bluuebunny/crossref_metadata_2025_split huggingface dataset:
Temporally different poly(adenosine diphosphate‐ribosylation) signals are required for DNA replication and cell division in early embryos of sea urchins
Authors: Imschenetzky María , Montecino Martín , Puchi Marcia 
Published: May 2005

AbstractTo analyze the temporal relationship of poly(adenosine diphosphate [ADP]‐ribosylation) signal with DNA replication and cell divisions, the effect of 3 aminobenzamide (3ABA), an inhibitor of the poly(ADP‐ribose)synthetase, was determined in vivo during the first cleavage division of sea urchins. The incorporation of 3H‐thymidine into DNA was monitored and cleavage division was examined by light microscopy. The poly(ADP‐ribose) neosynthesized on CS histone variants was measured by labeling with 3H‐adenosine during the two initial embryonic cell cycles and the inhibitory effect of 3ABA on this poly(ADP‐ribosylation) was determined. The results obtained indicate that the CS histone variants are poly(ADP‐ribosylated) de novo during the initial cell cycles of embryonic development. The synthesis of poly(ADP‐ribose) is decreased but not abolished by 20 mM of 3ABA. The incubation of zygotes in 3ABA at the entrance into S1 phase decreased 3H‐thymidine incorporation into DNA in phase S2, while S1 was unaltered. Alternatively, when the same treatment was applied to zygotes at the exit of S1 phase, a block of the first cleavage division and a retardation of S2 phase were observed. The inhibitory effect of 3ABA on both DNA replication and cell division was totally reversible by a release of the zygotes from this inhibition. Taking together these observations it may be concluded that the poly(ADP‐ribosylation) signals related to embryonic DNA replication are not contemporaneous with S phase progression but are a requirement before its initiation. These results also indicate that a poly(ADP‐ribosylation) signal is required for cell division; such signal is temporally different from that related to S phase initiation and occurs at the exit of S phase. © 1993 Wiley‐Liss, Inc.
Detection and characterization of low abundance cellular proteins that specifically increase upon loss of the metastatic phenotype
Authors: Nielsen‐Preiss Sheila M. , Quigley James P. 
Published: May 2005

AbstractHuman epidermoid carcinoma (HEp‐3) cells are highly tumorigenic and metastatic in vivo, but their metastatic phenotype is progressively and uniquely lost upon serial passage in vitro. The nonmetastatic phenotype is fully reversible to the highly metastatic state when HEp‐3 cells are passaged back in vivo.To study the complex process of metastasis and its possible negative regulation by specific gene products, the expression of specific proteins between the highly metastatic and nonmetastatic HEp‐3 cells was investigated by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) and subsequent computer assisted analyses. Concomitant with the in vitro loss of metastatic potential of HEp‐3 cells is the elevated expression of a subset of low abundance proteins detectable in 2D‐PAGE but not apparent in high resolution one dimensional PAGE. When the HEp‐3 cells revert to the metastatic state, the expression of these proteins declines. The increased abundance of four distinct proteins directly correlates with the loss of the metastatic phenotype: two of the four proteins are associated with isolated cellular membranes (36kD, pl 5.7; 22kDa, pl 5.6), on protein fractionates with the cytoplasm (65kD, pl 6.2), and one protein is enriched in the nuclei fraction (32kD, pl 5.8). These data indicate that computer‐assisted analysis of highly sensitive, large‐format, 2D‐PAGE can be used to identify specific proteins in subcellular compartments that are candidates for negative regulators of the metastatic process. © 1993 Wiley‐Liss, Inc.
Nature of DNA sequences at the attachment regions of genes to the nuclear matrix
Authors: Boulikas Teni 
Published: May 2005

