bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterogeneity and microtubule interaction of the CHO1 antigen, a mitosisspecific kinesin-like protein: Analysis of subdomains expressed in insect sf9 cells
Authors: Kuriyama Ryoko , Dragas-Granoic Sasa , Maekawa Takami , Vassilev Alexei , Khodjakov Alexey , Kobayashi Hitoshi 
Published: April 2021

ABSTRACT
The CHO1 antigen is a mitosis-specific kinesin-like motor located at the interzonal region of the spindle. The human cDNA coding for the antigen contains a domain with sequence similarity to the motor domain of kinesin-like protein (Nislow et al., Nature 359, 543, 1992). Here we cloned cDNAs encoding the CHO1 antigen by immunoscreening of a CHO Uni-Zap expression library, the same species in which the original monoclonal antibody was raised. cDNAs of CHO cells encode a 953 amino acid polypeptide with a calculated molecular mass of 109 kDa. The N-terminal 73% of the antigen was 87% identical to the human clone, whereas the remaining 27% of the coding region showed only 48% homology. Insect Sf9 cells infected with baculovirus containing the full-length insert produced 105 and 95 kDa polypeptides, the same doublet identified as the original antigen in CHO cells. Truncated polypeptides corresponding to the N-terminal motor and Cterminal tail produced a 56 and 54 kDa polypeptide in Sf9 cells, respectively. Full and N-terminal proteins co-sedimented with, and caused bundling of, brain microtubules in vitro, whereas the C-terminal polypeptide did not. Cells expressing the N terminus formed one or more cytoplasmic processes. Immunofluorescence as well as electron microscopic observations revealed the presence of thick bundles of microtubules, which were closely packed, forming a marginal ring just beneath the cell membrane and a core in the processes. The diffusion coefficient and sedimentation coefficient were determined for the native CHO1 antigen by gel filtration and sucrose density gradient centrifugation, respectively. The native molecular mass of overinduced protein in Sf9 cells was calculated as 219 kDa, suggesting that the antigen exists as a dimer. Intrinsic CHO1 antigen in cultured mammalian cells forms a larger native complex (native molecular mass, 362 kDa), which may suggest the presence of additional molecule(s) associating with the CHO1 motor molecule.
A maternal product of the Punch locus of Drosophila melanogaster is required for precellular blastoderm nuclear divisions
Authors: Chen Xiongying , Reynolds Elaine R. , Ranganayakulu Gogineni , O’Donnell Janis M. 
Published: April 2021

ABSTRACT
The Punch locus of Drosophila melanogaster encodes the pteridine biosynthesis enzyme guanosine triphosphate cyclohydrolase. One class of Punch mutants is defective for a maternal function that results in embryonic death. We demonstrate here that the embryos exhibit nuclear division defects during the precellular blastoderm stage of development. These defects include abnormal nuclear distribution, mitotic asynchrony, and persisting chromatin bridges. Daughter nuclei that do not complete chromosome separation nevertheless initiate new interphase and mitotic cycles. As a result, interconnected mitotic figures are observed. Mitotic spindles and nuclear envelopes appear essentially normal. A mutant phenocopy was induced in wild-type embryos by treatment with the guanosine triphosphate cyclohydrolase inhibitor, 2,4-diamino-6-hydroxypyrimidine, at a very early cleavage stage. Furthermore, an inhibitor of a terminal step in pteridine biosynthesis produced an identical phenotype. Immunolocalization experiments define expression of Punch protein in nurse cells during oogenesis. The protein is packaged into granules as it is transported into the oocyte cytoplasm. As syncytial blastoderm nuclear divisions proceed, Punch protein levels decrease and disappear by cellularization. Defects in the expression of the protein in Punch maternal effect mutants correlate well with the early phenotypes. These results show that a Punch product is directly involved in early nuclear divisions and suggest a possible role in chromosome separation.
Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle
Authors: Pasion Sally G. , Brown Grant W. , Brown Lisa M. , Ray Dan S. 
Published: April 2021

