Establishment and Maintenance of the Interferon-Induced Antiviral State: Studies in Enucleated Cells

Requirements for the physical presence of the cell's nucleus for the establishment and maintenance of the interferon-induced antiviral state were investigated. Enucleated chicken embryo fibroblasts were obtained by cytochalasin B treatment during centrifugation. The inhibition of vaccinia virus cytoplasmic DNA synthesis, monitored by autoradiography, was used to measure the antiviral activity resulting from interferon treatment. The antiviral state is not established in cells treated with interferon after removal of their nuclei. On the other hand, cells first treated with interferon for 6 or 12 h and then enucleated express the antiviral state. Furthermore, the antiviral state is maintained in enucleated cells for 16 h after enucleation. The antiviral state appears to be more stable in enucleates than in the residual nucleated cells found in the same cultures. Single cells of antiviral populations are found to be either fully permissive or fully restrictive to vaccinia DNA synthesis. The effect of an increasing intracellular multiplicity of infectious virus is to overcome the antiviral cell's block against viral DNA synthesis.     

The influence of enucleation on the ultrastructure of in vitro matured and enucleated cattle oocytes

The enucleation of recipient oocytes in nuclear transfer experiments is generally carried out by aspirating one third of the ooplasm adjacent to the first polar body. It was supposed that this enucleation step affects the ultrastructure of the remaining cytoplast, resulting in a decline or destruction of its cellular compartments. Even if the transferred nucleus had the potential to support the development of a single-cell nucleus transfer embryo to the blastocyst stage, meiotic division could be stopped at any stage if the destruction of the ultrastructure of host cytoplasm resulted in a limited metabolism. The present study was conducted to investigate the influence of the enucleation procedure on the ultrastructure of the remaining ooplast. In vitro matured oocytes; in vitro matured and enucleated oocytes; and in vitro matured and enucleated oocytes that were subsequently cultivated in vitro for additional 4 h were prepared for transmission electron microscopy (TEM). An examination of ultra-thin sections showed that the arrangement of organelles in all matured oocytes was in accordance with that already described for normal oocyte development. Immediately after enucleation no major differences in the arrangement of cortical granules, mitochondria, smooth endoplasmic reticulum (SER), lipid droplets and vacuoles were found compared with nonmanipulated oocytes. After enucleation and 4 h of culture, 24- and 36-h matured oocytes differed from each other in the arrangement of large aggregates of SER surrounded by a wall of mitochondria and lipid droplets. These complexes were still found in the 24-h but not in 36-h matured, enucleated and cultivated oocytes. Clusters of SER, mitochondria and lipid droplets were described by different authors as having metabolic activity. The results of this study in connection with results from nuclear transfer experiments suggest that these aggregates and their metabolic activity can be transferred with cytoplasm from 24- but not 36-h matured oocytes. Only cytoplasm from the 24-h matured oocytes showed a development-supporting effect when fused to enucleated recipient cells before nuclear transfer.     

Metabolic changes in the nucleus of the optic tract after monocular enucleation as revealed by cytochrome oxidase histochemistry

The histochemistry for the mitochondrial enzyme cytochrome oxidase (CO) was used to evaluate the levels of metabolic activity in neurons of the nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN) in the opossum (Didelphis aurita). The observations were performed in four groups: normal juveniles (4 months old), monocularly enucleated juveniles analysed when adults, normal adults (8 to 18 months old) and monocularly enucleated adults. CO labeled cells were observed to have a similar distribution along the NOT-DTN anteroposterior axis in both juvenile and adult normal animals. Monocular enucleation performed in adults produced a significant reduction of the reactive neuropil but not of the number of CO labeled cells in the deafferented NOT-DTN: the number of labeled neurons per section in the deafferented side matched those of the ipsilateral complex. In juveniles, however, this procedure caused a systematic reduction of the number of CO labeled cells in the contralateral NOT-DTN in comparison to the spared complex. The lack of reduction in the number of neurons found on the deafferented side of the NOT-DTN of monocularly enucleated adult opossums compared with the ipsilateral side might result from the presence of compensatory inputs to maintain their metabolic equivalence. However, when the monocular enucleation was performed in juvenile opossums, a statistically significant asymmetry of CO neurons in the NOT-DTN was observed. In other words, the compensatory mechanisms proposed for the adults were either absent or insufficient to achieve symmetry in juveniles, suggesting a more heavily reliance in the retinal input.     

