Morphological changes in frog pronephric cell surfaces after transformation by herpes virus.

A scanning electron-microscope study has established that there are major modifications in the surface membranes of malignant cells transformed from the pronephric kidney of the frog. Tumours were induced in vivo after exposure of embryos Lucké Herpes virus. A pronephric cell line from normal embryos and 3 tumour cell lines derived from experimentally induced adenocarcinomas of the pronephros were observed. The normal embryonic kidney cells have their surfaces covered with long fingerlike microvilli throughout the cell cycle. In contrast, the surface of the pronephric tumour cells are mainly covered by multiple, ruffled or parallel straight microridges and short, stubby microvilli. These comparisons were made on cells subject to similar culture conditions. The topographic changes in the tumour cells are considered significant since the characteristic microridges and stubby villi were consistently found in all 3 pronephric tumour lines.     

Improved system for capillary microinjection into living cells

The effect of inhibition of protein synthesis on the synthesis and processing of low molecular weight RNA (LMW RNA) hs been studied on CHO-tsH1, a mutant cell line in which protein synthesis is rapidly inhibited at non-permissive temperature by inactivation of the enzyme leucyl-tRNA synthetase. The increase in temperature results in an increase in uridine uptake and in the specific activity of UTP pool which is probably not related to the mutation. We report in this paper that there is no significant alteration in the synthesis of LMW RNA (including 5S ribosomal RNA (rRNA) and tRNA) except for the inhibition of synthesis of nucleolar RNA species A. Since, in a previous paper, it has been shown that the processing of preribosomal nucleolar RNA does not proceed at 39.5 degrees C in CHO-tsH1 cells, these results are consistent with the hypothesis that nucleolar RNA species A is involved in the processing of rRNA depends on its synthesis and maturation.     

Effects of insulin on the fine structure of hepatocytes from winter-acclimatized carps: studies on protein synthesis

Male and female winter-acclimatized carps were injected with insulin. This treatment resulted in a sharp decrease in the liver glycogen content. Although an increase in the ribosomal RNA level was also observed, a cell-free system obtained from the hormone-treated fish exhibited less amino acid incorporation activity as compared to the control fish. However, polysomes from insulin-treated fish exhibited a higher amino acid incorporating activity when a soluble fraction of untreated winter carps was used. Insulin induced a profound change in the cytoarchitecture of the winter carp hepatocyte. The cytoplasm and nuclei showed all the features of the summer carp liver cell. The nucleolar components were totally intermingled suggesting a high rate of gene expression as in the case of the summer-acclimatized fish.     

Cytoplasmic microtubules of normal and tumor cells of the leopard frog. Temperature effects

The cytoplasmic microtubule complex (CMTC) was examined in monolayer cultures of normal tadpole mesonephros, primary renal adenocarcinoma, and an established cell line derived from a pronephric renal adenocarcinoma (PNKT-4B) of the leopard frog, Rana pipiens. Immunocytochemistry revealed typical arrays of microtubules extending from the cytocentrum to the cell periphery in all three cell types when cultured at 28 degrees C; similar results were obtained at 20 degrees C. However, the CMTC was disorganized in both tumor types, in contrast to the retention of a typical CMTC in normal tissue cultured at 7 degrees C. The response of PNKT-4B cells differed from that of normal tadpole mesonephros when treated with the microtubule inhibitor drug nocodazole. At 28 degrees C, PNKT-4B and tadpole mesonephros cells lost their CMTC with nocodazole treatment, and both were able to reconstitute CMTC when nocodazole was removed. Similarly, both lost CMTC organization with nocodazole and culture at 70 degrees C. However, while normal cells could effect a recovery at 7 degrees C after the removal of nocodazole, PONKT-4B cells were unable to restructure CMTC under the same conditions. Metastasis in the frog renal adenocarcinoma is temperature-dependent, with an elevated prevalence of metastasis in tumor-bearing frogs maintained at 28 degrees C. Few metastatic colonies are detected in tumor-bearing frogs maintained at a low temperature (7 degrees C). Other studies have indicated that microtubules, which are essential for cell motility, play an important role in the invasion by tumor cells of normal tissue fragments in vitro. The effects of temperature on metastasis of the Lucke renal adenocarcinoma are consistent with temperature-mediated changes in tumor-cell CMTC.     

