bluuebunny/crossref_metadata_2025 huggingface dataset:
Real-time analysis of the transcriptional regulation of hiv and hcmv promoters in single mammalian cells
Authors: White Michael R. H. , Masuko Masayuki , Amet Lorene , Elliott Gill , Braddock Martin , Kingsman Alan J. , Kingsman Susan M.
Published: April 2021

The regulation of human cytomegalovirus (hCMV) and human immunodeficiency virus (HIV) gene expression has been studied in single intact mammalian cells. Viral promoters were placed upstream of the firefly luciferase reporter gene and the resulting hybrid reporter constructs were stably integrated into the HeLa cell genome. A highly sensitive photon-counting camera system was used to study the level of gene expression in single intact cells. Luciferase expression was studied in the absence of activators of viral gene expression, in the presence of the HIV-1 TAT trans-activator protein, or in the presence of sodium butyrate, a non-viral activator of gene expression. In the absence of any activator of gene expression, while expression was undetectable in most cells, significant levels of basal luciferase activity were observed in a few cells, indicating heterogeneity in gene expression in the cell population. In the presence of the general activator of viral gene expression, sodium butyrate, transcriptional activation from the viral promoters gave rise to significant and relatively homogeneous levels of luciferase expression in a majority of cells. The luciferase imaging technology was used for the real-time analysis of changes of gene expression within a single cell. This non-invasive reporter assay should become important for studies of the temporal regulation of gene expression in single cells.
Tenascin-c inhibits extracellular matrix-dependent gene expression in mammary epithelial cells: localization of active regions using recombinant tenascin fragments
Authors: Jones Peter Lloyd , Boudreau Nancy , Myers Connie A. , Erickson Harold P. , Bissell Mina J.
Published: April 2021

The physiological role of tenascin in vivo has remained obscure. Although tenascin is regulated in a stage and tissue-dependent manner, knock-out mice appear normal. When tenascin expression was examined in the normal adult mouse mammary gland, little or none was present during lactation, when epithelial cells actively synthesize and secrete milk proteins in an extracellular matrix/lacto-genic hormone-dependent manner. In contrast, tenascin was prominently expressed during involution, a stage characterized by the degradation of the extracellular matrix and the subsequent loss of milk production. Studies with mammary cell lines indicated that tenascin expression was high on plastic, but was suppressed in the presence of the laminin-rich, Engelbreth-Holm-Swarm (EHS) tumour biomatrix. When exogenous tenascin was added together with EHS to mammary epithelial cells, β-casein protein synthesis and steady-state mRNA levels were inhibited in a concentration-dependent manner. Moreover, this inhibition by tenascin could be segregated from its effects on cell morphology. Using two β-casein promoter constructs attached to the chloramphenicol acetyltransferase reporter gene we showed that tenascin selectively suppressed extra-cellular matrix/prolactin-dependent transcription of the β-casein gene in three-dimensional cultures. Finally, we mapped the active regions within the fibronectin type III repeat region of the tenascin molecule that are capable of inhibiting β-casein protein synthesis. Our data are consistent with a model where both the loss of a laminin-rich basement membrane by extracellular matrix-degrading enzymes and the induction of tenascin contribute to the loss of tissue-specific gene expression and thus the involuting process.
Nucleo-cytoplasmic distribution of human hnrnp proteins: a search for the targeting domains in hnrnp a1
Authors: Weighardt Florian , Biamonti Giuseppe , Riva Silvano
Published: April 2021

hnRNP A1 (34 kDa) is an RNA binding protein consisting of two tandemly arranged RNA binding domains C-terminally linked to a glycine-rich auxiliary domain (2×RBD-Gly). A1 belongs to the set of polypeptides that bind nascent hnRNA in the nucleus to form the so called hnRNP complexes. These complexes seem to be involved both in pre-mRNA processing and in the nuclear export of mRNA. In fact A1, along with other hnRNP proteins, is exported from the nucleus probably bound to mRNA and is immediately re-imported. A1 nuclear re-import, which requires active transcription, is not mediated by a canonical nuclear localisation signal (NLS).

To identify the determinants of A1 subcellular localisation we developed an expression vector for studying the localisation, in transiently transfected cells, of the different structural motifs of A1 fused to a small reporter protein (chloramphenicol acetyltransferase, CAT; 26 kDa). We demonstrate that a 30 amino acid sequence in the glycine-rich domain (YNDFGNYNNQSSNFGPMKGGNFG-GRSSGPY), which bears no resemblance to canonical NLS, is necessary and sufficient to target the protein to the nucleus. Our data suggest that this targeting sequence might act by mediating the interaction of A1 with a NLS-containing nuclear import complex. On the other hand, the nuclear export of A1 requires at least one RNA binding domain in accord with the hypothesis that A1 exits from the nucleus bound to mRNA.