AbstractMatrix‐attached regions (MARs) have been demonstrated to nest origins of replication and transcriptional enhancers. A set of 13 rules is proposed aimed at facilitating the classification of a DNA sequence as a matrix attachment region. These rules, which were deduced from a study of known MARs from other genes and some others identified in our laboratory, are (1) potential origin of replication are MARs; (2) the major class of MARs seclude clusters of AT‐rich motifs and may harbor topoisomerase II binding and cleavage sites; (3) the AT‐rich class of MARs may comprise the DNA sequence motifs ATTA and ATTTA representing core binding sites of homeotic proteins, implying that MARs may participate in the differential activation of origins of replication and in gene switch during development; (4) the habitat of MARs may include mass binding sites for protein transcription factors; even weak factor binding sites may lead to the formation of tight protein‐DNA supramolecular structures; (5) MARs may contain intrinsically curved DNA; one type is oligo(dA) stretches of 3 to 7 nucleotides spaced every 10.5 nucleotides; (6) a class of MARs may contain kinked DNA, formed by CA, TG, and TA dinucleotides at distances of 5 or 10.5 nucleotides from their centers; the same dinucleotides, known to be abundant in protein recognition sites, may be overrepresented in a special class of MARs; (7) the AT‐rich core of MARs may be flanked, at one or both sides, by sequences that can adopt the left‐handed or triple‐helical DNA structure; these include TG, TA, GC repeats and polypurine or polypyrimidine stretches; (8) palindromic (dyad symmetry) sequences, able to form cruciform structures when the DNA is under torsional strain may be found within MARs, and more so when the MAR is also an origin of replication; (9) transcriptional enhancers may be MARs; (10) a class of MARs may be composed of stretches of GA‐rich DNA alternating with CT‐rich stretches, 5–50 nucleotides long; (11) a class of MARs may be enriched in TG boxes, usually 6–12 nucleotides long, such as TGTTTTGGGG; this type of MAR occurs in the 3′‐untranslated region of several genes, builds up the chromosome telomeres, and is highly recombinogenic; (12) a small fraction of Alu sequences might have MAR activity. This might depend on the number and distance from one another of DNA sequence motifs representing protein binding sites; and (13) MARs may coincide with the DNAse I hypersensitive sites of chromatin.It is proposed here that MAR sequences can provide markers for mapping and sequencing the human, and other, genomes. Furthermore, it is proposed that large scale random cloning of MARs might advance our knowledge on the nature of DNA sequences that are used for the initiation of DNA replication, as transcriptional enhancers and as borders between chromatin domains.
Influence of electromagnetic fields on endochondral bone formation
Authors: Ciombor Deborah McK. , Aaron Roy K. 
Published: May 2005

AbstractEndochondral ossification is a basic physiological process in limb development and is central to bone repair and linear growth. Factors which regulate endochondral ossification include several biophysical and biochemical agents and are of interest from clinical and biological perspectives. One of these agents, electric stimulation, has been shown to result in enhanced synthesis of extracellular matrix, calcification, and bone formation in a number of experimental systems and is the subject of this review. The effects of electric stimulation have been studied in embryonic limb rudiments, growth plates, and experimental endochondral ossification induced with decalcified bone matrix and, in all these models, endochondral ossification has been enhanced. It is not known definitively whether electric fields stimulate cell differentiation or modulate an increased number of molecules synthesized by committed cell population and this is a fertile area of current study.
The identification of a naturally occurring cell surface growth inhibitor related to a previously described bovine sialoglycopeptide
Authors: Fattaey Heideh K. , Enebo Daniel J. , Moos Philip J. , Johnson Terry C. 
Published: May 2005

AbstractA 66‐kDa sialoglycoprotein has been identified as the parental membrane molecule of an earlier described sialoglycopeptide (SGP), an 18‐kDa molecule released by protease treatment of intact bovine cerebral cortex cells that was shown to be a potent inhibitor of cellular proliferation. The 66‐kDa parental sialoglycoprotein (p‐SGP) was purified approximately 2,400‐fold, to apparent homogeneity, from bovine cerebral cortex cell membranes by its release during incubation with 3 M NaCl, preparative isolelectric focusing and lectin affinity chromatography. Although a membrane‐associated molecule, the p‐SGP appeared to be tightly bound to the cell membrane, since it was not released during incubations in the absence of 3 M NaCl. Incubation of the membrane preparations with 3 M urea proved to be too harsh, and the antigenicity required to follow the purification of the p‐SGP was abolished. Analyses by SDS–PAGE, under reducing and nonreducing conditions, suggested that the p‐SGP membrane component was a single polypeptide without subunit structure. The p‐SGP was shown to be structurally related to the SGP fragment by immunoblots with IgG raised to the SGP inhibitor, and functionally related to the SGP by its ability to inhibit Swiss 3T3 proliferation at concentrations strikingly similar to that previous measured with the SGP fragment.
Signal transduction pathways and cellular intoxication with Clostridium difficile toxins
Authors: Shoshan Maria Caspar , Fiorentini Carla , Thelestam Monica 
Published: May 2005

