bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chick corneal development in vitro: Diverse effects of pH on collagen assembly*
Authors: Bard J. B. L. , Hulmes D. J. S. , Purdom I. F. , Ross A. S. A. 
Published: April 2021

ABSTRACT
In vivo, the embryonic chick corneal epithelium lays down a stroma of collagen and proteoglycans whose fibrils are unusual as their diameter distribution peaks sharply about a mean of 20 nm. Such epithelia cultured on Nuclepore filters will also lay down a stroma containing 20 nm diameter fibrils, although there is only limited orthogonal organisation. We report here that collagen fibril morphology is critically dependent on the pH of the medium in which the corneal epithelium is cultured and that normal 20 nm diameter fibrils only assemble in a narrow band around neutral pH (approx. 6.9-7.4). At higher pH (7.6-8.1), fibrils in the distal region of the stroma more closely resemble those seen in non-corneal stroma as their diameters can be up to 200 nm even though fibrils near the basal lamina are only about 10 nm in diameter. At low pH (approx. 6.5), there are again wide fibrils, but with the hieroglyphic cross-sections typical of those seen in heritable disorders of N-terminal procollagen processing. Biochemical analysis by SDS-PAGE and fluorography confirms that N-terminal procollagen processing is deficient at this pH. At very low pH (approx. 5.8-6.2), there is little processing of procollagen and the stroma comprises filamentous material with the occasional banded structures typical of those formed by unprocessed procollagen at high concentration. Gel electrophoresis and peptide mapping showed that the collagens produced by the corneal epithelium of the primary stroma included types I, II and V and that total collagen production, as assessed by incorporation of [3H]proline, increased with pH, although the relative amounts of the different collagens produced remained essentially unchanged. While the biochemical data can account for the altered morphologies in the pH range 5.8 to 7.0, the sensitivity of fibril diameter to small changes around neutral pH remains unexplained, but points to the subtle, chargebased interactions necessary for the formation of 20 nm diameter fibrils in the developing cornea.
Chicken filensin: a lens fiber cell protein that exhibits sequence similarity to intermediate filament proteins
Authors: Remington Susann G. 
Published: April 2021

ABSTRACT
Filensin, a 100 kDa, membrane-associated, cytoskeletal protein, is uniquely expressed in the lens fiber cell (Merdes, A., Brunkener, M., Horstmann, H., and Georgatos, S. D. (1991) J. Cell Biol. 115, 397-410). I cloned and sequenced a full-length chicken lens cDNA encoding filensin, also known as CP95 (Ireland, M. and Maisel, H. (1989) Lens and Eye Toxicity Research 6, 623638). The deduced amino acid sequence of 657 residues contained an internal 280 residue heptad repeat domain with sequence similarities to the rod domain of intermediate filament proteins. The putative filensin rod domain could be divided into three α-helical segments (1A, 1B and 2) separated by short, non-helical linkers. The sequence of the amino-terminal end of the filensin rod domain contained the highly conserved intermediate filament segment 1A motif (Conway, J. F. and Parry, D. A. D. (1988) Int. J. Biol. Macromol. 10, 79-98). Allowing conservative amino acid substitutions, the sequence of the carboxy-terminal end of the filensin rod domain was similar to that of the highly conserved intermediate filament rod carboxy terminus. The cx-helical segments of the shorter filensin rod domain aligned with the corresponding segments of intermediate filament proteins by allowing a gap of four heptad repeats in the amino-terminal half of filensin segment 2. Filensin rod segment 2 contained the characteristic stutter in heptad repeat phasing, nine heptads from the end of the intermediate filament rod. The overall sequence identity between the rod domains of filensin and individual intermediate filament proteins was 20 to 25%, approximately the level of sequence identity observed between intermediate filament proteins of different types.
The open reading frame of chicken filensin predicted a 657 amino acid protein with molecular mass of 76 kDa. Embryonic chicken filensin migrated in SDS-PAGE as a triplet of 102, 105 and 109 kDa, while rooster filensin migrated as a 105 and 109 kDa doublet. Antibodies to filensin labeled lens fiber cells but not lens epithelial cells. By immunofluorescence methods filensin was localized to the fiber cell plasma membranes, including the ends of elongated fiber cells.
Heparan sulfate proteoglycan and FGF receptor target basic FGF to different intracellular destinations
Authors: Reiland Jane , Rapraeger Alan C. 
Published: April 2021

