bluuebunny/crossref_metadata_2025_split huggingface dataset:
A human nuclear protein with sequence homology to a family of early s phase proteins is required for entry into s phase and for cell division
Authors: Todorov Ivan T. , Pepperkok Rainer , Philipova Radka N. , Kearsey Stephen E. , Ansorge Wilhelm , Werner Dieter 
Published: April 2021

ABSTRACT
Molecular cloning and characterisation of a human nuclear protein designated BM28 is reported. On the amino acid level this 892 amino acid protein, migrating on SDS-gels as a 125 kDa polypeptide, shares areas of significant similarity with a recently defined family of early S phase proteins. The members of this family, the Saccharomyces cerevisiae Mcm2p, Mcm3p, Cdc46p/Mcm5p, the Schizosaccha-romyces pombe Cdc21p and the mouse protein P1 are considered to be involved in the onset of DNA replication. The highest similarity was found with Mcm2p (42% identity over the whole length and higher than 75% over a conservative region of 215 amino acid residues), suggesting that BM28 could represent the human homologue of the S. cerevisiae MCM2.
Using antibodies raised against the recombinant BM28 the corresponding antigen was found to be localised in the nuclei of various mammalian cells. Microinjection of anti-BM28 antibody into synchronised mouse NIH3T3 or human HeLa cells presents evidence for the involvement of the protein in cell cycle progression. When injected in G1 phase the anti-BM28 antibody inhibits the onset of subsequent DNA synthesis as tested by the incorporation of bro-modeoxyuridine. Microinjection during the S phase had no effect on DNA synthesis, but inhibits cell division. The data suggest that the nuclear protein BM28 is required for two events of the cell cycle, for the onset of DNA replication and for cell division.
Histone h1 kinase activity, germinal vesicle breakdown and m phase entry in mouse oocytes
Authors: Gavin Anne-Claude , Cavadore Jean-Claude , Schorderet-Slatkine Sabine 
Published: April 2021

ABSTRACT
Meiotic reinitiation of the mouse oocyte is caracterized by a slow entry into metaphase I, beginning with germinal vesicle breakdown and ending with spindle formation. It is accompanied by a cascade of protein kinases and phos-phatases increasing protein phosphorylation. The activa-tion of histone H1 kinase and that of the mitogen-activated protein kinase p42 have been compared during sponta-neous or okadaic acid-induced meiotic reinitiation.
In spontaneously maturing oocytes, histone H1 kinase activity increases before germinal vesicle breakdown (2-fold), in a protein synthesis-independent manner. It is asso-ciated with the disappearance of the upper migrating form of p34cdc2, which, in our system, seems to represent the tyrosine phosphorylated form. Following germinal vesicle breakdown, histone H1 kinase activity culminates (8-fold) in metaphase I and requires protein synthesis. Activation by phosphorylation of p42MAPK is observed as a permanent shift upward-migrating form and by its myelin basic protein kinase activity. It occurs after germinal vesicle breakdown and depends on protein synthesis.
In contrast, no increase of histone H1 kinase is detectable in oocytes induced to reinitiate meiosis by a transient inhi-bition of okadaic acid-sensitive phosphatase(s), either before germinal vesicle breakdown or during the following 7 hours of culture. A slight increase is nevertheless evident after 17 hours, when oocytes are arrested with an abnormal metaphase I spindle. The upper migrating form of p34cdc2 is present for 8 hours. The activation of p42MAPK begins before germinal vesicle breakdown. Furthermore, when okadaic acid is applied to oocytes that have had meiosis reinitiation prevented by p13suc1 microinjection, neither increase of histone H1 kinase activity nor p34cdc2 dephos-phorylation is observed although germinal vesicle breakdown is induced; p42MAPK is activated.
Altogether these results suggest that germinal vesicle breakdown may or may not be associated with a detectable activation of histone H1 kinase, depending on the experi-mental conditions. Activation of p34cdc2 and p42MAPK are separable events. The effect of okadaic acid on mouse oocyte maturation is positive and negative. It induces meiotic events (germinal vesicle breakdown) independently of histone H1 kinase activation, perhaps through premature activation of p42MAPK. It interferes negatively with the meiotic post-germinal vesicle breakdown events, most probably through mechanisms inhibiting protein synthesis and preventing cell cycle progression. We propose that, in the mouse oocyte, okadaic acid might be able to activate an alternative pathway leading to germinal vesicle breakdown that is cdc2-independent and that involves p42MAPK activation ensuing maturation promoting factor-independent phosphorylations.
Characterization of the novel brown adipocyte cell line hib 1b adrenergic pathways involved in regulation of uncoupling protein gene expression
Authors: Klaus Susanne , Choy Lisa , Champigny Odette , Cassard-Doulcier Anne-Marie , Ross Susan , Spiegelman Bruce , Ricquier Daniel 
Published: April 2021