Nucleolar localization of GLTSCR2/PICT-1 is mediated by multiple unique nucleolar localization sequences

The human glioma tumor suppressor candidate region 2 gene product, GLTSCR2, also called 'protein interacting with carboxyl terminus 1' (PICT-1), has been implicated in the regulation of two major tumor suppressor proteins, PTEN and p53, and reported to bind the membrane-cytoskeleton regulator of cell signaling, Merlin. PICT-1 is a nucleolar protein, conserved among eukaryotes, and its yeast homolog has been functionally associated with ribosomal RNA processing. By means of confocal microscopy of EGFP and myc-tagged PICT-1 fusion proteins, we delineate that the nucleolar localization of PICT-1 is mediated by two independent nucleolar localization sequences (NoLS). Unlike most NoLSs, these NoLSs are relatively long with flexible boundaries and contain arginine and leucine clusters. In addition, we show that PICT-1 exhibits a nucleolar distribution similar to proteins involved in ribosomal RNA processing, yet does not colocalize precisely with either UBF1 or Fibrillarin under normal or stressed conditions. Identification of the precise location of PICT-1 and the signals that mediate its nucleolar localization is an important step towards advancing our understanding of the demonstrated influence of this protein on cell fate and tumorigenesis.     

Cytophotometric study of nuclear proteins during gametogenesis in Ascaris lumbricoides.

Reduction in the number of nucleoli/nucleus and increase in their size were usually observed in rat liver after partial hepatectomy. These changes of nucleoli were greatest 16–18 h after the operation, when RNA biosynthesis in the nucleoli is reported to be highest. Approx. 50% of the nuclei had one enlarged nucleolus at this time but after the increase in nuclear DNA synthesis less than 15% of the nuclei had one nucleolus, as in normal liver. Before the next peak of nuclear DNA synthesis, nucleolar changes appeared again, though less conspicuously.The enlarged nucleoli of regenerating liver were separated from smaller ones by discontinuous sucrose gradient centrifugation and the contents of nucleic acid and ribosomal cistrons in different-sized nucleoli were measured. The large nucleoli in regenerating liver were found to have increased DNA content, whereas smaller ones had the normal content. The total number of ribosomal cistrons in the enlarged nucleoli from regenerating liver was also increased roughly in proportion to the DNA content. No significant difference was found between the percentages of ribosomal cistrons in whole nuclear DNAs from regenerating and normal liver. Small but reproducible [³H]TdR incorporation into nucleolar DNA was observed and this was similar in normal liver and regenerating liver 12 h after partial hepatectomy. Therefore, the nucleolar changes in regenerating liver were not accompanied by any particular DNA synthesis in the nucleolus itself. These results suggest that in the nuclei of regenerating liver nucleolar chromatins may be redistributed and assembled into large nucleoli, rather than that any amplification of ribosomal cistrons occurs.     

Studies on the Growth in Culture of Plant Cells: VI. NUCLEIC ACID METABOLISM OF ACER PSEUDOPLATANUS L. CELL SUSPENSIONS

Changes in the nucleic acid content of cell suspension culturesof Acer pseudoplatanus, L. have been investigated. Both RNAand DNA contents rise sharply early in the period of cultureand before any significant increase in cell number. With theonset of rapid cell division there is a rapid decline in DNAcontent per cell to reach the value which persists during thesubsequent period of growth. The high level of RNA establishedat the beginning of the phase of rapid cell division, declinesgradually as growth proceeded. More detailed investigation ofthe RNA revealed no signficant changes, during the 3-week periodof culture, in either its base composition or in the relativeamounts of its different constituent molecular species.     

The relationship between cellular nucleic acids in the developing rat cerebral cortex

1. In the rat cerebral cortex net DNA synthesis ceases when the animal has reached about 25g. body weight (18 days of age). There is then little further change in the DNA content per cortex. 2. Nuclear and transfer RNA follow a similar pattern to DNA. 3. Microsomal and ribosomal RNA content increases up to 25g. body weight but then declines. The decrease in ribosomal and microsomal RNA content is associated with a change in RNA base composition. 4. Incorporation of [(14)C]orotic acid into nuclear RNA proceeds at a similar rate in 4-day-old and adult animals. However, there is a lag period of about 60min. in the young animals during which incorporation into the ribosome fractions proceeds slowly. In the adult animals the lag period is not seen.     

Post-mortem changes in human central nervous tissue and the effects on quantitation of nucleic acids and enzymes.

