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PubMed1999

Effect of rev on the cytoplasmic localization of intron-containing human immunodeficiency virus type 1 RNA.

Human immunodeficiency virus type 1 (HIV-1) proteins are expressed from both intron-containing and completely spliced RNAs. Rev, an HIV-1 regulatory protein, is necessary for the expression of intron-containing RNAs. The effect of Rev on the subcellular localization of intron-containing HIV-1 RNA was examined by in situ RNA hybridization. In the presence of Rev, intron-containing HIV-1 RNA accumulated at the nuclear membrane and within the cytoplasm of transfected cells. In the absence of Rev, intron-containing HIV-1 RNA accumulated within the nucleus. In approximately 20% of the cells transfected in the absence of Rev, intron-containing HIV-1 RNA was also found in the cytoplasm. Differences in the subcytoplasmic localization of intron-containing HIV-1 RNA in the presence and absence of Rev were not observed using in situ RNA hybridization. To determine the effect of Rev on RNA localization within the cytoplasm, an extensive fractionation protocol involving both hypotonic and detergent lysis was used. In the presence of Rev, 40.9 +/- 4.6% of the cytoplasmic intron-containing HIV-1 RNA was released by hypotonic lysis. A similar fractionation profile was seen for several other translated viral and cellular RNAs. However, in the absence of Rev, only 16.5 +/- 5.1% of the cytoplasmic intron-containing HIV-1 RNA was released on hypotonic lysis (P < 0. 005). Thus the cytoplasmic fractionation pattern of this RNA was altered in the absence of Rev.

Cell Line↗

PubMed1994

Characterisation and expression of the mitochondrial genome of a new type of cytoplasmic male-sterile sunflower.

A new cytoplasmic male sterile sunflower, CMS3 [44], was characterised in relation to the Petiolaris (PET1) cytoplasmic male-sterile sunflower, CMS89 [25]. Southern blot analysis showed that the mitochondrial genome of CMS3 contains unique rearrangements in at least five loci (atp6, atp9, atpA, nad1 + 5 and coxIII) compared to the PET1 sterile and the fertile cytoplasms. Transcripts of two (coxIII and atp6) of the five rearranged loci differed in CMS3 when compared to the corresponding loci in the PET1 and fertile cytoplasms. In organello protein synthesis experiments showed that the ca. 15 kDa mitochondrial polypeptide, characteristic of PET1, is not present in the CMS3 line. These data suggest that the molecular basis of male sterility in the CMS3 line differs from that of the PET1 cytoplasm. The nucleotide sequences of the coding and the immediate flanking regions of the coxIII and atp6 genes of CMS3 were compared to the corresponding regions from the fertile sunflower. In CMS3 the ORFB-coxIII locus is located immediately 3' to the atpA gene whereas in the fertile cytoplasm these two loci are ca. 60 kb apart. This DNA rearrangement probably involved a 265 bp repeat which may be implicated in the DNA recombination associated with PET1 CMS. The atp6 gene in CMS3 contains a 5'-terminal extention which results in an extended ORF. The potential involvement of the rearrangements associated with the coxIII and atp6 loci in relation to the CMS phenotype is discussed.

Amino Acid Sequence↗

PubMed1995

Migration of the yeast linear DNA plasmid from the cytoplasm into the nucleus in Saccharomyces cerevisiae.

The Kluyveromyces linear plasmids, pGKL1 and pGKL2, carrying terminal protein (TP), are located in the cytoplasm and have a unique gene expression system with the plasmid-specific promoter element termed UCS, which functions only in the cytoplasm. In this study we have developed an in vivo assay system in Saccharomyces cerevisiae which enables the detection of a rare migration of the yeast cytoplasmic plasmid to the nucleus, using a pGKL1-derived cytoplasmic linear plasmid pCLU1. pCLU1 had both the UCS-fused LEU2 gene (a cytoplasmic marker) and the native URA3 gene (a nuclear marker) and therefore its cytoplasmic-nucleo localized could be determined by the phenotypic analysis of the marker. The nuclearly migrated plasmids were often detected as linear plasmids having the telomere sequence of the host yeast at both ends, although circular plasmids were also found. The circular form was produced by the the terminal fusion of pCLU1. Insertion of a Ty element into a nuclearly migrated plasmid was observed, allowing the ROAM-regulated expression of the adjacent nuclearly silent UCS-fused LEU2 gene. The nuclearly located plasmids, whether linear or circular, were less sensitive to UV-mediated curing than pGKL and pCLU1.

