bluuebunny/crossref_metadata_2025 huggingface dataset:
Stable and slow-turning-over microtubules characterize the processes of motile epithelial cells treated with scatter factor
Authors: Prescott A. R. , Dowrick P. G. , Warn R. M.
Published: April 2021

The turnover of microtubules was studied in the processes of PtK2 cells, after treatment with the cytokine scatter factor (SF), using micro-injected biotin-tubulin as a reporter of new microtubule growth. Cells treated with SF became dispersed and fibroblast-like in morphology, showing one or more elongated processes. These processes contained bundles of microtubules, a significant proportion of which did not turn over during incubation times of up to an hour. Short broken pieces of microtubule were frequently found in all parts of the cell, particularly after longer incubation times, suggesting that more-stable microtubules were cut into pieces, which were subsequently degraded. From about half an hour after injection small tangles of stable microtubules were found. Some of these were clearly within the cell bodies. Others were usually larger in size and seemingly located outside the injected cells. These were considered to have formed part of small ‘feet’ presumed to be broken off during the retraction of trailing processes.

The microtubules within the processes were resistant to the effects of both microtubule-depolymerizing drugs and cold under conditions where the processes were maintained. When these microtubules disappeared as the result of longer drug treatment the processes were also lost although, rarely, short processes lacking microtubules were found. It is concluded that the stable microtubules have a major role in process maintenance, although one that is indirect rather than a structural relationship.
Retinoic acid receptor expression in human skin keratinocytes and dermal fibroblasts in vitro
Authors: Redfern Christopher P. F. , Todd Carole
Published: April 2021

Retinoic acid is essential for the normal differentiation of epithelia but its cellular function is obscure. The expression patterns of retinoic acid receptors (RARs) in skin cell types may give an insight into the role of retinoic acid in skin. We have compared the patterns of RAR expression in human keratinocytes and dermal fibroblasts in vitro, and studied the effects of retinoic acid on RAR expression. RAR-α and RAR-γ were expressed in keratinocytes and fibroblasts: RAR-γ was expressed at similar levels in both cell types but RAR-α was more abundant in fibroblasts. There were no differences in expression of either RAR-α or RAR-γ between stratifying (high-calcium medium) and proliferating (low-calcium medium) keratinocytes and expression of these RARs was unaffected by retinoic acid. RAR-β was undetectable in keratinocytes. In the majority of fibroblast cell lines, RAR-β transcripts were either undetectable or expressed at a low level. Retinoic acid at low concentrations (10−10 to 10−9 M) rapidly induced the expression of RAR-γ. Cyclic adenosine monophosphate (cAMP) analogues inhibit RAR-β induction in teratocarcinoma cells. However, dibutyryl-cAMP did not affect RAR-β induction in fibroblasts. Forskolin, an adenylate cyclase activator, and the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) decreased constitutive RAR-β mRNA levels but did not block induction of RAR-β by retinoic acid. Since intracellular cAMP levels were only increased detectably in response to forskolin, the reduction in constitutive levels of RAR-β mRNA may be mediated by mechanisms other than via cAMP.
Identification of a large pre-lysosomal compartment in the pathogenic protozoon Trypanosoma cruzi
Authors: Soares Maurilio J. , Souto-Padrón Thais , Souza Wanderley De
Published: April 2021

Epimastigote forms of the pathogenic parasite Trypanosoma cruzi were used to study the endocytic process in a protozoon. These elongated unicellular organisms are highly polarized cells: endocytosis occurs only at the anterior region through the cytostome and the flagellar pocket membrane, areas of the plasma membrane where the cell cytoskeleton, formed by sub-pellicular microtubules, is absent. When the cells were incubated at 4°C or 28°C with gold-labeled transferrin, fixed and processed for routine transmission electron microscopy our observations show that this ligand initially binds to the cytostome and the membrane lining the flagellar pocket and is subsequently ingested through a clathrin-independent receptor-mediated endocytotic process, with formation of uncoated pits and vesicles. Ingested complexes are carried in uncoated vesicles to the reservosomes, large membrane-bound organelles found mostly at the posterior end of the cell. Immunocytochemical data from Lowicryl-embedded cells demonstrated that the reservosomes are acidic compartments (pH 6.0, as shown using DAMP as a pH probe) with no acid phosphatase or typical lysosome-associated membrane proteins (LAMP 1, LAMP 2 and Igp 120), but rich in cysteine proteinase. These data suggest that the reservosome is a pre-lysosomal compartment. Since cysteine proteinase of T. cruzi contains no phosphorylated mannose residues and the cation-independent mannose 6-phosphate receptor could not be immunocytochemically detected in the reservosomes, it is possible that lysosomal enzymes in the epimastigote forms of T. cruzi are targeted to compartments related to the endocytic pathway through a mechanism different from that which occurs in other eukaryotic cells.
Vertebrate p34cdc2 phosphorylation site mutants: effects upon cell cycle progression in the fission yeast Schizosaccharomyces pombe
Authors: Krek Wilhelm , Marks John , Schmitz Nicole , Nigg Erich A. , Simanis Viesturs
Published: April 2021