A study of post-mortem changes in human central nervous tissue has shown that within 100 h of death, no significant change occurs in the amount of nerve cell DNA and nucleolar RNA nor in some membrane-associated enzymes such as succinate dehydrogenase, NADH and NADPH diaphorase, and cytochrome oxidase. Low molecular weight RNA species, probably transfer and messenger RNA are quickly lost, but there is little alteration in ribosomal RNA content. Cytoplasmic enzymes show variable changes; phosphofructokinase activity is rapidly decreased; hexokinase is unaltered but lactate dehydrogenase, pyruvate kinase and glucose-6-phosphate dehydrogenase initially show increases in activity which subsequently decline. Oxygen uptake diminishes quickly. These findings indicate that mechanical alterations in cell structure, following death, render organelles physiologically ineffective long before any significant changes in certain constituent biochemicals are detected. This report emphasizes the great importance necessary in the selection of appropriately time matched post-mortem tissues if accurate comparative studies of many of the cells constituents are to be made.     

Cytoplasmic microtubules of normal and tumor cells of the leopard frog

Abstract. The cytoplasmic microtubule complex (CMTC) was examined in monolayer cultures of normal tadpole mesonephros, primary renal adenocarcinoma, and an established cell line derived from a pronephric renal adenocarcinoma (PNKT-4B) of the leopard frog, Rana pipiens. Immunocytochemistry revealed typical arrays of microtubules extending from the cytocentrum to the cell periphery in all three cell types when cultured at 28° C; similar results were obtained at 20° C. However, the CMTC was disorganized in both tumor types, in contrast to the retention of a typical CMTC in normal tissue cultured at 7° C. The response of PNKT-4B cells differed from that of normal tadpole mesonephros when treated with the microtubule inhibitor drug nocodazole. At 28° C, PNKT-4B and tadpole mesonephros cells lost their CMTC with nocodazole treatment, and both were able to reconstitute CMTC when nocodazole was removed. Similarly, both lost CMTC organization with nocodazole and culture at 7° C. However, while normal cells could effect a recovery at 7° C after the removal of nocodazole, PNKT4B cells were unable to restructure CMTC under the same conditions. Metastasis in the frog renal adenocarcinoma is temperature-dependent, with an elevated prevalence of metastasis in tumor-bearing frogs maintained at 28° C. Few metastatic colonies are detected in tumor-bearing frogs maintained at a low temperature (7° C). Other studies have indicated that microtubules, which are essential for cell motility, play an important role in the invasion by tumor cells of normal tissue fragments in vitro. The effects of temperature on metastasis of the Lucke renal adenocarcinoma are consistent with temperature-mediated changes in tumor-cell CMTC.     

Elucidation of Motifs in Ribosomal Protein S9 That Mediate Its Nucleolar Localization and Binding to NPM1/Nucleophosmin

Biogenesis of eukaryotic ribosomes occurs mainly in a specific subnuclear compartment, the nucleolus, and involves the coordinated assembly of ribosomal RNA and ribosomal proteins. Identification of amino acid sequences mediating nucleolar localization of ribosomal proteins may provide important clues to understand the early steps in ribosome biogenesis. Human ribosomal protein S9 (RPS9), known in prokaryotes as RPS4, plays a critical role in ribosome biogenesis and directly binds to ribosomal RNA. RPS9 is targeted to the nucleolus but the regions in the protein that determine its localization remains unknown. Cellular expression of RPS9 deletion mutants revealed that it has three regions capable of driving nuclear localization of a fused enhanced green fluorescent protein (EGFP). The first region was mapped to the RPS9 N-terminus while the second one was located in the proteins C-terminus. The central and third region in RPS9 also behaved as a strong nucleolar localization signal and was hence sufficient to cause accumulation of EGFP in the nucleolus. RPS9 was previously shown to interact with the abundant nucleolar chaperone NPM1 (nucleophosmin). Evaluating different RPS9 fragments for their ability to bind NPM1 indicated that there are two binding sites for NPM1 on RPS9. Enforced expression of NPM1 resulted in nucleolar accumulation of a predominantly nucleoplasmic RPS9 mutant. Moreover, it was found that expression of a subset of RPS9 deletion mutants resulted in altered nucleolar morphology as evidenced by changes in the localization patterns of NPM1, fibrillarin and the silver stained nucleolar organizer regions. In conclusion, RPS9 has three regions that each are competent for nuclear localization, but only the central region acted as a potent nucleolar localization signal. Interestingly, the RPS9 nucleolar localization signal is residing in a highly conserved domain corresponding to a ribosomal RNA binding site.     

