bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional down-regulation of α5β1 integrin in keratinocytes is reversible but commitment to terminal differentiation is not
Authors: Hotchin Neil A. , Kovach Nicholas L. , Watt Fiona M. 
Published: April 2021

ABSTRACT
Extracellular matrix receptors of the integrin family have a dual role in the epidermis, regulating both adhesion and differentiation. Loss of contact with the extra-cellular matrix causes keratinocytes to become commit-ted to terminal differentiation, and results in a decrease in the ability of the α5β1 integrin to bind fibronectin. We have investigated whether the decrease in ligand-binding ability is reversible and, if so, whether commitment to terminal differentiation can also be reversed. Keratinocytes that had been placed in suspension for 5 hours to induce commitment were compared with the starting population (0 hour cells) in the presence or absence of 8A2, an activating anti-β 1 antibody. 8A2 IgG or FAb fragments increased the amount of α5β1 in cell extracts that bound to fibronectin-Sepharose and in the presence of 8A2 the amount of bound α5β1 in 0 hour and 5 hour extracts was equal. 8A2 also restored α5β1function in adhesion assays of intact 5 hour cells. Ca2+, Mg2+ and Mn2+ alone, at concentrations of up to 1 mM, did not increase the adhesiveness of 5 hour cells relative to 0 hour cells; however, the effect of 8A2 on keratinocytes was dependent on Ca2+. Although 8A2 restored 5 1 ligand-binding ability it did not prevent committed cells from withdrawing from the cell cycle and expressing involucrin, a differentiation marker. The results suggest that loss of matrix contact triggers two distinct events in keratinocytes: a reversible change in α5β1 conformation and generation of an irreversible signal through the receptor that culminates in terminal differentiation.
Kinesin-like molecules involved in spindle formation
Authors: Rodionov Vladimir I. , Gelfand Vladimir I. , Borisy Gary G. 
Published: April 2021

ABSTRACT
To study the possible involvement of kinesin-like mole-cules in mitosis a polyclonal antibody against the head domain of Drosophila kinesin heavy chain (HD anti-body) was microinjected into PtK1 cells at the prophase-prometaphase transition. Progress of the cell through mitosis was recorded for subsequent detailed analysis. Cells injected with pre-immune IgG progressed through mitosis at rates similar to those for noninjected cells. After HD antibody injections, chromosomes failed to congress to an equatorial plane and cells failed to form a bipolar spindle. Rather, the spindle poles came together, resulting in a monopolar-like configuration with chromosomes arranged about the poles in a rosette. Sometimes the monopolar array moved to the margin of the cell in a way similar to anaphase B movement in normal cells. Antibody-injected cells progressed into the next cell cycle as evidenced by chromosome decondensation and nuclear envelope reformation. Anti-tubulin immunofluorescence confirmed the presence of a radial monopolar array of microtubules in injected cells. HD antibody stained in a punctate pattern in interphase and the spindle region in mitotic PtK1 cells. The antibody also reacted with spindle fibers of isolated mitotic CHO spindles and with kinetochores of isolated CHO chro-mosomes. Immunoblotting indicated that the major component recognized by the antibody is the 120 kDa kinesin heavy chain. At higher protein loads the anti-body recognized also a 34 kDa polypeptide in PtK1 cell extracts, a 135 kDa polypeptide in a preparation of CHO spindles and a 300 kDa polypeptide in a preparation of CHO mitotic chromosomes. We conclude that a kinesin-like molecule is important for the formation and/or maintenance of the structure of mitotic spindle.
Molecular cloning and subcellular localization of three GTP-binding proteins of the rab subfamily
Authors: Olkkonen Vesa M. , Dupree Paul , Killisch Iris , Lütcke Anne , Zerial Marino , Simons Kai 
Published: April 2021

