bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylatioh of vertebrate smooth muscle and nonmuscle myosin heavy chains in vitro and in intact cells
Authors: Kelley Christine A. , Kawamoto Sachiyo , Conti Mary Anne , Adelstein Robert S.
Published: May 2013

In this article we summarize our recent experiments studying the phosphorylation of vertebrate myosin heavy chains by protein kinase C and casein kinase II. Protein kinase C phosphorylates vertebrate nonmuscle myosin heavy chains both in vitro and in intact cells. A single serine residue near the end of the helical portion of the myosin rod is the only site phosphorylated in a variety of vertebrate nonmuscle myosin heavy chains. There does not appear to be a site for protein kinase C phosphorylation in vertebrate smooth muscle myosin heavy chains. Casein kinase II phosphorylates a single serine residue located near the carboxyl terminus of the 204×103 Mr smooth muscle myosin heavy chain in vitro as well as in cultured smooth muscle cells. It does not phosphory-late the 200×103Mr smooth muscle myosin heavy chain. However, the site is present in vertebrate nonmuscle myosin heavy chains. The 204×103Mr myosin heavy chain of embryonic chicken gizzard smooth muscle is exceptional in not containing a site for casein kinase II phosphorylation.
A myosin-like protein from smooth muscle
Authors: Kohama Kazuhiro , Lin Yuan , Takano-Ohmuro Hiromi , Ishikawa Ryoki
Published: May 2013

A protein was purified from chicken gizzard smooth muscle. It bound ATP and actin. Actin activated the Mg2+-ATPase activity of this protein. The Ca2+-ATPase activity was lower than K+-EDTA ATPase activity. Thus, it appears that this protein is akin to myosin I rather than to conventional myosin. However, ATPase activities of the protein were much lower than those of myosin I. A protein cofactor, such as protein kinase, which would enhance these activities remains to be purified from the smooth muscle.
The use of native thick filaments in in vitro motility assays
Authors: Sellers James R. , Han Yung Jin , Kachar Bechara
Published: May 2013

Native thick filaments from the clam, Mercinaria mercinaria translocate actin filaments both toward and away from the center of the thick filament in an in vitro motility assay. The thick filaments from the adductor muscle are about 10 gm long whereas those from the catch muscle are 30-50 gm long. These thick filaments should prove useful in understanding the mechanism of myosin-dependent movement of actin filaments.
Protein engineering and the study of muscle contraction in Drosophila flight muscles
Authors: Sparrow John , Drummond Douglas , Peckham Michelle , Hennessey Emma , White David
Published: May 2013

We describe an experimental approach to the use of genetics to study muscle contraction in Drosophila melanogaster. Mutations induced by in vitro mutagenesis are inserted into the genome of flies using P-element mediated transformation, permitting the effects of the mutant genes to be studied in vivo in the indirect flight muscles (IFMs). Details of how mechanical experiments can be performed on skinned IFMs, despite their small size, are provided. The effects of two in vitro actin mutations, G368E and E316K, are described. The problems of performing biochemical and biophysical experiments on the IFMs and their myofibrillar proteins are described, together with indications as to how these may be overcome.
Bidirectional movement of actin filaments along tracks of heavy meromyosin and native thick filaments
Authors: Toyoshima Yoko Yano
Published: May 2013

Flexibility of the myosin molecule was studied by an in vitro motility assay in terms of the direction of actin movement. Actin filaments can move in both directions on tracks of heavy meromyosin made on a nitrocellulose surface, and, furthermore, along the native thick filaments passing over their central bare zone. These observations indicate that the myosin molecule has a considerable flexibility in interacting with actin filaments.
Kinesin interactions with membrane bounded organelles in vivo and in vitro
Authors: Brady Scott T. , Pfister K. Kevin
Published: May 2013

The ability of kinesin to interact with microtubules in a nucleotide-dependent manner and mediate microtubule-based motility has received the greatest amount of attention to date. Several lines of experimentation are now beginning to examine the interaction with membrane-bounded organelles. Immunochemical, biochemical and morphological approaches have shown that kinesin is associated with some, but not all, classes of membrane-bounded organelles found in cells. Similarly, evidence suggests that the distal portion of the rod and the tail portions of the kinesin heavy chain as well as the kinesin light chains may be important for the interaction with membrane surfaces. As a substantial amount of information about the molecular structure and biochemistry of kinesin has become available, the functional implications of interactions with membrane structures in vivo are being addressed.
Purification and assay of kinesin from sea urchin eggs and early embryos
Authors: Buster Dan , Scholey Jonathan M.
Published: May 2013

This paper describes the procedures used to purify the microtubule motor, kinesin, from mitotic cells, namely sea urchin eggs and cleavage stage embryos, and describes methods for assaying its motor activity.
Axonemal dynein from Tetrahymena
Authors: Crossley E. M. , Hyman S. C. , Wells C.
Published: May 2013

Axonemal dynein from Tetrahymena cilia can be separated on a sucrose gradient into two fractions, at least one of which appears to be polymorphic. We have been using immuno-electron microscopy in order to try and locate the different types of dynein molecules within the axonemal structure.
Preparation of marked microtubules for the assay of the polarity of microtubuie-based motors by fluorescence
Authors: Hyman A. A.
Published: May 2013

