bluuebunny/crossref_metadata_2025 huggingface dataset:
Suprabasal change and subsequent formation of disulfide-stabilized homo- and hetero-dimers of keratins during esophageal epithelial differentiation
Authors: Pang Yuk-Ying Susana , Schermer Alexander , Yu Jun , Sun Tung-Tien
Published: April 2021

Rabbit esophageal epithelium, a parakeratinized stratified epithelium, synthesizes as one of its major differentiation products a keratin pair consisting of a basic K4 (59 kDa) and an acidic K13 (41 kDa) keratin. Although immunohistochemical staining data suggest that in esophageal epithelia of some other species these two keratins are suprabasally located, antigenic masking of the epitopes in the basal cells has not been ruled out. Using several well-characterized monoclonal antibodies including AE8, which specifically recognizes K13, coupled with biochemical analysis of keratins of basal and suprabasal cells isolated from confluent rabbit esophageal epithelial culture, we have obtained direct evidence that K4 and K13 keratins are largely absent in the undifferentiated basal cells, but are present in large amounts in suprabasal cells. We also show that in the cornified cell layers that are formed during the terminal stage of esophageal epithelial differentiation, K4 and K13 keratins become disulfide-crosslinked to form three different dimers. Two of them (110 kDa and 100 kDa) are heterodimers and consist of equimolar amounts of K4 and K13; they presumably represent isomers crosslinked via different cysteine residues. The third dimer (90 kDa) was found to be a homodimer of the acidic K13 keratin. Trypsinization experiment established that at least some of the disulfide crosslinks in the K4/K13 heterodimer must involve cysteine residues residing in the trypsin-resistant rod domains of keratins. Air-oxidation of in vitro reconstituted filaments reproduced the two heterodimers, which most likely involve the crosslinking between type I and type II keratins of different coiled coils. The formation of these disulfidecrosslinked keratin dimers, instead of higher molecular mass oligomers or polymers as occurring in the epidermis and hair, may contribute to the formation of cornified cells with a physical stability and rigidity that are optimal for esophageal function. Our data also suggest that interactions involved in the formation of homodimers, thought to be metastable and unimportant during the initial step of filament assembly (i.e. tetramer formation), may actually play an important role in stabilizing a higher order structure in mature keratin filaments.
Defining E-cadherin-associated protein complexes in epithelial cells: plakoglobin β- and γ-catenin are distinct components
Authors: Piepenhagen Peter A. , Nelson W. James
Published: April 2021

Ca2+-dependent cell adhesion is mediated by a family of proteins termed cadherins, and is modulated by cytosolic proteins that include α-, β-, and γ-catenin and other cytoskeletal proteins that bind to the cytoplasmic domain of cadherins. Recent studies have suggested that either β- or γ-catenin may be identical to plakoglobin, a protein associated with adherens junctions. However, the relationship between these proteins, and their interaction with cadherins, are not well understood. In this study, we have further defined the relationship between plakoglobin and the catenins in complexes with E-cadherin in Madin-Darby canine kidney (MDCK) cells. Specific immunoprecipitations revealed that plakoglobin (86 kDa) and β-catenin (92 kDa) have different detergent extractabilities and apparent molecular weights in these cells; however, plakoglobin has an apparent molecular weight similar to that of γ-catenin (86 kDa). Immunoblotting of E-cadherin immunoprecipitates demonstrated that both plakoglobin and β-catenin co-immunoprecipitate with E-cadherin. Laserscanning confocal microscopy demonstrated temporally and spatially co-ordinate redistribution of plakoglobin and E-cadherin following induction of cell-cell contact in MDCK cells. Although plakoglobin comigrated with γ-catenin on SDS-PAGE, quantitative analysis of E-cadherin and plakoglobin immunoprecipitates revealed that plakoglobin accounted for of the γ-catenin signal. Two-dimensional gel electrophoresis resolved the β-catenin protein band into two proteins. One protein was identified as plakoglobin, based upon apparent molecular weight, immunoreactivity and isoelectric point (pI ∼ 6.1). The other protein comigrated with γ-catenin on SDS-PAGE, did not react with plakoglobin antibodies and had a pI of ∼4.25; we refer to this protein as γ-catenin to distinguish it from plakoglobin. Two-dimensional gel electrophoresis further revealed that plakoglobin comprised multiple isoelectric variants, but that, within the newly synthesized pool of plakoglobin, only the most basic of these variants co-immunoprecipitated with E-cadherin; phosphorylation did not account for the plakoglobin isoelectric variants seen by two-dimensional gel electrophoresis. These results demonstrate directly that plakoglobin associates and co-localizes with the E-cadherin in MDCK epithelial cells in a complex that contains α-, β-, and γ-catenin. Although plakoglobin shares sequence similarity with β-catenin, and comigrates with γ-catenin in SDS-PAGE, plakoglobin is distinct from the catenins. The asociation of plakoglobin with E-cadherin may be regulated by post-translational modifications of plakoglobin.
Fibronectin splice variants are differentially incorporated into the extracellular matrix of tumorigenic and non-tumorigenic hybrids between normal fibroblasts and sarcoma cells
Authors: Mardon Helen J. , Grant Richard P. , Grant Kate E. , Harris Henry
Published: April 2021