ABSTRACT
Small GTPases of the rab subfamily are involved in reg-ulation of intracellular membrane transport events. We recently used a PCR approach to isolate short cDNA fragments of a number of novel rab sequences. These PCR fragments have now been used with cDNA library screening and PCR-based techniques to clone the cDNAs encoding three of these proteins, rab12, rab22, and rab24. By northern blot analysis, the messages were found to be present in a wide variety of mouse tissues. However, quantitative differences in the mRNA levels between the tissues were detected. We determined the subcellular localization of the GTPases by expressing the c-myc epitope-tagged proteins with the Semliki Forest virus and the vaccinia T7 vector systems. Transiently expressed rab12 was localized to the Golgi complex. This localization was confirmed using a polyclonal anti-peptide antibody detecting the endogenous protein in BHK cells. rab22 expressed from the cDNA was local-ized to endosomal compartments and to the plasma membrane. After longer periods of expression, the protein was found on abnormally large perinuclear endo-somal structures, suggesting that it is a potent regulator of events in the endocytic pathway. Finally, rab24 was found in the endoplasmic reticulum/cis-Golgi region and on late endosomal structures. The localization of rab24 may indicate its involvement in autophagy-related processes.
Immunological properties and cDNA sequence analysis of an intermediate-filament-like protein from squid neuronal tissue
Authors: Adjaye James , Marsh Philip J. , Eagles Peter A. M. 
Published: April 2021

ABSTRACT
A cDNA library has been constructed in the expression vector gt11 from mRNA isolated from squid (Loligo forbesi) optic lobes. The library was screened with anti-bodies generated against purified squid neurofilaments. A positive clone was isolated, which harboured a gt11 recombinant having an insert size of 3.5 kb. Hybridiz-ation analysis by Southern and northern blotting showed that the corresponding protein is encoded by a single gene that gives rise to a transcript of 2.6 kb. Translation of the full nucleotide sequence of the gene revealed an open reading frame covering 557 amino acids. This squid-neurofilament-like protein, SNLK, bears the characteristic N-terminal head, rod and C-ter-minal tail domains present in all intermediate filament (IF) proteins. The rod has the classical heptad repeats indicating coiled-coil-forming ability, and the predicted lengths of the coils are similar to coils 1a, 1b and 2 of intermediate filaments. At the C-terminal end of the rod there is a strongly conserved IF epitope, and a fusion protein containing SNLK is recognised by the pan-specific intermediate filament antibody, IFA. A poly-clonal antibody raised against SNLK has been used to show that the protein is present only in neuronal tissues and that it is immunologically related to neurofilaments from Myxicola but not from mammals.
A small molecular mass inhibitor of growth of 3T3 cells and porcine aortic smooth muscle cells released from the macrophage cell line P388D1
Authors: McMurray H. F. , Proudfoot D. , Davis J. B. , Parrott D. P. , Bowyer D. E. 
Published: April 2021

ABSTRACT
Murine peritoneal macrophages and the macrophage-like cell line, P388D1, were found to release both mito-genic and inhibitory modulators of growth of cells in culture. These growth factors were effective against both murine Swiss 3T3 fibroblasts and porcine aortic smooth muscle cells as assessed by [3H]thymidine incorporation into DNA and by measurement of cell number.
Partial characterisation of the inhibitory activity demonstrated it to be lost on dialysis using a membrane with a 10 kDa cut-off, trypsin sensitive, heat stable, and slightly sensitive to freeze-thawing. The inhibitory activity not only affected cell growth but was found to change the morphology of porcine aortic smooth muscle cells. Gel permeation studies showed an estimated molecular mass in the range 2.5 to 6.5 kDa. The inhibitory activity could be partially purified using ion-exchange chromatography. Experiments with a neutralising anti-body against transforming growth factor beta (TGF-) showed that TGF-is not responsible for the activity observed. Indomethacin had no effect on the production of inhibitor suggesting that it is not an inhibitory prostanoid. The inhibitory activity was not due to a non-specific toxic mechanism as confirmed by a [3H]adenine release assay.
Incubation of P388D1 cells with cycloheximide pre-vented the release of inhibitory activity.
Regulation of pathways within cultured epithelial cells for the transcytosis of a basal membrane-bound peroxidase-polylysine conjugate
Authors: Taub Mitchell E. , Shen Wei-Chiang 
Published: April 2021

