Inhibition of in vitro RNA synthesis by hycanthone,oxamniquine and praziquantel

Summary The schistosomicides, hycanthone, oxamniquine and praziquantel, were found to inhibit the in vitro RNA synthesis using isolated hamster liver nuclei. Preincubation of the nuclei with these drugs revealed that the inhibitory effect of oxamniquine was irreversible and progressed with time, whereas that of hycanthone and parziquantel was reversible. On the other hand, hycanthone and praziquantel have a high affinity for DNA but oxamniquine does not. The data indicate that the mechanism of inhibition by oxamniquine is different from that of hycanthone and praziquantel.     

The localization of mutarotase in rat kidney.

The localization of mutarotase in rat kidney was investigated by fluorescein-labelled and peroxidase-labelled antibody techniques, and by method of isolation of the nuclei and cytoplasm in non-aqueous solvents. In these immunohistochemical studies, mutarotase was almost exclusively recognized in the nuclei of epithelial cells of renal tubules and glomeruli in rat. The specific activity of mutarotase was found to be 1.5 times higher in the nuclei (122 units/g dry wt) than that in the cytoplasm (80 units/g dry wt) isolated with non-aqueous solvents. These results suggest that mutarotase may be involved in the metabolism of D-glucose in nuclei.     

The localization of mutarotase in rat kidney

Summary The localization of mutarotase in rat kidney was investigated by fluorescein-labelled and peroxidase-labelled antibody techniques, and by method of isolation of the nuclei and cytoplasm in non-aqueous solvents. In these immunohistochemical studies, mutarotase was almost exclusively recognized in the nuclei of epithelial cells of renal tubules and glomeruli in rat. The specific activity of mutarotase was found to be 1.5 times higher in the nuclei (122 units/g dry wt) than that in the cytoplasm (80 units/g dry wt) isolated with non-aqueous solvents. These results suggest that mutarotase may be involved in the metabolism of D-glucose in nuclei.Acknowledgment. The authors thank Prof. Y. Nishizuka (Department of Biochemistry, School of Medicine, Kobe University) for his valuable advice.     

Chromatosomes are not produced from Tetrahymena chromatin by micrococcal nuclease digestion

The subnucleosomal organization of Tetrahymena chromatin, which has an unusual H1 histone, was investigated by NaCl extraction and micrococcal nuclease digestion of nuclei. It was found that Tetrahymena histone H1 is extracted with 0.35 M NaCl, whereas bovine thyroid H1 is not. Micrococcal nuclease digestion of Tetrahymena nuclei did not yield chromatosomes as a stable intermediate, whereas digestion of bovine thyroid nuclei did.     

Localization and analysis of nonpolar regions in onconase

A detailed analysis of the composition and properties of hydrophobic nuclei and microclusters has been carried out for onconase. Two main hydrophobic nuclei in the onconase structure were detected. Their composition and shape were found to be very similar to those of RNase A, in accordance with the predictions made. The nuclei in onconase are more compact, the side-chain atoms of residues included in the nuclei in onconase form more contacts with the environment than in RNase A. The hydrophobic nuclei should be considered as individual structural units along with elements of the secondary structure. Differences in composition and conformation of exposed loops between onconase and RNase A were found. The additional hydrophobic clusters attached to the nuclei in onconase might be involved in the fixation of an appropriate conformation of site(s) for manifestation of the biological activity of onconase. A comparison of amphibian representatives of the RNase A superfamily was also made. The results obtained suggest that the availability of nonpolar residues in established key positions of amino acid sequences determines the characteristic fold of homologous proteins and the structure of the active site cleft.     

The nature of DNA synthesis by isolated nuclei from cells of a rat tumour

Summary The DNA synthetic activity of nuclei isolated from a solid rat tumour was determined. The nuclei had DNA synthetic properties similar to nuclei from other sources but the time course of the reactions was different.     

Chromatosomes are not produced fromTetrahymena chromatin by micrococcal nuclease digestion

Summary The subnucleosomal organization ofTetrahymena chromatin, which has an unusual H1 histone, was investigated by NaCl extraction and micrococcal nuclease digestion of nuclei. It was found thatTetrahymena histone H1 is extracted with 0.35 M NaCl, whereas bovine thyroid H1 is not. Micrococcal nuclease digestion ofTetrahymena nuclei did not yield chromatosomes as a stable intermediate, whereas digestion of bovine thyroid nuclei did.     

Structural characteristics and distribution of satellite cells along crayfish muscle fibers.