We have used the fission yeast Schizosaccharomyces pombe to analyse the effects of in vitro mutagenesis of the four known phosphorylation sites in the chicken p34cdc2 protein, Thr14, Tyr15, Thr161 and Ser277, upon cell cycle progression. We have studied both the effect of overexpression of mutant proteins in a cdc2+ background and assayed their ability to rescue null and temperature-sensitive alleles of cdc2. Mutations of Thrl4 and Tyrl5 within the ATP binding domain of p34cdc2 that mimic constitutive phosphorylation cause dominant negative cell cycle arrest when overexpressed. In contrast, some substitutions that simulate permanent dephosphorylation of the corresponding sites advance mitosis. These data confirm the model that p34cdc2 function is negatively regulated by phosphorylation of residues in the ATP binding site. Mutagenesis of the conserved residue Thr161 functionally inactivates p34cdc2, and our data suggest that both phosphorylation and dephosphorylation events at Thr161 are required for progression through the cell cycle. Mutations at the fourth site of phosphorylation, Ser277, lead to coldsensitive cell cycle arrest, in minimal but not rich growth medium, suggesting that this site is involved in monitoring the nutritional status of the cell.
PCNA and total nuclear protein content as markers of cell proliferation in pea tissue
Authors: Citterio Sandra , Sgorbati Sergio , Levi Marisa , Colombo Bruno Maria , Sparvoli Elio
Published: April 2021

The identification of cell proliferation markers has been shown to be a useful tool with which to study basic mechanisms of cell cycle progression. The use of immunofluorescence techniques revealed the presence of the proliferating cell nuclear antigen (PCNA) in pea tissue, where we observed a high PCNA expression in proliferating cells of the root meristem compared to noncycling cells of the differentiated leaf. The presence of PCNA was monitored also during the time-course of seed germination, before, during and after the cell cycle resumption of the embryo cells. PCNA is present in embryo cells not only during and after resumption of the cell cycle but also before, when cells have not yet begun replicating their genome. A bivariate flow cytometric analysis of DNA and nuclear protein content was used to localize precisely the cells of the examined pea tissues in different cell cycle phase subcompartments. A high correlation was found between the degree of cell proliferation and the protein content of G1 nuclei, on the one hand, and the percentage of PCNA positive cells on the other.
Drosocrystallin, a major 52 kda glycoprotein of the Drosophila melanogaster corneal lens purification, biochemical characterization, and subcellular localization
Authors: Komori Naoka , Usukura Jiro , Matsumoto Hiroyuki
Published: April 2021

We have identified a 52 kDa protein, which is a potent substrate for cholera toxin-dependent ADP-ribosylation, in the compound eye preparation of the fruit fly, Drosophila melanogaster. We find that the 52 kDa protein is a glycoprotein and a Ca2+ binder bearing a high content of leucine, serine and glycine. By microsequencing we determined its 13 N-terminal sequence, AYL*PIDLNQLAK, with the asterisk representing an ambiguous signal. In order to study further the 52 kDa protein we have raised a polyclonal antibody against a synthetic oligopeptide representing the N-terminal 13 residues of the 52 kDa protein. By immunogold labelling with the antibody, the epitopes were localized at the EM level to the laminated corneal lens. The number of the gold particles per /un2 in the electron-dense layer of the corneal lens was 2.5 times higher than that of the electron-lucent layer. The pattern of the 52 kDa protein distribution in the corneal lens suggests that the 52 kDa protein is the major protein component that participates in the pattern formation of the alternate refractive indices of the D. melanogaster corneal lens. An X-ray dispersion analysis in situ revealed that the laminated corneal lens contained a higher concentration of Ca2+, supporting the hypothesis that the 52 kDa protein binds Ca2+in vivo. To the best of our knowledge, this is the first report that identifies the protein entity of an arthropod corneal lens. We propose to designate this 52 kDa protein drosocrystallin.
Distribution of a nuclear envelope antigen during the syncytial mitoses of the early Drosophila embryo as revealed by laser scanning confocal microscopy
Authors: Riparbelli Maria Giovanna , Callaini Giuliano
Published: April 2021

The changing distribution of a nuclear envelope antigen recognized by a monoclonal antibody raised against human fibroblast vimentin during the syncytial mitoses of the Drosophila embryo has been studied with a confocal laser scanning microscope. The antigen appears very early as irregular aggregates in the peripheral cytoplasm of the preblastoderm embryo. As the first nuclei reach the periplasm the antigen is localized on the nuclear envelope and the cytoplasmic staining decreases. In addition to the perinuclear labeling we observed intense midzone and polar staining during the mitotic cycle. A possible relationship between polar localization of the antigen and centrosome position is discussed.
Cytokeratins and retinal epithelial cell behaviour
Authors: Robey Helen L. , Hiscott Paul S. , Grierson Ian
Published: April 2021

The expression of cytokeratins 18 and 19 by human retinal pigment epithelial cells (HRPE) has been suspected of being associated with HRPE proliferation. We have investigated the involvement of these cytokeratin subtypes in the proliferative and migratory behaviour of cultured HRPE. Cell proliferation markers (bromodeoxyuridine and proliferating cell nuclear antigen) and the cytokeratins were identified using immunohistochemical techniques. In vitro, cytokeratins 18 and 19, as detected by the monoclonal antibodies RGE 53 and K4.62, were expressed in a subset of HRPE and this subset was significantly less likely to be proliferating. Micro-chemotaxis chambers were used to study migrating cells and immunohistochemical staining for cytokeratins 18 and 19 revealed that actively migrating cells always expressed these two cytokeratins, whereas stationary cells did not label for these cytokeratin subtypes. It was apparent that cytokeratins 18 and 19 were not markers of proliferation, but were involved in the mobility of HRPE in vitro. Cytokeratins 18 and 19 may be useful indicators of simple epithelial cell migration in tissues.
A functional model of adult human prostate epithelium the role of androgens and stroma in architectural organisation and the maintenance of differentiated secretory function
Authors: Hayward Simon W. , Buono Raffaele Del , Deshpande Nagesh , Hall Pete A.
Published: April 2021