Degradation of nuclear proteins: studies on transplanted B82 cell karyoplast proteins

Karyoplasts were prepared from B82 cells (thymidine kinase deficient mouse L cells) by cytochalasin B mediated enucleation. Morphological measurements show that the nucleus constitutes 89% of a karyoplast by volume. Homokaryons were obtained by Sendai virus mediated karyoplast-B82 cell fusion. Transplanted nuclei were not destroyed catastrophically but were maintained intracellularly for at least 140 h. Transplanted nuclear proteins were degraded with an average half-life of 84 +/- 7 h by processes partially sensitive to inhibition by NH4Cl (50%) and leupeptin (30%). The data therefore suggest that some nuclear proteins are translocated to the cytoplasm for lysosomal degradation.     

Accurate Differentiation of Neuronopathic and Nonneuronopathic Forms of Niemann-Pick Disease by Evaluation of the Effective Residual Lysosomal Sphingomyelinase Activity in Intact Cells

Abstract: Niemann-Pick disease types A and B are two clinical forms of an inherited lysosomal storage disorder characterized by accumulation of sphingomyelin due to deficient activity of the lysosomal enzyme, acid sphingomyelinase. Patients with both types have hepatosplenomegaly, but only those with type A have nervous system involvement leading to death in early infancy. The residual activities of lysosomal sphingomyelinase in types A and B have never been well characterized because of limitations in both in vitro enzymatic assays and loading tests on intact cells. To evaluate the effective level of sphingomyelinase activity, intact, living cultured Epstein-Barr virus-transformed lymphoid cells were incubated with a radiolabeled sphingomyelin that was first associated to human low-density lipoproteins. This lipoprotein-associated sphingomyelin was targeted to lysosomes, thereby permitting selective hydrolysis by the lysosomal sphingomyelinase. Short-term pulse-chase experiments allowed the determination of the initial rates of degradation; in normal cells, the half-time of sphingomyelin degradation averaged 4.5 h. Whereas cells from the severe neuronopathic type A form of Niemann-Pick disease exhibited about 0.15% residual sphingomyelinase activity, cells from patients with the visceral type B form exhibited about 4%, i.e., 27 times more. Cells from heterozygous Niemann-Pick subjects showed about 70% residual activity. These results provide the first approach to measuring the effective activity of a lysosomal enzyme and represent an accurate method for the differential diagnosis of Niemann-Pick disease types A and B. They also support the hypothesis of relationships among the effective in situ residual enzyme activity, the amount of stored substrate, and the severity of the ensuing lysosomal storage disorder.     

Reactivation of chick erythrocyte nuclei after fusion with enucleated cells.

Inactivated Sendai virus was used to fuse nucleated chick erythrocytes with mouse L and A9 cells which had been enucleated by centrifugation in the presence of cytochalasinB. The enucleation step removed the nuclei from more than 99% of the cells. During the fusion step, chick erythrocyte nuclei were introduced into 20% of the enucleated mouse cytoplasms. This resulted in the formation of a large number of "reconstituted cells" where practically all the cytoplasm originated from the mouse cell while the nucleus was of chick origin. The chick erythrocyte nuclei appeared to become well integrated into the mouse cytoplasms since they increased dramatically in size and dry mass, formed nucleolus-like bodies, and resumed RNA synthesis. This, however, did not prevent a gradual decrease in the rate of protein synthesis in the cytoplasm after the removal of the mouse nucleus. Protein synthesis decayed at a similar rate in both reconstituted and enucleated cells. The majority of these "cells" died within 48 h and all of them within 5 days after enucleation/fusion. By contrast, the small number of L cells which failed to become enucleated multiplied rapidly. The results obtained suggest that the reactivation of the chick erythrocyte nuclei is not fast enough to rescue the enucleated mouse cytoplasms.     

Effects of insulin on cardiac lysosomes and protein degradation

Hearts perfused in the absence of added insulin had 1) accelerated rates of protein degradation, as assessed by release of phenylalanine and tyrosine; 2) increased rates of release of seven other amino acids; 3) decreased lysosomal latency and sedimentable lysosomal enzyme activity; 4) increased numbers of autophagic vacuoles in cardiac muscle cells; and 5) decreased activity of beta-N-acetylglucosaminidase in dense lysosomes (1.06-1.09 g/ml), as compared to hearts perfused in the presence of the hormone. After 3 h of perfusion in the absence of insulin, the changes that developed in protein degradation, lysosomal latency, and sedimentability, and in enzyme activity in dense lysosomes, were reversed by insulin addition during 90 min of subsequent perfusion. These studies suggest a role for insulin in controlling the activity of the lysosomal system and the involvement of this system in protein degradation, particularly in insulin-deprived tissue.     