Recent reports have described transformation- and tumour-specific expression of fibronectin isoforms generated by alternative splicing of the fibronectin pre-mRNA. We have investigated the expression and distribution of EDIIIA+ and EDIIIB+ fibronectin splice variants in tumorigenic and non-tumorigenic somatic cell hybrids made by fusing fibrosarcoma-derived cells (HT1080) and normal fibroblasts (GM00097). Alternative splicing of EDIIIA and EDIIIB was assessed quantitatively by S1 nuclease analyses. The levels of EDIIIA+ and EDIIIB+ fibronectin mRNAs were similar in the parental and hybrid cells. Domain-specific monoclonal antibodies were used in immunohistochemical studies to identify EDIIIA+ and EDIIIB+ fibronectins in fixed cells. GM00097 and the non-tumorigenic hybrid (clone G3) showed high levels of both EDIIIA+ and EDIIIB+ fibronectin staining. The tumorigenic hybrid (clone C1) showed reduced amounts of EDIIIA+ fibronectin, but no detectable EDIIIB+ fibronectin. No fibronectin was detected on the surface of HT1080 cells. Western blots of protein extracted from culture supernatants and extracellular matrices revealed that GM00097 and G3 cells incorporated most of the EDIIIA+ and EDIIIB+ fibronectin into the extracellular matrix whereas C1 cells released a large proportion of the EDIIIA+ fibronectin, and almost all of the EDIIIB+ fibronectin, into the supernatant. We conclude that there are differences in the presence of EDIIIA+ and EDIIIB+ FNs on the surface of tumorigenic and non-tumorigenic cells and that these differences are due to differential incorporation of FN variants into the ECM.
Platelet shape change and protein phosphorylation induced by ADP and thrombin are not sensitive to short periods of microgravity
Authors: Schmitt Didier A. , Ohlmann Philippe , Gachet Christian , Cazenave Jean-Pierre
Published: April 2021

Recent experiments have shown that the stimulation of animal cells in vitro by direct protein kinase C (PKC) activators is significantly reduced under microgravity (μg). Platelets undergo protein phosphorylation and morphological changes a few seconds after stimulation by agonists such as phorbol esters which activate PKC. Therefore, taking advantage of parabolic plane flight to obtain short periods of microgravity, we studied phosphorylation of myosin light chain (20K), specific PKC-dependent phosphorylation of a 40,000 Mr protein, pleckstrin (40K) and platelet shape change. SDS-PAGE analysis and electron microscopy were performed on platelets subjected to 20 seconds microgravity as compared to normal gravity (1 g) conditions. These investigations showed that neither Ca2+-calmodulin-mediated activation nor the PKC-dependent pathways are inhibited during short periods of microgravity.
Mitotic Golgi clusters are not tubular endosomes
Authors: Pypaert Marc , Nilsson Tommy , Berger Eric G. , Warren Graham
Published: April 2021