We propose a mechanism for the nucleo-cytoplasmic shuttling of A1 that envisages a specific role for the different structural domains and can explain the dependence of nuclear import from active transcription.
A novel essential fission yeast gene Pad1+ positively regulates Pap1+-dependent transcription and is implicated in the maintenance of chromosome structure
Authors: Shimanuki Mizuki , Saka Yasushi , Yanagida Mitsuhiro , Toda Takashi
Published: April 2021

Fission yeast pap1+ gene encodes an AP-1-like transcription factor, whose overexpression can confer resistance to staurosporine, a protein kinase inhibitor. We have previously identified a target gene (p25) for pap1+, and shown that, crm1+, which is required for maintenance of higher order chromosome structure, negatively regulates pap1-dependent transcription. In this study, we have characterized a novel gene, pad1+, which was isolated as a multicopy plasmid capable of confering staurosporine-resistance. We showed that high copy pad1+ induces transcriptional activation of the p25 gene and that the induction by pad1+ is dependent on the pap1+ gene. Furthermore, a cis-element analysis of the 5′-region of the p25 gene showed that two elements (an AP-1 site and a 14 bp palindrome sequence) where pap1 binds in vitro is essential for the induction by pad1+. These results indicate that pad1 can positively regulate pap1-dependent transcription. Through an electromobility shift assay we showed that overexpression of pad1+ is not capable of enhancing the DNA-binding activity of pap1 directly. The pad1+ gene encodes a 35 kDa protein that has significant identity (68%) to Caenorhabditis elegans F37A4.5, and is also similar to mouse Mov34 and human C6.1A. Gene disruption experiments have demonstrated that pad1+ is essential for viability. A disruption mutant of pad1+ obtained after spore germination exhibited an elongated cell body with abberantly folded chromosomes. A mitotic plasmid loss experiment also produced similar cells having an abnormal chromosome structure. These suggest that pad1+ may play an important role in higher order chromosome structure. Taken concurrently with our previous results, two essential genes pad1+ and crm1+ regulate pap1-dependent transcription; pad1+ and crm1+ are positive and negative regulators, respectively.
Effects of cofilin on actin filamentous structures in cultured muscle cells: intracellular regulation of cofilin action
Authors: Nagaoka Rie , Kusano Ken-ichi , Abe Hiroshi , Obinata Takashi
Published: April 2021

The previous investigation (Abe et al. (1989)J. Biochem. 106, 696-702) suggested that cofilin is deeply involved in the regulation of actin assembly in developing skeletal muscle. In this study, to examine further the function of cofilin in living myogenic cells in culture, recombinant cofilin having extra Cys residues at the N terminus was produced in Escherichia coli and was labeled with tetramethylrhodamine-iodoacetamide (IATMR). When the cofilin labeled with IATMR (IATMR-cofilin) was introduced into myogenic cells, actin filaments in the cytoplasm or nascent myofibrils were promptly disrupted, and many cytoplasmic rods which contained both IATMR-cofilin and actin were generated. Sarcomeric myofibrillar structures were not disrupted but tropomyosin was dissociated from the structures by the exogenous cofilin, and the IATMR-cofilin became localized in I-band regions. 24 hours after the injection, however, the actin-cofilin rods disappeared completely and the IATMR-cofilin became diffused in the cytoplasm as endogenous cofilin. Concomitantly, actin filaments were recovered and tropomyosin was re-associated with sarcomeric I-bands. At this point, the IATMR-cofilin in the cells still retained the functional activity to form intranuclear actin-cofilin rods in response to stimulation by DMSO just as endogenous cofilin. FITC-labeled actin introduced into myogenic cells at first failed to assemble into filamentous structures in the presence of the exogenous cofilin, but was gradually incorporated into myofibrils with time. The drastic effects of the exogenous cofilin on actin assembly were suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). These results indicate that the exogenous cofilin is active and alters actin dynamics remarkably in muscle cells, but its activity in the cytoplasm gradually becomes regulated by the action of some factors including PIP2-binding.
Alteration of collagen-dependent adhesion, motility, and morphogenesis by the expression of antisense α2 integrin mrna in mammary cells
Authors: Keely Patricia J. , Fong Alan M. , Zutter Mary M. , Santoro Samuel A.
Published: April 2021

Although integrins are known to mediate adhesive binding of cells to the extracellular matrix, their role in mediating cellular growth, morphology, and differentiation is less clear. To determine more directly the role of the α2β1 integrin, a collagen and laminin receptor, in mediating the collagen-dependent differentiation of mammary cells, we reduced expression of the integrin by the well differentiated human breast carcinoma cell line, T47D, by stably expressing α2 integrin antisense mRNA. Flow cytometry demonstrated that the antisense-expressing clones had levels of α2β1 integrin on their surfaces that were decreased by 30-70%. Adhesion of antisense-expressing clones to both collagens I and IV was decreased relative to controls in a manner that correlated with the level of cell surface α2β1 integrin expression. Adhesion to fibronectin and laminin were not affected. Motility across collagen-coated filters in haptotaxis assays was increased for only those clones that exhibited intermediate levels of adhesion to collagen, suggesting that an intermediate density of cell-surface α2β1 integrin optimally supports cell motility. When cultured in three-dimensional collagen gels, T47D cells organized in a manner suggestive of a glandular epithelium. In contrast, antisense-expressing clones with decreased α2β1 integrin were not able to organize in three-dimensional collagen gels. The growth rate of T47D cells was reduced when the cells were cultured in three-dimensional collagen gels. Unlike adhesion, motility, and morphogenesis, growth rates were unaffected by reduction of α2β1 integrin expression. Our results suggest that adhesive interactions mediated by a critical level of surface α2β1 integrin expression are key determinants of the collagen-dependent morphogenetic capacity of mammary epithelial cells.
Evidence that tyrosine phosphorylation may increase tight junction permeability
Authors: Staddon James M. , Herrenknecht Kurt , Smales Caroline , Rubin Lee L.
Published: April 2021

