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PubMed2006

Injection of somatic cell cytoplasm into oocytes before intracytoplasmic sperm injection impairs full-term development and increases placental weight in mice.

This study investigated the effects on fertilized embryo development of somatic cytoplasm after its injection into intact mouse oocytes. Mature oocytes collected from female B6D2F1 mice were injected with cumulus cell cytoplasm of different volumes and from different mouse strains (B6D2F1, ICR, and C57BL/6), or with embryonic cytoplasm. After culture for 1 h, B6D2F1 sperm were injected into those oocytes by intracytoplasmic sperm injection (ICSI). The oocytes were examined for pre- and postimplantation developmental competence. Increases in the volume of the somatic cytoplasm from onefold to fourfold resulted in an impairment of blastocyst development and full-term development (28% and 7%, respectively, vs. 96% and 63%, respectively, in the control group; P < 0.01). An increase in the volume of somatic cytoplasm reduced the expression of POU5F1 (more commonly known as OCT4) in expanded blastocysts. The frequency of embryos that developed to the blastocyst stage did not differ when B6D2F1 or ICR somatic cytoplasm was injected, but injection of C57BL/6 somatic cytoplasm induced a two-cell block in embryo development. Injection of the cytoplasm from fertilized embryos did not reduce the frequency of embryos attaining full-term development. Interestingly, somatic cytoplasm significantly increased the placental weight of ICSI embryos, even the injection of onefold cytoplasm (0.20 +/- 0.02 [n = 32] vs. 0.12 +/- 0.02 in the control group [n = 87]; P < 0.01). These findings indicate that the injection of somatic cytoplasm into oocytes before ICSI causes a decrease in preimplantation development, clearly impairs full-term development, and causes placental overgrowth in fertilized embryos. To our knowledge, placental overgrowth phenotypes are only caused by interspecies hybridization and cloning, and in genetically modified mice. Here, we report for the first time that somatic cytoplasm causes abnormal placentas in fertilized embryos. This study suggests that somatic cell cytoplasmic material is one cause of the low rate of full-term development in cloned mammals.

Animals↗
PubMed1999

The cleavage and polyadenylation specificity factor in Xenopus laevis oocytes is a cytoplasmic factor involved in regulated polyadenylation.

During early development, specific mRNAs receive poly(A) in the cytoplasm. This cytoplasmic polyadenylation reaction correlates with, and in some cases causes, translational stimulation. Previously, it was suggested that a factor similar to the multisubunit nuclear cleavage and polyadenylation specificity factor (CPSF) played a role in cytoplasmic polyadenylation. A cDNA encoding a cytoplasmic form of the 100-kDa subunit of Xenopus laevis CPSF has now been isolated. The protein product is 91% identical at the amino acid sequence level to nuclear CPSF isolated from Bos taurus thymus. This report provides three lines of evidence that implicate the X. laevis homologue of the 100-kDa subunit of CPSF in the cytoplasmic polyadenylation reaction. First, the protein is predominantly localized to the cytoplasm of X. laevis oocytes. Second, the 100-kDa subunit of X. laevis CPSF forms a specific complex with RNAs that contain both a cytoplasmic polyadenylation element (CPE) and the polyadenylation element AAUAAA. Third, immunodepletion of the 100-kDa subunit of X. laevis CPSF reduces CPE-specific polyadenylation in vitro. Further support for a cytoplasmic form of CPSF comes from evidence that a putative homologue of the 30-kDa subunit of nuclear CPSF is also localized to the cytoplasm of X. laevis oocytes. Overexpression of influenza virus NS1 protein, which inhibits nuclear polyadenylation through an interaction with the 30-kDa subunit of nuclear CPSF, prevents cytoplasmic polyadenylation, suggesting that the cytoplasmic X. laevis form of the 30-kDa subunit of CPSF is involved in this reaction. Together, these results indicate that a distinct, cytoplasmic form of CPSF is an integral component of the cytoplasmic polyadenylation machinery.

Amino Acid Sequence↗
PubMed1985

A proton-translocating ATPase regulates pH of the bacterial cytoplasm.