A functional model of adult human prostate epithelium is described. This model shows that stromal cells, but not an androgenic stimuli, are required for architectural organisation of prostate epithelium. Within an organised structure, androgenic stimulation is required for the establishment of secretory epithelial cell morphology and associated function. In the absence of stromal cells but in the presence of androgens architectural organisation and secretory function are lost. Epithelial parenchymal units (organoids) from human prostate tissue were isolated, cultured within a three-dimensional collagen matrix, and xenografted subcutaneously into athymie mouse hosts. The grafted gels were rapidly invaded by host fibroblasts. Epithelial organisation initially disappeared but was re-established concurrently with the stromal cell invasion. In intact male hosts, cuboidal and columnar cells that expressed human prostate-specific secretory markers were found. In castrated male and in female hosts epithelial structures were lined with flattened epithelium with no secretory function. This phenomenon could be reversibly replicated by treating intact male hosts with the anti- androgen Flutamide. Gels containing organoids grafted within 0.45 /mi Millipore chambers were not invaded by stromal cells and rapidly lost all epithelial organisation and secretory function. When organoids cocultured with human foreskin fibroblasts were grafted within chambers, structural organisation of the epithelium was supported. These results indicate that both heterologous human fibroblasts and mouse stromal cells are capable of permissively supporting adult human prostate epithelial function.
Mechanisms of epibolic tissue spreading analyzed in a model morphogenetic system roles for cell migration and tissue contractility
Authors: Armstrong Margaret T. , Armstrong Peter B.
Published: April 2021

The processes responsible for epithelial spreading during wound healing and embryonic morphogenesis were investigated in an organ culture model in which an epithelial tissue (chick embryo pigmented retinal epithelium) spread over the surface of an aggregate of mesenchyme cells (chick embryo cardiac mesenchyme). The heart mesenchyme aggregate is differentiated into a core of stellate cells associated with a fibronectin-poor matrix surrounded by a cortical zone, 2-5 cells in thickness, of flattened cells embedded in a fibronectin-rich extracellular matrix. Envelopment of the mesenchyme aggregate is accompanied by a movement of the cells and the fibronectin-rich extracellular matrix of the cortex over the core tissue in advance of the spreading pigmented retina tissue. Three distinct processes were identified as contributing to epithelial spreading in this system: (1) active migration of the pigmented retinal epithelium; (2) active contraction of the cortical cells of the mesenchyme aggregate to tow the attached epithelial tissue over the mesenchyme aggregate; and (3) ingression of surface-located cells of the mesenchyme aggregate to decrease the exposed surface area by decreasing the number of cells at the surface.
Poly ADP-ribosylation: a histone shuttle mechanism in DNA excision repair
Authors: Us Felix R. Altha
Published: April 2021

In DNA excision repair of mammalian cells, the processing of ADP-ribose by the poly ADP-ribosylation system of chromatin is stimulated several thousand-fold. Most of this turnover is associated with the automodification reaction of the nuclear enzyme poly(ADP-ribose) polymerase and the degradation of polymerase-bound polymers by the enzyme poly(ADP-ribose) glycohydrolase. The automodification cycle catalyzes a temporary dissociation from and reassociation of histones with DNA. It is proposed that this mechanism, termed “histone shuttle”, may guide specific proteins to sites of repair. In addition, histone shuttling driven by the poly ADP-ribosylation system seems to be involved in nucleosomal unfolding of chromatin in DNA excision repair.
Synchronization of hair cell regeneration in the chick cochlea following noise damage
Authors: Stone Jennifer S. , Cotanche Douglas A.
Published: April 2021

Pure-tone overstimulation for prolonged time leads to hair cell death in frequency-specific regions of the cochlear epithelium. Unlike mammals, birds replace missing hair cells by stimulating mitosis in an uncharacterized precursor cell. Regenerated hair cells, initially identifiable by their immature stereocilia and small surface areas, differentiate into mature cells in a manner which parallels embryonic development. In the current study, we examined whether hair cell regeneration is initiated during noise exposure or after the end of acoustic trauma. We exposed 7- to 15-day-old chicks to a 1500 Hz pure tone at 120 dB SPL (re 20 μPa) for 4, 12, and 24 hours and examined the recovering cochlear epithelium with scanning electron microscopy to determine when regenerated hair cells were first identifiable. The earliest evidence of new hair cells appeared roughly 96 hours after the onset of 4-, 12-, and 24-hour exposures. Our previous studies initially identified new hair cells 96 hours after the start of a 48-hour exposure. Therefore, hair cell regeneration follows a similar time course relative to the onset of noise exposure, regardless of the ultimate duration of exposure. Since we estimate that hair cells take at least 48 hours after their genesis to form immature stereocilia, the signal which induces hair cell precursors to re-enter the cell cycle and to divide probably has its initial effects very early during the exposure period. (A previous report of these data was given at the 1991 American Society for Cell Biology conference.)
Antennapedia homeobox as a signal for the cellular internalization and nuclear addressing of a small exogenous peptide
Authors: Perez F. , Joliot A. , Bloch-Gallego E. , Zahraoui A. , Triller A. , Prochiantz A.
Published: April 2021