Tight junction permeability control is important in a variety of physiological and pathological processes. We have investigated the role of tyrosine phosphorylation in the regulation of tight junction permeability. MDCK epithelial cells and brain endothelial cells were grown on filters and tight junction permeability was determined by transcellular electrical resistance (TER). The tyrosine phosphatase inhibitor pervanadate caused a concentration- and time-dependent decrease in TER in both MDCK and brain endothelial cells. However, as expected, pervanadate resulted in the tyrosine phosphorylation of many proteins; hence interpretation of its effects are extremely difficult. Phenylarsine oxide, a more selective tyrosine phosphatase inhibitor, caused the tyrosine phosphorylation of relatively few proteins as analyzed by immunoblotting of whole cell lysates. This inhibitor, like pervanadate, also elicited a decrease in TER in the two cell types. In the MDCK cells, the action of phenylarsine oxide could be reversed by the subsequent addition of the reducing agent 2,3-dimercapto-propanol. Immunocytochemistry revealed that phenylar-sine oxide rapidly stimulated the tyrosine phosphorylation of proteins associated with intercellular junctions. Because of the known influence of the adherens junction on tight junctions, we analyzed immunoprecipitates of the E-cadherin/catenin complex from MDCK cells treated with phenylarsine oxide. This revealed an increase in the tyrosine phosphorylation of β-catenin, but not of α-catenin. However, the tight junction associated protein ZO-1 was also tyrosine phosphorylated after PAO treatment. These data indicate that tight junction permeability may be regulated via mechanisms involving tyrosine phosphorylation of adherens junction and tight junction proteins.
Identification of a novel ca2+-regulated protein that is associated with the marginal band and centrosomes of chicken erythrocytes
Authors: Zhu Jian , Bloom Stephen E. , Lazarides Elias , Woods Catherine
Published: April 2021

We have identified a novel Ca2+-regulated protein, p23, that is expressed specifically in avian erythrocyte and thrombocyte lineages. Sequence analysis of this 23 kDa protein reveals that it bears no homology to any known sequence. In mature definitive erythrocytes p23 exists in equilibrium between a soluble and a cytoskeletal bound pool. The cytoskeletal fraction is associated with the marginal band of microtubules, centrosomes and nuclear membrane under conditions of low free [Ca2+]. An increase in free [Ca2+] to10−6 M is sufficient to induce dissociation of >95% of bound p23 from its target cytoskeletal binding sites, yet this [Ca2+] has little effect on calmodulin-mediated MB depolymerization. Analysis of p23 expression and localization during erythropoiesis together with results from heterologous p23 expression in tissue cultured cells demonstrates that this protein does not behave as a bone fide microtubule-associated protein. In addition, the developmental analysis revealed that although p23 is expressed early in definitive erythropoeisis, its association with the MB, centrosome and nuclear membrane occurs only in the final stages of differentiation. This cytoskeletal association correlates with marked p23 stabilization and accumulation at a time p23 expression is being markedly downregulated. We hypothesize that the mechanism of p23 association to the MB and centrosomes may be induced in part by a decrease in intracellular [Ca2+] during the terminal stages of definitive erythropoiesis.
Identification and molecular evolution of new dynein-like protein sequences in rat brain
Authors: Tanaka Yosuke , Zhang Zhizeng , Hirokawa Nobutaka
Published: April 2021

RT-PCR cloning was performed to find unknown members of the dynein superfamily expressed in rat brain. Six kinds of degenerate primers designed for the dynein catalytic domain consensuses were used for extensive PCR amplifications. We have sequenced 550 plasmid clones which turned out to include 13 kinds of new dynein-like sequences (DLP1-8, 9A/B, 10-12) and cytoplasmic dynein heavy chain. In these clones, alternative splicing was detected for a 105 nt-domain containing the CFDEFNRI consensus just downstream of the most N-terminal P-loop (DLP9A and 9B). By using these obtained sequences, initial hybridization studies were performed. Genomic Southern blotting showed each sequence corresponds to a single copy of the gene, while northern blotting of adult brain presented more than one band for some subtypes. We further accomplished molecular evolutionary analysis to recognize their phylogenetic origins for the axonemal and non-axonemal (cytoplasmic) functions. Different methods (UPGMA, nJ and MP) presented well coincident phylogenetic trees from 44 partial amino acid sequences of dynein heavy chain from various eukaryotes. The trunk for all the cytoplasmic dynein heavy chain homologues diverged directly from the root of the phylogenetic tree, suggesting that the first dynein gene duplication defined two distinct functions as respective subfamilies. Of particular interest, we found a duplication event of the cytoplasmic dynein heavy chain gene giving rise to another subtype, DLP4, located between the divergence of yeast and that of Dictyostelium. Such evolutionary topology builds up an inceptive hypothesis that there are at least two non-axonemal dynein heavy chains in mammals.
Direct visualization of a vast cortical calcium compartment in Paramecium by secondary ion mass spectrometry (SIMS) microscopy: possible involvement in exocytosis
Authors: Stelly Nicole , Halpern Sylvain , Nicolas Gisèle , Fragu Philippe , Adoutte André
Published: April 2021

