bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adhesive properties and integrin expression profiles of two colonic cancer populations differing by their spreading on laminin
Authors: Simon-Assmann P. , Leberquier C. , Molto N. , Uezato T. , Bouziges F. , Kedinger M. 
Published: April 2021

ABSTRACT
The mostly undifferentiated parental HT29 (HT29p) human colonic adenocarcinoma cell line and a differentiated subpopulation selected by the anti-cancer drug 5-fluorouracil (HT29-Fu) (Lesuffleur et al. (1991)Int. J. Cancer 49, 721-730) display strikingly different behavior when grown on laminin coatings: the former grows as aggregates while the latter grows as monolayers. In an attempt to explain this difference, we performed a comparative study of cell adhesion properties and of expression, involvement and localization of the α6, β1 and β4 subunits constituting the integrin family among the two cell populations.
HT29p and HT29-Fu cells exhibited a similar adhesion pattern to laminin and laminin fragments E8 and P1. In both cell lines, cell adhesion could be blocked at about 90% with anti-α6 subunit antibodies and around 30-50% with anti-β1 antibodies; no inhibition of the cell adhesion was obvious when using anti-β4 antibodies. Immunoprecipitations of iodinated membrane-solubilized proteins and immunoblotting experiments showed that all α6 chains expressed in both HT29p and HT29-Fu cell populations exist as α6β4 integrins; β1 subunits are associated with α2 and α3 chains. When HT29p or HT29-Fu cells were injected subcutaneously in nude mice, a similar expression pattern of α6, β4 and β1 integrin subunits was noticeable in the resulting tumors: α6 and β4 subunits were localized at the basal surface of the tumor cells facing the stromal elements, and to a lesser extent at the cell-cell contacts within the tumor-cell clumps; β1 subunits were mainly found within the cytoplasm of the tumor cells.
Despite these overall similarities among the two cell lines, the following changes could account for their different behavior on laminin: less proteolytic processing of the β4 integrin subunit occurred in HT29-Fu cells yielding peptidic fragments of 175 kDa, which are absent from the parental cells; the immunostaining pattern of the various subunits demonstrated a segregation of α6, β4 and β1 integrin subunits on the basal side of the HT29-Fu cells when cultured on laminin to the detriment of their lateral location, a phenomenon that was not obvious in the parental cells.
Altogether, these results suggest that the distinct behavior of the undifferentiated versus differentiated HT29 cell populations on laminin is not related to altered adhesion properties of the cells but rather to a deficient stabilization of the adhesion leading to cell spreading. This difference is highlighted by a variable basal segregation potential of the laminin-binding integrins, which could be due to an altered form of the β4 subunits.
Analysis of the Schizosaccharomyces pombe cyclin puc1: evidence for a role in cell cycle exit
Authors: Forsburg Susan L. , Nurse Paul 
Published: April 2021

ABSTRACT
The puc1+ gene, encoding a G1-type cyclin from the fission yeast Schizosaccharomyces pombe, was originally isolated by complementation in the budding yeast Saccharomyces cerevisiae. Here, we report the molecular characterization of this gene and analyse its role in S. pombe. We fail to identify any function of this cyclin at the mitotic G1/S transition in S. pombe, but demonstrate that it does function in exit from the mitotic cycle. Expression of the puc1+ gene is increased during nitrogen starvation, and puc1 affects the timing of sexual development in response to starvation. Overexpression of the puc1 protein blocks sexual development, and rescues pat1ts cells, which would otherwise undergo a lethal meiosis. We conclude that puc1 contributes to negative regulation of the timing of sexual development in fission yeast, and functions at the transition between cycling and non-cycling cells.
Study of the higher eukaryotic gene function CDK2 using fission yeast
Authors: Paris Jeannie , Leplatois Pascal , Nurse Paul 
Published: April 2021