Regulatory mechanisms of cytoplasmic pH in Streptococcus faecalis with no respiratory chain were investigated. In a mutant defective in cytoplasmic alkalization conducted by a proton-translocating ATPase (H+-ATPase), the cytoplasmic pH is approximately 0.4 to 0.5 pH units lower than the medium pH, at pH 5.5 to 9.0. The cytoplasmic pH of the wild-type strain was always higher than that of the mutant at a pH below 8 and was the same as that of the mutant at an alkaline pH over 8. Thus, the cytoplasmic pH is regulated only by the cytoplasmic alkalization, and there is no regulation at alkaline pH in S. faecalis. A generation of the protonmotive force conducted by the H+-ATPase depended on the cytoplasmic pH rather than the medium pH, and the generation decreased rapidly when the cytoplasmic pH was increased over 7.7. The decrease at alkaline pH was not caused by increases in the rate of proton influx. These results suggest that cytoplasmic alkalization is diminished when alkaline pH of the cytoplasm is over 7.7, because of a low activity of proton extrusion by the H+-ATPase, and consequently, the cytoplasmic pH is regulated at about 7.7. The cytoplasmic pH was regulated at a high level in cells that had a high level of H+-ATPase. I conclude that in S. faecalis, the cytoplasmic pH is regulated by H+-ATPase.

Cytoplasm↗
PubMed1976

Aminoacylation of Phaseolus vulgaris cytoplasmic, chloroplastic and mitochondrial tRNAsPro and tRNAsLys by homologous and heterologous enzymes.

The cytoplasmic prolyl-tRNA synthetase can be separated by hydroxyapatite chromatography, from the enzyme present in the chloroplasts and in the mitochondria (organellar enzyme). The cytoplasmic lysyl-tRNA synthetase can also be separated from the organellar enzyme. There are two tRNAsPro in the cytoplasm; they can be charged by the cytoplasmic enzyme, but not by the organellar enzyme or the Escherichia coli enzyme. Chloroplasts contain, in addition to the two cytoplasmic tRNAsPro, one chloroplast-specific tRNAPro, which is not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E. coli enzyme. Mitochondria contain, in addition to the two cytoplasmic tRNAsPro, two mitochondria-specific tRNAsPro, which are not recognized by the cytoplasmic enzyme, but can be charged by the organellar or the E. coli enzyme. There are two tRNAsLys in the cytoplasm. Both can be charged by the cytoplasmic enzyme, but one can be charged by the organellar or E. coli enzyme. Chloroplasts contain in addition to one cytoplasmic tRNALys, one chloroplast-specific tRNALys which can only be charged by the organellar or E. coli enzyme. Mitochondria contain, in addition to one cytoplasmic tRNALys, one mitochondria-specific tRNALys which can only be charged by the organellar or E. coli enzyme.

Amino Acyl-tRNA Synthetases↗
PubMed1999

Subcellular trafficking of the cytoplasmic expression system.

Cationic liposomes have provided many advantages over viral vector formulations; however, the problem of inefficient gene expression remains. This is due in part to the nuclear membrane, which limits DNA entry into the nucleus. Cytoplasmic expression systems using T7 RNA polymerase have been developed to express genes in the cytoplasm and avoid the need for nuclear import of DNA. Although these systems show improved transgene expression, little is known about how they function in transfected cells. Direct comparisons between a cytoplasmic and nuclear expression system were carried out with a 293 cell line stably expressing T7 RNA polymerase. A formulation for optimal reporter gene expression was developed and used in conjunction with a variety of subcellular trafficking inhibitors to study the process of DNA endocytosis. Transfected cells were also studied at different stages of the cell cycle to determine the dependence of each system on mitosis. These results showed that cytoplasmic and nuclear expression systems utilize similar endocytosis pathways to the point of endosomal release. Once DNA is released into the cytoplasm, the cytoplasmic expression system shows immediate expression that is proportional to the amount of DNA released. In contrast, DNA targeted for nuclear expression requires additional time for nuclear entry. The level of nuclear expression is also restricted by the limited amount of DNA that is imported into the nucleus. Finally, mitosis is required for effective nuclear expression but not for cytoplasmic expression. Therefore, the cytoplasmic expression system has considerable advantages over traditional nuclear expression systems and may be an effective method for transfecting nondividing cells. Efficient expression of genes delivered by nonviral vectors is hindered owing to poor nuclear transport of plasmid DNA. A potential solution to this problem would be to use a cytoplasmic expression system. Previous studies have shown that this method produces enhanced gene expression when compared with traditional nuclear expression systems; however, the actual mechanisms by which the cytoplasmic expression system works remains unknown. This article focuses on a direct comparison between cytoplasmic and nuclear expression in terms of optimal DNA delivery formulations, intracellular trafficking of DNA, and cell cycle dependence. These results indicate that the cytoplasmic expression system has two primary advantages over nuclear expression in that it does not rely on nuclear DNA transport or mitosis for efficient expression.