Nonselective autophagy of cytosolic enzymes by isolated rat hepatocytes

Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.     

Study of germinal vesicle requirement for the normal kinetics of maturation/M-phase-promoting factor activity during porcine oocyte maturation

Mammalian immature oocytes contain large nuclei referred to as germinal vesicles (GVs). The translocation of maturation/M-phase promoting factor (MPF) into GVs just before the activation of MPF has been reported in several species. To examine whether the GV is required for MPF activation in mammalian oocytes, porcine immature oocytes were enucleated and their MPF activity and CCNB (also known as cyclin B) levels were investigated. The activation of MPF at the start of maturation was detected at normal levels in enucleated oocytes, whereas reactivation to induce the second meiosis was not observed. Although protein synthesis was found to be normal both qualitatively and quantitatively, even in the absence of the nucleus, CCNB1 did not sufficiently accumulate in the enucleated oocytes. The defects in the enucleated oocytes were reversed by the injection of GV material into the enucleated oocytes. Furthermore, the inhibition of CCNB1 degradation revealed drastic accumulation of CCNB1, indicating active synthesis of CCNB1 in enucleated oocytes. The mitogen-activated protein kinase cascade remained unaffected by enucleation. These results indicate that GV is not required for the activation of MPF during the first meiosis, but that it is required for the second meiosis because of its promotion of CCNB1 accumulation.     

CHANGES IN BRAIN ALCOHOL DEHYDROGENASE ACTIVITY DURING CHRONIC ETHANOL INGESTION AND WITHDRAWAL

Abstract— Chronic ethanol ingestion in rats leads to a slow rise in brain alcohol dehydrogenase activity which levels off after 2 weeks at approximately twice the initial activity. The half-time of the rise is approximately 8 days. Abrupt withdrawal of the ethanol is followed by a rapid decline of the brain alcohol dehydrogenase activity to the normal level with a half-time of approximately 15 h. The difference in time constants between the rise in enzyme activity during ethanol-feeding and its decline following withdrawal suggests that the increased enzyme activity is at least in part the result of a reduced rate constant of enzyme degradation in the presence of ethanol. The effect of ethanol on brain alcohol dehydrogenase activity is not altered by supplementation of the diet with carbohydrate or vitamins. The effect is seen only in the cerebral hemispheres and not in the brain-stem. Acquisition of tolerance to ethanol during chronic ethanol ingestion and its extinction following withdrawal follow almost the same time courses as the changes in brain alcohol dehydrogenase activity.     

Isolation of anucleate cells using a fluorescence activated cell sorter (FACS II)

Human fibroblasts or mouse teratocarcinoma cells were enucleated by density gradient centrifugation in the presence of cytochalasin B (CB). The resulting mixed population of nucleated and anucleate cells was further purified by flow sorting, using the dye Hoechst 33342 as a fluorescent label for the nucleated cells. The purity of the anucleate cells obtained with this technique was at least 99%, as was shown by histological staining of the sorted fractions. Sorted enucleated fibroblasts were shown to have an intact cell membrane as indicated by their ability to convert fluorescein diacetate into fluorescein and to accumulate this product. They were found to attach and spread when cultured and showed protein synthesis immediately after enucleation, evidenced by the incorporation of [³H]leucine. Sorted enucleated teratocarcinoma cells also had an intact cell membrane, but they did not attach when cultured.     

Interaction of the retina with suprachiasmatic pacemakers in the control of circadian behavior