HeLa cells were incubated with 15 nm BSA-gold for 1 or 2 hours to mark the endocytic pathway and mitotic cells were then isolated by shake-off. Thin, frozen sections were labelled with antibodies against two resident Golgi markers,β-(1,4)-galactosyltransferase and N-acetylglucosaminyltransferase I. Detection of the latter was aided by the use of a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. The secondary antibodies were coupled to either 5 or 10 nm gold so that the distribution of each of the three markers could be followed. Qualitative and quantitative stud-ies showed that there were two populations of clusters, those described by us earlier and termed Golgi clusters (Lucocq et al. (1987)J. Cell Biol. 104, 865-874), containing either or both Golgi markers, and clusters of tubular endosomes containing BSA-gold. There was very little overlap showing that Golgi clusters cannot be tubular endosomes as concluded by Tooze and Hollinshead (1992)Eur. J. Cell Biol. 58, 228-242.
An N-terminal truncation of the ncd motor protein supports diffusional movement of microtubules in motility assays
Authors: Chandra Rashmi , Endow Sharyn A. , Salmon Edward D.
Published: April 2021

The nonclaret disjunctional (ncd) protein is a kinesin-related microtubule motor protein that is encoded at the claret locus in Drosophila and is required for proper chromosome distribution in meiosis and early mitosis The protein contains a region with 41% amino acid sequence identity to kinesin heavy chain, but translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends The overall structure of ncd also differs from kinesin heavy chain, in that the proposed motor domain is present at the C terminus of the molecule instead of the N terminus, as in kinesin heavy chain In studies to define the molecular determinants of ncd function, we constructed and expressed a protein with a deletion of the N-terminal 208 amino acids of the non-motor region Analysis of the truncated protein shows that the protein exhibits microtubule-stimulated Mg2 +-ATPase activity and binds microtubules in pelleting assays In contrast to near fulllength ncd, the truncated protein does not support directional movement of microtubules in in vitro motility assays Instead, microtubules show nucleotide-sensitive binding to the truncated protein on glass surfaces and bound microtubules exhibit one-dimensional diffusional movement that is constrained to their longitudinal axis The diffusional movement reveals a weak binding state of the ncd motor that may represent a mechanochemical intermediate in its ATP hydrolysis cycle If diffusional movement is a characteristic intrinsic to the claret motor, it is likely to be important in the in vivo function of the protein.
Lymphocyte migration across high endothelium is associated with increases in α4β1 integrin (VLA-4) affinity
Authors: Hourihan Helen , Allen Terry D. , Ager Ann
Published: April 2021

The constitutive recirculation of lymphocytes between the widely distributed organs of the immune system is essential for host defence. We have developed an in vitro model of lymphocyte migration from the blood into lymph nodes which employs primary cultures of high endothelial cells (HEC). HEC-adherent lymphocytes adopt one of two distinct morphologies which correlates with their position in the endothelial layer; type I cells are bound to the surface of HEC and type II cells are underneath the endothelial layer. In a previous study we reported that the numbers of type I and type II cells are independently regulated, however the relationship between these two lymphocyte populations was not determined. In this study we have carried out detailed kinetic, phenotypic and functional analyses of type I and type II lymphocytes and determined their relationship. Using allotype marked lymphocytes from the PVG.RT7a and PVG.RT7b rat strains in a pulse-chase analysis, type I and type II lymphocytes were found to represent the same population of lymphocytes at different stages of interaction with the endothelial layer, rather than representing two independent lymphocyte populations. Migration was an irreversible event and the efficiency of migration (i.e. transition from type I to type II) was related to the concentration of lymphocytes plated on to the HEC layer. Following transmigration lymphocytes showed an increased ability to migrate across HEC layers and to bind to immobilised CS1 peptide. The increased binding to CS1 peptide was transient and fell to control levels over a 3 hour time period. The expression of 4 integrin subunit on lymphocytes was unchanged following migration which suggests that the affinity of the CS1 receptor, 4 1, is upregulated by interaction with HEC. Together these results suggest that transendothelial migration is regulated by increases in the affinity of 4 1 integrin on lymphocytes following contact with HEC.
Immunoelectron characterisation of the inter-endothelial junctions of human term placenta
Authors: Leach Lopa , Clark Peter , Lampugnani Maria-Grazia , Arroyo Alicia G. , Dejana Elisabetta , Firth J. Anthony
Published: April 2021

The molecular constituents of the paracellular clefts in human placental microvessels were investigated using antibodies against PECAM-1, pancadherin, A-CAM (N-cadherin), cadherin-5 and two types of integrins (those recognised by antibodies to the α1 chain and v 3). Ultrastructural localisation of these molecules in ultrathin frozen sections of human term placentae was attempted using colloidal gold immunocytochemistry, after establishing their presence by indirect immuno-fluorescence.