Base Sequence↗

PubMed1991

Ultrastructural study of a cytoplasmic bridge connecting a pair of erythroblasts in mice.

A unique cytoplasmic connection between erythroblasts was studied by electron microscopy in mouse hemopoietic tissues (fetal liver, fetal and neonatal spleen and adult bone marrow). Many pairs of interphase erythroblasts were connected by a "cytoplasmic bridge" that was very thin and sometimes long in comparison with telophase bridges. The stage of maturation of the cells in a pair was similar. Small numbers of microtubules ran along the cytoplasmic bridge; a mid-body was not seen. The plasma membrane at approximately the middle of the bridge bulged to form a ring-shaped ridge filled with dense amorphous substances; this was called a "bulging ring." Thus, the cytoplasmic bridge between erythroblasts did not morphologically correspond to the telophase bridge in the usual cytokinesis. Cytoplasmic bridges were observed in various differentiating stages of erythroblasts, whereas other cell types of the hemopoietic lineage did not have such a bridge. The cytoplasmic bridge is unique to erythroblasts and provides an evidence for the atypical cytokinesis of the erythroblastic lineage.

Animals↗

PubMed1995

Transgenesis in mice by cytoplasmic injection of polylysine/DNA mixtures.

Pronuclear injection is currently the most often used method to make transgenic animals, but in some animal species it is temporally restrictive due to difficulty in visualizing pronuclei. However, the injection of construct DNA into the cytoplasm does not result in transgenesis. The production of transgenic mice by a cytoplasmic microinjection technique of polylysine complexed DNA into pronuclear stage zygotes is described. Transgenic mice were produced from cytoplasmic microinjection of mixtures of a 5.3 kb linearized DNA and poly-L-lysine (degree of polymerization = 51). Effects on transgenic frequency of both the lysine to phosphate ratio of polylysine to DNA and DNA concentration were studied. About 12.8% of the pups born from zygotes cytoplasmically microinjected with a polylysine/DNA mixture having a lysine to phosphate ratio (L:P) of 1:1 at a DNA concentration of 50 micrograms ml-1 were transgenic. The transgenic frequency for the pronuclear microinjection positive control of DNA alone was 21.7%. No transgenic pups were born from microinjection of DNA alone into the cytoplasm. Complexes of polylysine/DNA were detected using agarose gel electrophoresis at the conditions which produced transgenic mice. The presence of polylysine with construct DNA altered the in vitro activities of restriction endonuclease and DNA ligase on the construct DNA. The production of transgenic animals using DNA and polylysine in the absence of any other signal protein suggests that a DNA/polylysine complex but not DNA alone can act as a substrate for transgenesis from the cytoplasm.

Animals↗

PubMed2001

Day-to-night variations of cytoplasmic pH in a crassulacean acid metabolism plant.

In crassulacean acid metabolism (CAM) large amounts of malic acid are redistributed between vacuole and cytoplasm in the course of night-to-day transitions. The corresponding changes of the cytoplasmic pH (pHcyt) were monitored in mesophyll protoplasts from the CAM plant Kalanchoe daigremontiana Hamet et Perrier by ratiometric fluorimetry with the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein as a pHcyt indicator. At the beginning of the light phase, pHcyt was slightly alkaline (about 7.5). It dropped during midday by about 0.3 pH units before recovering again in the late-day-to-early-dark phase. In the physiological context the variation in pHcyt may be a component of CAM regulation. Due to its pH sensitivity, phosphoenolpyruvate carboxylase appears as a likely target enzyme. From monitoring delta pHcyt in response to loading the cytoplasm with the weak acid salt K-acetate a cytoplasmic H(+)-buffer capacity in the order of 65 mM H+ per pH unit was estimated at a pHcyt of about 7.5. With this value, an acid load of the cytoplasm by about 10 mM malic acid can be estimated as the cause of the observed drop in pHcyt. A diurnal oscillation in pHcyt and a quantitatively similar cytoplasmic malic acid is predicted from an established mathematical model which allows simulation of the CAM dynamics. The similarity of model predictions and experimental data supports the view put forward in this model that a phase transition of the tonoplast is an essential functional element in CAM dynamics.