Anti-Bacterial Agents↗
PubMed2004

Clinical applications of antineutrophil cytoplasmic antibody testing.

PURPOSE OF REVIEW: Antineutrophil cytoplasmic antibodies are closely associated with Wegener granulomatosis, microscopic polyangiitis, and Churg-Strauss syndrome and have contributed to new pathogenetic concepts and improved nomenclature of systemic vasculitides (antineutrophil cytoplasmic antibody-associated vasculitides). However, the application of antineutrophil cytoplasmic antibody testing as a clinical diagnostic tool is still regarded as controversial. This review summarizes the most recent developments in the field, identifies areas of uncertainty, and gives practical guidelines. RECENT FINDINGS: The problems of antineutrophil cytoplasmic antibody testing include the diversity of antineutrophil cytoplasmic antibody target antigens, assay standardization and performance, the application of antineutrophil cytoplasmic antibody testing in a clinical setting with a low pretest probability, and, finally, the widespread assumption that antineutrophil cytoplasmic antibody titers alone may closely reflect disease activity and therefore may be used to guide therapy. SUMMARY: Recent findings demonstrate that the combined use of indirect immunofluorescence tests and solid phase assays to detect antineutrophil cytoplasmic antibody directed against myeloperoxidase and proteinase 3 can minimize the occurrence of false-positive antineutrophil cytoplasmic antibody results. Furthermore, the yield of antineutrophil cytoplasmic antibody testing can be improved by the use of a well-standardized test, adherence to published guidelines, and restricting the use of the tests to clinical situations with a rather high pretest probability for antineutrophil cytoplasmic antibody-associated vasculitides. However, treatment decisions should be based on the clinical presentation of the patient and histologic findings and not on the results of antineutrophil cytoplasmic antibody testing alone.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed2006

Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells.

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non-ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non-neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE2-M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1-5-microm inclusion bodies and were filled with electron-dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild-type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non-neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.

Animals↗
PubMed1993

Cortical cytoplasm, which induces dorsal axis formation in Xenopus, is inactivated by UV irradiation of the oocyte.

Localized maternal determinants control the formation of dorsal axial structures in Xenopus embryos. To examine the spatial distribution of dorsal determinants, we injected cytoplasm from various regions of the egg and 16-cell embryo into the ventral vegetal cells of a 16-cell recipient embryo. Cortical cytoplasm from the egg vegetal surface induced the formation of a secondary dorsal axis in 53% of recipients. In contrast, animal cortical, equatorial cortical and vegetal deep cytoplasm never induced secondary axis formation. We also compared the axis-inducing ability of animal versus vegetal dorsal cortical cytoplasm from 16-cell embryos. Significantly more dorsalizing activity was found in vegetal dorsal cytoplasm compared to animal dorsal cytoplasm at this stage. Previous work has shown that UV irradiation of the vegetal surface of either prophase I oocytes, or fertilized eggs, leads to the development of embryos that lack dorsal structures. Egg vegetal cortical cytoplasm was capable of restoring the dorsal axis of 16-cell recipient embryos derived from UV-irradiated oocytes or fertilized eggs. We also tested the axis inducing ability of cytoplasm obtained when UV-irradiated oocytes and eggs were treated as donors of cytoplasm. While vegetal cortical cytoplasm from UV-irradiated fertilized eggs retains its dorsalizing activity, cytoplasm obtained from eggs, UV irradiated as oocytes, does not. The egg vegetal cortex provides a suitable source for the isolation of maternal dorsal determinants. In addition, since UV irradiation of the oocyte vegetal surface destroys the dorsalizing activity of transferred cytoplasm, UV can be used to further restrict possible candidates for such determinants.