ABSTRACT
The HIB 1B cell line, derived from a brown fat tumor of a transgenic mouse, is the first established brown adipocyte cell line capable of expressing the brown fat-specific mitochondrial uncoupling protein (UCP). UCP gene expression, which was virtually undetectable under basic conditions, was stimulated by acute catecholamine or cyclic AMP treatment to levels comparable to primary cultures of brown adipocytes. Elevation of UCP mRNA levels following stimulation was very rapid but transient, decreasing after about 4 hours with a half-life between 9 and 13 hours. Immunoblotting showed the presence of UCP in HIB 1B mitochondria, but expression was much lower than observed in BAT or primary cultures of brown adipocytes. Upon transfection of HIB 1B cells with a reporter gene containing the UCP promoter, the activity of the transgene was regulatable by cAMP and norepineph-rine. Investigation of the possible adrenergic receptors involved in UCP stimulation showed that specific β3-adren-ergic agonists were much less effective than nonspecific β-adrenergic agonists and that mRNA levels of the atypical, fat-specific β3-adrenoceptor were lower than those observed in brown adipocytes differentiated in primary culture. From pharmacological evidence we conclude that β3-adrenergic receptors account for approximately 30-40% of catecholamine induced UCP gene stimulation, whereas about 60-70% is stimulated via the classical β1/2 adrenergic pathway. We conclude that HIB 1B cells represent a functional system for the study of mechanisms related to brown adipose thermogenesis.
Protein binding and cell adhesion properties of two laminin isoforms (amb1eb2e, amb1sb2e) from human placenta
Authors: Brown Judith C. , Wiedemann Hanna , Timpl Rupert 
Published: April 2021

ABSTRACT
Two isoforms of laminin were extracted from human placenta by neutral buffer containing EDTA, copurified through several steps and finally separated by Mono Q anion exchange chromatography. One variant consisted of disulphide-linked 340, 230 and 190 kDa subunits, which were identified by immunoblotting as Am, B1e and B2e chains. In the other variant, the B1e chain was replaced by B1s of 180 kDa. After rotary shadowing, both variants showed a similar cross-shaped structure. The nidogen content of these laminins was substoichiometric and variable (3-70%), indicating loss by endogenous proteolysis. Yet both human isoforms were able to bind mouse nidogen with an affinity (Kd∼0.5 nM) comparable to that of AeB1eB2e laminin from a mouse tumour. Since the binding site is known to be contributed by a single EGF-like motif of the B2e chain, this demonstrates that activity of this site is independent of chain assembly. Binding activity of both isoforms to collagen IV and the heparan sulphate proteo-glycan perlecan was correlated to the nidogen content and could be enhanced by adding nidogen. Binding to heparin was only partial and heparin did not inhibit perlecan binding. This indicated a crucial role for nidogen in mediating the integration of these laminin isoforms into basement membranes. Variant AmB1sB2e showed calcium-dependent binding to fibulin-1, while only a little activity was found for AmB1eB2e. Both isoforms promoted adhesion and spreading of several cell lines. Adhesion could be completely inhibited by antibodies to the integrin β1 subunit but not, or only weakly, by antibodies against β3,α2, α3, α5 and α6 subunits. No inhibition was observed with an Arg-Gly-Asp-containing peptide.
Characterisation of articular and growth plate cartilage collagens in porcine osteochondrosis
Authors: Wardale R. John , Duance Victor C. 
Published: April 2021