ABSTRACT
A conjugate of horseradish peroxidase (HRP) to poly(L-lysine) (PLL) was used as a non-specific adsorptive probe to study transcytosis in MDCK strain I and Caco-2 epithelial cells. As we have shown previously, HRP-PLL transcytosis proceeds via an intracellular, non-lyso-somal proteolytic compartment in MDCK cells; yet, this compartment is utilized for transcytosis only in the basal-to-apical direction (Taub, M. E. and Shen, W.-C. J. Cell. Physiol., 150, 283-290, 1992). Using size exclu-sion chromatography, we demonstrate that the PLL moiety of the conjugate is effectively cleaved during transcytosis, thus releasing free HRP from the apical surface of the cells. Pulse-chase studies indicate that ∼6% of basolateral surface-associated HRP-PLL conju-gate in Transwell-grown cell monolayers enters the basal-to-apical transcytotic pathway. Brief (1 hour) treatment with 160 nM phorbol ester (PMA), a protein kinase C stimulator, elicits a 2-fold increase in the rate and amount of HRP-PLL transcytosis following a 1 hour lag time. Treatment with 1.6 g/ml brefeldin A (BFA) inhibits HRP-PLL transcytosis by ∼30%; additionally, BFA is able to abolish completely the PMA stimulatory effect. Removal of BFA causes a re-establishment of the normal rate of transcytosis within 2 hours, demonstrat-ing the reversibility of BFA inhibition with respect to HRP-PLL transcytosis. Thus, PMA most likely elicits an increase in the amount of basally internalized conjugate delivered to BFA-sensitive transcytotic compartments. Leupeptin (50 g/ml), a protease inhibitor, which we have previously shown inhibits HRP-PLL transcytosis by ∼50%, fails to abolish the PMA effect, indicating that the protease involved in the PMA-enhanced pathway is either insensitive or inaccessible to basolaterally inter-nalized leupeptin. These results suggest that: (a) there exists more than one intracellular pathway for process-ing of endocytosed HRP-PLL conjugate; and (b) a strik-ingly similar mechanism for basal-to-apical transcytosis is utilized within two dissimilar species of polarized epithelium.
Orientation of macromolecules in the walls of elongating carrot cells
Authors: McCann M. C. , Stacey N. J. , Wilson R. , Roberts K. 
Published: April 2021

ABSTRACT
When round cells from a carrot cell suspension culture are diluted into fresh medium without auxin, the cells elongate to almost 50 times their original diameter within three days. This process of elongation is accom-panied by changes in both the composition and the orientation of cell wall polymers. We have obtained infor-mation on the orientation of wall polymers in elongating cells by two complementary techniques, one using microscopy and one spectroscopy. Images obtained by the fast-freeze, deep-etch, rotary-shadowed replica tech-nique show that walls of round carrot cells have no net orientation of cellulose microfibrils, and that many thin fibres can be seen cross-linking microfibrils. Walls of elongated carrot cells, in contrast, show a marked net orientation of microfibrils at right angles to the axis of elongation.
Fourier Transform Infrared (FTIR) spectra obtained from defined areas of single cell walls show that walls of round carrot cells contain more protein, esters and phenolics in a given area (10 m 10 m) than walls of elongated carrot cells, that contain proportionally more carbohydrate. The orientation of particular func-tional groups, with respect to the direction of elongation of the cell, can be determined by inserting a polariser into the path of the infrared beam, before it passes through a cell wall sample mounted on the stage of the microscope accessory. In the walls of elongated cells, ester bands, amide bands characteristic of proteins, and stretching frequencies in the carbohydrate region of the spectrum all show a net orientation transverse to the long axis of the cells. In the walls of round carrot cells, however, there is no such net orientation of polymers. Spectra obtained from 25 m-thick fresh sections of the etiolated stem of a carrot seedling show that different wall components are polarised in different tissue types. These techniques have therefore enabled us to define dif-ferences in both the composition and the architecture of walls of elongating cells at the level of a single cell, and to suggest that polymers not previously thought to be ordered, such as pectin and protein, are strictly oriented in some wall types.
Differential expression of sucrase-isomaltase in clones isolated from early and late passages of the cell line caco-2: Evidence for glucose-dependent negative regulation
Authors: Chantret Isabelle , Rodolosse Annie , Barbat Alain , Dussaulx Elisabeth , Brot-Laroche Edith , Zweibaum Alain , Rousset Monique 
Published: April 2021