Crassulaceae↗

PubMed1977

Nuclear and cytoplasmic RNase-activity in regenerating mouse liver.

Nuclear and cytoplasmic RNase activities at pH 5.0 and 7.6 were analyzed in regenerating mouse liver at 6, 12, 24, 48, and 72 h after partial hepatectomy. Two different nucleus-isolation methods were used, one in a EDTA-spermidine medium free from divalent cations, and one in a sucrose medium containing these ions. During regeneration, the cytoplasmic alkaline RNase activity in the sucrose medium was unchanged, but in the spermidine medium showed an increase toward the end of the period. Also the cytoplasmic acid RNase activity was unchanged in sucrose medium, whereas in the spermidine it slightly increased during regeneration. The nuclear alkaline RNase activity showed a notable peak 6 h after the operation and later decreased. Also the nuclear acid RNase activity displayed a similar marked peak 6 h after operation, then decreased, but remained high throughout the period. The nuclear RNase activities were about 1% of the corresponding cytoplasmic RNase activities. The absolute activities varied greatly according to the nucleus-isolation methods. In the controls, the absolute activity of nuclear alkaline RNase was slightly above (1.2 times) that of the corresponding acid activity after the spermidine method. After the sucrose method the nuclear alkaline activity was 2.7 times that of the acid activity. The absoluted activity of cytoplasmic alkaline RNase was slightly above (1.2 times) the acid activity after the spermidine method but after the sucrose method it was only 0.25 times that of the acid activity. In sham-operated animals, cytoplasmic acid and alkaline RNase activities generally were fairly similar to the normal value, but corresponding nuclear activities showed marked variations indicating an influence by anesthesia.

Animals↗

PubMed2000

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm.

The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (Cm) to monitor membrane changes in protoplasts from Vicia faba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 microM) at constant ambient osmotic pressure (pi omicron) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (fFM,i) increased with reference to the total fluorescence (fFM,t) by 7.4 x 10(-4) min(-1). This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of pi omicron by 200 mosmol kg(-1) caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time fFM,i/fFM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of Cm and total fluorescence of a protoplast (fFM,p) showed that an increase in pi omicron evoked a decrease in Cm but no change in fFM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 microm) with FM 1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area.

Cell Membrane↗

PubMed2005

Cytoplasmic expression of c-erbB2 in non-small cell lung cancers.

The aim of this study was to investigate the expression of c-erbB2 in non-small cell lung cancers (NSCLCs) with attention both to membranous and cytoplasmic reaction, and to try to elucidate the meaning of cytoplasmic expression of c-erbB2 in NSCLCs. Immunohistochemical c-erbB2 expression and related clinico-pathological features were examined in 312 surgically resected patient tissues of NSCLCs, including 175 cases of adenocarcinoma and 137 cases of squamous cell carcinoma. Immunostaining of inner- and ecto-domain of c-erbB2, mRNA expression and the quantitation of soluble c-erbB2 in cultured media were performed in five NSCLC cell lines. Cytoplasmic expression of c-erbB2 was observed more frequently than membranous, both in patient tissues and cell lines. Neither membranous nor cytoplasmic expression of c-erbB2 was significantly correlated with short outcome in NSCLCs. Membranous c-erbB2 was expressed by both inner and ecto-domain, while cytoplasmic c-erbB2 was expressed by either or both inner and ecto-domain. c-erbB2 mRNA was produced in most cell lines; however, the soluble form was only detectable in a cell line that only presented a membranous c-erbB2. In conclusion, cytoplasmic c-erbB2 of NSCLCs was not a full-length protein only expressed in cellular membrane, but reflected degenerated c-erbB2 fragments with less functional ability.