Animals↗
PubMed1997

Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells.

The existence of pronounced cytoplasmic pH gradients within the apices of tip-growing cells, and the role of cytoplasmic pH in regulating tip growth, were investigated in three different cell types: vegetative hyphae of Neurospora crassa; pollen tubes of Agapanthus umbellatus; and rhizoids of Dryopteris affinis gametophytes. Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual emission confocal ratio imaging of the pH-sensitive probe carboxy SNARF-1. Considerable attention was paid to the fine tuning of dye loading and imaging parameters to minimise cellular perturbation and assess the extent of dye partitioning into organelles. With optimal conditions, cytoplasmic pH was measured routinely with a precision of between +/-0.03 and +/-0.06 of a pH unit and a spatial resolution of 2.3 microm2. Based on in vitro calibration, estimated values of mean cytoplasmic pH for cells loaded with dye-ester were between 7.15 and 7.25 for the three cell types. After pressure injecting Neurospora hyphae with dextran-conjugated dye, however, the mean cytoplasmic pH was estimated to be 7.57. Dextran dyes are believed to give a better estimate of cytoplasmic pH because of their superior localisation and retention within the cytosol. No significant cytoplasmic pH gradient (delta pH of >0.1 unit) was observed within the apical 50 microm in growing cells of any of the three cell types. Acidification or alkalinisation of the cytoplasm in Neurospora hyphae, using a cell permeant weak acid (propionic acid at pH 7.0) or weak base (trimethylamine at pH 8.0), slowed down but did not abolish growth. However, similar manipulation of the cytoplasmic pH of Agapanthus pollen tubes and Dryopteris rhizoids completely inhibited growth. Modification of external pH affected the growth pattern of all cell types. In hyphae and pollen tubes, changes in external pH were found to have a small transient effect on cytoplasmic pH but the cells rapidly readjusted towards their original pH. Our results suggest that pronounced longitudinal gradients in cytoplasmic pH are not essential for the regulation of tip growth.

Benzopyrans↗
PubMed1990

[Histogenesis of the clear cytoplasm in the tumor cells of smooth muscle origin].

Perinuclear clear cytoplasm observed in the light microscopic specimens of the tumor cells of smooth muscle origin is in general, understood as the artefact caused by the formalin fixation. However, the precise mechanisms of the histogenesis of clear cytoplasm are still not clear. We observed the clear cytoplasm directly by mean of the electron microscopy of materials detached from light microscopic specimen. Furthermore, we observed the light microscopic specimens made by varying types of methods, examining whether the clear cytoplasm was present or not. The electron microscopy of materials detached from light microscopic specimens revealed the band-like defects of cytoplasm along the long axes of tumor cells. These defects were thought to result from the falling off of cytoplasm. The 1 mu section of the epon embedded block derived from the paraffin embedded block for light microscopic specimen presented no clear cytoplasm, suggesting that the cytoplasm falls off at the procedure of deparaffinization and staining. Although the specimen of conventional frozen section showed no clear cytoplasm, the specimen made by the frozen sectioning after formalin fixation revealed clear cytoplasm. Consequently, it is thought that the fixation of the tissue before the sectioning makes the cytoplasm fragile, thereafter, the cytoplasm falls off at the procedure of deparaffinization and staining.

Adult↗
PubMed1993

Prevalence and characterization of neutrophil cytoplasmic antibodies in autoimmune liver diseases.