ABSTRACT
Basic FGF is a prototype of a family of heparin binding growth factors that regulate a variety of cellular responses including cell growth, morphogenesis and differentiation. At least two families of receptors bind bFGF and could mediate its response: (1) tyrosine kinase-containing FGF receptors, designated FGFR-1 to FGFR-4, and (2) heparan sulfate proteoglycans that bind bFGF through their heparan sulfate chains. Both are known to undergo internalization and thus bFGF bound to the different receptors may be internalized via more than one pathway. It is not known whether the intracellular fate of bFGF differs depending upon which receptor binds it at the cell surface. To investigate the respective roles of these receptors in the intracellular targeting of bFGF, we utilized NMuMG cells that bind and internalize bFGF through their heparan sulfate proteoglycans, but do not express detectable levels of FGFRs nor respond to bFGF. Basic FGF conjugated to saporin (bFGF-saporin) was used as a probe to study targeting of bFGF by the different receptors. Saporin is a cytotoxin that has no effect on cells if added exogenously. However, it kills cells if it gains access to the cytoplasm. The NMuMG cells internalize bFGF-saporin but are not killed. Transfecting these cells with FGFR-1 results in bFGF-responsive cells, which bind and internalize bFGF through FGFR-1, and are killed. Removing the heparan sulfate from these cells eliminates killing by bFGF-saporin. Therefore, endocytosis of bFGF-saporin by these receptors can lead to two fates: (i) bFGFsaporin internalized by heparan sulfate proteoglycan, which is not targeted to the cytoplasm, and (ii) a bFGFsaporin internalized by the bFGF-saporin bound to a complex of heparan sulfate proteoglycan and FGFR-1 from which the saporin can gain access to the cytoplasm.
Biochemical and morphological characterization of the nuclear matrix during the synchronous cell cycle of Physarum polycephalum
Authors: Lang Siegfried , Decristoforo Thomas , Waitz Werner , Loidl Peter 
Published: April 2021

ABSTRACT
We have investigated biochemical and ultrastructural aspects of the nuclear matrix during the naturally synchronous cell cycle of Physarum polycephalum. The morphology of the in situ nuclear matrix exhibited significant cell cycle changes as revealed by electron microscopic examination, especially during the progression of nuclei through mitosis and S-phase. In mitosis the interchromatin matrix was found to be retracted to the nuclear periphery; during S-phase this interchromatin matrix gradually reassembled, concomitant with the reconstruction of a nucleolar remnant structure. During the G2-period no significant changes in matrix morphology were observed. The pattern of nuclear matrix proteins was invariant during the cell cycle; no cycle phase-specific proteins could be detected. In vivo labelling of plasmodia with [35S]methionine/cysteine showed that only a few proteins are synthesized and assembled into nuclear matrix structures in a cell cycledependent way; the majority of proteins were synthesized almost continuously. This was also shown for nuclear lamins homologues.
In contrast to bulk nuclear histones, those histones that remain tightly bound to the nuclear matrix were synthesized and assembled into nuclear structures in the very first hour of S-phase; assembly was terminated in mid-S-phase, indicating that nuclear matrix-bound chromatin is replicated early in S-phase. Comparison of the acetylation pattern of matrix-bound histone H4 with bulk nuclear H4 revealed a largely elevated acetate content of matrix H4. The percentage of acetylated subspecies was entirely different from that in bulk nuclear H4, indicating that matrix-associated histones represent a subpopulation of nuclear histones with distinct properties, reflecting specific structural requirements of matrix-attached chromatin.
Localization of 5 S genes and transcripts in Pisum sativum nuclei
Authors: Highett M. I. , Beven A. F. , Shaw P. J. 
Published: April 2021

ABSTRACT
We have investigated the position of the 5 S gene clusters in the nuclei of Pisum sativum root tip cells, and of their transcripts in the nucleolus, using in situ hybridization and confocal microscopy. Single-stranded RNA probes were produced by in vitro transcription, incorporating fluorescein or digoxygenin label. There are known to be 3 pairs of 5 S gene clusters in this species, which would be expected to give 6 sites of hybridization in G1 cells and 12 sites in G2 cells. In presumed G2 cells, many of the sites appeared as paired spots very close together, which we interpret as the sites on sister chromatids. A clear association of one or more clusters with the nucleolar periphery was observed, and it is possible that this has a functional significance. The transcript labelling within the nucleolus showed a reproducible and highly ordered pattern, consisting of cavities devoid of transcript labelling surrounded by sheets of bright labelling, making a characteristic and often very symmetrical cage-work structure. This labelling pattern may represent an underlying organization of transcript processing within the nucleolus, and is highly reminiscent of the distribution of a previously described nucleolar matrix protein.
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate blocks differentiation of HT-29 human colon cancer cells
Authors: García de Herreros Antonio , Fabre Myriam , Batlle Eduard , Balagué Cristina , Real Francisco X. 
Published: April 2021