In a previous study we demonstrated that a homeobox peptide corresponding to the 60 amino acid long DNA-binding region of the Drosophila antennapedia homeoprotein was capable of crossing the plasma membrane of cells in culture. This finding has led us to investigate whether chimeric molecules encompassing the homeobox would behave in a similar manner. We demonstrate here that a peptide of 93 amino acids composed of the homeobox and of the C terminus of Rab3, a small GTP-binding protein, crosses the membrane of myoblasts, myotubes and neurons and is conveyed to their nuclei.

This transport is highly efficient, is observed in all the cells present in the culture and occurs at 37°C and 12°C without quantitative peptide degradation. Beyond its theoretical implications for our current views on cellular interactions, this finding could have technical repercussions on the development of drugs with intracellular targets.
Stimulus-dependent relocation of the microtubule organizing center in human polymorphonuclear leukocytes
Authors: Chiplonkar Jayant M. , VandrÉ Dale D. , Robinson John M.
Published: April 2021

Polymorphonuclear leukocytes (PMNs) exhibit extensive directional migration (chemotaxis) and phagocytic activities. We have developed an in vitro model to evaluate the organization of the microtubule organizing center (MTOC) in PMNs as the latter interact with various substrata, including immobilized antigen-antibody complexes. PMNs were layered on poly-L-lysine substrata containing ferritin (PL+F) or ferritin-antiferritin complex (PL+F+AF) and the location of MTOCs was determined by indirect immunofluorescence of tubulin using conventional epifluorescence microscopy and confocal laser scanning microscopy. The MTOCs in the majority of the PMNs attached to PL+F occupied an apical location (81.29% ± 3.34%), while in the majority of PMNs layered onto PL+F+AF, a basal location (79.37% ± 5.26%) was observed. Following disruption of microtubules (MTs) by nocodazole before layering the cells on the substrata, the proportions of PMNs with apical MTOCs were 65.2% ± 6.27% for PL+F and 47.2% ± 4.1% for PL+F+AF substrata, while the proportions of PMNs with basal MTOCs were 26.11% ± 8.89% for PL+F and 39.6% ± 4.4 for PL+F+AF substrata. The results indicate that MTOCs in human PMNs in vitro (i) occupied a ‘pre-defined’ apical location; (ii) translocated to a ‘newly defined’ basal location upon stimulation with immobilized antigenantibody complex; (iii) and depended on intact MTs for placement of MTOCs in both situations.
Mammalian skeletal muscle C-protein: purification from bovine muscle, binding to titin and the characterization of a full-length human cDNA
Authors: FÜrst Dieter O. , Vinkemeier Uwe , Weber Klaus
Published: April 2021

We report a fast method for the isolation of homogeneous C-protein from bovine skeletal muscle. In electron micrographs C-protein appears as short rods with a relatively uniform length of about 50 nm. Protein sequencing shows a single N-terminal sequence. Radiolabelled C-protein strongly decorates titin II and myosin rods but not myosin heads. Binding to titin n is retained in preparations lacking titin-associated proteins. Antibodies to bovine C-protein were used to screen a λgtll cDNA library constructed from fetal human skeletal muscle. Clone HC38 is 3833 bp long and encodes a protein of 1138 amino acid residues. The start of the predicted sequence fits the N-terminal sequence of the bovine protein. All partial sequences obtained from the bovine protein (348 residues) and the sequence deduced from a partial chicken cDNA (Einheber and Fischman,1990can be aligned along the human sequence. The sequences of human and chicken C-proteins share 50% identity and 70% similarity. Along the repeat patterns of the human protein the fibronectin (Fn)-like domains are better conserved than the immunoglobulin (Ig)-like domains. Regions of strong divergence between chicken fast C-protein and human slow C-protein may represent differences in C-protein isoforms.
Inhibitors of topoisomerases do not block the passage of human lymphocyte chromosomes through mitosis
Authors: Sumner A. T.
Published: April 2021

Cultured human lymphocytes have been treated with a number of topoisomerase inhibitors, to see whether topoisomerase II is involved in the process of chromosome segregation at anaphase. Results were assessed by examination of cytogenetical preparations of spread chromosomes Four effects were observed, although no inhibitor produced all four effects. These effects were: inhibition of entry into mitosis; chromosome breakage and rearrangement; inhibition of chromosome condensation; and inhibition of chromosome segregation. Evidence for the last was ambiguous. Although there was evidence that separation of chromatids was affected when cells were treated with colchicine as well as topoisomerase II inhibitors (most notably with nalidixic acid, which resulted in complete fusion of the chromatids), no evidence was obtained to show that, in the absence of colchicine, cells treated with inhibitors could not proceed through anaphase normally. The topoisomerase I inhibitor, camptothecin, differed from the topoisomerase II inhibitors in not showing any effect on chromosome condensation or any significant effect on segregation.
Effects of okadaic acid on mitotic HeLa cells
Authors: Ghosh Sibdas , Paweletz Neidhard , Schroeter Dieter
Published: April 2021