Adult↗

PubMed2003

Impact of hypoosmotic challenges on spongy architecture of the cytoplasm of the giant marine alga Valonia utricularis.

The ultrastructure of the several micrometers thick cytoplasmic layer of the giant marine alga Valonia utricularis displays characteristics which are apparently linked with the capability of this alga to regulate turgor pressure. Transmission and scanning electron microscopy of cells prefixed in different ways, including a protocol that allows prefixation of the alga in a turgescent state, revealed a highly dendritic network of cytoplasmic strands connecting and enveloping the chloroplasts and the nuclei. Innumerable vacuolar entities are embedded in the network, giving the cytoplasm a spongy appearance. Vacuolar perfusion of turgor-pressure-clamped cells with prefixation solution containing tannic acid presented evidence that these vacuolar entities together with the huge central vacuole form a large unstirred continuum. In contrast to the tonoplast, the plasmalemma followed smoothly the lining of the cell wall, even at the numerous cell wall ingrowths. Sucrose, but not polyethylene glycol 6000, induced chloroplast clustering. Acute hypoosmotic treatment (established by reduction of external NaCl or by replacement of part of the external NaCl by equivalent osmotic concentrations of sucrose or polyethylene glycol 6000) resulted in a local relocation of the chloroplasts and cytoplasm towards the central vacuole. This effect did not occur when the relatively low reflection coefficients of these two osmolytes were taken into account. The increase in spacing between the spongy cytoplasm and the plasmalemma by chloroplast relocation (viewed by confocal laser scanning microscopy) was associated with a speckled appearance of the affected surface area under the light microscope. As indicated by electron microscopy, hypoosmotically induced chloroplast relocation resulted from disproportionate swelling of the vacuolar entities located close to the plasmalemma. The cytoskeleton in the cytoplasm and the mucopolysaccharide network in the central vacuole apparently resisted swelling of these compartments. This finding has the important consequence that relevant hydrostatic pressure gradients can be built up throughout the entire multifolded vacuolar space. This gradient could represent the trigger for turgor pressure regulation which is manifested electrically first in the tonoplast.

Chlorophyta↗

PubMed1991

The effect of cytoplasmic K+ on the activity of the Na+/K(+)-ATPase.

Experiments with the reconstituted (Na+ + K+)-ATPase show that besides the ATP-dependent cytoplasmic Na(+)-K+ competition for Na+ activation there is a high affinity inhibitory effect of cytoplasmic K+. In contrast to the high affinity K+ inhibition seen with the unsided preparation at a low ATP especially at a low temperature, the high affinity inhibition by cytoplasmic K+ does not disappear when the ATP concentration an-or the temperature is increased. The high affinity inhibition by cytoplasmic K+ is also observed with Cs+, Li+ or K+ as the extracellular cation, but the fractional inhibition is much less pronounced than with Na+ as the extracellular cation. The results suggest that either there are two populations of enzyme, one with the normal ATP dependent cytoplasmic Na(+)-K+ competition, and another which due to the preparative procedure has lost this ATP sensitivity. Or that the normal enzyme has two pathways for the transition from E2-P to E1ATP. One on which the enzyme with the translocated ion binds cytoplasmic K+ with a high affinity but not ATP, and another on which ATP is bound but not K+. A kinetic model which can accommodate this is suggested.

Adenosine Triphosphate↗

PubMed1978

Cytoplasmic DNA synthesis in rhinovirus type 14-inoculated KB cells.