ABSTRACT
Recently developed HT-29-derived cell lines, which display variable differentiated phenotypes provide an invaluable opportunity to analyze the mechanism by which cell differentiation is regulated in the intestine. We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in the differentiation phenotype of mucus-secreting (HT-29 M6) and absorptive (HT-29 M3) cells. TPA prevented the accumulation of differentiation markers such as dipeptidylpeptidase IV, villin or mucins, down-regulated the expression of these molecules in post-confluent differentiated cell cultures and induced the loss of the functional integrity of the tight junction in the monolayer (i.e. decreased transepithelial resistance and inhibited dome formation). These effects were mediated by activation of protein kinase C (PK-C), as demonstrated using the specific inhibitor GF109203x. Analysis by immunoblotting of the PK-C isoforms present in HT-29 M6 cells revealed that the most abundant TPA-sensitive isoform was PK-C ε, although low levels of cPK-C were also detected. Further studies are necessary to elucidate the role of the different PK-C isoforms in the differentiation of HT-29 cells.
Association of p34cdc2/cyclin B complex with microtubules in starfish oocytes
Authors: Ookata Kayoko , Hisanaga Shin-ichi , Okumura Eiichi , Kishimoto Takeo 
Published: April 2021

ABSTRACT
The microtubular cytoskeleton exhibits a dramatic reorganization, progressing from interphase radial arrays to a mitotic spindle at the G2/M transition. Although this reorganization has been suspected to be caused by maturation promoting factor (MPF: p34cdc2/cyclin B complex), little is known about how p34cdc2 kinase controls microtubule networks. We provide evidence of the direct association of the p34cdc2/cyclin B complex with microtubules in starfish oocytes. Anti-cyclin B staining of detergent-treated oocytes, isolated asters and meiotic spindles revealed fluorescence associated with microtubule fibers, chromosomes and centrosomes. Microtubules prepared from starfish oocytes were associated with cyclin B and p34cdc2 proteins. Microtubule-bound p34cdc2 and cyclin B were released from microtubules by a high-salt solution and possessed a complex form as shown by the adsorption to suc1-beads and by immunoprecipitation with the anti-cyclin B antibody. The p34cdc2/cyclin B complex associated to microtubules had high histone H1 kinase activity at meiotic metaphase. However, it was not necessary for the p34cdc2/cyclin B complex to be active for microtubule binding, as an inactive form in immature oocytes was also observed to bind to microtubules. The coprecipitation of suc1-column purified p34cdc2/cyclin B with purified porcine brain microtubules in the presence of starfish oocyte microtubule-associated proteins (MAPs) indicates that the association of p34cdc2/cyclin B with microtubules in vitro is mediated by MAPs.
Changes in the cytoskeletal structure of cultured smooth muscle cells induced by calyculin-A
Authors: Hosoya Natsumi , Mitsui Minori , Yazama Futoshi , Ishihara Hiroaki , Ozaki Hiroshi , Karaki Hideaki , Hartshorne David J. , Mohri Hideo 
Published: April 2021

ABSTRACT
Changes in the cytoskeletal structure of cultured A10 smooth muscle cells induced by calyculin-A (CL-A), a potent inhibitor of types 1 and 2A protein phosphatases, were analyzed using indirect fluorescence techniques. In the presence of 1×107 M CL-A the cells became round and subsequently detached from the substratum. The effect of CL-A was inhibited by a non-selective kinase inhibitor, K-252a, but not by EGTA. In rounded cells stress fibers were absent and staining for F-actin appeared in patches. Vinculin, one of the components of focal contacts, was localized at the periphery of control cells. CL-A treatment moved the focal contacts towards the inside of the cell along the stress fibers, and this was followed by the rounding up of the cell. In addition, rapid and marked changes in microtubule structure were observed in CL-A-treated cells. Many ‘nicks’ or ‘gaps’ were observed along the microtubules in the attached, spread cells. A filamentous network of microtubules was not observed in the detached cells, i.e. after longer exposure to CL-A. These results suggest that CL-A may change the structure of focal contacts, resulting in the rounding up of the cell, and inducing a microtubule-severing activity. These effects were independent of the external Ca2+ concentration. The changes in cytoskeletal structure may be caused by disturbing the balance of phosphorylation and dephosphorylation in the cell.
Macrophages, but not dendritic cells, accumulate colloidal carbon following administration in situ
Authors: Witmer-Pack Margit D. , Crowley Mary T. , Inaba Kayo , Steinman Ralph M. 
Published: April 2021