Mitotic HeLa cells were treated with different concentrations of okadaic acid (OA), known to inhibit phosphatase 1 and 2A activities. The cytological effects on the course of mitosis were studied at the light microscopic, immunofluorescence and electron microscopic levels. At the lowest concentration used (1 nM), OA did not show any effect on mitosis, but at higher concentrations it showed pronounced effects. The mitotic chromosomes became scattered, the mitotic spindle became deranged and the cells failed to enter anaphase. At the electron microscopic level formation of isolated microtubules and regular trilaminar kinetochores were observed. An extensive growth of the endoplasmic reticulum could be noted in these cells. Decondensation of chromatin and nuclear envelope re-formation could be seen only after withdrawal of OA. A high frequency of multinucleate cells could be found after 24 h of recovery. Cells treated with 100 nM OA for 3 hours showed diplochromosomes in over 50% of mitotic cells after 24 h recovery. These were presumably formed due to the failure of sister chromatid separation in the earlier mitosis in the presence of OA. At the electron microscopic level the diplochromosomes showed a quadruplet structure. The role of phosphatase 1 in controlling some late mitotic events, i.e. sister chromatid separation, MPF-inactivation and nuclear envelope re-formation etc., is discussed.
Nuclear F-actin: a functional component of baculovirus-infected lepidopteran cells?
Authors: Volkman Loy E. , Talhouk Salma N. , Oppenheimer Daniel I. , Charlton Carol A.
Published: April 2021

Cellular functions known to involve actin are thought to occur in the cytoplasm. Even though actin has frequently been found in the nucleus, systems well-suited for studying the function of such nuclear actin are rare. We observed filamentous (F) actin within nuclei of IPLB-Sf-21 cells infected with Autographa caUfornica M nuclear polyhedrosis virus (AcMNPV) as detected by laser confocal microscopy using fluorescent phalloidin probes. The nuclear F-actin co-localized with the major capsid protein of the virus during normal infections. Cytochalasin D, known to interfere with nucleocapsid morphogenesis, uncoupled the apparent co-localization of F-actin and the capsid protein and inhibited infectious progeny production. Inhibition was reversible throughout infection (even in the presence of a protein synthesis inhibitor) and the nuclear co-localization of F-actin and the capsid protein was re-established upon removal of the drug. These observations suggest that nuclear Factin plays a role in virus replication, and that AcMNPV-infected cells may constitute a useful system in which to expand our understanding of nuclear actin transport and function.
Tubulin in bovine retinal rod outer segments
Authors: Matesic Diane F. , Philp Nancy J. , Murray John M. , Liebman Paul A.
Published: April 2021

Bovine rod outer segment (ROS) preparations contain a major 58 kDa protein doublet that was identified by immunoblot as tubulin. Quantification by gel densitometry showed that the total amount of tubulin was 5- to 10-fold higher than that attributable to the rod axoneme, suggesting additional role(s) for tubulin in photoreceptor cells. Approximately 20% of this non-axonemal tubulin (15% of total tubulin) is tightly associated with outer segment membranes. This fraction remains membrane-associated after extensive low- or high-salt washing, requiring detergents or protein denaturants for release from ROS membranes. Unlike

ROS soluble tubulin it associates tightly with liposomes upon detergent solubilization and reconstitution. The ROS membrane-associated tubulin is highly enriched in isolated ROS plasma membrane fractions compared to the total outer segment membrane pool and can be localized to the plasma membrane but not to disks by immunofluorescent staining, suggesting a possible role in the structure or electrophysiology of the rod outer segment plasma membrane.
Cell guidance by micropatterned adhesiveness in vitro
Authors: Clark Peter , Connolly Patricia , Moores Geoffrey R.
Published: April 2021

Tracks of adhesiveness are believed to be involved in guiding morphogenetic cell migrations. Here, electronics microfabrication technology was used to manufacture patterns of alternating tracks of adhesive and non-adhesive substratum (untreated fused quartz and adjacent parallel tracks of hydrophobic treatment) of varying period (4, 6, 12, 24 and 50 μm). These experimental substrata were used to model, in vitro, possible differentially adhesive guidance cues. The effect of such patterned substrata was assessed using fibroblastic BHK cells and epithelial MDCK cells. Cells were oriented and elongated by these surfaces. Their responsiveness was dependent on cell type, cell-cell interactions, and the geometry of the patterns. Alignment of BHK cells increased with increasing pattern period.