AbstractIn cultured cells the cytopathic effects (CPE) of Clostridium difficile toxins A and B are superficially similar. The irreversible CPEs involve a reorganization of the cytoskeleton, but the molecular details of the mechanism(s) of action are unknown. As part of the work to elucidate the events leading to the CPE, cultured cells were preincubated with agents known to either stimulate or inhibit some major signal transduction pathways, whereupon toxin was added and the development of the CPE was followed. Both toxin‐induced CPEs were enhanced by phorbol esters and mezerein, which stimulate protein kinase C, while they were inhibited by the phospholipase A2 inhibitors quinacrine and 4‐bromophenacylbromide. Agents affecting certain G‐proteins, cGMP and cAMP levels, phosphatases, prostacyclin, lipoxygenase, and phospholipase C did not affect the development of the CPE of either toxin. Thus, the cytoskeletal effect induced by toxins A or B appears to require PLA2 activity and involves at least part of a protein kinase C‐dependent pathway, but not pertussis toxin‐sensitive G‐proteins, cyclic nucleotides, eicosanoid metabolites, or phospholipase C activity. In addition, both toxins were shown to activate phospholipase A2.
Effects of vitamin D3 on signaling by prostaglandin E2 in osteoblast‐like cells
Authors: Tokuda Haruhiko , Kotoyori Jun , Suzuki Atsushi , Oiso Yutaka , Kozawa Osamu 
Published: May 2005

AbstractWe investigated the effects of vitamin D3 on the signaling pathways by prostaglandin E2 (PGE2) in osteoblast‐like MC3T3‐E1 cells. The pretreatment with 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2D3), an active form of vitamin D3, significantly inhibited cAMP accumulation induced by 10 μM PGE2 in a dose‐dependent manner in the range between 1 pM and 1 nM. This effect of 1,25‐(OH)2D3 was dependent on the time of pretreatment up to 8 h. 1,25‐(OH)2D3 also inhibited the cAMP accumulation induced by NaF, a GTP‐binding protein activator, or forskolin which directly activates adenylate cyclase. On the other hand, 1,25‐(OH)2D3 significantly inhibited PGE2‐induced IP3 formation in a dose‐dependent manner between 10 pM and 1 nM. However, 1,25‐(OH)2D3 had little effect on NaF‐induced IP3 formation. The pretreatment with 24,25‐dihydroxyvitamin D3, an inactive form of vitamin D3, affected neither cAMP accumulation nor IP3 formation induced by PGE2. These results strongly suggest that 1,25‐(OH)2D3 modulates the signaling by PGE2 in osteoblast‐like cells as follows: the inhibitory effect on the cAMP production is exerted at a point downstream from adenylate cyclase and the inhibitory effect on the phosphoinositide hydrolysis is exerted at the point between the PGE2 receptor and GTP‐binding protein, probably Gi2.
Characterization of atrial natriuretic peptide degradation by cell‐surface peptidase activity on endothelial cells
Authors: Frost Stephen J. , Whitson Peggy A. 
Published: May 2005

AbstractAtrial natriuretic peptide (ANP) is a fluid‐regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I‐ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I‐ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 ± 60 nM and Vmax of 35 ± 14 pmol of ANP degraded/10 min/105 cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0–8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I‐ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell‐surface degradation of ANP was demonstrated when 125I‐ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin‐1, or substance P. The C‐terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell‐surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation‐independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.
Double immunofluorescent staining of α2,6 sialyltransferase and β1,4 galactosyltransferase in monensin‐treated cells: Evidence for different Golgi compartments?
Authors: Berger E. G. , Grimm K. , Bächi T. , Bosshart H. , Kleene R. , Watzele M. 
Published: May 2005