ABSTRACT
The dendritic cell system operates in situ to capture and present antigens in a form that is immunogenic to T cells. It is likely that dendritic cells require endocytic activity in order to process antigens. On the other hand, macrophages are considered to be the principal cells that internalize substrates in situ. We therefore investigated the phenotype of cells that scavenge the indigestible endocytic tracer, colloidal carbon, by phenotyping the endocytic cells with monoclonal antibodies that help distinguish macrophages from dendritic cells. Of some importance was the monoclonal N418, an antibody to the p150/90 leukocyte β2 integrin. FACS analyses on isolates from blood, spleen and peritoneal cavity showed that N418 reacts primarily with dendritic cells. N418 also stained dendritic profiles strongly in tissue sections of liver and spleen, but most of the cells that actively endocytosed carbon in both organs showed little or no N418 staining. Likewise, carbon could not be identified in cells that react with M342, which stains intracellular granules of dendritic cells. In contrast, the carbon-labeled cells in both liver and spleen were labeled with antibodies (SER-4, F4/80, FA11) that bind primarily to isolated macrophages. Therefore the clearance of colloidal carbon in situ reflects the scavenging activity of macrophages and not the endocytic activity that underlies the antigen presenting function of dendritic cells.
The product of the Drosophila segment polarity gene Armadillo is part of a multi-protein complex resembling the vertebrate adherens junction
Authors: Peifer Mark 
Published: April 2021

ABSTRACT
Sequence similarity between the Drosophila segment polarity protein Armadillo and the vertebrate adherens junction protein β-catenin raised the possibility that adherens junctions function in transduction of intercellular signals like that mediated by Wingless/Wnt-1. To substantiate the sequence similarity, properties of Armadillo were evaluated for consistency with a junctional role. Armadillo is part of a membrane-associated complex. This complex includes Armadillo, a glycoprotein similar in size to vertebrate cadherins, and the Drosophila homolog of α-catenin. Armadillo co-localizes with junctions that resemble vertebrate adherens junctions in morphology and position. These results suggest that Drosophila and vertebrate adherens junctions are structurally similar, validating use of Armadillo and its associated proteins as a model for vertebrate adherens junctions.
Loss of cytoplasmic basic fibroblast growth factor from physiologically wounded myofibers of normal and dystrophic muscle
Authors: Clarke Mark S. F. , Khakee Robert , McNeil Paul L. 
Published: April 2021

ABSTRACT
Using muscle as an in vivo model system, we have tested the hypothesis that basic fibroblast growth factor is released from a cytoplasmic storage site into the extracellular environment via diffusion through survivable, mechanically-induced plasma membrane disruptions. Normal and dystrophic (mdx) mouse muscle were studied. Strong immunostaining for bFGF was detected in the cytoplasm of myofibers of uninjured muscle fixed in situ by perfusion. By contrast, myofibers did not stain cytoplasmically for bFGF after suffering lethal disruptions of their plasma membranes caused by freezing and thawing followed by sectioning. Sub-lethal, transient disruptions of myofiber plasma membranes - termed plasma membrane ‘wounds’ - were shown to be induced by needle puncture or exercise of muscle. Quantitative image analysis revealed that these wounded fibers contained significantly reduced levels of bFGF. Dystrophic exercised and unexercised muscle was found to possess an ∼6-fold higher proportion of wounded myofibers than does normal muscle under equivalent conditions. Release of bFGF at a rate that is a direct function of the frequency of myofiber wounding may explain in part how a muscle adjusts its growth to meet changing mechanical demand as well as the pathological hypertrophy characteristic of certain stages of muscular dystrophy.
The expression of basic fibroblast growth factor and its receptor in cell lines derived from normal human mammary gland and a benign mammary lesion
Authors: Ke Youqiang , Fernig David G. , Wilkinson Mark C. , Winstanley John H. R. , Smith John A. , Rudland Philip S. , Barraclough Roger 
Published: April 2021