Rhinovirus type 14 (RV14) incuced a transient statistically significant stimulation in synthesis of DNA which appeared between 0 and 3 h post-inoculation in the cytoplasm of high density monolayer cultures of KB cells. Newly synthesized DNA was measured by incorporation of [3H] thymidine into acid-insoluble DNAase-sensitive material and the cytoplasmic location established by cell fractionation and electron microscope radioautographic methods. A minimum of 10 plaque-forming units per cell of RV14 was required to stimulate DNA synthesis which did not occur above 34.5 degrees C, a temperature optimal for virus replication. Cytoplasmic DNA taken from RV14-infected or control cells could be differentiated from the bulk of cell (nuclear) DNA by several criteria, including: (1) RV14 induction of synthesis; (2) lower buoyant density and greater heterogeneity in CsCl and ethidium bromide/CsCl gradients; and (3) a different kinetic complexity upon reannealing. The Cot 1/2 value of cytoplasmic DNA, calclated as 50--100 from reassociation profiles, was about 10-fold less complex than the Cot 1/2 value of nuclear DNA (800-1000). These data rule out the possibility that cytoplasmic DNA arises by random breakage of nuclear DNA during cell disruption and extraction and are compatible with the hypothesis that inoculation of KB cells with RV14 results in stimulation of synthesis of a specific class of cell DNA which is detected in the cytoplasm.

Cell Line↗

PubMed1984

Karyophobic proteins. A category of abundant soluble proteins which accumulate in the cytoplasm.

The cytoplasm of oocytes of Xenopus laevis is enriched in several soluble proteins which are either absent from the nucleus or are present there at very low concentrations. These molecules, collectively referred to as karyophobic (from the Greek verbs and which are meant here in the sense of "to be afraid of" or "to avoid") proteins represent more than 20% of the total soluble cytoplasmic proteins and include some of the most abundant soluble cellular components. They may be recovered from high-speed supernatant (S-100) fractions and, following sucrose gradient centrifugation, most of them appear in the form of complexes smaller than 8.5 S. On denaturation in urea and two-dimensional gel electrophoresis these proteins appear to be comprised of polypeptides of widely different sizes (ca Mr 15 000-230 000) and isoelectric points covering a broad range of pH values (4.2-8.0). Gel filtration and isoelectric focusing of native karyophobic proteins show that the majority occur in acidic complexes smaller than Mr 150 000, including one case of a small karyophobic protein (C9; Mr 30 000). In contrast to karyophilic proteins and proteins equilibrating between nucleus and cytoplasm karyophobic soluble proteins from [35S]methionine-labelled ooplasms, when injected into unlabelled oocytes, remain in the cytoplasm. Human proteins with a similar karyophobic behaviour have been identified in fractions of soluble proteins from HeLa cells; there, the major karyophobic protein (HCa Mr 36 000) is also one of the most abundant soluble proteins. We conclude that the specific nucleocytoplasmic compartmentalization of soluble proteins is governed not only by the principles of exclusion of large molecules from nuclear uptake and the existence of karyophilic signals in certain proteins but that a series of soluble, globular proteins exist in the cytoplasm, which have other molecular features which selectively exclude them from distribution over the nucleus. The possible functional role of the selective enrichment of these abundant proteins, which so far have escaped attention, in establishing a cytoplasmic milieu is discussed.

Animals↗

PubMed1991

Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli.

The very high concentration of macromolecules within cells can potentially have an overwhelming effect on the thermodynamic activity of cellular components because of excluded volume effects. To estimate the magnitudes of such effects, we have made an experimental study of the cytoplasm of Escherichia coli. Parameters from cells and cell extracts are used to calculate approximate activity coefficients for cytoplasmic conditions. These calculations require a representation of the sizes, concentrations and effective specific volumes of the macromolecules in the extracts. Macromolecule size representations are obtained either by applying a two-phase distribution assay to define a related homogeneous solution or by using the molecular mass distribution of macromolecules from gel filtration. Macromolecule concentrations in cytoplasm are obtained from analyses of extracts by applying a correction for the dilution that occurs during extraction. That factor is determined from experiments based upon the known impermeability of the cytoplasmic volume to sucrose in intact E. coli. Macromolecule concentrations in the cytoplasm of E. coli in either exponential or stationary growth phase are estimated to be approximately 0.3 to 0.4 g/ml. Macromolecule specific volumes are inferred from the composition of close-packed precipitates induced by polyethylene glycol. Several well-characterized proteins which bind to DNA (lac repressor, RNA polymerase) are extremely sensitive to changes in salt concentration in studies in vitro, but are insensitive in studies in vivo. Application of the activity coefficients from the present work indicates that at least part of this discrepancy arises from the difference in excluded volumes in these studies. Applications of the activity coefficients to solubility or to association reactions are also discussed, as are changes associated with cell growth phase and osmotic or other effects. The use of solutions of purified macromolecules that emulate the crowding conditions inferred for cytoplasm is discussed.