Abstractβ1,4 galactosyl‐ and α2,6 sialyltransferase (gal‐T EC 2.4.1.22 and sialyl‐T EC 2.4.99.1) sequentially elongate and terminate complex N‐glycan chains of glycoproteins. Both enzymes reside in trans Golgi cisternae; their ultrastructural relationship, however, is unknown. To delineate their respective Golgi compartment(s) we conducted a double label immunofluorescent study by conventional and confocal laser scanning microscopy in HepG2, HeLa, and other cells in presence of Golgi‐disturbing agents. Polyclonal, peptide‐specific antibodies to human sialyl‐T expressed as a β‐galactosidase‐sialyl‐T fusion protein in E. coli were developed and applied together with mABs to human milk gal‐T.In untreated HepG2 and HeLa cells Golgi morphology identified by immunofluorescent labeling of sialyl‐T and gal‐T, respectively, was nearly identical. Treatment of cells with brefeldin A (BFA) led to rapid and coordinated disappearance of immunostaining of both enzymes; after BFA washout, vesicular structures reappeared which first stained for gal‐T followed by sialyl‐T; in the reassembled Golgi apparatus sialyl‐T and gal‐T were co‐localized again. In contrast, monensin treatment produced a reversible swelling and scattering of gal‐T positive Golgi elements while sialyl‐T positive structures showed little change. Treatment with nocodazole led to dispersal of Golgi elements in which gal‐T and sialyl‐T remained co‐localized. Treatment with chloroquine affected Golgi structure less than monensin and led to condensation of gal‐T positive and to slight enlargement of sialyl‐T positive structures.Sequential recovery from BFA of gal‐T and sialyl‐T and their segregation by monensin suggest that these enzymes are targeted to different Golgi subcompartments.
Phorbol ester activation of functional rat protein kinase C β‐1 causes phenotype in yeast
Authors: Riedel Heimo , Hansen Hans , Parissenti Amadeo M. , Su Lihe , Sheih Hui‐Ling , Zhu Jianwei 
Published: May 2005

AbstractThe phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of various eukaryotic cellular signals. The molecular dissection of its mechanisms of action has been limited in part by the genetic inaccessibility and complexity of signaling in mammalian cells. Here we present a novel approach to study rat PKC β‐1 action in yeast, a simple lower eukaryotic genetic model. Expression of its cDNA in Saccharomyces cerevisiae introduces novel phorbol ester binding sites which stimulate a specific calcium‐ and phospholipid‐dependent catalytic activity in vitro consistent with a fully functional protein which phosphorylates cellular yeast proteins in vivo. Phorbol ester activation of PKC β‐1 in vivo results in biological responses which include stimulation of extracellular calcium uptake, changes in cell morphology, and an increase in the cell doubling time. These PKC functions are not affected by truncation of 12 amino terminal amino acids; however, they are completely abolished by truncation of 15 or more carboxyl terminal amino acids which likely result in inactivation of the kinase. The increase in the yeast doubling time caused by PKC β‐1 activation provides a phenotype which can be exploited as a screen for the activity of random PKC cDNA mutations. Our findings indicate that rat PKC β‐1 is functional in yeast and leads to biological responses which suggest compatible aspects of higher and lower eukaryotic signaling pathways and the feasibility of dissecting parts of the action of common signaling mediators in a simple genetic model.
Protein product of the somatic‐type transcript of the Hoxa‐4 (Hox‐1.4) gene binds to homeobox consensus binding sites in its promoter and intron
Authors: Wu Ke , Wolgemuth Debra J. 
Published: May 2005