At the light microscopical level, the endothelial paracellular clefts were found to be immunoreactive to the antibodies against PECAM-1, cadherin-5 and pan-cadherin, but not the integrins. The latter showed diffuse distribution in the endothelium and in the abluminal interstitial space. PECAM-1 and pan-cadherin were also seen in the cytoplasm and luminal surface of the endothelium. Immunoelectron studies revealed that the cadherins and PECAM-1 were present in the wide regions of the paracellular clefts, but not in tight junctional regions. Using immunocytochemistry, these wide junctional areas were found to be associated with the cytoskeletal linking molecules vinculin and α-actinin. These regions may therefore contain adherens-type junctions. Cadherin-5, localised by two different monoclonal antibodies, 7B4 and TEA, was the only antigen which was cleft-specific, the others also being seen in the cytoplasm of the microvascular endothelium. Cadherin-5 and pan-cadherin were colocalised in the same wide junction, but were usually seen to occupy different microdomains of, and different wide zones of, the same cleft. The cell adhesion molecules localised in the paracellular wide junctions of the human placental microvessels may play a role in maintaining the intercellular spacing between endothelial cells, and may be part of a paracellular “fibre matrix” with permeability-restricting properties.
Independent modes of propagation of calcium waves in smooth muscle cells
Authors: G. Mahoney Mỹ , Slakey Linda L. , Hepler Peter K. , Gross David J.
Published: April 2021

The purinergic agonist adenosine triphosphate (ATP) stimulates an initial transient followed by subsequent oscillations in cytosolic calcium ion concentration ([Ca2+]i) in individual porcine aortic smooth muscle cells. Using microinjection of fura-2 covalently coupled to dextran, we have analyzed in detail the spatial and temporal features of the oscillations. We have observed both cytoplasmic calcium waves and gradients within single cells. Single cells can contain multiple loci of initiation of oscillations. Independent oscillations in a single cell can have independent frequencies and these oscillations can propagate without interference across the same region of the cell, suggesting that they arise either from separately regulated stores of Ca2+ or a single Ca2+ store operated by two separate release mechanisms. The shape of the wave front and the manner of the wave’s decay can vary from one oscillation to the next. Ca2+ signaling in individual arterial smooth muscle cells thus displays complex spatial and temporal organization.
Glycosyl phosphatidylinositol membrane anchoring of melanotransferrin (p97): apical compartmentalization in intestinal epithelial cells
Authors: Alemany Ramón , Vilá M. Rosa , Francí Clara , Egea Gustavo , Real Francisco X. , Thomson Timothy M.
Published: April 2021

Melanotransferrin (p97) is an iron-binding membrane glycoprotein with 40% homology to transferrin and lactoferrin. It was first identified on the basis of its high level of expression in melanoma cells, as compared to normal melanocytes. It is also present in many cultured cell types. In normal tissues, p97 is expressed in fetal intestine, umbilical cord, sweat gland ducts and liver sinusoidal lining cells. Kinetic studies in melanoma cells have suggested that p97 plays a role in iron metabolism. We have examined expression of p97 in cell lines derived from human colorectal carcinomas which express a differentiated phenotype. When polarized, these cells showed a preferred apical distribution of p97, as demonstrated by immunohistochemistry, immune electron microscopy and domain-selective biotinylation. Correspondingly, p97 was only found on the apical brush border of epithelial cells in the fetal intestine. p97 was shown to be anchored to the membrane through a glycosyl phosphatidylinositol moiety by treatment with phophatidylinositol-specific phospholipase C (PI-PLC) and labeling with [14C]ethanolamine. These observations provide a basis for the elucidation of the physiological role of p97 in iron metabolism and its possible role in cell proliferation and malignant cell transformation.
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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.
Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae Study by fluorescence spectroscopy
Authors: Aubry Laurence , Klein Gérard , Martiel Jean-Louis , Satre Michel
Published: April 2021