ABSTRACT
In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.
Multiple unconventional myosin domains of the intestinal brush border cytoskeleton
Authors: Heintzelman Matthew B. , Hasson Tama , Mooseker Mark S. 
Published: April 2021

ABSTRACT
Representatives of class V and class VI unconventional myosins are identified as components of the intestinal brush border cytoskeleton. With brush border myosin-I and myosin-II, this brings to four the number of myosin classes associated with this one subcellular domain and represents the first characterization of four classes of myosins expressed in a single metazoan cell type. The distribution and cytoskeletal association of each myosin is distinct as assessed by both biochemical fractionation and immunofluorescence localization. Myosin-VI exists in both the microvillus and terminal web although the terminal web is the predominant site of concentration. Myosin-V is present in the terminal web and, most notably, at the distal ends of the microvilli, thus becoming the first actin-binding protein to be localized to this domain as assessed by both immunohistochemical and biochemical methods. In the undifferentiated enterocytes of the intestinal crypts, myosin-VI is expressed but not yet localized to the brush border, in contrast to myosin-V, which does demonstrate an apical distribution in these cells. An assessment of myosin abundance indicates that while myosin-II is the most abundant in the cell and in the brush border, brush border myosin-I is only slightly less abundant in contrast to myosins-V and -VI, both of which are two orders of magnitude less abundant than the others. Extraction studies indicate that of these four myosins, myosin-V is the most tightly associated with the brush border membrane, as detergent, in addition to ATP, is required for efficient solubilization.
Branching morphogenesis of human mammary epithelial cells in collagen gels
Authors: Berdichevsky Feodor , Alford Deborah , D’Souza Brendan , Taylor-Papadimitriou Joyce 
Published: April 2021

ABSTRACT
To study the morphogenesis of human epithelial cells in vitro we have used a three-dimensional collagen matrix and a newly developed mammary epithelial cell line, 1-7 HB2. In standard medium 1-7 HB2 cells formed compact balls/spheres inside collagen type I gels, while cocultivation with various fibroblast cell lines or growth in fibroblastconditioned media resulted in the appearance of branching structures. At least two different soluble factors secreted by fibroblasts were found to be implicated in the branching morphogenesis. Firstly, hepatocyte growth factor/scatter factor could induce branching in a concentrationdependent manner. Moreover, a polyclonal serum against hepatocyte growth factor/scatter factor completely inhibited the branching morphogenesis induced by medium conditioned by MRC-5 fibroblast cells. In contrast, a morphogenetic activity secreted by human foreskin fibroblasts was identified that appears to be different from hepatocyte growth factor/scatter factor and from a number of other well-characterized growth factors or cytokines. This model system has been used to examine the role of integrins in mammary morphogenesis. The expression of the α2β1, α3β1 and α6β4 integrins was decreased when cells were plated on collagen gels. The addition of specific blocking monoclonal antibodies directed to the α2and β1-integrin subunits to growth media impaired cell-cell interactions and interfered with the formation of compact structures inside collagen gels, suggesting that the α2β1 integrin can control intercellular adhesion in mammary morphogenesis. In contrast one of the blocking monoclonal antibodies against the α3-integrin subunit (P1B5) mimicked the effect of soluble ‘morphogens’. Our results suggest that the modulation of α3β1 activity may represent an important event in the induction of branching morphogenesis of human mammary epithelial cells.
E-cadherin mediates adherens junction organization through protein kinase C
Authors: Lewis Jani E. , Jensen Pamela J. , Johnson Keith R. , Wheelock Margaret J. 
Published: April 2021

ABSTRACT
Cultured human keratinocytes maintained in 30 μM Ca2+ do not form adherens junctions; however, when the extracellular Ca2+ concentration is raised to 1 mM, adherens junctions form very rapidly. The formation of a junction involves the coordinate organization of intracellular and extracellular components. Cadherins have been shown to mediate this coordinate organization. In this report we show that E-cadherin organizes the various junctional components by signalling through protein kinase C.
C/A dynein isolated from sea urchin sperm flagellar axonemes: Enzymatic properties and interaction with microtubules
Authors: Yokota Etsuo , Mabuchi Issei 
Published: April 2021