ABSTRACT
The articular and growth plate cartilages of osteochon-drotic pigs were examined and compared with those from clinically normal animals. Both types of osteochondrotic cartilage showed considerable localised thickening appar-ently due to a lack of ossification. Histological examination of cartilage lesions demonstrated a breakdown in the normal pattern of chondrocyte maturation. Articular cartilage lesions lacked mature clones of chondrocytes in the calcifying region. Growth plate cartilage showed an accumulation of disorganised hypertrophic chondrocytes rather than the well-defined columns seen in normal tissue.
The overall percentages of collagen in osteochondrotic lesions from both articular and growth plate cartilage were significantly reduced compared with levels in unaffected cartilage. There were substantial increases in the propor-tion of type I collagen in lesions from both osteochondrotic articular and growth plate cartilages and a reduction in the proportion of type II collagen. Type X collagen was detected in osteochondrotic but not normal articular cartilage. The proportion of type X collagen was unchanged in osteochondrotic growth plate cartilage.
The levels of the collagen cross-links, hydroxylysyl-pyridinoline, hydroxylysyl-ketonorleucine and dehydro-hydroxylysinonorleucine were radically reduced in samples from osteochondrotic growth-plate cartilage lesions when compared with normal tissue. Less dramatic changes were observed in articular cartilage although there was a significant decrease in the level of hydroxylysyl-ketonorleucine in osteochondrotic lesions.
Immunofluorescence examination of osteochondrotic lesions showed a considerable disruption of the organisa-tion of the collagenous components within both articular and growth-plate cartilages. Normal patterns of staining of types I and VI collagen seen at the articular surface in unaf-fected tissue were replaced by a disorganised, uneven stain in osteochondrotic articular cartilage lesions. Incomplete removal of cartilage at the ossification front of osteochon-drotic growth plate was demonstrated by immunofluores-cence staining of type IX collagen. Type X collagen was produced in the matrix of the calcifying region of osteo-chondrotic articular cartilage by small groups of hyper-trophic chondrocytes, but was not detected in normal articular cartilage. The distribution of type X collagen was unchanged in osteochondrotic growth plate cartilage.
A gradient method for the quantitative analysis of cell movement and tissue flow and its application to the analysis of multicellular Dictyostelium development
Authors: Siegert Florian , Weijer Cornelis J. , Nomura Atsushi , Miike Hidetoshi 
Published: April 2021

ABSTRACT
We describe the application of a novel image processing method, which allows quantitative analysis of cell and tissue movement in a series of digitized video images. The result is a vector velocity field showing average direction and velocity of movement for every pixel in the frame. We apply this method to the analysis of cell movement during different stages of the Dictyostelium developmental cycle. We analysed time-lapse video recordings of cell movement in single cells, mounds and slugs. The program can correctly assess the speed and direction of movement of either unlabelled or labelled cells in a time series of video images depending on the illumination conditions.
Our analysis of cell movement during multicellar devel- opment shows that the entire morphogenesis of Dic- tyostelium is characterized by rotational cell movement. The analysis of cell and tissue movement by the velocity field method should be applicable to the analysis of mor- phogenetic processes in other systems such as gastrulation and neurulation in vertebrate embryos.
Retention and retrieval: both mechanisms cooperate to maintain calreticulin in the endoplasmic reticulum
Authors: Sönnichsen Birte , Füllekrug Joachim , Van Phuc Nguyen , Diekmann Wilfried , Robinson David G. , Mieskes Gottfried 
Published: April 2021

