Thermal down-regulation of exportable rRNA in nuclei

The export of rRNP particles from nuclei isolated from Tetrahymena was investigated after preincubating the nuclei at different temperatures under nonpermissive export-conditions. We observed a new phenomenon: Temperature elevation from the sublethal cells' growth temperature, 8 degrees C, to the optimal temperature, 28 degrees C, lead to a gradual down-regulation in the maximal proportion of rRNP particles subsequently exported from nuclei at 28 degrees C. This thermal down-regulation is apparently not due to qualitative changes in the exported rRNP particles, a derangement in the gross nuclear organization, a degradation and/or nicking of the nuclear rRNA, a gross decomposition of the major nuclear proteins, a random cross-linking of nuclear components by disulfide bonds, or an elution of nuclear factors possibly required for rRNP export. Moreover, there is a corresponding thermal down-regulation in nuclear envelope-free nuclei. Our data indicate that nuclei possess a mechanism that regulates the number of potentially exportable rRNP particles at a level preceding the rRNP passage through the nuclear envelope.     

The retention of core ribonucleoproteins in the nuclear matrix isolated from nuclei treated with the phenantroline-copper complex

The nuclear matrix isolated from rat liver nuclei whose protein sulfhydryl groups were oxidised with the o-phenantroline-copper (OP-Cu) complex was enriched with a set of 32-44 kd polypeptides identified as core proteins of ribonucleoprotein particles (RNP). The most conspicuous protein in the nuclear matrix was a 36 kd protein present as a disulfide-linked homodimer. The propensity of protein 36 to be oxidised and form intermolecular associations suggests that it may contribute to the interaction of RNP particles with the nuclear matrix and thus to their spatial distribution in the nucleus.     

Potassium retention in membraneless thymus lymphocyte nuclei

Nonionic detergents, Triton X-100 and Brij 58, removed lipoid membranes of suspended thymus lymphocytes within 5 minutes. The mobilization and solubilization of cytoplasmic and nuclear proteins occurred much faster (less than 5 minutes) with Triton X-100 treatment than with Brij 58 treatment (less than 10 minutes). In Triton X-100 treated cells the loss of K+ was complete within 5 minutes whereas with Brij 58 treatment the K+ loss was not complete after 10 minutes. Thus, the high concentration of K+ and the low concentration of Na+ in the nuclei can remain near normal for minutes in the absence of membrane structures. If the ions were in free solution within the cells, disruption of membrane integrity should lead to equilibration of the ions with external media within seconds. The decrease of K+ in the Brij 58 treated cells with exposure time was correlated with the solubilization of the proteins. These results support the view that K+ and Na+ are not freely dissolved in the cellular water, but are co-compartmentalized with proteins inside the living cell.     

Protein- and carbohydrate-induced augmentation of whole body creatine retention in humans.

This study investigated the effect of creatine supplementation in conjunction with protein and/or carbohydrate (CHO) ingestion on plasma creatine and serum insulin concentrations and whole body creatine retention. Twelve men consumed 4 x 5 g of creatine on four occasions in combination with 1) 5 g of CHO, 2) 50 g of protein and 47 g of CHO, 3) 96 g of CHO, or 4) 50 g of CHO. The increase in serum insulin was no different when the protein-CHO and high-CHO treatments were compared, but both were greater than the response recorded for the low-CHO treatment (both P < 0.05). As a consequence, body creatine retention was augmented by approximately 25% for protein-CHO and high-CHO treatments compared with placebo treatment. The areas under creatine- and insulin-time curves were related during the first oral challenge (r = -0.920, P < 0.05) but not after the fourth (r = -0.342). It is concluded, first, that the ingestion of creatine in conjunction with approximately 50 g of protein and CHO is as effective at potentiating insulin release and creatine retention as ingesting creatine in combination with almost 100 g of CHO. Second, the stimulatory effect of insulin on creatine disposal was diminished within the initial 24 h of supplementation.     

Effect of hydrophobic compounds on rRNA synthesis in intact cells and isolated nuclei

Papaverine, cycloheximide, 2,4-dinitrophenol (DNP) and actinomycin D at low concentration have been shown to suppress selectively rRNA synthesis in Ehrlich ascite carcinoma cells. rRNA synthesis in isolated nuclei is not sensible to wide range of concentration of papaverine (0,005-0,1 mM), cycloheximide (0,5-100 micrograms/ml) and DNP (5-500 microM). Actinomycin D at low concentration does not act on the rRNA synthesis in vitro either. To suppress rRNA synthesis in this system much higher concentration of this agent (10 micrograms/ml) producing inhibition of all classes of rRNA synthesis in intact cells is required. Selective sensitivity of rRNA synthesis in the cells to papaverine, cycloheximide, DNP and low concentration of actinomycin D does not connect with their direct action on the apparatus of rRNA synthesis.     

