Comparison of biochemical properties of DNA-topoisomerase I from normal and regenerating liver.

Biochemical properties of topoisomerase I from normal and regenerating rat liver were analysed using crude or fractionated nuclear extracts. We could not detect significative change in topoisomerase I content or activity (magnesium stimulation and inhibition by ATP) during the course of liver regeneration. Topoisomerase I can be resolved into two species of 97 kDa and 100 kDa, with the same pI of 8.2-8.6 as shown by two dimensional gel electrophoresis. The two polypeptides contained a non-phosphorylated precursor and others forms with variable degrees of phosphorylation. In-vitro dephosphorylation with alkaline phosphatase leads to the disappearance of the phosphorylated forms and inactivation of the enzyme. The affinity of topoisomerase I for chromatin (measured by salt elution) differs markedly between normal and regenerating liver: nearly 50% of topoisomerase I remained bound to the chromatin from normal liver at 250 mM NaCl whereas it was completely eluted from 24-h-regenerating-liver nuclei. The biological significance of these results is discussed.     

Nucleosomes from normal and regenerating rat liver

Micrococcal-nuclease digestion of rat liver nuclei selectively released mononucleosomes associated with ADP-ribosylated [Caplan, Ord & Stocken (1978) Biochem. J.174, 475-483] histone H1. Two classes of mononucleosome were detected, those that leaked out during digestion and those that were subsequently released by 5mm-sodium phosphate buffer (pH6.8)/0.2mm-NaEDTA. The former, from which histone H1 had been dissociated, contained 140-base-pair-length DNA and core histones;the latter contained core particles and mononucleosomes with histone H1 and 200-base-pair-length DNA. When normal liver nuclei were phosphorylated with [gamma-(32)P]ATP, dissociated histone H1, which could be separated from core particles with Sephadex G-200, showed (32)P uptake. (32)P uptake into histones H2A and poly(ADP-ribosyl)ated H3 was appreciable in core particles, but was less evident in nucleosomes still containing histone H1. When [(3)H]-thymidine was given to partially hepatectomized rats in S-phase, 5-10min pulses in animals of over 300g body wt. showed the presence of high-specific-radioactivity DNA in released core particles and mononucleosomes compared with DNA retained in the nuclear pellets. Mononucleosomes from rat livers in S-phase with new, [(3)H]lysine-containing histones, had higher (32)P incorporation in histones H1 and their core histones, than for di- or tri-nucleosomes. Thermal-denaturation properties of control and phosphorylated mononucleosomes and core particles were very similar; removal of histone H1 and non-histone chromosomal proteins in 0.5m-NaCl markedly increased the proportion of DNA ;melting' below 70 degrees C.     

Cilia in normal and regenerating islet tissue

Summary Ciliated cells occasionally occur in pancreatic ductule cells and islet -cells of normal Chinese hamsters. In the regenerating pancreatic parenchyma of alloxan-treated Chinese hamsters an increased amount of cilia is observed in the ductule cells and islet -cells. No obvious cilia were found in the other pancreatic cell types of normal and alloxan-treated animals. One and the same ductule cell possesses one, two, or rather often many cilia protruding into the ductule lumen. In the islet -cells there are one or two cilia that often extend into intercellular spaces. The fibre arrangement varies in different parts of the cilia. The basic fibre pattern seems to be 9 + 2, the 9 peripheral fibres consisting of 2 subfibres, and the 2 central being single. The basal bodies (centrioles) consist of 9 groups of 2 or 3 aligned tubular elements. Filaments are associated with the centrioles. The functional significance of the cilia is discussed.This work was supported by grants from the Swedish Medical Research Council (Projects No. K67-12X-718-02 and K68-12X-718-03) and the Medical Faculty, University of Umeå.     

Hemidesmosomes of normal and regenerating mouse corneal epithelium

Hemidesmosomes of normal mouse corneal epithelium observed in tangential thin sections, occupy 14% of the basal plasma membrane. They consist of linear chains of densities with an orientation that is not random with respect to the radial axis of the cornea, tending to parallel it. During the repair of a small epithelial defect, cells of the corneal epithelium peripheral to the defect show chains of hemidesmosomes arranged parallel to the direction of migration of the epithelial sheet. This is parallel to the radius, like the orientation of the normal chains. Cells of the area that was denuded of epithelium, and is being resurfaced, show no hemidesmosomes. During repair of a large defect of the corneal epithelium hemidesmosomes are present on the cells covering the denuded area but they are small, few in number compared to the normal, and many are not arranged in chains. These small hemidesmosomes appear to be points of attachment of very fine basal filaments, possibly actin.     

Morphometry of normal, regenerating and cancerous hepatocytes

During the morphometric analysis of liver cell carcinomas arising in cirrhotic livers, the sizes and shapes of 2200 cancerous and 1800 regenerative hepatocytes were measured and compared to normal hepatocytes. The neoplastic population showed significantly higher polymorphism, nucleocytoplasmic ratio and the percentage of multinucleated cells, whereas the sizes of cancerous cells were the smallest. Values for regenerative cells were mainly between those of neoplastic and normal cells. The exception was constituted by the group of very large regenerative cells which met the criteria of large liver cell dysplasia (LLCD). Low nucleocytoplasmic ratio achieved by these cells is consistent with the hypothesis of regenerative character of LLCD.     

Studies on deoxyribonucleoproteins. IX. Subfractions of deoxyribonucleoproteins from rat and mouse liver.

By carrier-free continuous electrophoresis, deoxyribonucleoprotein from rat and mouse liver could be separated into two subfractions. The more anodic fraction (DNP I), comprising 5 - 8 per cent of the total, contains fewer proteins (two types of histones only). [3H]Cyclophosphamide caused in vivo a 2.5 times higher alkylation of the DNA in in DNP I than of the DNA in DNP II. These and additional results led to the suggestion of a structural model with DNP I as a spacer in the deoxyribonucleoprotein fiber.