The distribution of satellite cells (sc) in long-sarcomere muscle fibers from the carpopod extensor muscle of the crayfish (Astacus fluviatilis) has been studied electron-microscopically. The sc are spindle-shaped and are oriented parallel to the long axis of a fiber. The mean lengths of sc nuclei (17.00 microns) and that of myonuclei (18.35 microns) differ non-significantly. In older animals, the mean ratio of the number of sc nuclei to the total number of nuclei (sc nuclei + myonuclei) is 0.0716, 0.0848, and 0.034 for the tendon, central and shell segments, respectively. The corresponding values for younger animals are 0.158, 0.166, and 0.081. The mean numbers of sc nuclei per mm of a fiber are 94, 117, and 47 (older animals), and 164, 117, and 94 (younger animals) for the tendon, central and shell segments, respectively. The high incidence of sc per unit fiber length in crayfish may be related to the fact that crayfish muscle fibers have a much larger diameter than vertebrate muscle cells.     

Chromosomal territory segmentation in apoptotic cells

The nuclear architecture of selected chromosomes in apoptotic nuclei of human leukemic cells K-562 and HL-60 was investigated. Etoposide and prolonged confluence were used for the induction of apoptosis. DAPI as well as TUNEL labeling of apoptotic nuclear bodies was combined with visualization of chromosomal territories by the FISH technique. Simultaneous vital staining by annexin V, propidium iodide, and Hoechst 33342 was applied to distinguish apoptotic, necrotic, and intact cell fraction of tested populations. Our FISH analyses revealed that the three-dimensional (3D) structure of apoptotic nuclei as well as the 3D structure of apoptotic bodies is preserved in formaldehyde-fixed cells. High-molecular-weight DNA fragmentation was determined in apoptotic K-562 cells in contrast to oligonucleosomal cleavage observed in apoptotic HL-60 cells. In K-562 populations, chromosomal territories were located separately either in one apoptotic body or underwent disassembly into chromosomal segments dispersed into single and/or several apoptotic bodies. The apoptotic disorganization of chromosomal territories was irregular, leading mainly to chromosomal segments of different sizes and, consequently, chromosomal disassembly was not observed at specific sites. In comparison with the control, an increased number of centromeric FISH signals were observed in prolonged confluence-treated K-562 cells induced to apoptosis. This finding can be explained either as a consequence of apoptosis or by polyploidization. Sequential staining of the same apoptotic nuclei by the FISH and TUNEL techniques revealed that chromosomal territory segmentation precedes the formation of nuclear apoptotic bodies.     

Demonstration of "hydroxylase" and "epoxide hydrase" activities in preparations of nucleoli isolated from rat liver]

Aryl hydrocarbon hydroxylase (AHH) activity and epoxyde hydrase (EH) activity have been found in Rat liver nucleoli obtained from untreated (C) and methylcholanthrene (MC) pretreated Rats. Electron microscopic observations of nucleolar preparations did not reveal significant contamination either by intact nuclei or by nuclear membranes. Very low but detectable activity of NADPH cytochrome C reductase was found in the nucleoli. Nucleolar preparations revealed little AHH activity (12-18 pmoles/min/mg). AHH was inducible by MC in nuclei but not in nucleoli. The presence of EH in nucleoli was demonstrated with phenanthrene 9,10-oxide (550-620 pmoles/min/mg) and benzopyrene 4,5-oxide (92-116 pmoles/min/mg). These values were lower than those obtained using intact nuclei. The addition of TCPO (10(-4) M) inhibited EH activity.     

Similarity between the effects of suprachiasmatic nuclei lesions and of pinealectomy on gonadotropin release in ovariectomized,sulpiride-treated and melatonin-replaced rats

The aim of this study was to compare the effects of pineal indole treatments on LH and FSH release in pinealectomized and suprachiasmatic lesioned and ovariectomized rats rendered hyperprolactinemic by acute sulpiride treatment. pinealectomy or suprachiasmatic nuclei lesions in female rats both decreased plasma LH and FHS at 10, but not at 20 d after surgery, whereas the daily afternoon administration of melatonin effectively restored levels of both gonadotropins to control values. In ovariectomized rats, pinealectomy or suprachiasmatic nuclei lesions were ineffective in counteracting the high plasma levels of LH and FSH. However, sulpiride treatment in both pinealectomized and suprachiasmatic nuclei lesioned and castrated female rats significantly decreased the levels of LH and FSH, an effect which was counteracted by daily afternoon melatonin administration. Other pineal indoles tested, i.e., 5-hydroxy- and 5-methoxytryptophol, were ineffective in regulating gonadotropin levels. The results suggest that the pineal gland, through its hormone melatonin, can modulate gonadotropin secretion by acting on a dopamine mechanism independent of hypothalamic suprachiasmatic areas.     

Bestimmung von Glykolyse-Metaboliten in isolierten Zellkernen

Summary Isolated rat liver nuclei have been analyzed for the intranuclear concentration of 8 glycolytic metabolites, and in addition for the 3 adenine nucleotides, and for malate. All these substrates are to be found in nuclei in a range of concentration comparable to that in the whole tissue. The consequences of these findings with respect to the definite establishment of glycolysis in isolated nuclei, as well as for the intranuclear formation of energy-rich phosphate bonds, are discussed.

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Meinem Lehrer, Prof. Dr. Dr.K. Lang, in aller Verehrung und Dankbarkeit zum 60. Geburtstag gewidmet.     