ABSTRACT
The expression of the brush border-associated hydrolase sucrase-isomaltase was shown to increase from early to late passages of Caco-2 cells, concomitant with a decrease in the rates of glucose consumption. Twenty-six clones were isolated from early (P29) and late (P198) passages of the cell line. These clones show considerable and inverse differences in the levels of sucrase activities and rates of glucose consumption, without marked changes in other features of enterocytic differentiation of the cells (presence of an apical brush border, levels of expression of other brush border-associated hydrolases). Clones with low sucrase-isomaltase expression show a mosaic expression of the enzyme and a 38-fold higher rate of glucose consumption than clones with high sucrase-isomaltase expression. The clones with high expression show an homogeneous apical distribution of the enzyme and 70-fold and 35-fold higher levels of sucrase activities and sucrase-isomaltase mRNA, respectively. In contrast no differences were found from one clone to another in the enrichment of sucrase activity in brush border-enriched fractions as compared to cell homogenates. Switch to low glucose-containing medium (1 mM versus 25 mM in standard culture conditions) of cells with low sucrase-isomaltase results in an increased and more homogeneous expression of the enzyme and a tenfold augmentation of the levels of sucrase-isomaltase mRNA and sucrase activity. These results show that glucose interferes with the expression of sucrase-isomaltase in Caco-2 cells at the mRNA level.
The processing pathway of prelamin a
Authors: Sinensky M. , Fantle K. , Trujillo M. , McLain T. , Kupfer A. , Dalton M. 
Published: April 2021

ABSTRACT
The conversion of mammalian prelamin A to mature lamin A proceeds through the removal of 18 amino acids from the carboxyl terminus. The initial step in this processing is the isoprenylation of a CAAX box cysteine. This proteolytic event is distinctive for prelamin A among the known preny-lated mammalian proteins. Since the carboxyl terminus of prelamin A is removed during maturation, it is not obvious that this protein would undergo the two reactions subse-quent to prenylation observed in other CAAX box proteins-the endoproteolytic removal of the carboxyl-terminal 3 amino acids and the subsequent methylation of the now carboxyl-terminal cysteine.
To characterize the maturation of prelamin A further, we have developed a CHO-K1 cell line that possesses a dex-amethasone-inducible human prelamin A against a genetic background of high mevalonate uptake. Utilizing this cell line in association with antibodies specific to the transgenic prelamin A, we have been able to demonstrate directly in vivo that prelamin A undergoes farnesylation and car-boxymethylation prior to conversion to lamin A, as is the case for other prenylated proteins. We have demonstrated previously that in the absence of isoprenylation, conversion of prelamin A to lamin A is blocked, but that unprocessed prelamin A is transported to the nucleus where it can still undergo maturation. Consistent with the implications of these prior studies, we now demonstrate the presence of both subunits of farnesyl-protein transferase in the nucleus.
CaBP1, a calcium binding protein of the thioredoxin family, is a resident KDEL protein of the ER and not of the intermediate compartment
Authors: Füllekrug Joachim , Sönnichsen Birte , Wünsch Ulrike , Arseven Kamran , Van Phuc Nguyen , Söling Hans-Dieter , Mieskes Gottfried 
Published: April 2021

