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At least 253 records · Page 14Linked to original sources
PubMed2005

Cytoplasmic phospholipase A2 alpha overexpression in stromal cells is correlated with angiogenesis in human colorectal cancer.

In colorectal cancer, cyclooxygenase-2 (COX-2) overexpression in stromal cells induces angiogenesis through EP2 prostaglandin E2 receptor signaling. Cytoplasmic phospholipase A2 (PLA2) alpha preferentially hydrolyses arachidonic acid, which is the limiting substrate for prostaglandin production, from membrane phospholipids. We therefore investigated a possible relationship between cytoplasmic PLA2 and COX-2 overexpression in stromal cells, angiogenesis and microsatellite instability in 48 human colorectal adenocarcinomas. Cytoplasmic PLA2 and COX-2 expression in stromal cells and vascular endothelial growth factor (VEGF) expression in tumor cells were evaluated by immunohistochemistry. Microvessel density was assessed in 10 x 400 fields after CD31 staining. Microsatellite instability was evaluated by PCR and immunohistochemistry. A total of 16 tumors had microsatellite instability. We found an overexpression of cytoplasmic PLA2 in superficial stromal cells. These cells corresponded to fibroblasts and myofibroblasts. There was an association between the number of cytoplasmic PLA2 and COX-2-expressing cells (P=0.006). Cytoplasmic PLA2-positive stromal cells usually also expressed COX-2. A high number of cytoplasmic PLA2-positive stromal cells was correlated with a high microvessel density (P=0.002), a strong VEGF (P=0.01) and the absence of microsatellite instability (P=0.001). The coordinate overexpression of cytoplasmic PLA2 and COX-2 in stromal cells could lead to an important prostaglandin production. These results suggest that cytoplasmic PLA2 overexpression in these cells regulates COX-induced angiogenesis probably by providing arachidonic acid, which is the limiting factor for prostaglandin production. The lower number of cytoplasmic PLA2-positive stromal cells in carcinomas with microsatellite instability could be related to their lower microvessel density and VEGF expression.

Adult↗
PubMed1997

Modulation of AUUUA response element binding by heterogeneous nuclear ribonucleoprotein A1 in human T lymphocytes. The roles of cytoplasmic location, transcription, and phosphorylation.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) shuttles between the cytoplasm and nucleus and plays important roles in RNA metabolism. Whereas nuclear hnRNP A1 has been shown to bind intronic sequences and modulate splicing, cytoplasmic hnRNP A1 is associated with poly(A)+ RNA, indicating different RNA ligand specificity. Previous studies indicated that cytoplasmic hnRNP A1 is capable of high-affinity binding of reiterated AUUUA sequences (ARE) that have been shown to modulate mRNA turnover and translation. Through a combination of two-dimensional gel and proteolysis studies, we establish hnRNP A1 (or structurally related proteins that are post-translationally regulated in an identical manner) as the dominant cytoplasmic protein in human T lymphocytes capable of interacting with the ARE contained within the context of full-length granulocyte-macrophage colony-stimulating factor mRNA. We additionally demonstrate that cytoplasmic hnRNP A1 preferentially binds ARE relative to pre-mRNAs in both cross-linking and mobility shift experiments. RNA polymerase II inhibition increased the binding of ARE (AUBP activity) and poly(U)-Sepharose by cytoplasmic hnRNP A1, while nuclear hnRNP A1 binding was unaffected. Nuclear and cytoplasmic hnRNP A1 could be distinguished by the differential sensitivity of their RNA binding to diamide and N-ethylmaleimide. The increase in AUBP activity of cytoplasmic hnRNP A1 following RNA polymerase II inhibition correlated with serine-threonine dephosphorylation, as determined by inhibitor and metabolic labeling studies. Thus, cytoplasmic and nuclear hnRNP A1 exhibit different RNA binding profiles, perhaps transduced through serine-threonine phosphorylation. These findings are relevant to the specific ability of hnRNP A1 to serve distinct roles in post-transcriptional regulation of gene expression in both the nucleus and cytoplasm.

Adenosine↗
PubMed1991

Low viscosity in the aqueous domain of cell cytoplasm measured by picosecond polarization microfluorimetry.

Information about the rheological characteristics of the aqueous cytoplasm can be provided by analysis of the rotational motion of small polar molecules introduced into the cell. To determine fluid-phase cytoplasmic viscosity in intact cells, a polarization microscope was constructed for measurement of picosecond anisotropy decay of fluorescent probes in the cell cytoplasm. We found that the rotational correlation time (tc) of the probes, 2,7-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein (BCECF), 6-carboxyfluorescein, and 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) provided a direct measure of fluid-phase cytoplasmic viscosity that was independent of probe binding. In quiescent Swiss 3T3 fibroblasts, tc values were 20-40% longer than those in water, indicating that the fluid-phase cytoplasm is only 1.2-1.4 times as viscous as water. The activation energy of fluid-phase cytoplasmic viscosity was 4 kcal/mol, which is similar to that of water. Fluid-phase cytoplasmic viscosity was altered by less than 10% upon addition of sucrose to decrease cell volume, cytochalasin B to disrupt cell cytoskeleton, and vasopressin to activate phospholipase C. Nucleoplasmic and peripheral cytoplasmic viscosities were not different. Our results establish a novel method to measure fluid-phase cytoplasmic viscosity, and indicate that fluid-phase cytoplasmic viscosity in fibroblasts is similar to that of free water.