ABSTRACT
In the fission yeast Schizosaccharomyces pombe, cdc2 function is required both in G1 to enter the cell cycle and in G2 to initiate mitosis. In higher eukaryotes, these functions appeared to be shared between several cdc2-like genes including CDK2. Temperature-sensitive mutations in S. pombe cdc2 that arrest the cell cycle in both G1 and G2 phases are not complemented by CDK2. We have used S. pombe to investigate what functions CDK2 can perform. We found that overexpression of the human homologue (HsCDK2) caused cell cycle arrest in G2/M showing that HsCDK2 interfered with mitotic events. Xenopus CDK2 (XlCDK2) overexpression did not cause cell cycle arrest and could rescue the G1 block but not the G2 block of a cdc2-M26 ts strain. A mutant XlCDK2-R33, which is inactive as a kinase, failed to rescue the G1 block, suggesting that the protein kinase activity of CDK2 is required to enter the cell cycle in these circumstances.
We designed screens to select mutants that would require XlCDK2 expression for viability, hoping to isolate new gene functions interacting with, or that could be replaced by, XlCDK2 in G1, or new cdc2 mutants altered solely in their G1 role. From these screens several cell cycle mutants were selected that were XlCDK2-dependent. These were all cdc2 mutants altered only in their G2/M function. Therefore XlCDK2 can influence both the G1/S and G2/M transition points of cdc2 in S. pombe.
Cytoskeletal involvement in the modulation of cell-cell junctions by the protein kinase inhibitor H-7
Authors: Citi Sandra , Volberg Tova , Bershadsky Alexander D. , Denisenko Natasha , Geiger Benjamin 
Published: April 2021

ABSTRACT
The protein kinase inhibitor H-7 has been shown to block junction dissociation induced by low extracellular calcium in Madin Darby canine kidney epithelial cells (S. Citi, J. Cell Biol. (1992) 117, 169-178). To understand the basis of this effect, we have examined how H-7 affects the organization of junctions and the actin cytoskeleton in different types of epithelial cells in culture. Immunofluorescence microscopy showed that H-7 confers Ca2+ independence on cultured epithelial lens cells, which lack tight junctions and desmosomes but have microfilament-associated adherens junctions. In these cells, H-7 did not protect N-cadherin from trypsin digestion at low extracellular calcium, suggesting that H-7 does not stabilize the ‘active’ cadherin conformation. In cultured Madin Darby canine kidney cells, H-7 partially prevented the fall in transepithelial resistance induced by cytochalasin D, either alone or in conjunction with calcium chelators. Double-immunofluorescence microscopy showed that H-7 inhibits both the fragmentation of labeling for the tight junction protein cingulin and the condensation of actin into cytoplasmic foci induced by cytochalasin D. Taken together, these observations indicate that H-7 inhibits junction dissociation by affecting the contractility of the adherens junction-associated microfilaments following treatment with calcium chelators or cytochalasin D.
Exogenous histone H1 injection into mitotic cells disrupts synchronous progression of mitotic events by delaying chromosome decondensation
Authors: Matsuoka Yosuke , Takechi Shinji , Nakayama Tatsuo , Yoneda Yoshihiro 
Published: April 2021

ABSTRACT
At the end of open mitosis, chromosome decondensation, nuclear envelope re-formation and reassembly of interphase microtubules following mitotic spindle dissociation occur coordinately. To determine whether these events progress only synchronously in vivo, we delayed chromosome decondensation by injecting of exogenous proteins into the mitotic rat kangaroo kidney epithelium (PtK2) cells. When histone H1 purified from calf thymus was injected at prometaphase, chromosome condensation was prolonged for several hours, and sister chromatid separation and cytokinesis did not occur. However, interphase microtubules reassembled and lamin B-positive structures re-formed around the condensed chromosomes. Exactly the same results were obtained on injection of bacterially expressed H1. Kinetic experiments showed that there were two types of lamin B-positive structures. One type (type A) was stained uniformly with anti-lamin B antibodies. The other (type B) showed peripheral lamin B staining; that is, the normal interphase staining pattern, and was found to be competent for nuclear protein transport. As the chromosomes decondensed, the amount of type A decreased and that of type B increased. However, even cells containing highly condensed chromosomes had both type A and type B. From these results, we conclude that the re-formation of microtubules and reassembly of a nuclear transport-competent envelope do not depend on chromosome decondensation.
Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins
Authors: Milgram Sharon L. , Mains Richard E. 
Published: April 2021