Short microtubule seeds are constructed using heavily rhodamine-labeled tubulin. Polymerisation off the ends of these seeds is initiated using a mixture of 1:10 labeled and unlabeled tubulin, so that the new polymerisation is only dimly labeled. This is done in the presence of NEM tubulin, which inhibits growth from the microtubule minus ends. The polarity-marked microtubules are fixed at a desired length by adding taxol.
Dynamin: A microtubule-associated GTP-binding protein
Authors: Obar Robert A. , Shpetner Howard S. , Vallee Richard B.
Published: May 2013

We recently identified dynamin as a third nucleotide-sensitive microtubule-associated protein in brain tissue, in addition to kinesin and cytoplasmic dynein. Molecular cloning analysis has revealed that dynamin contains the three consensus elements characteristic of GTP-binding proteins, and biochemical results support a role for GTP in dynamin function. Dynamin is also homologous to the Mx proteins, involved in interferon-induced viral resistance, and the product of the yeast VPS1 gene, involved in vacuolar protein sorting. These results identify a novel class of GTP-utilizing proteins, with apparently diverse functions.
From genes to tensile forces: genetic dissection of contractile protein assembly and function in Drosophila melanogaster
Authors: Fyrberg Eric , Beall Clifford , Fyrberg Christine C.
Published: May 2013

Myoflbrils, the contractile organelles of skeletal muscle, are highly ordered and precisely regulated actomyosin networks. Investigations of myofibril assembly are revealing the cellular mechanisms by which contractile components are arranged and regulated. In order to facilitate this research we have developed formal molecular genetics for myofibrillar proteins of Drosophila flight muscle. Presently, mutations can be used systematically to perturb or eliminate any of the classical myofibrillar proteins within these fibers, and the in vivo consequences can be conveniently evaluated using protein electrophoresis, electron microscopy, or by assaying flight performance. Here we review some recent progress.
Differential effects of laminin and merosin on neurite outgrowth by developing retinal ganglion cells
Authors: Cohen James , Johnson Alan R.
Published: May 2013

In previous work, we showed that cultured avian embryonic retinal ganglion cells (RGC) extend neurites on EHS-laminin early in development, but lose this ability with maturation, as a result of a sharp decline in laminin receptor numbers. Here we show that EHS-laminin promotes neurite outgrowth also from embryonic mammalian RGC, in contrast to previous reports, and that these exhibit similar agedependent growth responses on laminin. Antibody blocking studies show that this behaviour is mediated in mouse RGC by 㯁6β1 integrin dimers. The laminin isoform merosin is also effective as a neurite outgrowth-promoting substrate for RGC but differs in its ability to elicit a response at advanced stages of development (up to hatching in the chick). Neurite outgrowth by RGC on merosin is inhibited, at all ages, by the function-blocking, anti-αβ integrin antibody, CSAT, suggesting that these neurons use alternative αβ1 dimers in their interactions with EHS-laminin and merosin. Together, these findings emphasise the generality of the responsiveness of vertebrate embryonic RGC to laminin during development, and reveal interesting differences in the effects of laminin variants on CNS axon growth and regeneration.
Microtubule protein phosphorylation in neuroblastoma cells and neurite growth
Authors: DÍAz-Nido J. , Armas-Portela R. , Correas I. , Dominguez J. E. , Montejo E. , Avila J.
Published: May 2013

The development of highly asymmetrical neurones from undifferentiated neuroblasts involves the extension of processes (axon and dendrites), that depends on the assembly of an inner microtubule scaffolding. Clonal cell lines of neuronal origin, N2A and NIE-115 neuroblastoma cells, have been chosen as model systems to study the modifications of microtubule protein which accompany the outgrowth of axon-like processes (neurites). Neuroblastoma cells grow as proliferating and undifferentiated cells in standard culture medium but can be considered as committed neuronal precursors. Thus, they are characterized by a high content of tubulin, including the minor neuronal-specific isoform, and of MAPs including MAP1B and tau-like proteins. Serum withdrawal from the culture medium results in the extension of axon-like processes which is paralleled by a net increase in the amount of assembled tubulin. However, there is not any increase in the total amount of either tubulin or major MAPs which suggests an involvement of other regulatory factors in the promotion of microtubule assembly. Of relevance in this respect is the fact that j83-tubulin, MAP1B, and tau-like proteins become phosphorylated during neurite extension.