To evaluate the diagnostic significance of neutrophil cytoplasmic antibodies in chronic liver diseases, we assessed the prevalence of neutrophil cytoplasmic antibodies in autoimmune liver diseases, in particular in primary sclerosing cholangitis, autoimmune chronic active hepatitis and primary biliary cirrhosis, and we also determined the specificity of perinuclear-pattern neutrophil cytoplasmic antibodies for these autoimmune liver diseases by testing sera from patients with nonautoimmune chronic liver diseases. Neutrophil cytoplasmic antibodies were detected in 79% of sera from patients with primary sclerosing cholangitis (n = 24), in 88% of sera from patients with autoimmune chronic active hepatitis (n = 24) and in 28% of sera from patients with primary biliary cirrhosis (n = 25). The presence of neutrophil cytoplasmic antibodies in these diseases correlated significantly (p < 0.008) with the presence of cirrhosis. Neutrophil cytoplasmic antibodies were not detected in nonautoimmune liver diseases. All neutrophil cytoplasmic antibody-positive sera produced a perinuclear fluorescence pattern on ethanol-fixed granulocytes. On neutrophils fixed with paraformaldehyde, a granular cytoplasmic immunofluorescence pattern was observed, demonstrating the cytoplasmic nature of the antigen or antigens involved. Further characterization studies showed that neutrophil cytoplasmic antibodies in autoimmune liver diseases are not directed against myeloperoxidase, proteinase 3 or elastase, the neutrophil cytoplasmic antibody specificities associated with necrotizing vasculitis, glomerulonephritis or both. On Western blots neutrophil cytoplasmic antibodies in autoimmune liver diseases showed reactivity with either lactoferrin, a 67/66-kD protein combination or a 40-kD polypeptide. Reactivity with either of these proteins was observed in sera from patients with primary sclerosing cholangitis (38%), autoimmune chronic active hepatitis (17%) and primary biliary cirrhosis (20%).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1987

Cytoplasmic genetic effects on preweaning growth and milk yield in Hereford cattle.

Performance records on Hereford cattle raised in two herds were used to evaluate cytoplasmic genetic effects on preweaning growth and milk production. Animals were traced through maternal lineage to foundation females to form cytoplasmic lines. Growth records were available on 1,189 calves at Raleigh and 1,599 at Plymouth representing 27 and 15 cytoplasmic lines, respectively. Milk records were available on 418 cows at Raleigh and 522 cows at Plymouth, representing 20 and 13 cytoplasmic lines. After adjustment for sire, cytoplasmic effects were significant for birth weight (BWT), average daily gain (ADG) and 205-d weight (WT205) in both herds. Cytoplasm accounted for 2, 5 and 5% of the variance for BWT, ADG and WT205 at Raleigh; and 1, 2 and 2% of the variance at Plymouth. After addition of maternal grandsire to the model, cytoplasm was still significant; however, variances were reduced at Plymouth. Cytoplasmic effects for milk yield were important at Raleigh (P less than .01) but marginal at Plymouth (P = .10). Variance components for cytoplasm accounted for 4 and 1% of the variance for milk yield at Raleigh and Plymouth, respectively. Ranges for least-squares constants for cytoplasmic lines corresponded to one of two standard deviations. Correlations among least-squares constants for ADG, WT205 and milk yield were high, suggesting that cytoplasmic effects were mediated through milk production. More research is needed to confirm these results before cytoplasmic inheritance is considered in breeding programs for beef cattle.

Animals↗
PubMed1983

Regulation of cytoplasmic streaming in Vallisneria mesophyll cells.

