bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oscillator control of cell division in Euglena: cyclic AMP oscillations mediate the phasing of the cell division cycle by the circadian clock
Authors: Carré Isabelle A. , Edmunds Leland N. 
Published: April 2021

ABSTRACT
The achlorophyllous ZC strain of Euglena gracilis exhibits a circadian rhythm of cell division in constant darkness (DD). Mitosis occurs during a restricted part of the circadian cycle, corresponding to the dark intervals in a light-dark cycle comprising 12 h of light and 12 h of darkness. We have demonstrated that division-phased cultures also exhibit bimodal, circadian changes of cyclic AMP level. Maximum cyclic AMP levels occurred at the beginning of the light period (CT (circadian time) 00-02), and at the beginning of darkness (CT 12-14). These variations persisted in cultures that had been transferred into DD and appeared to be under the control of the circadian oscillator rather than to be cell division cycle (CDC)-dependent, since they continued in cultures that had reached the stationary phase of growth. In the experiments reported in this paper, we tested for the possible role of this periodic cyclic AMP signal in the generation of cell division rhythmicity by examining the effects of exogenous cyclic AMP signals and of forskolin, which permanently increased the cyclic AMP level, on the cell division rhythm.
Perturbations of the cyclic AMP oscillation by exogenous cyclic AMP resulted in the temporary uncoupling of the CDC from the circadian timer. The addition of cyclic AMP during the subjective day resulted in delays (up to 9 h) of the next synchronous division step. In contrast, mitosis was stimulated when cyclic AMP was administered in the middle of the subjective night. Measurement of the DNA content of cells by flow cytometry indicated that cyclic AMP injected at CT 06-08 delayed progression through S phase, and perhaps also through mitosis. When added at CT 18-20, cyclic AMP accelerated the G2/M transition. The circadian oscillator was not perturbed by the addition of exogenous cyclic AMP: the division rhythm soon returned to its original phase. On the other hand, the permanent elevation of cyclic AMP levels in the presence of forskolin induced a rapid loss of cell division rhythmicity. These findings are consistent with the hypothesis that cyclic AMP acts downstream from the oscillator and that the cyclic AMP oscillation is an essential component of the signaling pathway for the control of the CDC by the circadian oscillator.
The receptors for cyclic AMP in Euglena have been shown to be two cyclic AMP-dependent kinases (cPKA and cPKB). Pharmacological studies using cyclic AMP analogs suggested that cPKA mediates cyclic AMP effects during the subjective day, and cPKB during the subjective night. On the basis of these results, we propose a model for the control of the CDC by the circadian clock.
A study on nucleolar DNA: isolation of DNA from fibrillar components and ultrastructural localization of different DNA probes*
Authors: Hozák Pavel , Schöfer Christian , Sylvester James , Wachtler Franz 
Published: April 2021

ABSTRACT
The nature and localization of DNA contained in the fibrillar centres and the dense fibrillar component (the fibrillar complex) in the nucleoli, was studied in human LEP cells, Sertoli cells, spermatogonia A and in mitotic chromosomes of stimulated lymphocytes. A novel procedure for isolating the intact fibrillar complex from LEP cells was used; the complex contains DNA that hybridizes to secondary constrictions of mitotic chromosomes and to 28 S rDNA sequences, on Southern blots. Electron microscopic DNA-DNA in situ hybridization was performed, with (a) a probe prepared from DNA extracted from the fibrillar complex of LEP cells, (b) a probe for human total genomic DNA, and (c) a probe for the transcribed part of human rDNA. On the basis of the results obtained we conclude that the ribosomal RNA genes in human Sertoli cells and spermatogonia A are predominantly associated with the dense fibrillar component, including the border region between fibrillar centres and the dense fibrillar component. The ribosomal RNA genes are the main, if not exclusive, DNA type present in the fibrillar complex in the studied cell types.
The isolation and culture of microvascular endothelium
Authors: Scott Prudence A. E. , Bicknell Roy 
Published: April 2021