A casein kinase Il-like enzyme may be involved in some of these phosphorylation events. This enzyme is primarily localized to the nuclei in undifferentiated neuroblastoma cells, whereas a wider distribution of the enzyme between the nucleus and the cytoplasm is found in differentiating neuroblastoma cells. It thus appears plausible that a modified sorting of casein kinase II into the nucleus and the cytoplasm may be involved in the triggering of the phosphorylation of microtubule proteins during neuroblastoma cell differentiation.
Identification of Fos target genes by the use of selective induction systems
Authors: Braselmann Sylvia , Bergers Gabriele , Wrighton Christopher , Graninger Paula , Superti-Furga Giulio , Busslinger Meinrad
Published: May 2013

c-Fos is a major component of the transcription factor AP-1 which has been implicated in the control of cell proliferation and differentiation as well as in transformation. In order to identify Fos target genes involved in these processes, we have taken advantage of the regulatory properties of the hormone-binding domain of the human estrogen receptor to develop transcriptional and post-translational induction systems, both of which allow selective elevation of Fos activity within a cell. Using this approach we have searched for Fos-responsive genes in rat fibroblasts and PC12 cells. Here we describe the identification and regulation of five Fosresponsive genes encoding a transcription factor (Fra-1), a secreted protein (Fit-1), a biosynthetic enzyme (ODC) and two membrane-associated proteins (annexin II and V), respectively. The post-translational induction system was also used to study the Fos-mediated block of neuronal differentiation of PC12 cells. These experiments demonstrate that Fos activity is dominant over NGF function and interferes with the expression of late NGF-inducible genes.
The role of the transcriptional activator protein DBP in circadian liver gene expression
Authors: Wuarin JÉRÔMe , Falvey Eileen , Lavery Dan , Talbot Dale , Schmidt Ed , Ossipow Vincent , Fonjallaz Philippe , Schibler Ueli
Published: May 2013

DBP, a liver-enriched transcriptional activator protein of the leucine zipper protein family, accumulates according to a very strong circadian rhythm (amplitude approx. 1000-fold). In rat parenchymal hepatocytes, the protein is barely detectable during the morning hours. At about 2 p.m., DBP levels begin to rise, reach maximal levels at 8 p.m. and decline sharply during the night. This rhythm is free-running: it persists with regard to both its amplitude and phase in the absence of external time cues, such as daily dark/light switches. Also, fasting of rats for several days influences neither the amplitude nor the phase of circadian DBP expression. Since the levels of DBP mRNA and nascent transcripts also oscillate with a strong amplitude, circadian DBP expression is transcriptionally controlled. While DBP mRNA fluctuates with a similar phase and amplitude in most tissues examined, DBP protein accumulates to high concentrations only in liver nuclei. Hence, at least in nonhepatic tissues, cyclic DBP transcription is unlikely to be controlled by a positive and/or negative feedback mechanism involving DBP itself. More likely, the circadian DBP expression is governed by hormones whose peripheral concentrations also oscillate during the day. Several lines of evidence suggest a pivotal role of glucocorticoid hormones in establishing the DBP cycle.

Two genes whose mRNAs and protein products accumulate according to a strong circadian rhythm with a phase compatible with regulation by DBP encode enzymes with key functions in cholesterol metabolism: HMG-coA reductase is the rate-limiting enzyme in cholesterol synthesis; cholesterol 7-a hydroxylase performs the rate-limiting step in the conversion of cholesterol to bile acid. DBP may thus be involved in regulating cholesterol homeostasis.
Characterization of recombinant E-cadherin (uvomorulin) expressed in insect cells
Authors: Herrenknecht Kurt , Kemler Rolf
Published: May 2013

Cadherins are Ca2+-dependent cell adhesion molecules that mediate cell adhesion by homophilic binding. Structural and functional analysis of the extracellular part of cadherins that mediates this binding has often been hampered by the availability of sufficient amount of protein. Therefore, we have expressed the extracellular region of E-cadherin (uvomorulin) using the baculovirus expression vector system (BEVS). A recombinant baculovirus was generated that encodes the signal peptide, the precursor region and the extracellular part of the mature protein, under the control of the promotor for polyhedrin. Infection of insect cells with recombinant virus led to the expression of about 40 mg of the E-cadherin fragment per 2×109 infected cells. About half of the protein synthesized was secreted, either as mature protein or in its unprocessed form. The precursor peptide was removed by trypsin treatment in the presence of Ca2+ and recombinant protein was purified to homogeneity. Biochemical characterization of the recombinant protein revealed a high degree of similarity with the mouse wild-type protein. Recombinant protein exhibited the known resistance to trypsin in the presence of Ca2+ and was recognized by two different conformation-sensitive monoclonal anti-E-cadherin antibodies. Rabbit antibodies made against the recombinant protein recognized E-cadherin from different species. In spite of the high degree of structural resemblance recombinant E-cadherin was not able to inhibit E-cadherin mediated cell-cell adhesion.
Patches and fences: probing for plasma membrane domains
Authors: Edidin Michael
Published: May 2013

Cell plasma membranes appear to be composed of domains, patches whose composition and function differ from the average for an entire membrane surface. Proteins and lipids may be segregated into domains by different mechanisms. Some of these mechanisms are discussed, followed by a summary of the evidence for membrane domains obtained in my laboratory. This evidence is largely based on measurements of the lateral diffusion of membrane proteins and lipids. Recent new approaches to the interpretation of lateral diffusion measurements, consideration of so-called fractal or long time-tails promise to give new insights into the stability and lifetime of membrane domains.
Responses to Wnt signals in vertebrate embryos may involve changes in cell adhesion and cell movement
Authors: Moon Randall T. , DeMarais Alyce , Olson Daniel J.
Published: May 2013