The plasma membrane of ciliates is underlaid by a vast continuous array of membrane vesicles known as cortical alveoli. Previous work had shown that a purified fraction of these vesicles actively pumps calcium, suggesting that alveoli may constitute a calcium-storage compartment. Here we provide direct confirmation of this hypothesis using in situ visualization of total cell calcium on sections of cryofixed and cryosubstituted cells analyzed by SIMS (secondary ion mass spectrometry) microscopy a method never previously applied to protists. A narrow, continuous, Ca-emitting zone located all along the cell periphery was observed on sections including the cortex. In contrast, Na and K were evenly distributed throughout the cell. Various controls confirmed that emission was from the alveoli, in particular, the emitting zone was still seen in mutants totally lacking trichocysts, the large exocytotic organelles docked at the cell surface, indicating that they make no major direct contribution to the emission. Calcium concentration within alveoli was quantified for the first time in SIMS microscopy using an external reference and was found to be in the range of 3 to 5 mM, a value similar to that for sarcoplasmic reticulum. After massive induction of trichocyst discharge, this concentration was found to decrease by about 50%, suggesting that the alveoli are the main source of the calcium involved in exocytosis.
Cell cycle-dependent alterations of a highly phosphorylated nucleolar protein p130 are associated with nucleologenesis
Authors: Pai Chi-Yun , Chen Hung-Kai , Sheu Huey-Ling , Yeh Ning-Hsing
Published: April 2021

We identified a novel human nucleolar phosphoprotein p130 (130 kDa) using a strategy for selecting monoclonal antibodies against nuclear proteins which oscillate in the cell cycle. p130 is localized in interphase nucleoli in a dotted manner. Complete extraction of p130 required a high concentration of salt (0.5 M NaCl) indicating that it binds firmly to the nucleolar components via ionic interaction. p130 is heavily phosphorylated, since alkaline phosphatase treatment converted the purified p130 into a 95 kDa product; this was further supported by the in vitro demonstration that cellular phosphatase and casein kinase II activities were responsible for the interchange of these two forms. Extracts of mitotic cells had lower concentrations of p130 compared to those of interphase cells suggesting that a proportion of p130 might be degraded during mitosis. Moreover, all the remaining p130 in mitotic cells was further phosphorylated, likely by a cdc2 kinase, resulting in increase in its solubility, and its dispersion throughout the entire cytoplasm. Thus, p130 in metaphase and anaphase cells was unable to be detected by immunofluo rescence microscopy. At telophase, p130 reappeared and aggregated into a granular structure, resembling the prenucleolar bodies. These granules migrated from the nucleoplasm to the nucleoli in early Gɪ-phase. Actinomycin D was able to induce segregation of p130-containing granules into the nucleoplasm, similar to the well-known behavior of the fibrillarin-containing granules, indicating that p130 is localized in the dense fibrillar component, a subnucleolar region for pre-rRNA synthesis and processing. The cDNA sequence of p130 revealed a remarkable feature, that a serine-rich stretch interspersed with acidic residues is repeated ten times. Such a characteristic is shared with a rat nucleolar phosphoprotein Nopp140, which is thought to shuttle between the nucleolus and the cytoplasm. Although p130 shows 74% identity to Nopp140, our observations suggest that during mitosis the functions of p130 are related to nucleologenesis.
Cellular growth and survival are mediated by β1 integrins in normal human breast epithelium but not in breast carcinoma
Authors: Howlett Anthony R. , Bailey Nina , Damsky Caroline , Petersen Ole W. , Bissell Mina J.
Published: April 2021

We previously established a rapid three-dimensional assay for discrimination of normal and malignant human breast epithelial cells using a laminin-rich reconstituted basement membrane. In this assay, normal epithelial cells differentiate into well-organized acinar structures whereas tumor cells fail to recapitulate this process and produce large, disordered colonies. The data suggest that breast acinar morphogenesis and differentiation is regulated by cell-extracellular matrix (ECM) interactions and that these interactions are altered in malignancy. Here, we investigated the role of ECM receptors (integrins) in these processes and report on the expression and function of potential laminin receptors in normal and tumorigenic breast epithelial cells. Immmunocytochemical analysis showed that normal and carcinoma cells in a three-dimensional substratum express profiles of integrins similar to normal and malignant breast tissues in situ. Normal cells express α1, α2, α3, α6, β1 and β4 integrin subunits, whereas breast carcinoma cells show variable losses, disordered expression, or downregulation of these subunits. Function-blocking experiments using inhibitory anti-integrin subunit antibodies showed a >5-fold inhibition of the formation of acinar structures by normal cells in the presence of either anti-β1 or anti-α3 antibodies, whereas anti-α2 or -α6 had little or no effect. In experiments where collagen type I gels were used instead of basement membrane, acinar morphogenesis was blocked by anti-β1 and -α2 antibodies but not by anti-α3. These data suggest a specificity of integrin utilization dependent on the ECM ligands encountered by the cell. The interruption of normal acinar morphogenesis by anti-integrin antibodies was associated with an inhibition of cell growth and induction of apoptosis. Function-blocking antibodies had no inhibitory effect on the rate of tumor cell growth, survival or capacity to form colonies. Thus under our culture conditions breast acinar formation is at least a two-step process involving β1-integrin-dependent cellular growth followed by polarization of the cells into organized structures. The regulation of this pathway appears to be impaired or lost in the tumor cells, suggesting that tumor colony formation occurs by independent mechanisms and that loss of proper integrin-mediated cell-ECM interaction may be critical to breast tumor formation.
Possible continuity of subplasmalemmal cytoplasmic network with basement membrane cord network: ultrastructural study
Authors: Inoue Sadayuki
Published: April 2021