ABSTRACT
Endothelial cells are intimately involved in a wide range of biological processes including reproduction, development and wound healing (Folkman, 1992), as well as pathological processes such as inflammatory disorders of the skin and joints (Abbot et al., 1992), diabetic retinopathy and tumour invasion (Folkman, 1992). This has led to major efforts over the past twenty years to isolate and culture endothelial cells from both animal and human tissues, in order to investigate their role further. The most common isolation has been from the human umbilical vein, largely because of the ease with which the umbilici can be obtained. Isolation involves cannulation of the vein and introduction of a proteolytic enzyme, followed by a five to fifteen minute incubation before flushing to yield an isolate of endothelial cells (Jaffe et al., 1973). Alternatively, it is possible to obtain endothelial cells from large vessels, including arteries, by gentle scraping of the intimal surface with, for example, a cotton-wool tip.
A 300 bp 5’ -upstream sequence of a differentiation-dependent rabbit K3 keratin gene can serve as a keratinocyte-specific promoter
Authors: Wu Ren-Long , Galvin Sharon , Wu Shao-Kee , Xu Cong , Blumenberg Miroslav , Sun Tung-Tien 
Published: April 2021

ABSTRACT
Keratinocytes of the suprabasal compartment of many stratified epithelia synthesize as a major differentiation product a keratin pair, consisting of an acidic and a basic keratin, which accounts for 10-20% of the newly synthesized proteins. While genes of several differentiation-related keratins have been cloned and studied, relatively little is known about the molecular basis underlying their tissue-specific and differentiation-dependent expression. We have chosen to study, as a prototype of these genes, the gene of K3 keratin, which has the unique property of being expressed in the majority of corneal epithelial basal cells but suprabasally in peripheral cornea, the site of corneal epithelial stem cells. Using a monoclonal antibody, AE5, specific for K3 keratin, and a fragment of human K3 gene as probes, we have isolated several cDNA and genomic clones of rabbit K3 keratin. One genomic clone has been sequenced and characterized, and the identity of its coding sequence with that of cDNAs indicates that it corresponds to the single, functional rabbit K3 gene. Transfection assays showed that its 3.6 kb 5 -upstream sequence can drive a chloramphenicol acetyl transferase (CAT) reporter gene to express in cultured corneal and esophageal epithelial cells, but not in mesothelial and kidney epithelial cells or fibroblasts, all of rabbit origin. Serial deletion experiments narrowed this keratinocyte-specific promoter to within 300 bp upstream of the transcription initiation site. Its activity is not regulated by the coding or 3 -noncoding sequences that have been tested so far. This 300 bp 5 -upstream sequence of K3 keratin gene, which can function in vitro as a keratinocyte-specific promoter, contains two clusters of partially overlapping motifs, one with an NFkB consensus sequence and another with a GC box. The combinatorial effects of these multiple motifs and their cognate binding proteins may play an important role in regulating the expression of this tissue-restricted and differentiation-dependent keratin gene.
Centromeric DNA cloned from functional kinetochore fragments in mitotic cells with unreplicated genomes
Authors: Ouspenski Ilia I. , Brinkley B. R. 
Published: April 2021

ABSTRACT
Treatment of cells arrested in the cell cycle at the G1/S-phase boundary with 5 mM caffeine induces premature mitosis, resulting in chromosomal fragmentation and detachment of centromere-kinetochore fragments, which are subsequently attached to the mitotic spindle and segregated in anaphase. Taking advantage of this in vivo separation of the centromere, we have developed a procedure for isolation of a centromere-enriched fraction of mitotic chromatin. Using this method, we have isolated and cloned DNA from the centromere-enriched material of Chinese hamster cells. One of the clones thus obtained was characterized in detail. It contains 6 kb of centromere-associated sequence that exhibits no recognizable homology with other mammalian centromeric sequences and is devoid of any extensive repetitive structure. This sequence is present in a single copy on chromosome 1 and is species-specific. Distinctive features of the clone include the presence of several A+T-rich regions and clusters of multiple topoisomerase II consensus cleavage sites and other sequence motifs characteristic of nuclear matrix-associated regions. We hypothesize that these features might be related to the more compact packaging of centromeric chromatin in interphase nuclei and mitotic chromosomes.
Expression of the cystic fibrosis gene and the major pancreatic mucin gene, MUC1, in human ductal epithelial cells
Authors: Chambers Julie A. , Harris Ann 
Published: April 2021