Wnt genes encode secreted glycoproteins, and, because of their homology with the Drosophila segment polarity gene wingless, are likely to play important roles as modulators of local intercellular signalling during embryonic development. Although little is known of the mechanisms by which Wnts signal in an autocrine or paracrine manner, it is increasingly clear that cells can respond rapidly to Wnt signals in the absence of transcription, and that these responses may include changes in cell adhesion and cell movement. We review recent evidence from studies on Xenopus laevis and other sys-terns, which demonstrate that (1) a subset of Wnts modulate gap junctional permeability, which may be a reflection of changes in cadherin-mediated cell adhesion, (2) embryos express β-catenin and plakoglobin, which are homologs of the armadillo gene products, known to be involved in the wingless signalling pathway, and known to be found at cell junctions, and (3) overexpression of specific Wnts in Xenopus embryos leads to clear changes in cell behavior and movement.
Defective acidification of the biosynthetic pathway in cystic fibrosis
Authors: Barasch Jonathan , Al-Awqati Qais
Published: May 2013

Cystic fibrosis is associated with defective epithelial sodium chloride and fluid secretion in epithelia. In addition, there is widespread reductions in sialylation of secreted proteins and increases in the sulfation and fucosylation of mucus glycoproteins. The major morbidity in the disease is due to the colonization of respiratory epithelia by Pseudomonas. The cystic fibrosis gene (CFTR) is a cyclic AMP activated Cl channel, which when mutated is retained in the endoplasmic reticulum. We postulate that this Cl channel is responsible for effective acidification of the Golgi. In CF cells, we demonstrate the Golgi pH is higher than in normal cells and suggest that the abnormalities in glycoprotein biosynthesis is due to changes in the kinetics of sialyl transferase, a pH sensitive enzyme. Defects in sialylation also result in decreased sialylation of glycolipids and asialogangliosides are potential Pseudomonas receptors.
Phenotypic conversions in renal development
Authors: Herzlinger D. , Abramson R. , Cohen D.
Published: May 2013

The transporting epithelia of the kidney are derived from an embryonic rudiment containing two distinct cell populations: ureteric bud epithelia and mesenchymal cells of the metanephric blastema. The ureteric bud is a caudal outgrowth of the Wolffian Duct and gives rise to the renal collecting system by branching morphogenesis. The metanephric blastema gives rise to diverse cells of the nephron after receiving an inductive stimulus. It has been proposed that mesenchymal progenitors of the metanephric blastema derive directly from intermediate mesoderm, although this hypothesis has never been tested directly. Utilizing direct lineage analysis techniques we demonstrate, in an organ culture system, that mesenchymal nephron progenitors are immediate descendants of ureteric bud epithelia. Ureteric bud epithelia can give rise to mesenchymal nephron progenitors that populate the metanephric blastema by undergoing an epithelial-to-mesenchymal transition followed by delamination. If this process occurs in vivo, renal morphogenesis can be characterized by two phenotypic conversions: an epithelial-to-mesenchymal transition leading to the generation of mesenchymalnephron progenitors, followed by a mesenchymal-to-epithelial transition leading to the generation of diverse nephron epithelial cell types. We have immortalized an embryonic renal mesenchymal cell line and demonstrate that the clonal cell line, RSTEM-1, undergoes phenotypic conversions in vitro, providing a suitable model to study the regulation of the epithelial phenotype.
Recognition and processing of damaged DNA
Authors: Lindahl Tomas
Published: May 2013

Base excision-repair, which is required for correction of spontaneous hydrolytic and oxidative damage to DNA as well as lesions inflicted by alkylating agents, is a relatively well understood repair pathway. Mammalian factors involved in this pathway are reviewed, with emphasis on current uncertainties.

Most DNA replication and repair enzymes in mammalian cell nuclei, e.g. DNA polymerases α, β, δ, and ε, have direct counterparts in yeast. In contrast, the abundant enzymes in mammalian cell nuclei that bind and are activated specifically by DNA strand interruptions, poly(ADP-ribose) polymerase and DNA-dependent protein kinase, have not been detected in yeast; nor has p53, which is elevated in response to DNA strand breaks. We have found a family of four distinct DNA ligases in human cell nuclei, whereas only a single DNA ligase has been detected in yeast. It would appear that the cellular responses to DNA strand breaks may differ markedly between higher and lower eukaryotes.
Appearance of apparently ubiquitin-conjugated lκB-α during its phosphorylation-induced degradation in intact cells
Authors: Traenckner E. Britta-Mareen , Baeuerle Patrick A.
Published: May 2013