The ultrastructure of the subplasmalemmal cytoplasm of the cell and the associated basement membrane as well as the area of the cell-basement membrane border were observed with high resolution electron microscopy after preparation of the tissues with cryofixation or glutaraldehyde fixation followed by freeze substitution. The subplasmalemmal cytoplasm of the smooth muscle cells of rat epididymal tubules and the podocyte processes of the mouse glomerular visceral epithelium were found to be composed of a fine network of irregular anastomosing strands. This network closely resembled the previously characterized cord network of the basement membrane. The cords are known to be composed of a 1.5 to 3 nm thick core filament made up of type IV collagen which is surrounded by an irregular ‘sheath’ of other components. The strands in the subplasmalemmal network showed ultrastructural features similar to those of the cord network. Ribbon-like, 4.5 nm wide heparan sulfate proteoglycan ‘double tracks’ were previously reported to be associated with the cord network. Structures similar in size and appearance to the double tracks were also found in the subplasmalemmal network. At the cell-basement membrane border, the lamina densa of the basement membrane was in contact with the cell without the intervening space of a lamina lucida which was recently found to be an artefact caused by conventional tissue processing. Furthermore, the subplasmalemmal network appeared to be continuous, through the plasma membrane, with the cord network of the basement membrane. These results raise the possibility that the subplasmalemmal region of the cell and the associated basement membrane is occupied by a seemingly ultrastructurally unified network in spite of the fact that two such areas have been known to be composed of bichemically distinct components.
Role of the heparin binding domain of fibronectin in attachment and spreading of human bone-derived cells
Authors: Dalton B. Ann , McFarland Clive D. , Underwood P. Anne , Steele John G.
Published: April 2021

Human bone-derived cells are known to attach and spread on surfaces which have been precoated with fibronectin, but the contributions made by specific domains of the molecule have not yet been defined. Here we refer to the osteoblast-like cells as human bone cells. We have determined the relevance of separate regions of fibronectin, particularly the heparin-binding region, for the initial attachment and spreading of these cells.

Human bone cells attached to fragments from each of the cell- and heparin-binding regions of fibronectin, but failed to attach to a fragment from the gelatin-binding region. Bovine corneal epithelial cells, which were included as an example of an alternative primary cell strain, attached to the cell-binding fragment but showed no specific shortterm attachment to the heparin or gelatin-binding fragments. Monoclonal antibody MAb17, which binds to the cell binding region of fibronectin, partially inhibited the attachment of both human bone cells and corneal epithelial cells to intact fibronectin when present at 50 µg/ml and reduced human bone cell attachment to the cell-binding region fragment of fibronectin. Monoclonal antibody, MAb 32, which binds to the heparin-binding region of fibronectin, failed to inhibit attachment of the human bone cells to fibronectin but reduced the attachment of these cells to the heparin-binding region fragment. Heparin and chondroitin sulphate were able to inhibit human bone cell attachment to the heparin-binding fragment of fibronectin but had no effect on their attachment to intact fibronectin or the cell-binding region of fibronectin.

Immunofluorescent staining and confocal microscopy showed extensive spreading and actin filament formation when human bone cells were cultured on intact fibronectin. Cells cultured on the heparin-binding fragment showed only minimal spreading coinciding with less extensive actin filament organisation. On the cell-binding fragment of fibronectin more spreading was seen than on the heparin-binding fragment but it was not as extensive as on intact fibronectin.

Taken together, these data suggest that human bone cells, unlike bovine corneal epithelial cells, have an attachment mechanism for the heparin-binding region of fibronectin. Attachment to this region is probably mediated by cell surface proteoglycans. However, interaction with the cell-binding domain is required for effective cell spreading of human bone cells on fibronectin during the first 90 minutes after seeding into culture.
RNAs radiate from gene to cytoplasm as revealed by fluorescence in situ hybridization
Authors: Dirks Roeland W. , Daniël Kurt C. , Raap Anton K.
Published: April 2021

Genes for Epstein-Barr virus, human cytomegalovirus immediate early antigen and luciferase are abundantly transcribed in Namalwa, rat 9G and X1 cells, respectively. The EBV transcripts and HCMV-IE transcripts are extensively spliced, while in the luciferase transcript only a small intron sequence has to be spliced out. EBV transcripts are hardly localized in the cytoplasm while the luciferase and HCMV-IE transcripts are present in the cytoplasm and translated into proteins. We have correlated these characteristics with nuclear RNA distribution patterns as seen by fluorescence in situ hybridization. Transcripts of the HCMV-IE transcription unit were shown to be present in a main nuclear signal in the form of a track or elongated dot and as small nuclear RNA signals that radiate from this site towards the cytoplasm. A similar distribution pattern of small RNA signals was observed for transcripts of the luciferase gene, whereas the main nuclear signal was always observed as a dot and never as a track or elongated dot. In Namalwa cells, EBV transcripts were only present as track-like signals. The results suggest that when the extent for splicing is high, unspliced or partially spliced mRNAs begin to occupy elongated dot or track-like domains in the vicinity of the gene. When the extent of splicing is low, splicing is completed co-transcriptionally, leading to a bright dot-like signal. The presence of small nuclear spots in addition to the main signal correlates with cytoplasmic mRNA expression. The small spots most likely represent, therefore, mRNAs in transport to the cytoplasm.
Perlecan is a component of cartilage matrix and promotes chondrocyte attachment
Authors: SundarRaj Nirmala , Fite Dana , Ledbetter Steven , Chakravarti Shukti , Hassell John R.
Published: April 2021