Single MDCK cells aligned to all pattern periods, their elongation being period-dependent, whereas colonies were mainly unaffected. These in vitro data have important implications, which are discussed with regard to in vivo guidance cues. The ability of cells to bridge over non-adhesive regions will influence the effectiveness of linear cues, and will be important for guideposting. The geometry of patterns of differential adhesion is here shown to be an important factor in determining the precision with which local guidance of cells may be controlled.
The role of the cytoskeleton and intercellular junctions in the transcellular membrane protein polarity of bovine aortic endothelial cells in vitro
Authors: Stolz Donna Beer , Bannish Gregory , Jacobson Bruce S.
Published: April 2021

This project examines the transcellular membrane protein polarity of bovine aortic endothelial cell (BAEC) monolayers in vitro with respect to the roles that intercellular junctions (as defined by comparing confluent and subconfluent monolayers) and the submembranous cytoskeleton play in controlling this phenomenon. Plasma membrane (PM) proteins obtained from apical (AP) and basolateral (BL) PM domains of confluent BAEC monolayers were isolated using the cationic colloidal silica technique and resolved by twodimensional gel electrophoresis (2-D PAGE). To facilitate the identification of domain-specific PM proteins, an isoelectric point/molecular weight database of the proteins from AP and BL PM domains was constructed. Domain-specific PM proteins were assessed for their interaction with the cytoskeleton by determining whether they co-isolated with a Triton X-100 detergentresistant cytoskeletal/extracellular matrix fraction. The maintenance of polarized PM protein segregation by intercellular junctional complexes was determined by comparing AP and BL protein patterns of confluent monolayers with patterns generated by subconfluent monolayers, which lack such junctional structures. Proteins isolated from AP and BL PM domains from both confluent states were immunoblotted with antibodies to angiotensin-converting enzyme (ACE) and collagen receptors (CR). ACE was restricted exclusively to the AP PM domain in the subconfluent condition, even though no apparent cytoskeletal interaction was observed. CRs, found to interact with the cytoskeleton in either confluence state, were predominantly segregated to the BL PM domain regardless of the presence or absence of cell-cell contact. Membrane proteins found by 2-D PAGE to be asymmetrically distributed in the absence of intercellular junctions were assessed for cytoskeletal interaction by their inability to be extracted by Triton X-100 from monolayers in the subconfluent state. Computer cross-referencing of 2-D PAGE peak lists and immunodetection generated from the above fractionation protocols identifies a set of four proteins associated with the cytoskeleton that remain segregated in the proper domain, and five proteins associated with the cytoskeleton that become equally distributed between AP and BL PM domains in the absence of intercellular junctions. Additionally, six proteins not associated with the cytoskeleton remain asymmetrically distributed to the AP domain in the subconfluent state. The data suggest that BAEC monolayers have unknown mechanisms, apart from intercellular junctions expressed at confluency or cytoskeletal binding, for maintaining transcellular PM protein polarity.
Distribution of desmosomal proteins in F9 embryonal carcinoma cells and epithelial cell derivatives
Authors: Trevor Katrina T. , Steben Lara S.
Published: April 2021

In diverse epithelia, cytoskeletal keratin intermediate filaments (IFs) associate with the cytoplasmic face of intercelluar junctional desmosomes. The processes underlying desmosome formation and keratin IF interactions remain unclear. We have examined F9 embryonal carcinoma (EC) cell differentiation as a model for embryonic development of epithelial surface desmosomes. As determined by immunofluorescence microscopy and biochemical protein techniques, F9 EC cells, which lack surface desmosomes and keratin IFs, express the desmosomal proteins desmoplakins I and II (DP I/II), desmoglein I (DG I) and plakoglobin (PK). DP I/II are present at low levels and are relatively soluble in buffer containing Triton X-100. Immunofluorescence localizes DP l/n to the juxtanuclear, centrosomal region. Species of DG I and PK are detected in both the Triton X-100-soluble and-insoluble protein fractions. DG I appears dispersed throughout the cell while PK resides at cell-cell boundaries. In epithelial cell cultures induced by retinoic acid (RA) treatment, each of the desmosomal proteins is organized into punctate desmosome-like structures with the appearance of simple epithelial K8/K18 IFs. The steady-state levels of DP l/ll and PK increase with a partitioning of the majority of the desmosomal components into the insoluble fraction. In epithelial cells which lack distinct surface desmosomes, an intracellular association of keratin bundles with DP I/II is observed, suggesting that keratin filaments may facilitate the translocation of these desmosomal components to the cell surface. Parietal endoderm-like cells, derived by treatment with RA and dibutyryl cAMP, are analogous to F9 EC cells in that the cells express desmosomal components and do not display surface desmosomes. Moreover, K8 and K18 do not form distinct filaments, and the protein and RNA levels of K8 are low relative to epithelial cells induced by RA alone. The F9 system appears to be a relevant model for studies of desmosome assembly and the potential interactions of desmosomal proteins and keratin IFs in embryonic epithelial cell types.
The involvement of discrete genome regions in post-mitotic chromosome decondensation and in G1 timing in Allium cepa L. meristematic cells
Authors: Gonzalez-Fernandez A. , Sans J. , Aller P. , Torre C. De La
Published: April 2021

The role of DNA regions replicated at different times during the S period in the process of chromatin decondensation that takes place in the next G1 period, as well as in the timing of the G1 to S transition, was analyzed in synchronous populations of cells in Allium cepa L. root meristems. For this analysis, DNA bromosubstitution (10 −7 M 5-bromo-2 ′-deoxyuridine feeding) was carried out at similar times corresponding to the first, middle and last thirds of the S period prior to telophase when anoxic 313 nm irradiation was carried out.

Evaluation, after Feulgen staining, of the chromatin pattern corresponding to G0 and G1 phenotypes in these synchronous cells showed that chromatin decondensation after mitosis appeared to be under the control of a double negative/positive regulatory mechanism related, respectively, to early and late replicating sequences.