ABSTRACT
C/A dynein is a novel dynein isolated from sea urchin sperm flagellar axonemes. It is composed of C and A heavy chains and some additional lower molecular mass polypeptide chains. The characterization of ATPase activity and the interaction of this dynein with microtubules polymerized from calf brain tubulin were investigated in this study. The ATPase activity of C/A dynein (0.3-0.4 μmol Pi/min per mg) was about one half that of outer arm 21 S dynein (0.6-0.8 μmol Pi/min per mg) at 25°C. Vanadate inhibited the ATPase activity with a half-maximal inhibition at 1 μM. C/A dynein adsorbed to the glass surface was able to translocate the microtubules towards its plus end. The velocity of the microtubule movement in the presence of 1 mM ATP was 4.0 to 4.5 μm/s at 22°C. C/A dynein binds to and bundles the microtubules even in the presence of ATP. Cross-bridges were found between adjacent microtubules in the bundle with an axial periodicity of about 24 nm. The ATPase activity of C/A dynein was enhanced up to several-fold by the microtubules at concentration as low as 1 mg/ml. On the other hand, 21 S dynein bound to the micro-tubules with 24 nm axial periodicity only in the absence of ATP. Its ATPase activity was not activated by the micro-tubules. From these results, it is concluded that the manner of interaction with microtubules of C/A dynein is different from that of the outer arm dynein.
Developmental expression of mucin genes MUC1 and MUC2
Authors: Chambers Julie A. , Hollingsworth Michael A. , Trezise Ann E. O. , Harris Ann 
Published: April 2021

ABSTRACT
The mucin gene MUC1, is expressed in a number of human ductal epithelia in vivo including those within the pancreas, mammary gland, kidney and genital ducts. Further it is expressed at a high level in certain tumours and tumour-derived cell lines. MUC2 was initially isolated from a human jejunum cDNA library and is thought to be one of the major intestinal mucin genes, though it is also expressed in the trachea. We have examined the develop-mental expression of these two mucin genes in human tissues. High level expression of MUC1 has been seen by 12.5 weeks of gestation in the epithelia of the distal respiratory tract and the collecting ducts in the kidney. By 18 weeks MUC1 mRNA is detectable in the colon but pancreatic expression of MUC1 is not seen until late in gestation. MUC2 mRNA is seen by 12 weeks of gestation in the jejunum, ileum and colon, and in large bronchioles of the lung by 18 weeks. The pattern of expression of MUC1 suggests that this mucin may not be involved in early ductal obstruction in the CF pancreas, but both MUC1 and MUC2 may play a role in the development of intestinal disease and MUC1 in early respiratory disease associated with CF.
Analysis of HPV16 e6 and mutant p53-transfected keratinocytes in reconstituted epidermis suggests that wild-type p53 inhibits cytokeratin 19 expression
Authors: Molès Jean-Pierre , Schiller John T. , Tesniere Anne , Leigh Irene M. , Guilhou Jean-Jacques , Basset-Séguin Nicole 
Published: April 2021