ABSTRACT
Vesicular transport within the secretory pathway can be arrested by incubating cells at 15°C or 20°C to block exit from the endoplasmic reticulum or trans-Golgi network, respectively. Using this powerful tool we have compared the intracellular sites of endoproteolytic processing of proopiomelanocortin and two prohormone processing enzymes in AtT-20 mouse pituitary corticotrope tumor cells. For comparison, proopiomelanocortin processing was also evaluated in primary neurointermediate pituitary cultures. AtT-20 cells synthesize and store endogenous proopiomelanocortin and prohormone convertase 1; AtT-20 cells expressing high levels of integral membrane or soluble peptidylglycine α-amidating monooxygenase were generated by stable transfection. Cells were incubated with [35S]methionine and chased at 4°C, 15°C, 20°C or 37°C. The endoproteolytic processing of peptidylglycine α-amidating mono-oxygenase, prohormone convertase 1, and proopiomelanocortin was compared following immunoprecipitation. Endoproteolytic processing of integral membrane and soluble peptidylglycine α-amidating monooxygenase proteins was completely blocked by incubation of cells at 20°C. In contrast, prohormone convertase 1 processing from the 87 kDa precursor to the 81 kDa intermediate proceeded to completion at both 15°C and 20°C, while cleavage to generate the 63 kDa prohormone convertase 1 protein was completely blocked at 20°C. In AtT-20 cells and neurointermediate pituitary cultures, generation of β-lipotropin from proopiomelanocortin continued at a slow but significant rate at 20°C, while processing of β-lipotropin to β-endorphin was blocked. Thus prohormone convertase 1 processing begins in the endoplasmic reticulum and is not completed until after the trans-Golgi network, while peptidylglycine α-amidating monooxygenase processing begins after the trans-Golgi network. Selected proopiomelanocortin cleavages begin before entry into immature granules.
Endocrine and mucous differentiation by a cloned human rectal adenocarcinoma cell line (HRA-19) in vitro: inhibition by TGF-β1
Authors: Kirkland Susan C. , Henderson Karen 
Published: April 2021

ABSTRACT
Colorectal epithelium is composed of absorptive, mucous and endocrine cells, all of which are considered to arise from a common stem cell located in the crypt base. However, the factors controlling the commitment to differentiate are poorly understood. This is partly due to the lack of in vitro model systems for the study of differentiation in colorectal epithelium. The HRA-19 cell line, established from a human rectal adenocarcinoma, has been shown to have multipotential characteristics with cloned HRA-19 cells able to differentiate into absorptive, mucous and endocrine cells when grown as xenografts. The lack of such differentiated cells in HRA-19 monolayers in vitro suggests that differentiation is controlled by extracellular matrix, stromal cells and/or soluble factors. Such observations show that differentiation in HRA-19 cells can be controlled by extrinsic factors and therefore provide a model system for studying control of differentiation in colorectal epithelium. Unfortunately, the restriction of differentiation to xenografts of the cell line limits the degree to which this differentiation can be manipulated.
In this study, the possibility that HRA-19 cells could be induced to differentiate in vitro under appropriate conditions has been investigated. Endocrine and mucous cells were identified by immunocytochemistry with differentiation-related antibodies and histology of monolayers. Preconfluent HRA-19 cells grown in 10% foetal calf serum formed a well polarised monolayer with apical tight junctions and sparse microvilli, but cells with mucous or endocrine phenotypes were only very occasionally observed. However, endocrine and mucous cells could reproducibly be demonstrated in postconfluent monolayers grown in 1% foetal calf serum. The numbers of endocrine and mucous cells were quantified in monolayers, grown on coverslips, and this formed the basis for a differentiation assay. In this assay, TGF-β inhibited HRA-19 cell proliferation and also inhibited differentiation into both endocrine and mucous cells.
Therefore, HRA-19 cells provide a useful model system for identifying factors controlling differentiation in colorectal epithelial cells.
Cytoskeletal proteins of insect muscle: location of zeelins in Lethocerus flight and leg muscle
Authors: Ferguson C. , Lakey A. , Hutchings A. , Butcher G. W. , Leonard K. R. , Bullard B. 
Published: April 2021