Aggrecan, a chondroitin/keratan sulfate-containing pro teoglycan, is a major component of cartilaginous tissues. Immunolocalization studies, using antibodies directed to perlecan, a heparan sulfate proteoglycan first detected in basement membranes, and laminin (another major component of basement membranes), indicate that perlecan and laminin are also present in the matrices of hyaline cartilage in the nasal septum, the articular surface of the bone and the growth plate of the developing bone. Consequently, we used antibodies to both aggrecan and perlecan to characterize their synthesis and secretion by primary cultures of chondrocytes derived from the rat chondrosarcoma. Chondrocytes were pulsed for 20 minutes with [35S]methionine and then chased for up to six hours. The radiolabeled perlecan and aggrecan were immunoprecipitated and analyzed by SDS-PAGE. The results show that chondrocytes synthesize precursor proteins to both proteoglycans, but that only the aggrecan precursor protein is secreted as a proteoglycan. Perlecan was also secreted but with less posttranslational modifica tions than aggrecan. Northern blot analyses of the RNAs from immortalized rat chondrocytes indicated that the major mRNA encoding for perlecan was approximately 13 kb in length, similar in size to that expressed by other cell types, which synthesize 400 kDa core protein perlecan. Analyses of the proteoglycan fractions from the extracts of bovine articular surface indicated that perlecan in this tissue contains both chondroitin and heparan sulfate side chains. Purified perlecan and laminin were found to promote attachment of immortalized rat chondrocytes in vitro. These studies indicated that perlecan, once thought to be a unique component of the basement membranes, is more widely distributed and is an important component of the cartilage matrix, where it may provide for cell adhesion to the matrix.
Myogenic conversion of mammalian fibroblasts induced by differentiating muscle cells
Authors: Salvatori G. , Lattanzi L. , Coletta M. , Aguanno S. , Vivarelli E. , Kelly R. , Ferrari G. , Harris A. J. , Mavilio F. , Molinaro M. , Cossu G.
Published: April 2021

Somite-derived skeletal myoblasts are supposed to be the sole source of muscle fibre nuclei during pre- and postnatal development, but evidence is accumulating for unorthodox contributions to muscle fibre nuclei from other cell types. For example, in tissue culture, fibroblasts can fuse with dysgenic myoblasts and restore correct membrane function.

We report here the results of a series of experiments investigating this phenomenon and its possible mechanism. 10T1/2 cells, infected with a replication defective retrovirus encoding the bacterial enzyme β-galactosidase, fused to form β-galactosidase positive, differentiated myotubes when cocultured with differentiating uninfected C2C12 or primary myogenic cells, but this did not occur when they were cocultured with other cells such as 3T3 fibroblasts or PC12 pheochromocytoma cells. Myogenic conversion ranged from 1 to 10% of the 10T1/2 cell population and required close cell interaction between the different cells types: it was not induced by conditioned medium or extracellular matrix deposited by C2C12 cells. Myogenic conversion was also observed in vivo, after injection of similarly infected 10T1/2 cells into regenerating muscle. Conversion was seen also after coculture of uninfected 10T1/2 cells with primary chick myoblasts, thus demonstrating that it was not dependent upon viral infection and that there is no species or class barrier in this phenomenon. Primary fibroblasts, isolated from different organs of transgenic mice carrying a Lac Z marker under the control of a musclespecific promoter, restricting β-galactosidase expression to striated muscle cells, also underwent myogenic conversion, when cocultured with C2C12 myoblasts. The efficiency of this conversion varied with their embryological origin, being common in cells with a dorsal mesoderm lineage but rare in cells of ventral mesoderm origin. These experiments demonstrate that myogenic conversion is a true embryological feature of mammalian mesodermal cells. Conversion of mononucleated cells was also observed, showing that fusion is not a pre-requisite for myogenic differentiation and may indeed be a consequence of differentiation induced by short-range local signalling. We conclude that a proportion of adult cells of mesodermal origin may conserve a bi-or multi-potential state of determination throughout the life of an animal, enhancing the regenerative capacity of the tissues in which they reside.
A role for interleukin-1 in epidermal differentiation: Regulation by expression of functional versus decoy receptors
Authors: Eller Mark S. , Yaar Mina , Ostrom Kristin , Harkness Daniel D. , Gilchrest Barbara A.
Published: April 2021