NF-κ B is a dimeric protein that serves to initiate gene transcription in higher eukaryotic cells in response to mainly pathogenic stimuli. Its activity is controlled by a third inhibitory subunit, called IκB. When IκB is bound, NF-κB cannot bind to DNA or enter the nucleus but is stored in a latent cytoplasmic form. Upon stimulation of cells IκB is released, which allows the activation of NF-κB. We have analyzed the molecular mechanism underlying the removal of IκB-α. Distinct extracellular stimuli lead to a phosphorylation of IκB-α on serines 32 and 36 by a yet unidentified kinase. These modifications do not directly dissociate IκB from NF-κB but render the inhibitor highly susceptible for proteolytic degradation by, presumably, the proteasome. In this paper, we report for the first time that higher molecular mass forms of IκB-α occur under conditions that lead to a phosphorylation of IκB-α and activation of NF-KB. These IκB-α variants had discrete molecular masses and were most prominent in cells overexpressing IκB-α, suggesting the covalent modification of IκB-α by ubiquitin conjugation. The proteasome inhibitor Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (PSI), which stabilizes the phospho form of IκB-α, only slightly increased the amount of conjugates indicating that the conjugation of IκB-α with ubiquitin was the rate-limiting step in I κ B-α degradation, and not its phosphorylation or proteolysis. Our data suggest that conjugation of IκB-α with ubiquitin is an intermediate reaction in the phosphorylation-controlled degradation of IκB-α and the subsequent activation of NF-κB.
Regulation of cell proliferation and differentiation by Myc
Authors: Hopewell Robert , Li Linheng , MacGregor Douglas , Nerlov Claus , Ziff Edward B.
Published: May 2013

Myc is a nuclear phosphoprotein which controls cellular proliferation, most likely by regulating gene activity. The finding that the neuronal model cell line PC12 lacks the Myc DNA binding partner, the Max protein, and the demonstration that Myc is a repressor of gene activity as well as a transactivator, lead to models for Myc action in regulating cell growth.
Regulation of transcription by E2F1/DP1
Authors: Martin Klaus , Trouche Didier , Hagemeier Christian , Kouzarides Tony
Published: May 2013

The E2F1 transcription factor, in co-operation with DPI, controls the expression of several S-phase specific genes. This activity is most likely responsible for the oncogenic and S-phase inducing properties of E2F1, suggesting that this transcription factor plays a key role in regulating the cell cycle. The transcriptional activation functions of E2F1 are resident in a small C-terminal domain which can inde-pendently activate transcription. Here we review the protein-protein interactions which impinge upon and regulate this activation domain and put forward some models on their mechanism of action.
Does the Wilms’ tumour suppressor gene, WT1, play roles in both splicing and transcription?
Authors: Charlieu Jean-Paul , Larsson Stefan , Miyagawa Kiyoshi , Heyningen Veronica van , Hastie Nicholas D.
Published: May 2013

The Wilms’ tumour suppressor gene (WT1) encodes a protein(s) with 4 zinc fingers that is essential for the development of the genitourinary system. A considerable body of evidence exists to support the idea that WT1 binds DNA and functions as a transcription factor. However, we have shown recently by confocal microscopy and immunoprecipitation studies that a significant proportion of WT1 is associated with splice factors in kidney cell lines, fetal tissues and transfected Cos cells. Different isoforms of WT1 are produced by an alternative splice that leads to the presence or absence of a 3 amino acid insertion (KTS) between zinc fingers 3 and 4. We have shown that these different forms localise differently in the nucleus. The +KTS form mainly localises with splice factors, the −KTS form mainly with transcription factors. Here we propose a model to account for these different localisations. Also, we discuss the possible significance of these findings.
Protein kinases in the control of mitosis: focus on nucleocytoplasmic trafficking
Authors: Gallant P. , Fry A. M. , Nigg E. A.
Published: May 2013

The eukaryotic cell nucleus is a highly dynamic organelle. This is illustrated most dramatically during mitosis, when the nuclear envelope breaks down, the nuclear lamina disassembles, chromosomes condense, and a microtubulebased spindle apparatus distributes sister chromatids to the dividing daughter cells. Many of these dramatic changes in nuclear architecture and microtubule organization are controlled by phosphorylation and dephosphorylation events. Whereas the cardinal role of cyclin-dependent kinases (CDKs) in the regulation of mitosis is well established, there is now clear evidence for the requirement of additional mitotic protein kinases. Studies into the regulation of CDKs and other mitotic kinases have revealed that these enzymes undergo cell cycle dependent changes in subcellular distribution, suggesting that localization may contribute to regulating their activities. This article describes some recent findings relating to the nucleocytoplasmic translocation of CDK/cyclin complexes at the onset of mitosis. In addition, it summarizes recent information on two novel human protein kinases which have been implicated in the control of mitotic progression.
Analysis of the temporal program of replication initiation in yeast chromosomes
Authors: Friedman Katherine L. , Raghuraman M. K. , Fangman Walton L. , Brewer Bonita J.
Published: May 2013