ABSTRACT
Many soluble resident proteins of the endoplasmic reticulum share a COOH-terminal Lys-Asp-Glu-Leu (KDEL) sequence. Current opinion favours a model in which these proteins can escape from the endoplasmic reticulum (ER) by bulk flow and are recognized and sorted in the Golgi apparatus by binding to a specific KDEL- receptor, which returns them to the ER. Through bio- chemical, morphological and mutational analysis we have studied the mechanisms that determine the localization of calreticulin, a soluble 60 kDa KDEL-protein of the ER. Immunogold labelling established the ER localization of calreticulin in transfected and nontransfected COS cells. Although the ER cisternae in transfected cells were enormously dilated and heavily labelled by gold particles we found no significant label in any other compartment. In vivo pulse chase experiments with [35S]methionine followed by biochemical fractionation of calreticulin overexpressing COS cells (50- to 100-fold) revealed that only a minor part of labelled calreticulin leaves the ER. Retrieval from the Golgi was confirmed by a partial redistribution of the endogenous KDEL-receptor as shown by double immuno- fluorescence. These data suggest a KDEL-independent retention of calreticulin in the ER. Further supporting evidence has come from morphological in vivo studies using calreticulin-transfected and vesicular stomatitis virus (ts045)-infected COS cells. Stimulation of vesicular transport from the ER by releasing the temperature- dependent transport block for the viral G-protein resulted in a small but significant appearance of calreticulin in a post-ER compartment. In contrast a calreticulin mutant, which lacked the Ca2+-binding domain but included the KDEL sequence, could escape from the ER to a much higher extent. Secretion of the nonmutated calreticulin was very low (1-2% of total calreticulin in 3 hours) compared to the mutated form (18% in 3 hours). Deletion of the KDEL sequence led to an increase in secretion to 29% over a 3 hour period, which is much less than expected for a secretory protein. Taken together these results strongly support the hypothesis of two independently operating retention/retrieval mechanisms for calreticulin: one providing for direct retention in the ER with a very high capacity and having Ca2+-dependent properties; the other a KDEL-based retrieval system for escaped calreticulin present in the Golgi apparatus.
Ultrastructure of the proteoliaisin-ovoperoxidase complex and its spatial organization within the Strongylocentrotus purpuratus fertilization envelope
Authors: Mozingo Nancy M. , Somers Cynthia E. , Chandler Douglas E. 
Published: April 2021

ABSTRACT
Ovoperoxidase is a cortical granule-derived enzyme that hardens the sea urchin fertilization envelope by catalyzing the formation of dityrosine residues. Ovoperoxidase works in concert with a second protein, proteoliaisin, which anchors ovoperoxidase to the nascent fertilization envelope in a divalent cation-dependent manner. In this study, we examined the Ca2+-dependent interaction of proteoliaisin with ovoperoxidase in rotary-shadowed Pt replicas. Ovoperoxidase, a uniformly sized globular molecule, binds to a distal portion of rod-shaped proteoliaisin when low concentrations of Ca2+ are present. Higher Ca2+ concentrations lead to the formation of extended proteoliaisin strands that are decorated along their lengths with ovoper-oxidase. Using immunogold labeling, we also examined the assimilation of these two proteins into the fertilization envelope in quick-frozen, deeply etched samples. Both proteins are abundant in the fertilization envelope as early as one minute after fertilization. Coincident with paracrystalline coating of the envelope, the labeling density is markedly reduced, suggesting that antigenic sites may be masked by the paracrystalline coat. This suggests that the ovoperoxidase-proteoliaisin complex resides within the central portion of the fertilization envelope, rather than in the paracrystalline coat.
The fission yeast cdc19+ gene encodes a member of the MCM family of replication proteins
Authors: Forsburg Susan L. , Nurse Paul 
Published: April 2021

ABSTRACT
We have cloned and characterized the fission yeast cdc19+ gene. We demonstrate that it encodes a structural homologue of the budding yeast MCM2 protein. In fission yeast, the cdc19+ gene is constitutively expressed, and essential for viability. Deletion delays progression through S phase, and cells arrest in the first cycle with an apparent 2C DNA content, with their checkpoint control intact. The temperature-sensitive cdc19-P1 mutation is synthetically lethal with cdc21-M68. In addition, we show by classical and molecular genetics that cdc19+ is allelic to the nda1+ locus. We conclude that cdc19p plays a potentially conserved role in S phase.
Dinoflagellates have a eukaryotic nuclear matrix with lamin-like proteins and topoisomerase II
Authors: Mínguez Ana , Franca Susana , Díaz de la Espina Susana Moreno 
Published: April 2021