Heterogeneous nuclear RNA-protein fibers in chromatin-depleted nuclei

The heterogeneous nuclear RNA-protein (hnRNP) fibers in HeLa cell nuclei are visualized by a nuclear subfractionation technique which removes 96% of the chromatin in a single step and 99% in a two-step elution but leaves the bulk of the hnRNA complexed with the remnant nuclear structure or lamina. Both steady-state and newly synthesized (approximately 15-s label) hnRNA are associated with the remnant nuclei to about the same extent. This association does not appear to depend on the presence of chromatin and exists in addition to any possible association of hnRNP with chromatin itself. Electron microscopy of partially purified nuclear hnRNA complexes shows that the hnRNP fibers form a ribonucleoprotein network throughout the nucleus, whose integrity is dependent on the RNA. Autoradiography confirms that hnRNA is a constituent of the fibers. The RNA network visualized in these remnant nuclei may be similar to RNA networks seen in intact cells. The hnRNA molecules appear to be associated with the nuclear lamina, at least in part, by unusual hnRNA sequences. More than half of the recovered poly(A) and double-stranded hnRNA regions remains associated with the nuclear structures or the laminae after digestion with RNase and elution with 0.4 M ammonium sulfate. In contrast, the majority of oligo(A), another ribonuclease resistant segment, is released together with most of the partially digested but still acid-precipitable single- stranded hnRNA and the hnRNP proteins not eluted by the ammonium sulfate alone. These special RNA regions appear to be tightly bound and may serve as points of attachment of the hnRNA to nuclear substructures. It is suggested that hnRNA metabolism does not take place in a soluble nucleoplasmic compartment but on organized structures firmly bound to the nuclear structure.     

Status of the nuclear matrix in mature and embryonic chick erythrocyte nuclei

The adult chicken erythrocyte nucleus was found to lack an internal nuclear matrix: even milder extraction procedures resulted in the production of empty shells of pore complex-lamina together with loose aggregates of core histone. In contrast, rat liver nuclei showed a typical intranuclear salt-resistant skeleton. These results show that an internal matrix is not an obligatory nuclear component, and is not required for the spatial organization of chromatin. 5-day-old embryonic erythrocytes did, however, contain an interchromatinic nuclear matrix, suggesting a correlation between the presence of matrix structures, and nuclear 'activity'.     

Rapid translocation of NTF2 through the nuclear pore of isolated nuclei and nuclear envelopes

The mechanism by which macromolecules are translocated through the nuclear pore complex (NPC) is little understood. However, recent measurements of nuclear transport in permeabilized cells showed that molecules binding to phenylalanine-glycine-rich repeats (FG repeats) in NPC proteins were translocated much faster through the NPC than molecules not interacting with FG repeats. We have studied that substrate preference of the NPC in isolated oocyte nuclei and purified nuclear envelopes by optical single transporter recording. NTF2, the transport receptor of RanGDP, was exported ~30 times faster than green fluorescent protein, an inert molecule of approximately the same size. The data confirm that restricted diffusion of inert molecules and facilitated transport of FG-repeat binding proteins are basic types of translocation through the NPC, demonstrating that the functional integrity of the NPC can be conserved in isolated nuclei and nuclear envelopes and thus opening new avenues to the analysis of nucleocytoplasmic transport.     

Release of rRNA from liver nuclei during the early stages of the acute-phase reaction

Liver nuclei isolated from rats 5 h after turpentine injection show an increased release of rRNA, of the transport-related nucleoside-triphosphatase activity and of the amount of nuclear RNA; RNA methylation is also likely to undergo some activation. These changes occur when RNA synthesis is still normal.     

Detection of chick rRNA in the cytoplasm of heterokaryons containing reactivated chick red cell nuclei

Collagen fiber formation in vitro was morphologically investigated on 22 epithelial-like cell lines originating from normal rat livers mainly by using specific histochemical stainings. All the cell lines, eight of which were cloned formed typical “collagenous” and/or “reticulin” fibers in the histological sense when they had been left as a full sheet for more than 3 weeks without subculture. Only one of five fibroblastic cell lines serving as control formed fibers to a lesser degree. Different patterns were recognized in fibers appearing between the liver cell cultures and the fibroblast cultures.     

v-mos oncoproteins affect the nuclear retention and reutilization of glucocorticoid receptors

Expression of the p85gag-mos oncoprotein in temperature sensitive transformed 6m2 cells results in desensitization of glucocorticoid induction of metallothionein-1 mRNA. Indirect immunofluorescence analyses demonstrate that hormone insensitivity in v-mos transformed cells is associated with inefficient nuclear retention of glucocorticoid receptor (GR) protein. Desensitized receptors that accumulate in the cytoplasm of transformed 6m2 cells do not regain the capacity for hormone-dependent nuclear translocation after turnover of the thermo-labile p85gag-mos oncoprotein. Although ligand induced down-regulation of immunoreactive GR protein occurs in transformed 6m2 cells, desensitized receptors appear to retain some capacity to bind hormone in vivo. Thus alterations in the intracellular partitioning of GR protein in v-mos-transformed cells result in the generation of a novel desensitized receptor that is apparently trapped in the cytoplasm and incapable of being reutilized.     

DNA and nuclear protein measurement in isolated nuclei of human endometrium

Feulgen-DNA and nuclear light green-protein measurements have been performed in isolated nuclei of normal (nonmalignant) and malignant human endometrial homogenates. The DNA content of the G0/G1 fraction of malignant endometrium showed much overlap with that of normal endometrium, or was slightly increased. Two of the 18 carcinomas were clearly aneuploid. No correlation was found between the histological grade and the DNA content. The tumors of clinical stage II and higher all had a higher DNA content than that of normal endometrium. The percentage of cells present in the proliferative fraction was higher in proliferative endometrium than in secretory and post-menopausal atrophic endometrium. For malignant endometrium, percentages were found comparable to that of normal endometrium or higher. No correlation was found with the histological grade. Tumors of stage II and higher had intermediate values compared to those of carcinomas below stage II. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. However, for post-menopausal women, most values of the carcinomas exceeded that of normal, atrophic, endometrium. Within the tumor population, no correlation was found with the histological grade. Higher values were found with tumors of clinical stage II and higher.