Thus, such decondensation was favoured when bromosubstitution took place during the first part of the S period, but delayed when bromosubstitution took place during the third part or the whole of the S period. Nevertheless, bromosubstitution at both the early and late stages of the S period delayed the timing of the G1 to S transition. In this way, the initiation of replication appears to be positively controlled by the function of early replicating DNA. Finally, the DNA sequences replicated in the middle third of the S period did not modify the decondensing capacity of the chromatin in G1 or the timing of the G1 to S transition.
Dolichol delays G1-arrest for one cell cycle in human fibroblasts subjected to depletion of serum or mevalonate
Authors: Larsson Olle , Wejde Johan
Published: April 2021

It is well-established that some product(s) or metabolite(s) of mevalonate is (are) critical for growth of mammalian cells. In the search for this (these) compound(s) it seems meaningful to distinguish between compounds needed for cell cycle progression in proliferating cells and compounds needed for growth activation of arrested cells. By using time-lapse video recording we have studied the possible regulatory role of cholesterol, dolichol and mevalonate in the cell cycle of human diploid fibroblasts (HDF). HDF, which are serum-dependent, were rapidly growth-arrested in the first part of G1 upon removal of serum factors. They also responded to mevinolin (an HMG CoA reductase inhibitor) by a similar G1-block, indicating that a mevalonate-derived product is involved in the G1-located cell cycle control of HDF. Interestingly, dolichol counteracted the G1-block caused by both types of treatment. Hence, the early G1-cells could traverse the remainder of the cell cycle and divide despite depletion of serum or mevalonate. We also demonstrated that addition of dolichol resulted in a significant decrease in the rate of protein degradation. This protein stabilizing effect may constitute the mechanism by which dolichol delays the G1-arrest of HDF.
Cell phenotype, binding affinity and promoter structure modulate transactivation by HNF1 and LAP
Authors: Angrand Pierre-Olivier , Rousset Jean-Pierre , Weiss Mary C.
Published: April 2021

To evaluate the importance of the transcription factors known to bind to the albumin promoter as well as the parameters involved in their activity, we have used cotransfections with an albumin promoter-cat plasmid combined with expression vectors driving the expression of cDNAs coding for liver-enriched factors known to interact with this promoter. We describe the characteristics of a set of clones of hepatic origin: well differentiated, partial variants or pleiotropic dedifferentiated variants. These lines have been characterized for the accumulation of RNAs corresponding to each of the albumin promoter-binding factors. Only HNF1, and to a lesser extent C/EBP, show differences depending upon the differentiation state of the cells. Overexpression of exogenous HNF1 in these cells reveals that this factor is able to transactivate the albumin promoter only in variant cells where the endogenous protein is limiting. By contrast, if the HNF1-binding site is of weak affinity, overexpresion of exogenous HNF1 stimulates the albumin promoter even in the HNF1-rich differentiated cells. Overexpression of exogenous LAP strongly transactivates an artificial promoter containing one LAP-binding site, but surprisingly in all the cell lines, it has little effect upon the albumin promoter. These results demonstrate that the transactivation potential of a given transcription factor depends on the degree of differentiation of the recipient cells, on the promoter structure, and on the affinity of the binding site for this factor.
Chondrons from articular cartilage: V.* Immunohistochemical evaluation of type VI collagen organisation in isolated chondrons by light, confocal and electron microscopy
Authors: Poole C. Anthony , Ayad Shirley , Gilbert Raymond T.
Published: April 2021

The pericellular microenvironment around articular cartilage chondrocytes must play a key role in regulating the interaction between the cell and its extracellular matrix. The potential contribution of type VI collagen to this interaction was investigated in this study using isolated canine tibial chondrons embedded in agarose monolayers. The immunohistochemical distribution of an anti-type VI collagen antibody was assessed in these preparations using fluorescence, peroxidase and gold particle probes in combination with light, confocal and transmission electron microscopy. Light and confocal microscopy both showed type VI collagen concentrated in the pericellular capsule and matrix around the chondrocyte with reduced staining in the tail region and the interconnecting segments between adjacent chondrons. Minimal staining was recorded in the territorial and interterritorial matrices. At higher resolution, type VI collagen appeared both as microfibrils and as amorphous deposits that accumulated at the junction of intersecting capsular fibres and microfibrils. Electron microscopy also showed type VI collagen anchored to the chondrocyte membrane at the articular pole of the pericellular capsule and tethered to the radial collagen network through the tail at the basal pole of the capsule. We suggest that type VI collagen plays a dual role in the maintenance of chondron integrity. First, it could bind to the radial collagen network and stabilise the collagens, proteoglycans and glycoproteins of the pericellular microenvironment. Secondly, specific cell surface receptors exist, which could mediate the interaction between the chondrocyte and type VI collagen, providing firm anchorage and signalling potentials between the pericellular matrix and the cell nucleus. In this way type VI collagen could provide a close functional interrelationship between the chondrocyte, its pericellular microenvironment and the load bearing extracellular matrix of adult articular cartilage.
Multiplicity of clathrin light-chain-like polypeptides from developing pea (Pisum sativum L.) cotyledons
Authors: Lin Hong-Bo , Harley Suzanne M. , Butler Juliet M. , Beevers Leonard
Published: April 2021