ABSTRACT
The main pathology of cystic fibrosis results from obstruction of ducts in several organs by mucous secretions. The cause of this obstruction remains unclear. We have examined expression of the cystic fibrosis transmembrane conductance regulator (CFTR) and of the major pancreatic mucin, MUC1, in primary pancreatic duct and vas deferens epithelial cells, and in pancreatic duct cell lines. MUC1 is expressed at a high level in the primary ductal epithelial cells and at variable levels in different pancreatic adenocarcinoma cell lines. How-ever, although the pancreatic duct is one of the sites in vivo where CFTR transcription is at its highest level, the majority of cell lines examined no longer express CFTR. Only one pancreatic duct cell line, Capan 1, expresses CFTR at a significant level; further, the level of expression is dependent on confluency. We have shown that salt stress alone is not sufficient to account for the build-up of mucous secretions in CF ducts.
Alveolar type II cell-fibroblast interactions, synthesis and secretion of surfactant and type I collagen
Authors: Griffin Miriam , Bhandari Rena , Hamilton Graham , Chan Yiu-Che , Powell Janet T. 
Published: April 2021

ABSTRACT
During alveolar development and alveolar repair close contacts are established between fibroblasts and lung epithelial cells through gaps in the basement membrane. Using co-culture systems we have investigated whether these close contacts influence synthesis and secretion of the principal surfactant apoprotein (SP-A) by cultured rat lung alveolar type II cells and the synthesis and secretion of type I collagen by fibroblasts.
The alveolar type II cells remained cuboidal and grew in colonies on fibroblast feeder layers and on Matrigel-coated cell culture inserts but were progressively more flattened on fixed fibroblast monolayers and plastic. Alveolar type II cells cultured on plastic released almost all their SP-A into the medium by 4 days. Alveolar type II cells cultured on viable fibroblasts or Matrigel-coated inserts above fibroblasts accumulated SP-A in the medium at a constant rate for the first 4 days, and probably recycle SP-A by endocytosis. The amount of mRNA for SP-A was very low after 4 days of culture of alveolar type II cells on plastic, Matrigel-coated inserts or fixed fibroblast monolayers: relatively, the amount of mRNA for SP-A was increased 4-fold after culture of alveolar type II cells on viable fibroblasts.
Co-culture of alveolar type II cells with confluent human dermal fibroblasts stimulated by 2- to 3-fold the secretion of collagen type I into the culture medium, even after the fibroblasts’ growth had been arrested with mitomycin C. Collagen secretion, by fibroblasts, also was stimulated 2-fold by conditioned medium from alveolar type II cells cultured on Matrigel. The amount of mRNA for type I collagen increased only modestly when fibroblasts were cultured in this conditioned medium. This stimulation of type I collagen secretion diminished as the conditioned medium was diluted out, but at high dilutions further stimulation occurred, indicating that a factor that inhibited collagen secretion also was being diluted out. The conditioned medium contained low levels of IGF-1 and the stimulation of type I collagen secretion was abolished when the conditioned medium was pre-incubated with antibodies to insulin-like growth factor 1 (IGF-1).
There are important reciprocal interactions between alveolar type II cells and fibroblasts in co-culture. Direct contacts between alveolar type II cells and fibroblasts appear to have a trophic effect on cultured alveolar type II cells, increasing the levels of mRNA for SP-A. Rat lung alveolar type II cells appear to release a factor (possibly IGF-1) that stimulates type I collagen secretion by fibroblasts.
Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal
Authors: Frangioni John V. , Neel Benjamin G. 
Published: April 2021

ABSTRACT
We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting. The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK). The vector has been engineered such that any fusion construct can be sub-cloned into the versatile pJxΩ family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents. In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C). We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus.
Involvement of annexin II in DNA replication: evidence from cell-free extracts of Xenopus eggs
Authors: Vishwanatha Jamboor K. , Kumble Sarita 
Published: April 2021