Induction and cessation of the rotational cytoplasmic streaming in Vallisneria mesophyll cells could be controlled by external stimuli. In cells that had been kept in darkness the cytoplasm remained quiescent. However, when the cells were treated in the dark with EGTA solution (10 mM or 20 mM buffered with 10 mM-Tris-maleate at pH 7.0), rotational cytoplasmic streaming was induced. When the cells were transferred again to artificial pond water in the dark, the induced streaming was inhibited; that is, only 50% of the observed cells exhibited active streaming after 2 h. When the cells were irradiated continuously with far-red light (lambda max = 750 nm, 0.4 W/m2) in the same external medium, the induced streaming was inhibited almost completely within 2 h. The relative quantum effectiveness of monochromatic light (450--800 nm) in producing cessation of streaming was also investigated. Irradiation with light of 450, 550 and 600 nm was almost as effective as darkness. Light of 500 and 650 nm was less effective than dark exposure. Only irradiation at 750 nm stopped streaming in almost all cells. But when calcium was excluded from the external medium, the effect of far-red light decreased to almost the dark control level. Light of 800 nm also inhibited the streaming but the effect was much less than that of far-red light. Microfilaments in bundles with the long axis parallel to the streaming direction were localized in the vicinity of the cell membrane. Their configuration, localization and distribution were the same in the present experimental system irrespective of whether the cytoplasm was streaming or quiescent. Intracellular calcium was examined by electron microscopic cytochemistry and X-ray microanalysis. In cells with streaming induced by EGTA, only a small amount of calcium-containing precipitates formed in the cytoplasm in the presence of antimony. A few precipitates were found in the chloroplasts, the middle lamella of the cell wall and at the border between the cytoplasm and the cell wall. On the other hand, in cells treated with EGTA and subsequently irradiated with far-red light in artificial pond water, many precipitates were observe in the cytoplasm, chloroplasts, mitochondria and endoplasmic reticulum. The middle lamella was also heavily stained. On the basis of these observations, it was concluded that rotational cytoplasmic streaming in Vallisneria cells can be induced when the free calcium concentration in the cytoplasm decreases and that the induced streaming is arrested when the free calcium concentration in the cytoplasm increases. Far-red light accelerates the increase of calcium in the cytoplasm.

Calcium↗
PubMed2001

Yeast cytoplasmic and mitochondrial methionyl-tRNA synthetases: two structural frameworks for identical functions.

The yeast Saccharomyces cerevisiae possesses two methionyl-tRNA synthetases (MetRS), one in the cytoplasm and the other in mitochondria. The cytoplasmic MetRS has a zinc-finger motif of the type Cys-X(2)-Cys-X(9)-Cys-X(2)-Cys in an insertion domain that divides the nucleotide-binding fold into two halves, whereas no such motif is present in the mitochondrial MetRS. Here, we show that tightly bound zinc atom is present in the cytoplasmic MetRS but not in the mitochondrial MetRS. To test whether the presence of a zinc-binding site is required for cytoplasmic functions of MetRS, we constructed a yeast strain in which cytoplasmic MetRS gene was inactivated and the mitochondrial MetRS gene was expressed in the cytoplasm. Provided that methionine-accepting tRNA is overexpressed, this strain was viable, indicating that mitochondrial MetRS was able to aminoacylate tRNA(Met) in the cytoplasm. Site-directed mutagenesis demonstrated that the zinc domain was required for the stability and consequently for the activity of cytoplasmic MetRS. Mitochondrial MetRS, like cytoplasmic MetRS, supported homocysteine editing in vivo in the yeast cytoplasm. Both MetRSs catalyzed homocysteine editing and aminoacylation of coenzyme A in vitro. Thus, identical synthetic and editing functions can be carried out in different structural frameworks of cytoplasmic and mitochondrial MetRSs.

Acylation↗
PubMed1991

Characterization of the cytoplasm of Escherichia coli K-12 as a function of external osmolarity. Implications for protein-DNA interactions in vivo.