Although human epidermis contains levels of interleukin-1 (IL-1) up to 100 times higher than other tissues, the role of this cytokine in epidermal biology is unknown. Here, we show that interleukin-1 regulates the expression of mRNAs for two proteins associated with the differentiated phenotype of human keratinocytes, cellular retinoic acidbinding protein type II (CRABP II) and small, proline rich protein 1 (SPRR1). The ability of IL-1 to induce these transcripts correlates directly with keratinocyte expression of the IL-1 receptor type I (IL-1 RI) during differentiation and inversely with the expression of the type II IL-1 receptor (IL-1 RII), shown in other cell types to be a non-functional, decoy receptor. Furthermore, addition to ker-atinocyte cultures of an IL-1 RI-blocking, but not an IL-1 RII-blocking, antibody reduces the level of CRABP II and SPRR1 mRNAs in these cells. These data suggest that epidermal IL-1 functions to promote keratinocyte differentiation and that a change in the IL-1 receptor profile of these cells initiates this IL-1 response through a relative enhanced expression of functional IL-1 receptors.
Guidance of oligodendrocytes and their progenitors by substratum topography
Authors: Webb Anna , Clark Peter , Skepper Jeremy , Compston Alastair , Wood Andrew
Published: April 2021

Oligodendrocyte progenitors arise in subventricular zones and migrate extensively during development before differentiating into mature oligodendrocytes, which myelinate nerve tracts in the central nervous system. We have used microfabricated substrata, containing periodic patterns of contours similar to those of central nervous system axons to assess the influence in vitro of substratum topography on oligodendrocytes isolated from 7 day rat optic nerve. Antiganglioside antibody A2B5 positive oligodendrocyte-type 2 astrocyte progenitors, and galactocerebroside positive and myelin basic protein positive oligodendrocytes, were highly aligned by surface contours as small as 100 nm depth and 260 nm repeat spacing. Rat optic nerve astrocytes also aligned on surface contours, but rat hippocampal and cerebellar neurons were unresponsive. Oligodendrocytes demonstrated enhanced parallel extension of their processes on narrow repeating topography in an arrangement similar to that found in the intact optic nerve. This is in marked contrast to the phenotype displayed by this cell type on planar substrata. Neither oligodendrocytes nor oligodendrocyte-type 2 astrocyte progenitors showed high-order Factin cytoskeletal networks; thus their alignment on gratings is unlikely to result from deformation of actin cables and focal contacts. In contrast, aligned astrocytes showed striking arrangements of actin stress fibres. These results establish glial cells as potentially the most topographically sensitive cell types within the central nervous system. Furthermore, the topographical pattern inducing maximal alignment of oligodendrocyte lineage cells corresponds to the diameters of single axons within the 7 day optic nerve. Thus the migration of oligodendrocyte-type 2 astrocyte progenitors and axonal ensheathment by oligodendrocytes may be guided by axonal topography within the developing nerve.
Microtubules released from the neuronal centrosome are transported into the axon
Authors: Ahmad Fridoon J. , Baas Peter W.
Published: April 2021

There is controversy concerning the source of new microtubules required for the development of neuronal axons. We have proposed that microtubules are released from the centrosome within the cell body of the neuron and are then translocated into the axon to support its growth. To investigate this possibility, we have developed an experimental regime that permits us to determine the fate of a small population of microtubules nucleated at the neuronal centro-some. Microtubules within cultured sympathetic neurons were depolymerized with the anti-microtubule drug noco-dazole, after which the drug was removed. Microtubules rapidly and specifically reassembled from the centrosome within three minutes of nocodazole removal. At this point, low levels of vinblastine, another anti-microtubule drug, were added to the culture to inhibit further microtubule assembly while not substantially depolymerizing the small population of microtubules that had already assembled at the centrosome. Within minutes, released microtubules were apparent in the cytoplasm, and many of these had already translocated to the cell periphery by ten minutes. By one hour, virtually all of the microtubules had been released from the centrosome and were concentrated at the cell periphery. With increasing time, these microtubules appeared within and progressively farther down developing axons. Nonneuronal cells within the culture also reassembled microtubules at the centrosome, but only a small portion of these microtubules were released. These observations indicate that microtubules are released from the neuronal centrosome and transported into growing axons, and that microtubule release and relocation from the centrosome are especially active in neurons compared to nonneuronal cells.
Galectin-3 expression and effects on cyst enlargement and tubulogenesis in kidney epithelial MDCK cells cultured in three-dimensional matrices in vitro
Authors: Bao Qi , Hughes R. Colin
Published: April 2021

Galectin-3 is a member of a closely related family of β-galactoside-binding soluble proteins found in many vertebrate epithelial and myeloid cell types. The developmentally regulated presence of galectin-3 in tissues, for example kidney, and an affinity for many cell-surface and matrix glycoproteins indicate its importance in extracellular biological processes. Since a polarised expression and secretion of galectin-3 was observed in monolayer-cultured MDCK cells, an understanding of the secretion and distribution of this lectin in a three-dimensional in vitro model would help to uncover its role(s) in the interplay between cell-surface adhesion molecules and extracellular matrix components occurring during cell aggregation and polarisation in tissue formation. In this study, the cellular distribution and secretion of galectin-3 were examined in MDCK cells cultured within a gel matrix. MDCK cells were cultured within type I collagen or Matrigel to obtain multicellular cysts, and tubule formation was induced in collagen gels with hepatocyte growth factor. Immunofluo-rescent staining of these structures using antibodies against galectin-3 and other cell-surface domain markers was carried out either in situ or on cryosections and was visu-alised by confocal and conventional epifluorescence microscopy. Our results show that MDCK cells suspended in hydrated collagen gels or Matrigel exhibit differential and polarised galectin-3 expression on the baso-lateral surface domains of cells lining the cysts. The lectin is colocalised with laminin on the basal surface. In tubuleforming cysts, galectin-3 is excluded from the initial spikes and the progressing tips of the tubules although its baso-lateral expression on the cyst body remains. Galectin-3 added exogenously to cultures, as well as antibodies against laminin subunits and integrin β1 subunit, exerted an inhibitory effect on cyst enlargement of MDCK cells in 3D Matrigel while galectin-3-specific antibodies could promote this process. The results suggest that galectin-3 exerts its effect on MDCK cells in a three-dimensional environment through modulation of both cell-cell and cell-substratum adhesions, and the interplay between these adhesions is important in the growth of multicellular aggregates and extensions occurring during normal kidney tubulogenesis.
Immunocytochemical localization of β-COP to the ER-Golgi boundary and the TGN
Authors: Griffiths Gareth , Pepperkok Rainer , Locker Jacomine Krijnse , Kreis Thomas E.
Published: April 2021