THE INTRACELLULAR DISTRIBUTION AND HETEROGENEITY OF RIBONUCLEIC ACID IN STARFISH OOCYTES

A study has been made of the content and composition of RNA in cytoplasm, nucleoplasm, and nucleoli from growing oocytes of the starfish Asterias rubens. The determinations were carried out, using ultramicrochemical methods, on units isolated by microdissection from fixed sections. Macrochemical and interferometric control experiments show that RNA can be quantitatively evaluated in this way. The results show that the growing oocyte represents a system in which the relations between the quantities of nucleolar, nucleoplasmic, and cytoplasmic RNA undergo great changes. These changes are continuous for nucleolar and cytoplasmic RNA so that their amounts may be predicted from the size of the cell. Nucleoplasmic RNA, on the other hand, shows great variations among different cells, independent of cell size. Purine-pyrimidine analyses show that each cell component contains an RNA which differs significantly from that of the other two. Cytoplasmic and nucleolar RNA are closely related, the only difference being a slightly higher guanine/uracil quotient for the nucleolar RNA. They are both of the usual tissue RNA type, i.e., they show a preponderance of guanine and cytosine over adenine and uracil. Nucleoplasmic RNA deviates grossly from the RNA of the other two components. Here the concentrations of adenine and uracil are higher than those of guanine and cytosine, respectively. This RNA consequently shows some resemblance to the general type of animal DNA although the purine/pyrimidine ratio is far from unity. Our data favor a nucleolar origin for the stable part of the ribosomal RNA and a nucleoplasmic one for the unstable part (the messenger RNA).     

Diacridines: bifunctional intercalators. III. Definition of the general site of action.

Electron microscopy of HeLa cells exposed to spermine diacridine shows nucleolar distortions which disappear after several days despite the persistence of the metabolic changes promoted by spermine diacridine. This compound inhibits ribosomal RNA synthesis and appears to act independently of any particular phase of the cell cycle. The DNA content of the HeLa cells remains unchanged and the cell distribution is not significantly disturbed from its normal distribution in the various phases of the cell cycle. Spermine diacridine and other diacridines inhibit primarily chain initiation but also chain elongation by DNA-directed RNA polymerase of Azotobacter vinelandii.     

Nucleolar Arf tumor suppressor inhibits ribosomal RNA processing

The p19(Arf) tumor suppressor, a nucleolar protein, binds to Mdm2 to induce p53-dependent cell cycle arrest. Arf also prevents the proliferation of cells lacking Mdm2 and p53, albeit less efficiently. We show that p19(Arf) inhibits production of ribosomal RNA, retarding processing of 47/45S and 32S precursors. These effects correlate with but do not strictly depend upon inhibition of rRNA biosynthesis or cell cycle arrest, are not mimicked by p53, and require neither p53 nor Mdm2. Arf mutants lacking conserved amino acid residues 2-14 do not block rRNA synthesis and processing or inhibit cell proliferation. Evolution may have linked a primordial nucleolar Arf function to Mdm2 and p53, creating a more efficient checkpoint-signaling pathway for coordinating ribosomal biogenesis and cell cycle progression.     

AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS DURING SECONDARY RHIZOID CELL DIFFERENTIATION IN PTERIS VITTATA

The pre- and postmitotic stages of rhizoid cell differentiation in Pteris vittata L. were pulsed in radioactive uridine for 30 min and the data analyzed quantitatively by autoradiography. The total grain count rises from 594 in the nondifferentiating cell to 1,369 in the corona stage; increases to 2,745 in the internally segmented rhizoid cell (IRC) and to 2,779 in the wheel; and then drops to 1,306 in the protrusion and to 1,261 in the early rhizoid. The significant increase in nucleolar labeling in the corona reflects an increase in ribosomal RNA synthesis for mitosis-associated syntheses. The two-fold increase in grains over the cellular compartments of the IRC and wheel reflects an increase in RNA synthesis in these postmitotic stages. The similarity of labeling profiles between these stages suggests that they are not far enough separated to show a difference in their roles. The decline in total grain counts in the protrusion and early rhizoid stages to half the maximum levels suggests that an overall decrease in RNA synthesis is attended by a shift from a differentiating phase to a steady-state growth system.     

Effects of camptothecin, an inhibitor of DNA topoisomerase I on ribosomal gene structure and function in TG cells.

The effects of camptothecin treatment and topoisomerase I inhibition on ribosomal gene structure and function were investigated in TG cells, a human tumour cell line. 90- and 180-min treatments with 25 microM camptothecin resulted in an increased DNA fragmentation and decreased activity of topoisomerase I in cell extracts. After 180-min treatment, the incorporation of labelled uridine into total cell RNA was reduced to 39% and the ribosomal RNA synthesis to 10%, as compared to values of control cells. At the ultrastructural level, the nucleolar components appeared to be segregated; after selective DNA staining, with osmium-amine complex, a part of the nucleolar chromatin of treated cells showed the presence of thin, extended DNA filaments, superimposable to those present in control cells.