ABSTRACT
mRNA for basic Fibroblast Growth Factor (bFGF) was expressed in a series of SV40-transformed human mammary cell lines as molecules of 7.1, 3.6, 2.0 and 1.2 kb. This expression was much weaker in those lines of epithelial morphology than in myoepithelial-like cell lines derived from them. It was confirmed, using northern hybridization to single-stranded RNA probes, that the multiple mRNAs were transcribed from the coding strand for bFGF. bFGF activity was detected in extracts of the cells and the relative amounts of activity corresponded in general to the amounts of mRNA found. Similar results were obtained from spontaneously transformed cell lines derived from a human benign breast lesion. The presence of bFGF protein in the extracts was confirmed by western blotting, which showed a band of 18-19 kDa, migrating in the same position as authentic
bFGF; in addition, the myoepithelial-like cells showed prominent bands of bFGF at 24 and 26 kDa. No FGF receptor was detectable by the binding of 125I-bFGF to the SV40-transformed cell lines or to the epithelial cell lines from the benign breast lesion, but both high- and low-affinity receptors were found on myoepithelial-like cells derived from the latter. The results indicate that differentiation to the human myoepithelial-like phenotype in culture is associated with the enhanced expression of bFGF, and it is suggested that bFGF, immunocytochemically detected in the basement membrane of the human breast, may arise, at least in part, from the myoepithelial cells of the mammary parenchyma.
In vivo observation of the puff-specific protein no-on transient a (nona) in nuclei of drosophila embryos
Authors: Buchenau Peter , Arndt-Jovin Donna J. , Saumweber Harald 
Published: April 2021

ABSTRACT
The spatial distribution of no-on transient A (NONA), a protein associated with specific puffs on polytene chromosomes, was followed in nuclei of living Drosophila embryos by microinjection of fluorescently labeled monoclonal antibody to NONA. The injected antibodies remained active until the larval stage, revealing the distribution of the NONA protein throughout embryogenesis. Most injected animals completed embryonic development and hatched as normal larvae. NONA was restricted to the cytoplasm until the end of cycle 11. We document an active uptake of the NONA-antibody complex into early interphase nuclei from nuclear cycle 14 onwards, following each mitosis. Significant differences in the distribution of the protein between fixed and living embryos were apparent, particularly at high resolution. The NONA protein was localized in the nuclei of living embryos at discrete sites, most of which lay at the periphery and some of which were tightly clustered. The constellation of sites changed with time; in some nuclei these changes were fast whereas in other nuclei the pattern was quite stable. These data suggest that specific protein complexes associated with active interphase chromatin, and possibly chromatin in general, are mobile in the living organism.
A mutation in the α1-tubulin gene of Chlamydomonas reinhardtii confers resistance to anti-microtubule herbicides
Authors: James Steven W. , Silflow Carolyn D. , Stroom Pamela , Lefebvre Paul A. 
Published: April 2021

ABSTRACT
A mutation in the α1-tubulin gene of Chlamydomonas reinhardtii was isolated by using the amiprophosmethyl-resistant mutation apm1-18 as a background to select new mutants that showed increased resistance to the drug. The upA12 mutation caused twofold resistance to amiprophos-methyl and oryzalin, and twofold hypersensitivity to the microtubule-stabilizing drug taxol, suggesting that the mutation enhanced microtubule stability. The resistance mutation was semi-dominant and mapped to the same interval on linkage group III as the α1-tubulin gene. Two-dimensional gel immunoblots of proteins in the mutant cells revealed two electrophoretically altered α-tubulin isoforms, one of which was acetylated and incorporated into microtubules in the axoneme. The mutant isoforms co-segregated with the drug-resistance phenotypes when mutant upA12 was backcrossed to wild-type cells. Two-dimensional gel analysis of in vitro translation products showed that the non-acetylated variant α-tubulin was a primary gene product. DNA sequence analysis of the α-tubulin genes from mutant and wild-type cells revealed a single missense mutation, which predicted a change in codon 24 from tyrosine in wild type to histidine in mutant upA12. This alteration in the predicted amino acid sequence corroborated the ∼+1 basic charge shift observed for the variant α-tubulins. The mutant allele of the cx1-tubulin gene was designated tua1-1.