The water-accessible volumes, the amounts of all significant osmolytes, and the protein concentration in the cytoplasm of aerobically grown Escherichia coli K-12 have been determined as a function of the osmolarity of the minimal growth medium. The volume of cytoplasmic water (Vcyto) decreases linearly with increasing osmolarity from 2.23(+/- 0.12) microliters/mg dry weight in cells grown at 0.10 OSM to 1.18(+/- 0.06) microliters/mg dry weight at 1.02 OSM. Above 0.28 OSM, growth rate decreases linearly with increasing osmolarity. The growth rate extrapolates to zero at an osmolarity of approximately 1.8, corresponding to an estimated Vcyto of 0.5(+/- 0.2) microliters/mg dry weight. Measurements of Vcyto in titrations of non-growing cells with the plasmolyzing agent NaCl were used to obtain volumes of "bound" water (presumably water of macromolecular hydration) and cytoplasmic osmotic coefficients for cells grown in medium of low (0.10 OSM) and moderate (0.28 OSM) osmolarity. The volume of bound water Vb is similar in the two osmotic conditions (Vb = 0.40(+/- 0.04) microliters/mg dry wt), and corresponds to approximately 0.5 g H2O/g cytoplasmic macromolecule. Since Vcyto decreases with increasing osmolarity, whereas Vb appears to be independent of osmolarity, water of hydration becomes a larger fraction of Vcyto as the osmolarity of the growth medium increases. Growth appears to cease at the osmolarity where Vcyto is approximately equal to Vb. K+ and glutamate (Glu-) are the only significant cytoplasmic osmolytes in cells grown in medium of low osmolarity. The amount of K+ greatly exceeds that of Glu-. Analysis of cytoplasmic electroneutrality indicates that the cytoplasm behaves like a concentrated solution of the K+ salt of cytoplasmic polyanions, in which the amount of additional electrolyte (K+ Glu-) increases with increasing osmolarity. As the osmolarity of the growth medium becomes very low, the cytoplasm approaches an electrolyte-free K+-polyanion solution. In vivo osmotic coefficients were determined from the variation of Vcyto with external osmolarity in plasmolysis titrations of non-growing cells. The values obtained (phi = 0.54(+/- 0.06) for cells grown at 0.10 OSM and phi = 0.71(+/- 0.11) at 0.28 OSM) indicate a high degree of non-ideality of intracellular ions arising from coulombic interactions between K+ and cytoplasmic polyanions. Analysis of these osmotic coefficients using polyelectrolyte theory indicates that the thermodynamic activity of cytoplasmic K+ increases from approximately 0.14 M in cells grown at an external osmolarity of 0.10 OSM to approximately 0.76 M at 1.02 OSM.(ABSTRACT TRUNCATED AT 400 WORDS)

Biophysical Phenomena↗
PubMed2003

p53-associated 3'-->5' exonuclease activity in nuclear and cytoplasmic compartments of cells.

The tumor suppressor protein p53 plays an important role in maintenance of the genomic integrity of cells. p53 possesses an intrinsic 3'-->5' exonuclease activity. p53 was found in the nucleus and in the cytoplasm of the cell. In order to evaluate the subcellular location and extent of p53-associated 3'--> 5' exonuclease activity, we established an in vitro experimental system of cell lines with different nuclear/cytoplasmic distribution of p53. Nuclear and cytoplasmic extracts obtained from LCC2 cells (expressing a high level of cytoplasmic wild-type p53), MCF-7 cells (expressing a high level of wild-type nuclear p53), MDA cells (expressing mutant p53) and H1299 cells (p53-null) were subjected to the analysis of exonuclease activity. Interestingly, 3'-->5' exonuclease was predominantly cytoplasmic; the nuclear extracts derived from all cell lines tested, exerted a low level of exonuclease activity. Cytoplasmic extracts of LCC2 cells, with a high level of wild-type p53, showed an enhanced exonuclease activity in comparison to those expressing either a low level of wild-type p53 (in MCF-7 cells) or the mutant p53 (in MDA cells). Evidence that exonuclease function detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: First, this activity closely parallels with levels and status of endogenous cytoplasmic p53. Second, immunoprecipitation of p53 from cytoplasmic extracts of LCC2 cells markedly reduced the exonuclease activity. Third, the observed 3'-->5' exonuclease in cytoplasmic fraction of LCC2 cells displays identical biochemical properties characteristic of recombinant wild-type p53. The biochemical functions include: (a) substrate specificity; exonuclease hydrolyzes single-stranded DNA in preference to double-stranded DNA and RNA/DNA template-primers, (b) efficient excision of 3'-terminal mispairs from DNA/DNA and RNA/DNA substrates, (c) the preferential excision of purine-purine mispairs over purine-pyrimidine mispairs and (d) functional interaction with exonuclease-deficient DNA polymerase, for example, murine leukemia virus reverse transcriptase (representing a relatively low fidelity enzyme), thus enhancing the fidelity of DNA synthesis by excision of mismatched nucleotides from the nascent DNA strand. Taken together, the data demonstrate that wild-type p53 in cytoplasm, in its noninduced state, is functional; it displays intrinsic 3'-->5' exonuclease activity. The possible role of p53-associated 3'-->5' exonuclease activity in DNA repair in nucleus and cytoplasm is discussed.

