej. Od samego zarania prawo stoi na straży współżycia międzyludzkiego, które ze swej natury narażone jest na działanie destrukcyjnych konfliktów. Najbardziej źródłowa relacja łączy zatem prawo nie z jego formalną organizacją, ale z samym życiem wspólnoty. To właśnie w tym pierwotnym i radykalnym znaczeniu należy rozumieć immunizującą rolę prawa: podobnie jak w sferze biologiczno‑medycznej w odniesieniu do ludzkiego organizmu, gwarantuje ono wspólnocie przetrwanie w sytuacji śmiertelnego zagrożenia. Chroni ją i przedłuża jej życie, oddalając od niej widmo niechybnej śmierci. Odniesienie do obszaru semantycznego medycyny pozwala uchwycić bardziej złożone i niepokojące znaczenie immunizacji prawnej. Tak jak w przypadku ludzkiego ciała, immunizująca funkcja prawa wobec wspólnoty nie może spełnić się w sposób bezpośredni i – by tak rzec – afirmatywny. Przeciwnie: droga wiodąca do celu jest niebezpośrednia, pokrętna i na swój sposób perwersyjna. Celu nie da się bowiem osiągnąć inaczej, jak tylko za pomocą środka, który – choć tylko częściowo i czasowo – kłóci się z nim, gdyż zawiera w sobie cząstkę tej samej substancji, przed którą ma chronić.
Aetiopathogenesis of sandcrack in cattle and its treatment
Authors: Murray Richard D , Williams Helen J , Bardell David A
Published: May 2013

Sandcrack is the most acutely painful claw horn defect that affects dairy and beef cattle. It is the most common lesion associated with cattle lameness that is referred by practising veterinary surgeons to the University of Liverpool's Farm Animal Hospital. The key to its successful treatment is excellent restraint and analgesia that enables precise surgical removal of all under-run and abnormal growing horn from surrounding necrotic laminae and corium.