AbstractThe murine Hoxa‐4 gene encodes a protein with a homeodomain closely related to those produced by the Antennapedia‐like class of Drosophila genes. Drosophila homeodomain proteins can function as transcription factors, binding to several specific DNA sequences. One sequence that is frequently encountered contains a core ATTA motif within a larger consensus sequence, such as CAATTAA. The in vitro synthesized protein product of Hoxa‐4 was shown to bind to a subset of restriction fragments of the Hoxa‐4 gene itself as determined by gel retardation experiments. Direct examination of the sequences of the fragments bound by Hoxa‐4 protein revealed the presence of four regions containing the core ATTA motif. Two regions contained sequences of the CAATTAA class and were located ∼ 1 kb upstream from the putative somatic Hoxa‐4 promoter and within the intron. Two additional binding sites containing the consensus target sequence involved in autoregulation of Drosophila Deformed gene were identified: one immediately downstream of the putative embryonic transcription start site and one within the intron, respectively. Specific binding of the in vitro produced Hoxa‐4 protein to oligonucleotides corresponding to these sequences was observed in gel retardatin assays. The same results were obtained with Hoxa‐4 protein produced in a Baculovirus expression system. Experiments using oligonucleotides containing base substitutions in positions 1, 3, 4, and 5 in the sequence CAATTAA showed severely reduced binding. The use of truncated mutant Hoxa‐4 proteins in gel retardation assays and in transient co‐transfection experiments revealed that the intact homeodomain was required for the binding. These results also suggested that the Hoxa‐4 gene has the potential to auto‐regulate its expression by interacting with the homeodomain binding sites present in the promoter as well as in the intron.
Multitude of inverted repeats characterizes a class of anchorage sites of chromatin loops to the nuclear matrix
Authors: Boulikas Teni , Kong C. F. 
Published: May 2005

AbstractIn order to understand the nature of DNA sequences that organize chromatin into domains or loops, we have cloned the nuclear matrix DNA (1.7% of the total DNA) from human myelogeneous leukemia cells in culture. Nuclear matrix is formed by interactions between specific stretches of DNA of about 0.1 to 5.0 kb with protein transcription factors, nuclear enzymes, and structural proteins. Nuclear matrix is believed to be the exclusive nuclear microenvironment in which initiation of DNA replication, transcription, and repair take place. The matrix attachment regions (MARs) of DNA have transcriptional enhancer activity, harbor the origins of replication of the human genome, and define the borders between neighbouring chromatin loops. In this study we report the sequence of the human MAR fragment 19.2 of a size of 542 bp. Hum. MAR 19.2 is composed of TG‐, CA‐, CT‐, and GA‐rich blocks and shows 8 perfect and imperfect inverted repeats. Thus, we have identified a novel class of MARs with sequence characteristics divergent from the AT‐rich class of MARs. The inverted repeats of the 19.2 sequence might be stabilized into their cruciform configuration by torsional strain and by specific transcription/replication protein factors. This MAR might function in the initiation of replication of the flanking chromatim domain and in the regulation of the transcriptional activity of the gene(s) that reside in this domain.
Evidence against a role for alkaline phosphatase in the dephosphorylation of plasma membrane proteins: Hypophosphatasia fibroblast study
Authors: Fedde Kenton N. , Michel Michelle P. , Whyte Michael P. 
Published: May 2005

AbstractA major impasse to understanding the physiologic role(s) of alkaline phosphatase (ALP) is uncertainty as to its natural substrates. Various in vitro studies have led other investigators to suggest that ALP functions as a plasma membrane phosphoprotein phosphatase, consistent with our demonstration of ecto‐topography of ALP in a variety of cell types. Thus, we compared the phosphorylation of plasma membrane proteins from control fibroblasts to those from profoundly ALP‐deficient fibroblasts of hypophosphatasia patients. Fibroblasts from 3 controls and 3 hypophosphatasia patients (ALP activity < 4% of control) were biosynthetically labeled with 32Pi for 2 h. 32P incorporation into total trichloracetic acid (TCA)‐precipitable material was not significantly different in control and patient cells. Plasma membranes were prepared from these cells by hypotonic shock, solubilized, and subjected to two‐dimensional (2‐D) gel electrophoretic separation. Video densitometric analysis of silver‐stained 2‐D gels failed to reveal any consistent difference in the protein profile between patient vs. control fibroblasts (i.e., unique species, altered pls, or increased abundance). Autoradiography of individual 2‐D gels demonstrated 63 plasma membrane phosphoproteins with molecular weights ranging from 15 to 152 kDa and predominantly acidic pls. Although several of these phosphoproteins appeared to have had donor‐specific labeling, none was unique or especially abundant in the hypophosphatasia group. Thus, in ALP‐deficient fibroblasts, normal incorporation of 32P into total cellular protein and into all identifiable plasma membrane phosphoproteins indicates that ALP does not modulate the phosphorylation of plasma membrane proteins.