L'absorption du facteur «L.E.» par des noyaux cellulaires isolés

Summary The serum containing the L.E. factor loses its activity after having been put in contact with isolated cell nuclei. The electrophoretic examination shows a clear diminution of the gamma globulin and a slight diminution of the globulin alpha 2. That the factor responsible for the L.E. phenomenon can be absorbed by isolated cell nuclei is an argument in favor of the hypothesis that this factor is a substance having the character of an antinuclear anti-body, without one being able to exclude with certitude the possibility that this factor is an enzyme.     

Effects of lesions of the anterior raphe nuclei on the self-administration of d-amphetamine in the rat : considerable increase of desire for the poison

The acquisition of the intravenous self-administration of d-amphetamine was studied after separate lesions of anterior raphe nuclei (dorsal or median). Every lever-press delivered 2.5 microliter of a d-amphetamine solution dosed at 7.5 microgram/kg. Lesion of anterior raphe nuclei produced an hyper-sensitivity to d-amphetamine as indicated by a dramatic increase in self-administration by experimental Rats compared to the controls. Moreover this enhanced self-administration behavior is observed with a low blood concentration of d-amphetamine. The greater increase was obtained for median raphe lesioned Rats. These effects are interpreted in terms of 5HT-DA balance and could provide an experimental model for neurobiological bases of drug addiction studies.     

Arrest of somatic cells at G2 by Sevin (pesticide)

Summary The treatment of onion root meristems with the pesticide Sevin [carbaryl (1-naphthyl n-methyl carbamate)] induces an accumulation of interphase nuclei with a larger diameter, and a subsequent decrease in the index of smaller nuclei. It is concluded that Sevin depresses mitosis by arresting cells at G₂ without affecting DNA synthesis.The authors are grateful to Prof. K. S. Bilgrami, Head of the Postgraduate Department of Botany, Bhagalpur University, for providing facilities and to Prof. B.N. Mookerjee for his valued suggestions.     

Large scale preparation of calf liver nuclei by continuous flow centrifugation

Summary Large scale purification and preparation of calf liver nuclei was accomplished by high speed centrifugation of a fraction enriched in nuclei (nuclear homogenate) through 1.8 M sucrose by means of a Beckman CF-32 Ti continuous flow rotor. In comparison with methods involving the use of conventional high capacity rotors, larger volumes of homogenate could be processed. This method was used to prepare nuclei from calf liver for the preparation of DNA-dependent RNA polymerases. The use of continuous flow ultracentrifugation avoids time-consuming manipulations, thus allowing handling of large quantities of tissue.Acknowledgments. The authors wish to thank Professor J. Brachet for his interest and fruitful discussion. They also thank Ms J. Gilder for secretarial assistance.     

High salt effect on RNA synthesis in Krebs ascites tumor cells

The incubation of Krebs ascites tumor cells in medium with a high salt concentration resulted in a partial inhibition of nuclear RNA synthesis. The residual RNA polymerase activity in such nuclei was only slightly inhibited by low concentrations (50 nM) of alpha-amanitin. This finding suggested an inhibition of RNA polymerase II activity under conditions of medium hypertonicity. Indeed, RNA polymerase II, isolated from the nuclei of cells exposed to hypertonicity, revealed only half of the control activity. On the other hand, RNA polymerase I was not affected by hypertonicity. Moreover, chromatin fractions isolated from cells incubated in hypertonic or isotonic medium were equally template-active in RNA synthesis.     

Structural characteristics and distribution of satellite cells along crayfish muscle fibers

The distribution of satellite cells (sc) in long-sarcomere muscle fibers from the carpopod extensor muscle of the crayfish (Astacus fluviatilis) has been studied electron-microscopically. The sc are spindle-shaped and are oriented parallel to the long axis of a fiber. The mean lengths of sc nuclei (17.00 m) and that of myonuclei (18.35 m) differ non-significantly. In older animals, the mean ratio of the number of sc nuclei to the total number of nuclei (sc nuclei + myonrclei) is 0.0716, 0.0848, and 0.034 for the tendon, central and shell segments, respectively. The corresponding values for younger animals are 0.158, 0.166, and 0.081. The mean numbers of sc nuclei per mm of a fiber are 94, 117, and 47 (older animals), and 164, 117, and 94 (younger animals) for the tendon, central and shell segments, respectively. The high incidence of sc per unit fiber length in crayfish may be related to the fact that crayfish muscle fibers have a much larger diameter than vertebrate muscle cells.     

Intranuclear crystalloids in arcuate nucleus neurons after clomiphene citrate administration

The action of high doses of clomiphene citrate on the nuclei of hypothalamic arcuate neurons of male cats has been studied. Clomiphene produces an accumulation of typical crystalloid material in the nuclei. After administration of a protein-synthesis inhibitor, no such material was observed in clomiphene-treated animals. These ultrastructural features could possibly be due to a more intense protein synthesis in the hypothalamic arcuate neurons.