ABSTRACT
A cDNA encoding rat CaBP1 has been isolated and sequenced. The deduced polypeptide chain consists of 440 amino acids including two internal thioredoxin-like domains and a C-terminal KDEL retention/retrieval signal. Regarding the high degree of identity to the hamster protein P5, CaBP1 is considered to be the homologous rat protein. Previous work has suggested that CaBP1 is a resident luminal protein of the intermediate compartment (Schweizer, A., Peter, F., Nguyen Van, P., Söling, H. D. and Hauri, H. P. (1993) Eur. J. Cell Biol. 60, 366-370). Our con-clusion that CaBP1 is a resident protein of the endoplasmic reticulum and not of the intermediate compartment is based on three different approaches: subcellular fractionation, indirect immunofluorescence and overexpression of CaBP1. Subcellular fractionation of Vero cells in a velocity controlled step gradient led to copurification of CaBP1-containing vesicles and several marker proteins for the ER including calreticulin and α-SSRP. The intermediate compartment, as defined by a monoclonal antibody against the marker protein p53 (ERGIC-53), could be separated from these ER markers. Double immunofluorescence analysed by laser scanning microscopy showed no significant colocalization between CaBP1 and p53, but between CaBP1 and calreticulin. In additional experiments, Vero cells were infected with VSV tsO45. At 15°C the VSV-G protein accumulated in punctuate structures representing the intermediate compartment, while CaBP1 maintained its original reticular localization. Even after high-level overexpression in COS cells, CaBP1 was not detected in the intermediate compartment, but was efficiently retained in the ER as judged by light microscopy.
Expression of N-terminally truncated cyclin B in the Drosophila larval brain leads to mitotic delay at late anaphase
Authors: Rimmington Georgina , Dalby Brian , Glover David M. 
Published: April 2021

ABSTRACT
We have introduced an N-terminally truncated form of cyclin B into the Drosophila germ-line downstream of the yeast upstream activator that responds to GAL4. When such lines of flies are crossed to lines in which GAL4 is expressed in imaginal discs and larval brain, the majority of the resulting progeny die at the late pupal stage of development. Very rarely (&lt;0.1% of progeny) adults emerge that have a mutant phenotype typical of flies with mutations in genes required for the cell cycle; they have rough eyes, deformed wings, abnormal bristles, and die within hours of emergence. The brains of third instar larval progeny show an abnormally high proportion of mitotic cells containing overcondensed chromatids that have undergone anaphase separation, together with cells that cannot be assigned to a particular mitotic stage. Immunostaining indicates that these anaphase cells contain moderate levels of cyclin B, suggesting that persistent p34cdc2 kinase activity can prevent progression from anaphase into telophase.
Role of integrins in melanocyte attachment and dendricity
Authors: Hara Masahiro , Yaar Mina , Tang Aimin , Eller Mark S. , Reenstra Wende , Gilchrest Barbara A. 
Published: April 2021

ABSTRACT
Integrins are a family of proteins known to mediate attach-ment of cells to extracellular matrix materials. The sub-stratum specificity and cation dependence of specific integrin heterodimers have been extensively characterized, and to a lesser degree specialized roles in cell attachment versus dendricity have been defined in some cell types. In the past decade, melanocyte attachment rate and morphology have been found to have strong substratum dependence, suggesting a major role for integrins in these processes. In order to investigate this aspect of pigment cell biology, human newborn melanocytes were subjected to flow cytometry analysis and plated on a variety of substrata under conditions known to promote or block the binding of specific integrin pairs. Melanocyte attachment to laminin and type IV collagen was promoted by Mg2+ and Mn2+ but not by Ca2+, in the range of concentrations examined. However, dendrite outgrowth from melanocytes already attached on laminin or type IV collagen was promoted by Ca2+ to a far greater degree than by Mg2+, and Mn2+ had no effect on dendrite outgrowth. Flow cytometry analysis revealed that melanocytes expressed β1, α2, α3, α5, α6 and αv integrin subunits as well as the αvβ3 heterodimer. The influence of substratum on the profile of integrin expression was minimal, but α6 and β1 integrins were observed by confocal microscopy to be expressed over the entire cell surface, while α2, α5 and αVβ3 integrins localized along dendritic processes or at their tips. In accordance with the implications of these distribution patterns, anti-β1 and anti-α6 integrin monoclonal antibodies blocked melanocyte attachment to laminin, while anti-α2, anti-α5 and anti-αvβ3 inhibited dendrite outgrowth but did not block substratum attachment on either laminin or type IV collagen. On the basis of these data and the known char-acteristics of integrin molecules, we conclude that melanocyte attachment to laminin is mediated primarily by α6β1 integrin in a Ca2+-independent, Mg2+- and/or Mn2+-dependent manner, while dendrite outgrowth on laminin and type IV collagen requires extracellular Ca2+ and is mediated by αvβ3 as well as α2 and α5 integrins.