Cytoplasm↗

PubMed1982

Physicochemical properties of the cytoplasmic glucocorticoid receptor complex in HeLa S3 cells.

The physiochemical properties, size, shape, and surface charge, have been determined for the cytoplasmic glucocorticoid receptor complex (GR) in randomly growing HeLa S3 cells. Sucrose density gradient and gel exclusion chromatographic analysis have shown GR to undergo a marked reduction in size and shape when analyzed under increasing KCl concentration conditions. Analysis of GR prepared in a hypotonic buffer solution revealed a large, 7-8 S species with Ve/Vo ratio of 1.15, and a Stokes radius of congruent to 95 A. Increasing the KCl concentration in the analysis buffer to 0.15 resulted in reduction in GR size and shape to a 4.5 S species with the following properties: Ve/Vo = 1.38. Stokes radius = 69 A. and calculated Svedberg molecular weight = 132,000. A limit to the observed decrease in GR size and shape was obtained under hypertonic, 0.4 M KCl, conditions; a 3.75 S form was observed to elute from the gel exclusion column with a Ve/Vo = 1.40. Stokes radius = 65 A. and a calculated Svedburg molecular weight = 102,000. Exposure to higher KCl conditions, 0.6 M and 0.8 M, resulted in no further decrease in GR size or shape. Ion exchange chromatographic analysis of cytoplasmic GR revealed heterogeneous populations of GR with apparent differences in surface charge. GR binding to DEAE cellulose revealed a predominant form which eluted at 0.15 M KCl (Form I). Under both hypotonic and hypertonic conditions small populations of GR forms were observed to elute from DEAE at or approximately 0.1 M KCl (Form III), and or approximately 0.3 M KCl (Form II), respectively. Binding of GR to hydroxylapatite (HAP) confirmed the heterogenous nature of GR. Cytoplasmic GR partitioned into at least 3 forms om HAP. The predominant GR form eluted from HAP at 0.1 M K2HPO4 (Form I); secondary, and tertiary forms eluted at 0.125 M (Form II), and 0.15 M K2POH4 (Form III), respectively. Glucocorticoid receptors were observed to elute from columns of phosphocellulose, and DNA cellulose in the void volume. Two receptor forms were observed with isoelectric focusing of HeLa S3 cytoplasm in Sephadex IEF G-75 gels. The primary, and secondary species had pI values of 7.5 and 6.3, respectively. These results demonstrate that randomly growing HeLa S3 cells contain heterogeneous population of GR in the cytoplasmic compartment. Randomly growing HeLa S3 cell cytoplasm appears to contain two "unactivated" receptor species (Forms I and II) that differ in their overall surface charge properties; a third activated species (Form III) appears to arise from heat induced "activation" during cell homogenization, and is more basic in nature.

Cell Cycle↗

PubMed1984

Autoradiographic study of thermo-dependent nucleo-cytoplasmic distribution of aldosterone binding sites in intact target cells.

Autoradiographic studies of [3H]aldosterone [( 3H-A] and [3H]dexamethasone binding sites in intact target cells (isolated collecting tubules of rabbit and rat kidney) revealed an almost exclusive nuclear localization of the hormone-receptor complexes. In the present work we compared the nucleo-cytoplasmic repartition of [3H]A-receptor complexes studied in parallel by biochemical and autoradiographic methods. In addition, the thermo-dependency of the nuclear translocation was examined. Kidney pyramids were incubated in vitro with [3H]A (2 X 10(-9) M) in the presence or absence of a 100-fold excess unlabelled A, at 30 degrees C for 1 h or 4 degrees C for 2 h. Then tissue was processed for isolation of nuclear and cytoplasmic fractions, on the one hand, or for obtention of microdissected tubular segments on which autoradiographs on dry films were performed. Autoradiographs showed that the specific labelling was almost exclusively nuclear without significant cytoplasmic labelling, at both 30 or 4 degrees C. This indicates that almost all binding sites migrated rapidly into nuclei, and that this translocation did not depend on temperature. In contrast, parallel biochemical experiments yielded classical results, that is, at 30 degrees C, the presence of specific binding sites in both cytoplasm and nuclei with a predominance in cytoplasm. At 4 degrees C, the cytoplasmic binding was unchanged, but nuclear binding was drastically reduced, indicating thermodependency of nuclear translocation, when studied by biochemical methods including cell disruption. Autoradiographic results thus questioned the classical notion of thermo-dependent nuclear translocation of aldosterone-receptor complexes, based on results obtained by biochemical methods.