ABSTRACT
Unicellular Dinoflagellates represent the only eukaryotic Phylum lacking histones and nucleosomes. To investigate whether Dinoflagellates do have a nuclear matrix that would modulate the supramolecular organization of their non-nucleosomal DNA and chromosomes, cells of the free- living unarmored Dinoflagellate Amphidinium carterae were encapsulated in agarose microbeads and submitted to sequential extraction with non-ionic detergents, nucleases and 2 M NaCl. Our results demonstrate that this species has a residual nuclear matrix similar to that of vertebrates and higher plants. The cytoskeleton-nuclear matrix complex of A. carterae shows a relatively intricate polypeptide pattern. Immunoblots with different antibodies reveal several intermediate filament types of proteins, one of which is immunologically related to vertebrate lamins, confirming that these proteins are ancestral members of the IF family, which is highly conserved in eukaryotes. A topoi- somerase II homologue has also been identified in the nuclear matrix, suggesting that these structures could play a role in organizing the Dinoflagellate DNA in loop domains. Taken together our results demonstrate that the nuclear matrix is an early acquisition of the eukaryotic nucleus, independent of histones and nucleosomes in such a way that the mechanisms controlling the two levels of organization in eukaryotic chromatin would be molecularly and evolutionarily independent.
The effects of the neuN and neuT genes on differentiation and transformation of mammary epithelial cells
Authors: Lucassen E. , Andres A.-C. , Reichmann E. , Entwistle A. , Noble M. 
Published: April 2021

ABSTRACT
Overexpression of the proto-oncogene product, p185neuN, in a non-tumorigenic mammary epithelial line (31E) facilitates aspects of lactogenic differentiation. Formation of branching cords and induction of β-casein synthesis by 31E cells normally require co-culture of these cells with fibroblasts, or the presence of collagen or fibronectin. In contrast, 31E cells expressing p185neuN spontaneously form branching cords when grown on tissue culture plastic and can synthesize β-casein in the absence of exogenous substrates or feeder layers. Under these conditions, the cells deposit laminin and fibronectin, indicating a possible role for p185neuN in the deposition of extracellular matrix proteins. Overexpression of the corresponding oncogene product, p185neuT, has markedly different effects. Expression of p185neuT does not facilitate the formation of branching cords or the synthesis of β-casein when grown on tissue culture plastic, although these cells do deposit laminin and fibronectin. Confocal microscopy indicates a significant difference in the distribu-tion of laminin and fibronectin in 31E cells expressing p185neuT compared to those expressing p185neuN. The effects of p185neuN and p185neuT expression on cell transformation depend on cell type. Expression of both p185neuN and p185neuT increases anchorage-independent growth of 31E cells, but only p185neuT induces anchorage-independent growth of NIH 3T3 fibroblasts. This lineage specificity in the action of p185neuN may be related to obser-vations that overexpression of p185c-erbB-2 (the human homologue of p185neuN) is only associated with the devel-opment of human epithelial cancers. The effects of p185neuN on laminin deposition by 31E cells may be relevant to the transforming ability of p185neuN, since laminin can induce anchorage-independent growth of mouse mammary cells. These results suggest that p185neuN and p185neuT could exert their effects on differentiation and transformation of mammary epithelial cells in part by promoting the deposition of extracellular matrix proteins.
Function of the syndecans - a family of cell surface proteoglycans
Authors: Elenius Klaus , Jalkanen Markku 
Published: April 2021

ABSTRACT
Cell surface proteoglycans are membrane-anchored glycoproteins that contain covalently linked glycosaminoglycan sidechains. They interact via their extracellular part with various effector molecules such as growth factors, extracellular matrix components, other cell surface molecules and proteins involved in the regulation of blood coagulation. This together with numerous examples of strictly regulated expression patterns during embryogenesis and malignant transformation has lead to the attribution of several specific functions to cell surface proteoglycans. Molecular biology techniques that have made it possible to identify and characterize individual proteoglycans, may also turn out to be crucial in determining the relevance of the several suggested functions in vivo. This review will mainly focus on the functional aspects of one well-characterized group of cell surface proteoglycans, the members of the syndecan gene family.