The multiple origins of eukaryotic chromosomes vary in the time of their initiation during S phase. In the chromosomes of Saccharomyces cerevisiae the presence of a functional telomere causes nearby origins to delay initiation until the second half of S phase. The key feature of telomeres that causes the replication delay is the telomeric sequence (C1-3A/G1-3-3T) itself and not the proximity of the origin to a DNA end. A second group of late replicating origins has been found at an internal position on chromosome XIV. Four origins, spanning ∼140 kb, initiate replication in the second half of S phase. At least two of these internal origins maintain their late replication time on circular plasmids. Each of these origins can be separated into two functional elements: those sequences that provide origin function and those that impose late activation. Because the assay for determining replication time is costly and laborious, it has not been possible to analyze in detail these ‘late′ elements. We report here the development of two new assays for determining replication time. The first exploits the expression of the Escherichia coli dam methylase in yeast and the characteristic period of hemimethylation that transiently follows the passage of a replication fork. The second uses quantitative hybridization to detect two-fold differences in the amount of specific restriction fragments as a function of progress through S phase. The novel aspect of this assay is the creation in vivo of a non-replicating DNA sequence by site-specific pop-out recombination. This non-replicating fragment acts as an internal control for copy number within and between samples. Both of these techniques are rapid and much less costly than the more conventional density transfer experiments that require CsCl gradients to detect replicated DNA. With these techniques it should be possible to identify the sequences responsible for late initiation, to search for other late replicating regions in the genome, and to begin to analyze the effect that altering the temporal program has on chromosome function.
On the structure of replication and transcription factories
Authors: Hughes Thomas A. , Pombo Ana , McManus John , Hozák Pavel , Jackson Dean A. , Cook Peter R.
Published: May 2013

Recent experiments suggest that active polymerases are concentrated in large structures, ‘factories’, within eukaryotic nuclei. Data concerning the structure of these factories is reviewed.
Stepwise assembly of initiation complexes at budding yeast replication origins during the cell cycle
Authors: Diffley John F. X. , Cocker Julie H. , Dowell Simon J. , Harwood Janet , Rowley Adele
Published: May 2013

DNA replication is a pivotal event in the cell cycle and, as a consequence, is tightly controlled in eukaryotic cells. The initiation of DNA replication is dependent upon the completion of mitosis and upon the commitment to complete the cell cycle made during G1. Characterisation of the protein factors required for initiating DNA replication is essential to understand how the cell cycle is regulated. Recent results indicate that initiation complexes assemble in multiple stages during the cell cycle. First, origins are bound by the multisubunit origin recognition complex (ORC) which is essential for DNA replication in vivo. ORC, present at little more than one complete complex per replication origin, binds to origins immediately after initiation in the previous cell cycle. ORC binding occurs by the recognition of a bipartite sequence that includes the essential ARS consensus sequence (ACS) and the functionally important Bl element adjacent to the ACS. A novel pre-replicative complex (pre-RC) assembles at origins at the end of mitosis in actively cycling cells and remains at origins until DNA replication initiates. Finally, Dbf4, which is periodically synthesised at the end of Gi, interacts with replication origins. Dbf4-origin interaction requires an intact ACS strongly suggesting that interaction occurs through ORC. Dbf4 interacts with and is required for the activation of the Cdc7 protein kinase and together, Dbf4 and Cdc7 are required for the Gi-S transition. Separate regions of Dbf4 are required for Cdc7- and origin-interaction suggesting that Dbf4 may act to recruit Cdc7 to replication origins where phosphorylation of some key component may cause origin firing.
Nature in our memory
Authors: Puura Ivar
Published: May 2013

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Wspólnota, immunizacja, życie – o filozofii politycznej Roberta Esposita
Authors: Burzyk Mateusz , Sawczyński Piotr
Published: May 2013

Community, Immunization, Life – On the Political Philosophy of Roberto Esposito The article aims to present and to problematize the political philosophy of a representative of the reviving Italian thought – Roberto Esposito. The most fundamental question is his original understanding of the concept of the community. Its meaning, which is derived from Latin communitas, bases on a particular combination of gift, office and obligation. Such an understanding of the term is radically opposed to the all conceptualisations of the social contract: the community is rather a deprivation and a suppression of what is individual than a rationally chosen and additionally possessed form of one’s own existence. As a counterweight to the risk of the common, the logic of immunization (exception to the gift‑giving obligation) is portrayed. Last two parts focus on the issue of biopolitics. Using the reflections of Foucault, Derrida, Agamben, Deleuze & Guattari, Negri & Hardt as a theoretical background, the stance of Esposito is derived: his interpretations of both Nazism (thanatopolitics) and natalism are confronted. Thus, the possibility of the community positively immunized could be sketched.
Wspólnota i nihilizm
Authors: Esposito Roberto
Published: May 2013

Jaki związek zachodzi pomiędzy tymi pojęciami? Odpowiedź, jaką dają na to pytanie rozmaite filozofie wspólnoty – a także szeroko rozpowszechniona interpretacja nihilizmu – prowadzi w stronę radykalnego przeciwieństwa. Nihilizm i wspólnota nie stoją wobec siebie w relacji zwykłej odmienności, lecz otwartego antagonizmu, który nie pozwala na zaistnienie między nimi żadnych punktów stycznych – żadnych płaszczyzn nakładania się na siebie. Terminy te wzajemnie się wykluczają: tam, gdzie (lub kiedy) jest pierwszy z nich, tam nie ma miejsca na drugi i odwrotnie. Niezależnie od tego, czy opozycję tę umieścimy na osi synchronicznej czy diachronicznej, jej istotę stanowić będzie wyraźna granica alternatywy, dzieląca przeciwległe, nieprzystające do siebie w żaden sposób bieguny.
Giovanni Preziosi i Generalny Inspektorat do spraw Rasowych (1944-1945)
Authors: Sondel-Cedarmas Joanna
Published: May 2013