ABSTRACT
Using a reconstituted skin culture model we have analysed the effects of oncogenic human papillomavirus (HPV) and mutant TP53 genes on the proliferation and differentiation of human keratinocytes. Immortal cell lines generated by transfection of early passage normal human keratinocytes with HPV16 E7 plus mutant human TP53 (KN #1), HPV16 E7/E6 (KN #2), or HPV16 E7 plus murine p53 (KN #3) were examined. KN #1 and KN #2 behaved identically, reconstructing a tumor-like epidermis characterized by the lack of differentiation and the presence of an aberrant epidermal architecture. In contrast, KN #3 reconstructed an epidermis that was more similar to that obtained with normal keratinocytes. KN #1 and KN #2 were further characterized by the inversion of the proliferative compartment and the abnormal expression of cytokeratin 19 (CK19). Because p53 function is reduced in these cells, either by heterocomplex formation between endogenous wild-type p53 and transfected mutant p53 or by E6-induced degradation of wild-type p53, we hypothesized that CK19 expression may be normally repressed by wild-type p53. This hypothesis was supported by the strict correlation observed between TP53 mutation and CK19 expression in a set of human skin tumors. CK19 was detected in all eight carcinomas containing a mutated TP53 gene but in none of the 16 carcinomas containing only wild-type TP53. These results illustrate the utility of the in vitro reconstituted skin model for investigating the consequences of genetic alterations in human keratinocytes.
Constitutive expression of mhc and adhesion molecules by alveolar epithelial cells (type ii pneumocytes) isolated from human lung and comparison with immunocytochemical findings
Authors: Cunningham Anne C. , Milne Debra S. , Wilkes Julie , Dark John H. , Tetley Teresa D. , Kirby John A. 
Published: April 2021

ABSTRACT
Highly purified populations of alveolar epithelial cells (type II pneumocytes) were isolated from human lung specimens. These cells were characterised histochemically, by demonstrating the presence of intracellular alkaline phosphatase, and morphologically, by electron microscopic demonstration of lamellar bodies and microvilli. Expression of the epithelial glycoprotein HEA-125, of MHC class I and class II (HLA-DR, -DP and -DQ) antigens and of the intercellular adhesion molecules ICAM-1, VCAM-1, LFA-3 and B7 was quantified by flow cytometry. Comparison was made between the expression of these molecules by isolated type II cells and by alveolar epithelium in normal human lung tissue after immunocytochemical staining of frozen sections of donor lung.
Isolated type II pneumocytes expressed HEA-125 and class I MHC molecules and the class II MHC molecules HLA-DR and -DP; HLA-DQ was not detected. The inter-cellular adhesion molecule ICAM-1 was expressed constitutively at low levels but there was minimal expression of VCAM-1, LFA-3 and B7.
It was not possible to differentiate type II cells from the predominant type I pneumocytes on frozen sections. Alveolar epithelium expressed HEA-125, class I MHC antigens, the class II molecules HLA-DR, and -DP and the intercellular adhesion molecule LFA-3. Expression of the adhesion molecules ICAM-1, VCAM-1 and B7 was variable. As with the isolates, HLA-DQ was not observed on alveolar epithelium.
In conclusion, a reproducible method for the isolation of pure populations of human type II pneumocytes has been developed. These cells were not damaged by the isolation procedure. It is not known whether alveolar epithelium can present antigens to T lymphocytes. However, the expression of potentially immunogenic class II MHC antigens and of intercellular adhesion molecules may facilitate pulmonary inflammation.
Cell cycle redistribution of U3 snRNA and fibrillarin: presence in the cytoplasmic nucleolus remnant and in the prenucleolar bodies at telophase
Authors: Azum-Gélade Marie-Claude , Noaillac-Depeyre Jacqueline , Caizergues-Ferrer Michèle , Gas Nicole 
Published: April 2021

ABSTRACT
The distribution of the U3 small nuclear RNA during the cell cycle of the CHO cell line was studied by in situ hybridization using digoxigenin-labelled oligonucleotide probes. The location of the hybrids by immunofluorescence microscopy and at the ultrastructural level was correlated with the distribution of two nucleolar proteins, nucleolin and fibrillarin. The U3 snRNA molecules persist through-out mitosis in close association with the nucleolar remnant. U3 snRNA is present in the prenucleolar bodies (PNBs) and could participate in nucleologenesis in association with several nucleolar proteins such as nucleolin and fibrillarin. The interaction of U3 snRNP with the 5′ external spacer of pre-RNA newly synthesized by active NORs is proposed to be the promoting event of nucleologenesis.
Induction of fibroblast 92 kDa gelatinase/type iv collagenase expression by direct contact with metastatic tumor cells
Authors: Himelstein Bruce P. , Canete-Soler Rafaela , Bernhard Eric J. , Muschel Ruth J. 
Published: April 2021