ABSTRACT
Asynchronous insect flight muscles produce oscillatory contractions and can contract at high frequency because they are activated by stretch as well as by Ca2+. Stretch activation depends on the high stiffness of the fibres and the regular structure of the filament lattice. Cytoskeletal proteins may be important in stabilising the lattice. Two proteins, zeelin 1 (35 kDa) and zeelin 2 (23 kDa), have been isolated from the cytoskeletal fraction of Lethocerusflight muscle. Both zeelins have multiple isoforms of the same molecular mass and different charge. Zeelin 1 forms micelles and zeelin 2 forms filaments when renatured in low ionic strength solutions. Filaments of zeelin 2 are ribbons 10 nm wide and 3 nm thick.
The position of zeelins in fibres from Lethocerusflight and leg muscle was determined by immunofluorescence and immunoelectron microscopy. Zeelin 1 is found in flight and leg fibres and zeelin 2 only in flight fibres. In flight myofibrils, both zeelins are in discrete regions of the A- band in each half sarcomere. Zeelin 1 is across the whole A-band in leg myofibrils. Zeelins are not in the Z-disc, as was thought previously, but migrate to the Z-disc in glycerinated fibres. Zeelins are associated with thick filaments and analysis of oblique sections showed that zeelin 1 is closer to the filament shaft than zeelin 2.
The antibody labelling pattern is consistent with zeelin molecules associated with myosin near the end of the rod region. Alternatively, the position of zeelins may be determined by other A-band proteins. There are about 2.0 to 2.5 moles of myosin per mole of each zeelin. The function of these cytoskeletal proteins may be to maintain the ordered structure of the thick filament.
Fission yeast protein kinase C gene homologues are required for protoplast regeneration: a functional link between cell wall formation and cell shape control
Authors: Kobori Hiromi , Toda Takashi , Yaguchi Hiroko , Toya Mika , Yanagida Mitsuhiro , Osumi Masako 
Published: April 2021

ABSTRACT
Two novel protein kinase C (nPKC) gene homologues, pck1+and pck2+were isolated from the fission yeast Schizosaccharomyces pombe(Toda et al. (1993)EMBO J. 12, 1987). We examined the functional differences of pck1+and pck2+in cell wall formation and actin organization of S. pombe. Regenerating protoplasts of a wild-type strain, single gene disruptants of pck1+(Δpck1) and pck2+(Δpck2) were used as a simple model to examine the functional links between PKC, cell wall formation and actin organization. Protoplasts of the wild-type strain and those of Δpck1reverted to intact cells in osmotically stabilized liquid medium. A close spatial association between new cell wall formation and actin was observed in these two strains. In Δpck2, protoplasts did not revert to intact cells: (1) scarcely any new cell wall material was formed; (2) actin was not reorganized; and (3) nuclear division and an increase in the amount of cytoplasm were observed in the regenerating protoplasts. These findings demonstrate that the pck2+gene has a function essential for protoplast regeneration but the pck1+gene does not. Involvement of nPKCs in cell wall formation and actin organization was also clarified. The effect of staurosporine (a potent inhibitor of protein kinases) on regenerating protoplasts of the three strains confirmed the assumption that the pck2 protein is an in vivo target of staurosporine in the fission yeast.
Macrophage recruitment during limb development and wound healing in the embryonic and foetal mouse
Authors: Hopkinson-Woolley James , Hughes Derralynn , Gordon Siamon , Martin Paul 
Published: April 2021

ABSTRACT
Macrophages play a pivotal role in the adult inflammatory response to wounding. They are directly responsible for cellular débridement and, by providing a source of growth factors and cytokines, they recruit other inflammatory and fibroblastic cells and influence cell proliferation and tissue remodelling. In this paper we investigate the role of macrophages in clearing areas of programmed cell death in the developing embryo and also their role in embryonic and foetal wound healing. Immunocytochemistry using the monocyte/macrophage-specific monoclonal antibody, F4/80, reveals a close association between areas of programmed cell death in the remodelling interdigital regions of the mouse footplate and of F4/80-positive cells, suggesting that monocyte-derived macrophages, and not locally recruited fibroblastic cells, as previously believed, are responsible for phagocytosing and clearing areas of interdigital apoptosis. Our studies of wound healing reveal that macrophages are not recruited to, and therefore cannot be playing an active role in the healing of, excisional wounds made in the mouse embryo at any stage up until E14.5. Beyond this transition stage we see a significant recruitment of macrophages within 12 hours of wounding. We find that macrophages can be attracted to wounds in earlier embryos if the wound results in significant cell death such as after burning.
p53 expression in cultured cells following radioisotope labelling
Authors: Dover Robin , Jayaram Yogi , Patel Kirtika , Chinery Rebecca 
Published: April 2021