Giovanni Preziosi and the General Inspectorate of Race (Ispettorato Generale Della Razza) (1944‑ 1945)
This article is devoted to the analysis of the ideas of Giovanni Preziosi (1881‑1945), an Italian journalist and director of the main anti‑Semitic journal in Italy, “La Vita Italiana”, in circulation from 1921 to 1945. He also served as Minister of State from 1942‑1943, as well as the head of the General Inspectorate of Race in the period of the Italian Social Republic. He is regarded as one of the foremost theorists of Italian anti‑Semitism in the XX century, as well as one of the main representatives of Fascism in the period preceding the March on Rome. Preziosi also played a leading role in the implementation of racist policies in Italy in the years 1938‑1945. In his famous Memoriale, which he developed in January 1944 and then sent to Mussolini and Hitler, he blames Jews and Masons for the fall of Fascism on 25 July 1943 and proclaims the “final resolution of the Jewish problem” in Italy. As the head of the General Inspectorate of Race in the years 1944‑1945, he attempted to adopt the German model in Italy, particularly in the project of bills he wrote in May 1944, which were based on the Nuremburg Laws from 1935 and which were to be a revision of the decrees from 1938‑1939.
Separate, Equal, or Separate-But-Equal? The Changing Image of Race in the U.S. Supreme Court’s Decisions
Authors: Laidler Paweł
Published: May 2013

There is no doubt that the United States were not created as a purely democratic state. On the one hand, it established basic rules and principles of democratic government such as free elections, sovereignty of the nation, fundamental rights and freedoms of individuals or independent judiciary. All of these principles were, however, enjoyed only by the part of American society: free elections were guaranteed for white men, excluding women and blacks; sovereignty belonged to the nation, i.e. white women and men, because slaves were not considered citizens; fundamental rights and freedoms were guaranteed only for whites; institutional independence of the judicial branch did not prevent the system from injustice towards the blacks. Furthermore, one of the most important values of democratic society, equal protection of law, was absent in the original constitutional document of 1787, as well as the provisions of Bill of Rights. The clause became part of U.S. constitutional reality yet in 1868 when the Fourteenth Amendment was enacted, as a direct result of social and political changes caused by the civil war. After introducing the Thirteenth Amendment in 1865, which abolished slavery, the government took a step forward by making all citizens equal in 1868 and by providing black Americans with suffrage rights in 1870. For former slaves it meant a milestone step in their fight to destroy the social and political boundaries which limited their basic rights and freedoms. However, before the above mentioned events took place, the period of injustice and exploitation occurred with the U.S. Supreme Court in the middle of social and political tensions. The main purpose of the article is to show the changing attitude of the U.S. Supreme Court with regard to the social and political status of African‑Americans. This attitude influenced historical and contemporary social relations among the American society proving one of the most controversial aspects of U.S. democracy.
„Człowiek szalony powiedział sobie, w swym sercu, że nie ma takiej rzeczy, jak sprawiedliwość…”
Authors: Podniesiński Michał
Published: May 2013

“The fool hath said in his heart, there is no such thing as justice…” In Favor of Transcendental State. Several other Arguments in Dispute with Hobbes This paper is one of several articles focused on the theory of contract and the foundations of the modern liberal society. It is a continuation of the pervious paper: Why the Anglosaxon do not like Foucault? The text consists of two major parts. First is an analysis of a modern attempt to defend contractarianism by such authors as D. Gauthier, G. Kavka, J. Hampton, K. Hoekstra, M. Oakeschott, S.M. Brown and their argumentation, especially an attempt to interpret Hobbes in game theory categories. It is discredited as full of logical errors. In the second part, two new arguments against the theory of contract are introduced: (I) historical one – reality proved that human act rather conversely than Hobbes wanted them to, and (II) logical – based on the priority of mutuality rule as fundamental in human cognition.
Zasada rządów prawa w koncepcji Alberta Venn Diceya
Authors: Zabdyr-Jamróz Michał
Published: May 2013

The principle of the rule of law in Albert Venn Dicey’s theory
The aim of this paper is to present the original theory of the English rule of law developed by Victorian‑ era constitutional scholar, Albert Venn Dicey. The uniqueness of this theory will be presented, as well as its historical and doctrinal context, and theoretical implications. Dicey’s legal positivism identifies the rule of law as one of the two basic principles of the English unwritten constitution (together with the principle of sovereignty of Parliament). The rule of law itself consists of three components – the ideas of legal freedom, legal equality and predominance of the legal spirit. The latter is due to the inductive nature of the British constitution and in practice results in a strong emphasis on the institutional guarantees of the rule of law. The second idea – legal equality – in Dicey’s opinion interfered with the concept of administrative law, as it was developed on the European continent thus making it manifestly contrary to the English rule of law.
Życie codzienne i rewolucja według Henriego Lefebvre’a
Authors: Winkler Anna
Published: May 2013