Aldosterone↗

PubMed1987

The transforming protein of avian reticuloendotheliosis virus is a soluble cytoplasmic protein which is associated with a protein kinase activity.

We have identified the product (p57v-rel) of the transforming gene, v-rel, of avian reticuloendotheliosis virus (REV-T) using antisera generated against nonoverlapping sequences representing the middle and carboxy-terminal regions of the v-rel protein expressed in Escherichia coli (N.K. Herzog and H.R. Bose, Jr., 1986, Proc. Natl. Acad. Sci. USA 83, 812-816). The amino-terminal region of the v-rel protein was also expressed in E. coli and used to generate antisera. The immunoglobulin-enriched fractions of these antisera were used to determine the subcellular location of p57v-rel in REV-T transformed lymphoid cells. Cells were fractionated into nuclear, mitochondrial, microsomal, and cytoplasmic fractions. The majority of p57v-rel was found in the cytoplasm. Examination of REV-T transformed lymphoid cells labeled with 32Pi revealed that the majority of the phosphorylated form of the v-rel protein was also found in the cytoplasm. Indirect immunofluorescence of REV-T transformed cells gave a diffuse cytoplasmic pattern indicating that p57v-rel was not associated with any discrete cellular organelle. The distribution of p57v-rel was similar in REV-T transformed lymphoid cells labeled with [35S]methionine for short and long periods of time, suggesting that p57v-rel is a soluble cytoplasmic protein throughout its lifetime. The v-rel protein was phosphorylated when immune complexes precipitated from transformed cells with the immunoglobulin fractions obtained from antisera against the amino-terminal, middle, and carboxy-terminal regions of v-rel were incubated with [gamma-32P]ATP and Mn2+. The phosphorylation of p57v-rel in the in vitro immune complex kinase assay was inhibited when the immunoglobulin-enriched fraction of these antisera was preincubated with the homologous v-rel fusion proteins. Preincubation with heterologous proteins did not block the phosphorylation of p57v-rel. These observations suggest that p57v-rel is associated with a protein kinase activity. Most of the kinase activity was found in the soluble cytoplasmic fraction of transformed cells. The transforming protein encoded by v-rel is a relatively stable protein with a half-life of approximately 7 to 8 hr in transformed lymphoid cells.

Cell Transformation, Viral↗

PubMed1984

Clonal aging in Paramecium tetraurelia. Absence of evidence for a cytoplasmic factor.

Cells of Paramecium tetraurelia show clonal aging with characteristics comparable to the aging seen in tissue culture cell lines. An investigation is underway to determine the relative contributions, if any, of the cytoplasm and the macronucleus to the onset of senescence. Using a microinjection protocol, the importance of the cytoplasm to aging was tested. Cytoplasm was transferred from young cells into old cells to see if the mean age of death of the injected cell lines could be increased, compared with uninjected controls and sham injected controls. Cytoplasm from old cells was transferred into young cells to see if the mean age of death of the injected cell lines could be decreased, compared with sham injected and uninjected controls. In neither case was there any statistically significant change in mean ages of death. Furthermore, no changes can be demonstrated even with a protocol which transplanted 3 times the normal cytoplasmic volume. Thus, a cytoplasmic effect upon vegetative aging in P. tetraurelia cannot be demonstrated. Tests of a macronuclear influence are now underway.

Aging↗