ABSTRACT
p53 inhibits division following cellular damage. Cultured cells were found to express p53 protein following pulse labelling with radioisotopes, even at low doses normally used for growth and metabolic labelling studies. Some stem cells are exquisitely sensitive to radiation and thus p53 may have evolved as a major regulator of stem cell function. Therefore anygenetic damage may be able to induce p53 expression, which in turn will affect the biochemical outcome of many experiments by both cell cycle arrest and other mechanisms. In some cases the use of radioisotopes may directly change the results of the experiment. This will require a careful re-evaluation of the current literature and experimental protocols utilising radioisotopes.
17-β-Estradiol induced alterations of cell-matrix and intercellular adhesions in a human mammary carcinoma cell line
Authors: DePasquale Joseph A. , Samsonoff William A. , Gierthy John F. 
Published: April 2021

ABSTRACT
The MCF-7 human mammary carcinoma cell line undergoes morphological differentiation in vitro when treated with 17-β-estradiol. A prominent feature of this process is the postconfluent development of multicellular, three-dimensional nodules that rise above the surrounding monolayer. Formation of the nodules suggests that changes in cellular adhesion occur during this cellular overgrowth. Therefore changes in the distribution of cell-matrix and cell-cell adhesion plaque proteins were examined with respect to estradiol induction of nodule development. Estradiol treatment of the carcinoma cell line had the following effects: (1) vinculin- and talin-rich cell-matrix adhesion plaques were reduced in overall number and size in confluent and postconfluent cultures. No overt change in distribution or morphology of adhesion plaques was observed in subconfluent cultures. (2) Staining for vinculin was reduced in cell-cell adhesions situated at the apical region of subconfluent, confluent and postconfluent mono- layers. Staining for F-actin and plakoglobin was retained at this region in estradiol-induced cells. (3) vinculin was not detected in intercellular adhesions of nodule cells although intense labelling for both F-actin and plakoglobin was observed. In addition, in untreated monolayer cells, both F-actin and plakoglobin were concentrated in a subapical/basolateral location, as a vesicle-like pattern, which corresponded to intercellular spaces observed with phase-contrast microscopy. Treatment with estradiol caused the rearrangement of subapical/ basolateral F-actin and plakoglobin staining into a more uniform pattern. The findings of this study show that estradiol induces changes in both cell-matrix and cell-cell adhesions in an estrogen- responsive carcinoma cell line. The gradual loss of vinculin from cell-matrix and cell-cell adherens junctions of the monolayer could be a potential factor in the capacity of these cells to form multilayers or nodules in postconfluent growth. Furthermore, the development of the nodules in response to estradiol may provide a useful system in which to study steroid hormone regulation of adhesion and the cytoskeleton in responsive tumor cells.
Two proteins associated with secretory granule membranes identified in chicken regulated secretory cells
Authors: Jean-Léon Thomas , Stieber Anna , Gonatas Nicholas 
Published: April 2021

ABSTRACT
Lately, we have identified two polypeptides of 92-94 kDa (GRL1) and 45-60 kDa (GRL2), expressed in cytoplasmic granules of chicken granulocytes and thrombocytes.
Here, we report that GRL1 and GRL2 are widely distributed in all exocrine and several endocrine cell types, but not in neurons of the central nervous system, during late stages of embryonic development, as well as in newly hatched and two-month-old chickens. Immunogold studies in ultrathin frozen sections of pancreatic acinar cells show that GRL1 and GRL2 are co-localized at the periphery of zymogen granules, in granules fused with apical acinar membranes and on apical membranes of acini, while the pregranular compartments of the secretory pathway are weakly or not labeled. Semiquantitative morphometric studies indicate that GRL1 and GRL2 are equally distributed in secretory granules.
A variety of physical and metabolic studies reveal that GRL2, a highly N-glycosylated polypeptide, is an intrinsic membrane protein, while GRL1 is a peripheral membrane polypeptide released by Na2CO3treatment of granulocyte membranes. In all hematopoietic, exocrine or endocrine cells examinated, GRL1 shows identical electrophoretic patterns, while GRL2 is identified as a diffuse band, at 40-65 kDa, in hematopoietic and pancreatic cells. Taken together, the morphological and biochemical studies indicate that GRL1 and GRL2 are components of the secretory granule membrane in chicken exocrine, endocrine and hemopoietic cell types.