ABSTRACT
Cell-free extracts of Xenopus eggs efficiently initiate and complete semiconservative DNA replication of exogenously added plasmid DNA. DNA replication in such extracts can be neutralized by a monoclonal antibody (D1/274.5) against human annexin II. Specific immunodepletion of Xenopus annexin II from the egg extracts results in loss of DNA replicative ability. Immunodepletion of annexin II does not prevent nuclear assembly, a stringent requirement for DNA synthesis on exogenous DNA in this system. Replicative ability can be restored to the immunodepleted extracts by the addition of purified human annexin II. These results demonstrate that annexin II is involved in chromosomal DNA replication and has a role in the cell cycle of higher eukaryotes.
Visualization of replication sites in unfixed human cells
Authors: Hassa A. Bassim , Cook Peter R. 
Published: April 2021

ABSTRACT
Sites of DNA replication in nuclei are focally concentrated, suggesting that an underlying structure organizes the activity of many polymerases. As fixation could induce aggregation into foci, we examined the distribution of replication sites in unfixed nuclei. HeLa cells were encapsulated in agarose microbeads, permeabilized in a ‘physiological’ buffer, their DNA polymerizing activity characterized, and replication sites directly labelled by incubation with fluorochrome-dUTP conjugates. Using conventional and digital fluorescence microscopy, 80-250 foci were seen in these unfixed cells.
These foci are unlikely to be formed by the aggregation of separate polymerases as most replication activity found in vivo is retained throughout these procedures. Although commonly used fixation methods collapsed or dispersed their periphery, the central core was very stable. Foci remained when ∼90% chromatin was removed, suggesting they were attached to an underlying structure.
F-actin content and spatial distribution in resting and chemoattractantstimulated human polymorphonuclear leucocytes: Which role for intracellular free calcium?
Authors: Zaffran Yona , Lepidi Hubert , Bongrand Pierre , Mege Jean-Louis , Capo Christian 
Published: April 2021

ABSTRACT
Intracellular free calcium concentration ([Ca2+]i) plays a pivotal role for many responses in polymorphonuclear leucocytes (PMNs) stimulated by chemoattractants such as N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). The importance of [Ca2+]i in the morphological polarization was investigated by using calcium-manipulated PMNs. We loaded human PMNs with BAPTA/AM to buffer or chelate [Ca2+]i in the presence or the absence of extracellular calcium by using fluo-3/AM as calcium indicator. The shape changes of PMNs were determined by microscopic examination, and membrane ruffling by right-angle light-scatter changes. Actin polymerization and F-actin distribution were recorded by staining PMNs with bodipy-phallacidin and quantified by quantitative fluorescence microscopy.
We found that calcium-free incubation of PMNs loaded or not with 50 M BAPTA/AM did not modify morphological polarization, membrane ruffling, actin assembly and F-actin distribution of PMNs stimulated with fMet-Leu-Phe, suggesting that these responses were probably functionally linked. It should be noted that incubation of PMNs in calcium-free conditions resulted in a radial distribution of F-actin and a moderate polymerization of actin, but not in morphological polarization of PMNs. Moreover, both calcium-sensitive and calcium-insensitive mechanisms of actin polymerization were additive, and inhibitable by 5 μg/ml cytochalasin B.
The matrix secreted by 804G cells contains laminin-related components that participate in hemidesmosome assembly in vitro
Authors: Langhofer Mikayo , Hopkinson Susan B. , Jones Jonathan C. R. 
Published: April 2021

ABSTRACT
Hemidesmosomes are important adhesion devices found in epithelial cells. They connect the intermediate filament cytoskeleton network with components of the basement membrane zone. 804G cells are an unusual epithelial cell line, since they form bona fide hemidesmosomes when plated on glass or plastic. In this study we tested an hypothesis: that this ability is a consequence of an extracellular component produced by the 804G cells. As probes for our study we generated a rabbit antiserum (J18) and monoclonal antibodies against components of urea-solubilized 804G matrix. Antibodies in the J18 serum recognize major lectin-binding polypeptides of 150, 140 and 135 kDa in the 804G matrix. A monoclonal antibody (5C5) that shows reactivity with the 150 and 135 kDa polypeptides in western immunoblots immunoprecipitates all three molecular mass species, indicating that these polypeptides are part of a matrix complex. Moreover, one, at least, of these matrix elements is immunologically related to laminin, since J18 antibodies selected on fusion protein fragments of a newly characterized laminin variant, laminin B2t (Kallunki et al., J. Cell Biol., 119, 679-694, 1992), react with the 140 kDa polypeptide component of the 804G cell matrix.
To undertake functional analyses of 804G matrix, cells of the human epidermal carcinoma line SCC12, which do not assemble bona fide hemidesmosomes in vitro, were cultured on 804G matrix for 24 h and then analysed by confocal immunofluorescence and electron microscopy. In SCC12 cells maintained on 804G cell matrix, hemidesmosomal antigens localize in a distinctive leopard spot pattern that mirrors the distribution of 804G matrix elements. Furthermore, ultrastructural analysis reveals that the 804G cell matrix supports the formation of ‘mature’ hemidesmosomes by SCC12 cells. Thus 804G cell matrix is a remarkable tool for hemidesmosome studies and it will now be of great importance to determine the exact composition of the 804G matrix, especially its structural and antigenic relationship to laminins.
Characterization of calpain II in the retina and photoreceptor outer segments
Authors: Azarian Sassan M. , Schlamp Cassandra L. , Williams David S. 
Published: April 2021