The evolution of endo-lysosomal pH in Dictyostelium dis -coideum amoebae was examined during fluid-phase endocytosis. Pulse-chase experiments were conducted in nutritive medium or in non-nutritive medium using fluorescein labelled dextran (FITC-dextran) as fluid-phase marker and pH probe. In both conditions, efflux kinetics were characterized by an extended lag phase lasting for 45-60 min and corresponding to intracellular transit of FITC-dextran cohort. During the chase period, endosomal pH decreased during ∼20 min from extracellular pH down to pH 4.6-5.0, then, it increased within the next 20-40 min to reach pH 6.0-6.2. It was only at this stage that FITC-dextran was released back into the medium with pseudo first- order kinetics. A vacuolar H+-ATPase is involved in endosomal acidification as the acidification process was markedly reduced in mutant strain HGR8, partially defective in vacuolar H+-ATPase and in parent type strain AX2 by bafilomycin A1, a selective inhibitor of this enzyme. Our data suggest that endocytic cargo is channeled from endosomes to secondary lysosomes that are actively linked to the plasma membrane via recycling vesicles.
α Melanocyte stimulating hormone (αmsh) stimulates normal human melanocyte growth by binding to high-affinity receptors
Authors: Luca Michele De , Siegrist Walter , Bondanza Sergio , Mathor Monica , Cancedda Ranieri , Eberle Alex N.
Published: April 2021

The combined action of cholera toxin (CT)-dependent activation of the adenylate cyclase signaling pathway, stimulation of protein kinase C, and activation of the tyrosine kinase activity of cell surface receptors and proto-oncogene products, have been shown to stimulate melanocyte proliferation. However, natural factors responsible for the optimal stimulation of normal human melanocyte growth, either isolated or co-cultured with keratinocytes, remain largely unknown. aMSH (α melanocyte stimulating hormone) has previously been shown to bind to murine and human melanoma cells and to stimulate their adenylate cyclase and tyrosinase activity. In contrast, very little is known about the presence and function of αMSH receptors in normal human melanocytes. We now report that aMSH: (i) binds to normal human melanocytes through a single class of high-affinity receptors; (ii) does not induce per se melanocytes to enter the S-phase of the cell cycle; (iii) does indeed stimulate melanocyte proliferation in a dose-dependent fashion; but its stimulatory effect requires bFGF and/or the activation of protein kinase C.
Six git genes encode a glucose-induced adenylate cyclase activation pathway in the fission yeast Schizosaccharomyces pombe
Authors: Byrne Susan M. , Hoffman Charles S.
Published: April 2021

An important eukaryotic signal transduction pathway involves the regulation of the effector enzyme adenylate cyclase, which produces the second messenger, cAMP. Previous genetic analyses demonstrated that glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene requires the function of adenylate cyclase, encoded by the git2 gene. As mutations in git2 and in six additional git genes are suppressed by exogenous cAMP, these ‘upstream’ git genes were proposed to act to produce a glucose-induced cAMP signal. We report here that assays of cAMP levels in wild-type and various mutant S. pombe cells, before and after exposure to glucose, show that this is the case. The data suggest that the cAMP signal results from the activation of adenylate cyclase. Therefore these ‘upstream’ git genes appear to encode a glucose-induced adenylate cyclase activation pathway. Assays of cAMP on a strain carrying a mutation in the git6 gene, which acts downstream of adenylate cyclase, indicate that git6 may function to feedback regulate adenylate cyclase activity. Thus git6 may encode a cAMP-dependent protein kinase.
Transcriptionally active minichromosomes are attached transiently in nuclei through transcription units
Authors: Jackson D. A. , Cook P. R.
Published: April 2021

Viral minichromosomes provide simple models for chromatin domains. The sequences attaching them to larger nuclear structures were mapped; attachments were defined operationally by their ability to prevent chromatin fragments electroeluting from nuclei. Cells encapsulated in agarose microbeads were lysed and analysed subsequently in a ‘physiological’ buffer. Non-transcribed minichromosomes eluted from nuclei but transcriptionally active ones did not. Cutting attached minichromosomes with HaeIII enabled most of the resulting ∼400 bp fragments to elute. Analysis of residual fragments showed that no single sequence was responsible for attachment; rather, minichromosomes were attached at only one or two points through a promoter or part of a transcription unit. This suggests that RNA polymerases mediate attachments that change dynamically as the template slides past the attached enzyme. As, under optimal conditions, polymerases continue to elongate roughly at the rate found in vivo, these attachments are unlikely to be generated artifactually.