Recent data strongly suggest that the coatomer (COP) complex is involved in membrane transport between the ER and Golgi complex. This vesicular coat has been implicated in ER to Golgi, in intra Golgi as well as in Golgi to ER traffic. In this study we present a detailed immunocytochemical analysis of the distribution of β-COP in different tissue culture cells. Our results extend previous studies by showing, using electron microscopy, that β-COP accumulates on vesicular profiles and buds in the intermediate compartment (IC) under conditions that block ER to Golgi transport (15°C). Importantly, under these conditions β-COP co-localizes on these structures with a passenger protein, the membrane glycoprotein of vesicular stomatis virus (ts-O45-G). Furthermore, quantitative immunofluorescence microscopy of cells with ts-045-G accumulated in the ER, IC and trans-Golgi network, shifted briefly to the permissive temperature, showed that β-COP was associated with many of the putative transport intermediates containing the viral glycoprotein which is in transit between the ER/IC and the cis-Golgi. The simplest interpretation of these data is that COP-coated vesicles are involved in anterograde transport of ts-045-G from the IC to the Golgi complex. Since many putative COP vesicles lacked the G protein following release of the 15°C block this pool could be involved in retrograde transport. We also show that β-COP is present on the membranes of the transGolgi network. However, in contrast to the ER-Golgi boundary, we could find no convincing evidence that this pool of β-COP is associated with buds or trans-Golgi network-derived transport vesicles.
Inactivation of Cdc2 increases the level of apoptosis induced by DNA damage
Authors: Ongkeko Weg , Ferguson David J. P. , Harris Adrian L. , Norbury Chris
Published: April 2021

A number of lines of evidence have suggested a possible involvement of the mitosis-promoting protein kinase Cdc2 in the process of apoptotic cell death, and one recent study concluded that premature activation of Cdc2 is required for apoptosis. Here we have used a temperature-sensitive murine Cdc2 mutant cell line and Cdc2 inhibitor compounds to study the effect of inhibition of this protein kinase on apoptosis induced by DNA-damaging drugs. Inhibition of Cdc2 activity before or during exposure to DNA strand break-inducing drugs had the effect of increasing the level of subsequent apoptosis, as assessed by electron microscopy and flow cytometry. We conclude that, far from being required for cell death, a form of mammalian Cdc2 suppresses apoptosis induced by DNA damage. This form of Cdc2 appears to be active in G2-arrested cells and is therefore presumably distinct from the mitosis-promoting Cdc2-cyclin B heterodimer.
Identification of the nuclear localization signal of mouse DNA primase: nuclear transport of p46 subunit is facilitated by interaction with p54 subunit
Authors: Mizuno Takeshi , Okamoto Tomoko , Yokoi Masayuki , Izumi Masako , Kobayashi Akio , Hachiya Takahisa , Tamai Katsuyuki , Inoue Tadashi , Hanaoka Fumio
Published: April 2021

DNA polymerase α-primase is a replication enzyme necessary for DNA replication in all eukaryotes. Mouse DNA primase is composed of two subunits: a 46 kDa protein (p46), which is the catalytic subunit capable of RNA primer synthesis, and a 54 kDa protein (p54), whose physiological role is not clear. To understand the structure-function relationship of DNA primase, we set out to characterize these two subunits individually or in combination using a cDNA expression system in mammalian cultured cells, and determined the subcellular distribution of ectopically expressed DNA primase. The p54 expressed in COS-1 cells after transfection was predominantly localized in the nucleus, whereas p46 was retained in the cytoplasm as shown by indirect immunofluorescence analysis. Using several mutant proteins with deletions or substitutions as well as chimeric constructs, we identified the nuclear localization signal of p54 as RIRKKLR, encoded near the amino terminus (residues 6-12). Furthermore, co-expression of both p46 and p54 subunits markedly altered the subcellular distribution of p46; co-expressed p46 was transported into the nucleus as efficiently as p54. These results demonstrate that p54 has a nuclear localization signal and is able to be translocated into the nucleus independently of DNA polymerase α subunits. In contrast, p46 lacks a nuclear localization signal, and its nuclear translocation is facilitated by interaction with p54. We present here first evidence for a novel role of p54 in the nuclear translocation process, and a piggy-back binding transport mechanism of mouse DNA primase.