A comparative study has been made of clathrin-coated vesicles from developing pea (Pisum sativum L.) cotyledons and bovine brains in order to characterize the clathrin light chains from a plant system. Four polypeptides of 31 kDa, 40 kDa, 46 kDa and 50 kDa are considered as candidates for clathrin light chains in the developing pea cotyledons. The 31 kDa, 40 kDa, 46 kDa and 50 kDa polypeptides, together with the 190 kDa heavy chain, are dissociated as triskelions when coated vesicles of developing pea cotyledons are treated with 2 M urea. Partially purified 46 kDa and 50 kDa polypeptides have been demonstrated to bind to purified clathrin heavy chains. The 40 kDa, 46 kDa and 50 kDa polypeptides are sensitive to elastase. They are readily solubilized by neutralization of 10% trichloroacetic acid precipitates of clathrin. The 50 kDa polypeptide of plant clathrin-coated vesicles is heat-stable as are the light chains from bovine brains, while the heat stability of the 31 kDa, 40 kDa and 46 kDa polypeptides of plants is dependent on pH and ionic strength. The 40 kDa, 46 kDa and 50 kDa polypeptides bind calmodulin. The calcium binding properties of these polypeptides are ambiguous. The 40 kDa and 46 kDa polypeptides can be phosphorylated more extensively than the 31 kDa in vitro in the presence of polylysine, as can the smaller light chain of brains. The 50 kDa polypeptide can also be phosphorylated, even without the addition of polylysine. Unlike brain light chains, phosphorylation of the 31 kDa, 40 kDa, 46 kDa and 50 kDa polypeptides from peas is greatly reduced by N-ethylmaleimide (NEM). Our findings contrast with earlier reports of clathrin light chains of 30 and 38 kDa from zucchini and 57 and 60 kDa from carrots, respectively.
Cadherin-mediated adhesion in pancreatic islet cells is modulated by a cell surface N-acetylgalactosaminylphosphotransferase
Authors: Bauer G. Eric , Balsamo Janne , Lilien Jack
Published: April 2021

Rat pancreatic islet cells and RINm5F cells, an islet derived cell line, have at their cell surface an N-acetylgalactosaminylphosphotransferase (GalNAcPTase) similar to that found at the surface of chick neural retina cells and at the rat neuromuscular junction. On islet cells and RINm5F cells the GalNAcPTase is stably associated with cadherin cell-cell adhesion molecules. The effect of antibodies directed against the GalNAcPTase on homophilic, cadherin mediated adhesion was analyzed by measuring their effect on adhesion of islet and RINm5F cells to an immobilized anti-cadherin antibody.

In this experimental paradigm anti-GalNAcPtase antibodies completely inhibit cadherin mediated adhesion. Furthermore, cadherin and GalNAcPTase co-distribute in islet and non-islet tissue. We conclude that pancreatic islet cell-cell adhesion is cadherin mediated and under the control of a tightly associated, cell surface GalNAcPTase.
Expression of naked plasmids by cultured myotubes and entry of plasmids into T tubules and caveolae of mammalian skeletal muscle
Authors: Wolff Jon A. , Dowty Martin E. , Jiao Shoushu , Repetto Gabriella , Berg Randi K. , Ludtke James J. , Williams Phillip , Slautterback David B.
Published: April 2021

Plasmid DNA or artificial mRNA injected intramuscularly into skeletal muscle via a 27 g needle expressed transgenes at relatively efficient levels in skeletal myofibers and cardiac cells. In the present study, several approaches were used to determine the mechanism of cellular uptake. After exposure of naked plasmid DNA, primary rat muscle cells in vitro expressed transgenes to a much greater extent than other types of immortalized or primary cells. In vivo light microscope studies showed that intramuscularly injected plasmid DNA was distributed throughout the muscle and was able to diffuse through the extracellular matrix, cross the external lamina, and enter myofibers. Electron microscope studies showed that colloidal gold conjugated to plasmid DNA traversed the external lamina and entered T tubules and caveolae, while gold complexed with polylysine, polyethylene glycol or polyglutamate primarily remained outside of the myofibers. The results indicate that it is highly unlikely that the plasmid DNA enters the myofiber simply by the needle grossly disrupting the sarcolemma. In addition, transient membrane disruptions do not appear to be responsible for the uptake of DNA. Furthermore, no evidence for endocytosis could be found. The possible uptake of plasmid DNA by some type of cell membrane transporter, in particular via potocytosis, is discussed.
Haemocyte heterogeneity in the cockroach Periplaneta americana analysed using monoclonal antibodies
Authors: Chain Benjamin M. , Ø Rland Karen Leyshon-S , Siva-Jothy Michael T.
Published: April 2021

A panel of monoclonal antibodies (mAbs) raised against whole haemocytes from Periplaneta americana has revealed two broad categories of immunogens.

Three mAbs showed reactivity with membrane-bound antigens on the three major morphological classes of haemocyte as well as the basement membrane of tissues in contact with the haemocoel. Moreover, this reactivity showed heterogeneity within morphotypes: this heterogeneity was quantified using flow cytometry.

In contrast, two other mAbs did not show reactivity with tissues other than haemocytes: both mAbs appeared to label unique intracellular components of the haemocytes.

We have demonstrated that mAbs can be used to define haemocyte subpopulations in P. americana that are not recognisable on morphological criteria alone. We believe this heterogeneity underlies the mechanisms responsible for the identification and neutralisation of non-self in the ‘immune’ response of P. americana.