The suprachiasmatic nucleus (SCN) is the central circadian pacemaker governing the circadian rhythm of locomotor activity in mammals. The mammalian retina also contains circadian oscillators, but their roles are unknown. To test whether the retina influences circadian rhythms of locomotor behavior, the authors compared the activity of bilaterally enucleated hamsters with the activity of intact controls held in constant darkness (DD). Enucleated hamsters showed a broader range of free-running periods (tau) than did intact hamsters held for the same length of time in DD. This effect was independent of the age at enucleation (on postnatal days 1, 7, or 28). The average tau of intact animals kept in DD from days 7 or 28 was significantly longer than that of intact animals kept in DD from day 1 or any of the enucleated groups. This indicates that early exposure to light-dark cycles lengthens the tau and that the eye is required to maintain this effect even in DD. These data suggest that hypothalamic circadian pacemakers may interact continuously with the retina to determine the tau of locomotor activity. Enucleation caused a large decrease in glial fibrillary acidic protein in the SCN but has no (or slight) effects on calbindin, neuropeptide Y, vasopressin, or vasoactive intestinal polypeptide, which suggests that enucleation does not produce major damage to the SCN, an interpretation that is supported by the fact that enucleated animals retain robust circadian rhythmicity. The presence of an intact retina appears to contribute to system-level circadian organization in mammals perhaps as a consequence of interaction between its circadian oscillators and those in the SCN.     

Lysosomal degradation of ribonuclease A and ribonuclease S-protein microinjected into the cytosol of human fibroblasts

We have analyzed the subcellular localization of 125I-labeled ribonuclease A and ribonuclease S-protein (residues 21-124) after erythrocyte-mediated microinjection into confluent cultures of IMR-90 human lung fibroblasts. Microinjected cells were fractionated by two consecutive Percoll gradients, and the distribution of radioactive ribonuclease A and S-protein was compared to patterns for known enzyme markers. Ribonuclease A is localized in the cytosol immediately after microinjection, but thereafter a portion of the microinjected enzyme is associated with lysosomes. We obtained similar results for ribonuclease S-protein except extensive association with a nonlysosomal intracellular structure is also evident. The effects of ammonium chloride on proteolysis indicate that ribonuclease A and ribonuclease S-protein are degraded at least in part by lysosomal pathways. Degradation of long-lived cellular proteins is inhibited by 17% in the presence of serum and by 35% in the absence of serum. The effects of ammonium chloride on catabolism of microinjected proteins are more variable. Inhibition in the presence and absence of serum ranged between 43 and 64% for both ribonuclease A and ribonuclease S-protein. To quantitatively assess the role of lysosomal and cytosolic pathways in the degradation of microinjected proteins, we have tagged proteins with the inert trisaccharide, [3H] raffinose. The radioactive degradation products of such proteins are completely retained within lysosomes since the lysosomal membrane is impermeable to [3H] raffinose coupled to lysine or small peptides. These studies show that ribonuclease A and S-protein are degraded almost entirely by lysosomes while bovine serum albumin is degraded principally in the cytosol. A mixture of rat liver cytosolic proteins is degraded approximately 60% in the cytosol and 40% by lysosomes confirming that both lysosomal and nonlysosomal pathways of proteolysis are important in confluent human fibroblasts.     

Ultrastructural evidence of maintenance of concanavalin A binding sites in enucleated mammalian cells

Mouse L cells were enucleated by centrifugation in cytochalasin B. Following enucleation, the enucleated cells were incubated in fresh medium for 30 min, 4, 20, or 24 h before being fixed for electron microscopy. After fixation the cells were incubated in concanavalin A and then horseradish peroxidase was bound to the ConA. Electron microscopy of these enucleates revealed that the concanavalin A-binding sites (CABS) are present on the cell surface of the enucleates even at 24 h after enucleation. Although the method does not detail the number of sites present, the inherent distribution of sites appears similar in normal and enucleated cells. Furthermore, the sites are still functional in that the live enucleated cells are agglutinated by ConA to the same extent as are normal L cells—about 80% agglutination in each instance. The results of this study indicate that surface CABS are maintained in the absence of a nucleus and they are still present even after the Golgi apparatus is morphologically disrupted. Turnover of these sites and their relationship to nuclear function are discussed.     

Fusion with enucleated fibroblasts corrects “I-cell” defect

Fusions have been carried out between fibroblasts from patients with “I-cell” disease and enucleated human fibroblasts with a single lysosomal enzyme deficiency derived from patients with G_M1-gangliosidosis, Sandhoff disease and mannosidosis. Pure cytoplasts were obtained using cytochalasin B treatment followed by fluorescence activated cell sorting. After fusion with whole “I-cells”, the cybrid populations showed a restoration of deficient lysosomal enzyme activity and also the abnormal electrophoretic pattern characteristic for the residual hexosaminidase activity in “I-cells” was found to be corrected. The results described in this paper indicate that the defective post-translational modification, which is responsible for the multiple lysosomal enzyme deficiency, can be corrected by a factor that is stable for at least three days in enucleated cells. During this period the cytoplasmic factor can act without the need of de novo synthesis but the absence of correction in in vitro experiments shows that cellular integrity is required.     