Henri Lefebvre’s concepts of everyday life and revolution
The aim of this article is to analyse the concept of everyday life, which was used by Henri Lefebvre to build his theory of overcoming the alienation – both on the individual (as the theory of moments) and collective (in his concept of revolution as a popular festival) level. In the basic structures of everyday life Lefebvre saw the fundaments of spontaneity, human creative power that is capable of forcing its way through the alienating structures and that makes the total subjugation impossible. Moreover, placing the theory of revolution inside the concept of everyday life allows to draw particular attention to the importance of human consciousness in a revolutionary struggle. In the end, however, it seems that the categories introduced by Lefebvre, even though they create a good fundament for the discussion about the possibility of the human emancipation, cannot fully explain the phenomenon of the revolution.
Edward Dembowski – życie i twórczość „czerwonego kasztelanica”
Authors: Jezioro Artur
Published: May 2013

Edward Dembowski – life and work of “the red castellan’s‑ son”

Edward Dembowski is one of the most controversial figures of Polish history in the nineteenth century. Outstanding conspirator, totally dedicated to the democratic and revolutionary ideals, tirelessly tried to trigger off another national uprising, combined with the social revolution. He was primarily a man of action, but also left a number of writings, showing his achievements in the field of philosophy, literature and journalism. Dembowski’s figure is surrounded by legend, also because of his tragic death at a young age. But in fact, the “red castellan” is surrounded by two legends – the black one and the white one. According to the first of them, the character of the following article was an insane communist and revolutionary, guilty of bloodshed in the name of utopian ideals. The second legend, positive, presents his person as the heroic envoy, one of the national heroes of the nineteenth‑century, totally dedicated to the national and social causes, finally dying in the name of these ideals. Which of the two legends is closer to the truth? I hope that this article, which aims to provide an overview of the most important facts about the life of Edward Dembowski and to present his political, social and philosophical views, will help readers find the answer to this question. The first part of the paper primarily describes the life and activity of Edward Dembowski, while the second one focuses on his philosophical views.
Complementary Goods: Creating, Capturing, and Competing for Value
Authors: Yalcin Taylan , Ofek Elie , Koenigsberg Oded , Biyalogorsky Eyal
Published: May 2013

This paper studies the strategic interaction between firms producing strictly complementary products. With strict complements, a consumer derives positive utility only when both products are used together. We show that value-capture and value-creation problems arise when such products are developed and sold by separate firms (“nonintegrated” producers). Although the firms tend to price higher for given quality levels, their provision of quality is so low that, in equilibrium, prices are set well below what an integrated monopolist would choose. When one firm can mandate a royalty fee from the complementor producer (as often occurs in arrangements between hardware and software makers), we find that the value-capture problem is mitigated to some extent and consumer surplus rises. However, because royalty fees greatly reduce the incentives of the firm paying them to invest in quality, the arrangement exacerbates the value-creation problem and leads to even lower total quality. Surprisingly, this result can reverse with competition. Specifically, when the firm charging the royalty fee faces a vertically differentiated competitor, the value-creation problem is greatly reduced—opening the door for the possibility of a Pareto-improving outcome in which all firms and consumers benefit. It is worth noting that this outcome cannot be achieved by giving firms the option of introducing a line of product variants; competition serves as a necessary “commitment” ingredient.
Case report: bovine congenital erythropoietic protoporphyria in a pedigree Limousin herd
Authors: Truyers Isabelle , Ellis Kathryn
Published: May 2013

Bovine congenital erythropoietic protoporphyria (BCEPP), a disease caused by a decrease in activity of ferrochelatase enzyme, is most commonly characterised by photosensitisation and seizures. BCEPP is inherited as a simple, autosomal recessive defect, with only homozygote individuals exhibiting clinical signs, and occurs mainly in pure Limousin cattle. There is no specific treatment and the disease can be prevented by avoiding mating of carriers of the defective gene; these can be identified using DNA analysis. This report describes two cases of BCEPP in a pedigree Limousin herd and discusses the investigation of the condition and implications for the herd.
Equine respiratory tract — intra-sinus progressive ethmoidal haematoma
Authors: Barakzai Safia
Published: May 2013

Unilateral epistaxis in the horse presents a diagnostic challenge in order to differentiate between facial trauma, progressive ethmoidal haematoma and more serious conditions such as guttural pouch mycosis or sinus neoplasia. Clinical examination, endoscopy per nasum, oral examination, radiography, sinoscopy and in some cases, scintigraphic examination or computed tomography can be required to make a definitive diagnosis. Treatment options for ethmoidal haematoma cases include surgical removal, laser vaporisation and repeated intra-lesional injection with formalin. In this case, surgical removal was performed and the horse recovered well.
Seeking America in America
Authors: Wilson Karen S.
Published: May 2013

This article presents the California Gold Rush as an ideological battleground. The 1848 discovery of gold in California and the 1848 Revolutions in France launched an influx of French argonauts with expectations of equal opportunities and individual rights common to both nations’ ideologies. In the accounts of four bourgeois French observers of the Gold Rush, this article finds America’s promise falling short of practice as nativism barred European foreigners from the equal fruits of free enterprise.