ABSTRACT
Previous studies have correlated release of the 92 kDa type IV collagenase/gelatinase by tumor cells in culture with metastatic potential. We have now demonstrated that the ability of tumor cells that do not express the 92 kDa gelatinase to induce release of this metalloproteinase from normal fibroblasts may also be associated with the metastatic phenotype.
A transformed rat embryo cell line, 2.8, failed to release the 92 kDa gelatinase alone in culture, but gave rise to metastatic tumors whose explants contained the 92 kDa gelatinase. In contrast, a non-metastatic transformed cell line, RA3, did not express the 92 kDa gelatinase alone in culture or in tumor explants. To explore the mechanisms that might govern host-tumor cell interactions in this system, we have studied the effects of co-culture of these transformed cell lines with rat embryo fibroblasts (REF) in culture. 92 kDa gelatinase expression was induced by coculture of 2.8 with REF, but co-culture of the non-metastatic line RA3 with REF did not result in induction of the 92 kDa gelatinase. The 92 kDa gelatinase in these co-cultures was released by the fibroblasts; methanol-fixed 2.8 cells induced 92 kDa gelatinase expression in REF, but fixed REF cells did not induce enzyme expression in 2.8 cells. This suggested that cell contact was required for induction, which was confirmed by showing that 92 kDa gelatinase induction in co-culture was abolished by separating REF from 2.8 by solute-permissive membranes. In addition, REF could not be stimulated to produce the 92 kDa gelatinase by 2.8-derived conditioned medium, by 2.8-derived extracellular matrix, or by isolated matrix components. These data indicate that metastatic tumor cells can induce 92 kDa gelatinase expression in fibroblasts through a mechanism dependent upon cell contact.
In situ hybridization of nude mouse tumors derived from these transformed cell lines revealed 92 kDa gelatinase expression in the stroma of tumors from 2.8, but not in tumors from RA3. Therefore, the experiments based on in vitro co-culture of tumor cells and fibroblasts, together with the in situ localization of mRNA to host cells, suggest that host production of the 92 kDa gelatinase may occur in response to direct contact with metastatic tumor cells.
Integrin expression and localization in normal MDCK cells and transformed MDCK cells lacking apical polarity
Authors: Schoenenberger Cora-Ann , Zuk Anna , Zinkl Gregory M. , Kendall Donna , Matlin Karl S. 
Published: April 2021

ABSTRACT
Epithelial cells polarize in response to contacts with the extracellular matrix and with neighboring cells. Interac-tions of cells with the extracellular matrix are mediated mainly by the integrin family of receptors. To begin to understand the role of integrins in polarization, we have investigated the expression and localization of three integrin families in the polarized Madin-Darby canine kidney (MDCK) epithelial cell line and in transformed MDCK cells lacking apical polarity. We find that MDCK cells express several β1 integrins, including α2β1, α3β1, and an unidentified integrin designated αxβ1. The β1 integrins are the major receptors for collagens I and IV and laminin in MDCK cells, since a blocking anti-β1 antibody almost totally abolishes adhesion to these proteins. They also express a vitronectin receptor tentatively identified as αvβ3, and the epithelial-specific integrin α6β4. The latter is not a laminin receptor in MDCK cells because a function blocking anti-α6 antibody has no effect on cell adhesion to laminin. All three integrin families are expressed exclu-sively on both the basal and lateral surfaces, as determined by immunofluorescence microscopy and surface biotinyla-tion. Transformed MDCK cells express β1 integrins as well as αvβ3 and α6β4, but show alterations in the β1 family. Expression of αx is lacking, and the relative amount of the β1 subunit is diminished, resulting in the accumulation of Endo-H-sensitive α3. In addition, surface biotinylation and immunofluorescence indicate that significant amounts of both α2β1 and α3β1 appear on not only the basolateral but also the apical plasma membrane. These results indicate that integrins are the major receptors for the extracellular matrix in MDCK cells, and that they may affect epithelial cell polarization by mediating not only cell-substratum but also cell-cell contacts.