THE EFFECTS OF ENUCLEATION ON THE CYTOPLASMIC MEMBRANES OF AMOEBA PROTEUS

The dependence of cytoplasmic membranes upon the nucleus was studied by examining enucleated amebae with the electron microscope at intervals up to 1 wk after enucleation. Amebae were cut into two approximately equal parts, and the fine structure of the enucleated portions was compared with that of the nucleated parts and starved whole cells which had been maintained under the same conditions. Golgi bodies were diminished in size 1 day after enucleation and were not detected in cells enucleated for more than 2 days. The endoplasmic reticulum of enucleated cells appeared to increase in amount and underwent changes in its morphology. The sparsely scattered short tubules of granular endoplasmic reticulum present in unmanipulated amebae from stock cultures were replaced in 1–3-day enucleates by long narrow cisternae. In 3–7-day enucleates, similar cisternae of granular endoplasmic reticulum encircled areas of cytoplasm partially or completely. It was estimated that in most cases hundreds of these areas encircled by two rough membranes were formed per enucleated cell. The number of ribosomes studding the surface of the endoplasmic reticulum decreased progressively with time after enucleation. In contrast, the membranes of nucleated parts and starved whole cells did not undergo these changes. The possible identification of membrane-encircled areas as cytolysomes and their mode of formation are considered. Implications of the observations regarding nuclear regulation of the form of the Golgi apparatus and the endoplasmic reticulum are discussed.     

Enucleation of phagocytic cells with adenine, guanine, and their nucleosides in combination with centrifugation

Several purine compounds, such as adenine, guanine, adenosine, guanosine, and their related compounds, exhibited enucleation activity on adherent mouse peritoneal exudate cells (macrophages) during centrifugation at 25,000 and 35,000 g for 60 min at 34 degrees-36 degrees C in medium containing one of these compounds. Enucleation activity, however, did not occur in cells treated with adenine nucleotides, inosine, xanthine, or any of the tested pyrimidines. The purine compounds also had enucleation activity on mouse macrophage-like cell lines (P388D1 and RAW 264) and mouse polymorphonuclear leukocytes, but not on other typical cell lines such as a human epithelial cell line (HeLa S-3) or a mouse fibroblast cell line (L929). Cytochalasin B (CB) treatment, however, resulted in the enucleation of all cell types tested, even at a centrifugal force as low as 5,000 g. The process of macrophage enucleation was observed by both light microscopy and scanning electron microscopy. In enucleated macrophages that had been treated with purine compounds, but not with CB, a newly formed cytoplasmic crater-like structure (about 3-9 microns in diameter) was observed at the original site of the nucleus. Surface structures, such as microvilli and membrane ruffles, remained relatively intact in macrophages that had been enucleated by treatment with purine compounds. By contrast, these surface structures were markedly changed in CB-treated macrophages. Purine compounds may affect cytoskeletal elements in ways similar to the well characterized effects of CB, and thus result in the enucleation of phagocytes. However, the characteristic differences in the enucleation activity exhibited by purine compounds and CB may indicate that purines have a mechanism of action different from that of CB.     

Similarities in ornithine decarboxylase regulation in intact and enucleated 3T3 cells

The role of the cytoplasm in the regulation of ornithine decarboxylase (ODC) has been examined in enucleated 3T3 cells (cytoplasts). ODC activity can be increased 15–25-fold by a cytoplasmic mechanism(s) in enucleates prepared from growing cells by treatment which lead to 50–75-fold increases in intact growing cells. Since the enzyme activity simultaneously becomes less stable in these cytoplasts as in whole cells, it appears this increase is due either to activation of pre-existing enzyme or increased synthesis. A biphasic increase during the first 20 h after stimulus is seen in cytoplasts prepared from growing cells which have been stimulated for the prior 5 h. The second increase in activity does not appear to be due to decreased degradation or conversion to a more active form. These results are analogous to those reported previously for intact growing cells in experiments which employed metabolic inhibitors, and similarly suggest that there is cytoplasmic control of ODC synthesis. Medium polyamines reduce ODC activity in cytoplasts with kinetics and characteristics similar to those previously reported for intact cells. These data are also most consistent with regulation of synthesis at the translational level.