We’ve got obtained a polyclonal antibody that acknowledges a serious polypeptide element of hen mitotic chromosome scaffolds. This polypeptide migrates in SDS PAGE with Mr 170,000. Oblique immunofluorescence and subcellular fractionation experiments affirm that it’s current in each mitotic chromosomes and interphase nuclei. Two strains of proof counsel that this protein is DNA topoisomerase II, an plentiful nuclear enzyme that controls DNA topological states: anti-scaffold antibody inhibits the strand-passing exercise of DNA topoisomerase II; and each anti-scaffold antibody and an impartial antibody raised in opposition to purified bovine topoisomerase II acknowledge equivalent partial proteolysis fragments of the 170,000-mol-wt scaffold protein in immunoblots. Our outcomes counsel that topoisomerase II could also be an enzyme that can also be a structural protein of interphase nuclei and mitotic chromosomes.
Monoclonal and polyclonal L1 antibodies react by oblique immunofluorescence with the cell floor of cultured tetanus toxin-positive neurons from post-natal cerebella of mice, however not with glial fibrillary acidic protein-positive astrocytes, O4 antigen-positive oligodendrocytes or fibronectin-positive fibroblasts or fibroblast-like cells. Throughout cerebellar improvement L1 antigen is detectable on tetanus toxin-positive cells as early as embryonic day 13 after three days in tradition. In sections of the early post-natal cerebellum, L1 antigen is discovered on pre-migratory neurons within the inside, however not within the exterior a part of the exterior granular layer. Within the grownup cerebellum, L1 antigen is predominantly localized within the molecular layer and round Purkinje cells. Fibers in white matter and the granular layer are additionally L1 antigen-positive.
Granule cell our bodies and synaptic glomeruli are weakly antigen-positive. A number of cell strains derived from neuroblastoma C1300 additionally specific L1 antigen. The antigen will not be detectable by enzyme-linked immunosorbent assay in tissue homogenates of liver, kidney, lung, coronary heart, sperm or thymus. With polyclonal L1 antibodies, cross-reactive determinants are present in brains of rat, guinea pig, hamster, hen, rabbit and man, however not in frog, whereas monoclonal antibody reacts detectably solely with mouse mind. The molecular species acknowledged by each monoclonal and polyclonal antibodies show two outstanding bands by SDS-PAGE beneath lowering and non-reducing situations with obvious mol. wts. of 140 and 200 kd.
L1 antigen remoted from cultured cerebellar cells consists primarily of a band within the 200-kd vary and a faint one at 140 kd. L1 antigen from neuroblastoma N2A reveals two bands with barely increased obvious mol. wts. All molecular types of L1 antigen might be labeled by [3H]fucose and [3H]glucosamine. Ca2+-independent re-aggregation of cerebellar cells from early post-natal C57BL/6J mice and of the continual cell line N2A derived from the murine neuroblastoma C1300 is inhibited by Fab fragments of the polyclonal, however not of monoclonal antibody, each of that are recognized to react with the floor membrane of those cells.
Samples of lung, liver, kidney, pancreas, spleen, and lymph node from pigs with postweaning multisystemic losing syndrome from California (USA) and samples of mesenteric lymph nodes from equally diseased pigs from Brittany (France) have been examined by gentle microscopy, in situ hybridization (ISH), and/or virus isolation. Entire genomic probes for porcine circovirus (PCV) and hen anemia virus (CAV) have been used for ISH. Tissue homogenate supernatants have been inoculated onto PK/15 cells for virus isolation, and the presence of viral antigen and viral particles was verified by oblique immunofluorescence, ISH, and electron microscopy.
Histologic examination of lung from pigs from California revealed interstitial pneumonia, alveolar epithelial hyperplasia, and basophilic nuclear and cytoplasmic inclusions in mononuclear cell infiltrates and numerous pulmonary epithelial cells. Granulomatous lymphadenitis with syncytial cells typified the lesions seen within the pigs from France. PCV-like nucleic acid was detected by ISH in lung, pancreas, lymph node, kidney, and liver in pigs from California. Constructive sign was additionally obtained in lymph node sections from pigs from France. Probes for CAV have been constantly destructive.
PK/15 cell cultures inoculated with lung preparations from diseased California pigs and mesenteric lymph node preparations from pigs from France had constructive fluorescence by oblique staining for PCV utilizing pooled polyclonal pig sera and hyperimmune rabbit serum and had variable staining with a panel of seven monoclonal antibodies particular for cell tradition contaminant PCV. PCV-like nucleic acid was additionally detected by ISH in cell cultures. Cytopathic impact was not noticed. Electron microscopic examination of inoculated cell cultures revealed 17-nm viral particles morphologically in keeping with PCV.
No different virus particles have been noticed. Though genomic evaluation for the definitive identification of those viral isolates stays to be completed, the proof supplied strongly means that these tissue isolates are intently associated to, though antigenically distinct from, the unique PCV cell tradition contaminant. As well as, binding of antibodies to 140Ok, alpha-actinin, and fibronectin was excluded from vinculin-rich focal adhesion websites on the mobile periphery.
The attainable position of a 140Ok membrane-associated protein complicated (140Ok) in fibronectin-cytoskeleton associations has been examined. The 140Ok was recognized by the monoclonal antibody JG22E. Monoclonal and polyclonal antibodies to the 140Ok confirmed equivalent patterns of binding to the cell membranes of fastened and permeabilized hen embryonic fibroblasts; localization was diffuse, however with marked focus in cell-to-extracellular matrix contact websites.
Correlative localization with interference reflection microscopy and double-label or triple-label immunofluorescence confirmed that 140Ok co-distributed with extracellular fibronectin fibrils and intracellular alpha-actinin in microfilament bundles at extracellular matrix contact websites however tended to not co-localize with tropomyosin current in bundles at websites farther from adhesion websites. A progressive improvement of cell floor alpha-actinin-140Ok-fibronectin associations was noticed in early spreading cells. The anti-140Ok monoclonal antibody JG22E inhibited the attachment and spreading of each regular and Rous sarcoma virus-transformed hen embryonic fibroblasts to a fibronectin substratum.
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Nevertheless, the anti-140Ok monoclonal antibody turned a constructive mediator of cell attachment and spreading if it was adsorbed or cross-linked to the substratum. Our outcomes present the primary description of a membrane-associated protein complicated that co-localizes with fibronectin and microfilament bundles, they usually counsel that the 140Ok complicated could also be a part of a cell floor linkage between fibronectin and the cytoskeleton.