ABSTRACT
Calpain II was purified to apparent homogeneity from bovine neural retinas. It was found to be biochemically similar to brain calpain II, purified by the same procedure, with respect to: subunit mobility in SDS-polyacrylamide gel electrophoresis; Ca2+ sensitivity; inhibition by calpeptin and other cysteine protease inhibitors; and optimal pH. Semithin cryosections were immunolabeled with antibodies specific for the catalytic subunit of calpain II. Calpain II was detected in most layers of the retina, with the most pronounced label present in the plexiform layers (synaptic regions) and the photoreceptor outer segments. In dark-adapted retinas, the label was distributed throughout the outer segments. In light-adapted retinas, outer segment labeling was concentrated in the connecting cilium, and the inner segments were labeled. A partially pure preparation of calpain II from isolated rod outer segments was found to have the same biochemical characteristics as calpain II prepared in the same way from the whole retina. The enzyme was distributed fairly evenly between the cytosolic and cytoskeletal fractions of isolated rod outer segments. Immunoblots of the rod outer segment cytoskeleton were used to determine the susceptibility of known components of the actin-based cytoskeleton to proteolysis by calpain II in vitro. Actin was not proteolyzed at all, α-actinin was only slowly degraded, but myosin II heavy chain was rapidly proteolyzed. Actin filaments have been shown previously to be associated with myosin II and α-actinin in a small domain within the connecting cilium, where they play an essential role in the morphogenesis of new disk membranes. The localization of calpain II in the connecting cilium after light exposure, combined with the in vitro proteolysis of myosin II, suggests that calpain II could be involved in light-dependent regulation of disk membrane morphogenesis by proteolysis of myosin II.
cis-Golgi resident proteins and O-glycans are abnormally compartmentalized in the RER of colon cancer cells
Authors: Egea Gustavo , Franci Clara , Gambús Gemma , Lesuffleur Thecla , Zweibaum Alain , Real Francisco X. 
Published: April 2021

ABSTRACT
Neoplastic transformation is commonly associated with altered glycosylation of proteins and lipids. To understand the basis for altered mucin glycosylation, we have examined the distribution of RER markers, a cis-Golgi resident protein, and the GalNAc α-O-Ser/Thr epitope (Tn) in human colon cancer cells and in normal colon. In cultured mucin-producing colon cancer cells, Gal-NAc α-O-Ser/Thr was found in mucin droplets and in RER cisternae. In addition, the Golgi apparatus was disorganized in a proportion of cells and a 130 kDa cis-Golgi resident protein was also abnormally redistributed to the RER. The distribution of the MUC2 intestinal apomucin, protein disulphide isomerase, Gal-NAc α-O-Ser/Thr, and the 130 kDa cis-Golgi resident protein was analysed in normal colon and in colon cancer tissues. In normal colon, MUC2 apomucin and protein disulphide isomerase were located in the RER, whereas the cis-Golgi resident protein and GalNAc α-O-Ser/Thr were detected only in the cis-Golgi compartment. In contrast, the two Golgi markers colocalized with the MUC2 apomucin and protein disulphide isomerase in the RER of colon cancer cells. On the basis of these results, we propose that in colon cancer cells a redistribution of molecules normally present in the Golgi apparatus takes place; this alteration may contribute to the abnormal glycosylation of proteins and lipids associated with neoplastic transformation.