Breast Neoplasms↗
PubMed2000

Role of cytoplasmic dynein in melanosome transport in human melanocytes.

Cytoplasmic dynein is a microtubule-associated retrograde-directed motor molecule for transport of membrane-bound organelles. To determine whether cytoplasmic dynein is expressed in melanocytes, we performed reverse transcriptase polymerase chain reaction using melanocyte cDNA and primers complementary to human brain cytoplasmic dynein heavy chain. A polymerase chain reaction product of the expected molecular size was generated and the identity was confirmed by sequence analysis. Western blotting of total melanocyte proteins reacted with an anti-intermediate chain cytoplasmic dynein antibody identified the appropriate 74 kDa band. To determine whether cytoplasmic dynein plays a role in melanosome transport, duplicate cultures were treated with cytoplasmic dynein antisense or sense (control) oligodeoxynucleotides and the cells were observed by high-resolution time-lapse microscopy, which allows visualization of melanosomal aggregates and individual melanosomes. Antisense-treated melanocytes demonstrated a strong anterograde transport of melanosomes from the cell body into the dendrites, whereas melanosome distribution was not affected in sense-treated melanocytes. To determine whether ultraviolet irradiation modifies cytoplasmic dynein expression, melanocyte cultures were exposed to increasing doses of solar-simulated irradiation, equivalent to a mild to moderate sunburn exposure for intact skin. Within 24 h, doses of 5 and 10 mJ per cm2 induced cytoplasmic dynein protein, whereas doses of 30 mJ per cm2 or more were associated with decreased levels of cytoplasmic dynein compared with sham-irradiated controls. Our data show that cytoplasmic dynein participates in retrograde melanosomal transport in human melanocytes and suggest that the altered melanosomal distribution in skin after sun exposure is due, at least in part, to decreased cytoplasmic dynein levels resulting in augmented anterograde transport.

Base Sequence↗
PubMed1997

A conserved sequence motif in the integrin beta3 cytoplasmic domain is required for its specific interaction with beta3-endonexin.

Integrin signaling is mediated by interaction of integrin cytoplasmic domains with intracellular signaling molecules. Recently, we identified a novel 111-amino acid polypeptide, termed beta3-endonexin, which interacts selectively with the integrin beta3 cytoplasmic domain. In the present study we conducted a systematic mutational analysis of both the integrin beta3 cytoplasmic domain and beta3-endonexin to map sites required for interaction. The interaction of the full-length beta3 integrin subunit with beta3-endonexin in vitro required the beta3 cytoplasmic domain. In a yeast two-hybrid system, both membrane-proximal and membrane-distal residues of the beta3 cytoplasmic domain were necessary for interaction with beta3-endonexin. In particular, the membrane-distal NITY motif at beta3 756-759 was critical for the interaction. Exchange of beta3 residues 756-759 (NITY) for the corresponding residues in beta1 (NPKY) endowed the beta1 cytoplasmic domain with the ability to interact with beta3-endonexin. Conversely, exchange of the NPKY motif at beta1 772-775 for the NITY motif in beta3 abolished interaction of this chimeric cytoplasmic domain with beta3-endonexin. Because the NITY motif is present in the beta3 but not the beta1 cytoplasmic domain, these results explain the selective interaction of this cytoplasmic domain with beta3-endonexin. In addition, deletional analysis suggested that a core 91-residue sequence of beta3-endonexin is sufficient for specific binding to the beta3 cytoplasmic domain. These studies have identified a cytoplasmic domain sequence motif that specifies an integrin-specific protein-protein interaction.

Amino Acid Sequence↗