Animals↗
PubMed2002

The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import.

The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin alpha/beta- or transportin-dependent import.

Animals↗
PubMed1973

The contractile basis of amoeboid movement. I. The chemical control of motility in isolated cytoplasm.

Cytoplasm has been isolated from single amoeba (Chaos carolinensis) in physiological solutions similar to rigor, contraction, and relaxation solutions designed to control the contractile state of vertebrate striated muscle. Contractions of the isolated cytoplasm are elicited by free calcium ion concentrations above ca. 7.0 x 10(-7) M. Amoeba cytoplasmic contractility has been cycled repeatedly through stabilized (rigor), contracted, and relaxed states by manipulating the exogenous free calcium and ATP concentrations. The transition from stabilized state to relaxed state was characterized by a loss of viscoelasticity which was monitored as changes in the capacity of the cytoplasm to exhibit strain birefringence when stretched. When the stabilized cytoplasm was stretched, birefringent fibrils were observed. Thin sections of those fibrils showed thick (150-250 A) and thin (70 A) filaments aligned parallel to the long axis of fibrils visible with the light microscope. Negatively stained cytoplasm treated with relaxation solution showed dissociated thick and thin filaments morphologically identical with myosin aggregates and purified actin, respectively, from vertebrate striated muscle. In the presence of threshold buffered free calcium, ATP, and magnesium ions, controlled localized contractions caused membrane-less pseudopodia to extend into the solution from the cytoplasmic mass. These experiments shed new light on the contractile basis of cytoplasmic streaming and pseudopod extension, the chemical control of contractility in the amoeba cytoplasm, the site of application of the motive force for amoeboid movement, and the nature of the rheological transformations associated with the circulation of cytoplasm in intact amoeba.

Adenosine Triphosphate↗
PubMed1975

Cytoplasmic type 80S ribosomes associated with yeast mitochondria. IV. Attachment of ribosomes to the outer membrane of isolated mitochondria.

Growing yeast spheroplasts were shown to have, on the average, four times the number of cytoplasmic ribosomes in contact with the outer mitochondrial membrane compared to starved spheroplasts. Ribosomes in contact with mitochondria in the growing spheroplast preparation, like free cytoplasmic ribosomes, exist primarily as polysome structures. In the starved spheroplast preparation, both mitochondria-bound and free cytoplasmic ribosomes exist primarily as monosomes. Mitochondria isolated from growing spheroplasts in a medium containing lmM Mg++ have cytoplasmic ribosomes bound directly to the outer membrane. These ribosomes can be quantitatively removed by washing the mitochondria with 2 mM EDTA. Mitochondria from starved spheroplasts are capable of accepting either free cytoplasmic polysomes or cytoplasmic polysomes extracted from mitochondria. However, the extent of polysome binding to mitochondria was shown to be a direct function of the Mg++ concentration; a smaller percentage of the input polysomes bind as the Mg++ concentration is lowered. At 1 mM Mg++, neither free cytoplasmic nor mitochondria-bound polysomes bind to mitochondria. Nevertheless, when growing spheroplasts are broken and mitochondria isolated in medium containing 1 mM Mg++, the mitochondria are seen to have cytoplasmic ribosomes firmly attached to the outer membrane. This result, in addition to our earlier data (Kellems, R. E., and R. A. Butow. 1974. J. Biol. Chem. 249:3304-3310), support the view that cytoplasmic ribosomes attached to the outer membrane of purified mitochondria were attached in vivo. In preparations of mitochondria isolated from growing spheroplasts, ribosomes appear to be found to specific regions of the outer membrane, namely those regions which are in close association or in contact with the inner mitochondrial membrane. This is particularly evident with mitochondria in a condensed configuration. This finding suggests a mechanism whereby cytoplasmically synthesized mitochondrial protein could be transferred by a process of vectorial translation across both membranes of the organelle.

Cell Division↗
PubMed2003

Nuclear-cytoplasmic interactions affect in utero developmental capacity, phenotype, and cellular metabolism of bovine nuclear transfer fetuses.

We generated a clone of bovine somatic cell nuclear transfer embryos using oocyte pools from defined maternal sources to study nuclear-cytoplasmic interactions. Nucleocytoplasmic hybrids were reconstructed with Bos taurus (Brown Swiss) granulosa cells and oocytes that contained B. taurus A (Simmental), B. taurus B (Simmental), or Bos indicus (Dwarf Zebu) cytoplasm. Another set of embryos was reconstructed with randomly selected Brown Swiss (B. taurus R) oocytes. Embryo transfer resulted in nine (12.5%), nine (13.8%), three (50%), and 11 (16.7%) Day 80 fetuses, of which eight (11.1%), three (4.6%), three (50%), and 10 (15.2%) were viable, respectively. The proportion of viable fetuses was affected by cytoplasm (likelihood ratio test, P < 0.02) and was higher for embryos with B. indicus cytoplasm than for the B. taurus A (P < 0.05) and B (P < 0.01) groups. Furthermore, the proportion of surviving Day 80 fetuses was reduced for B. taurus B as compared with B. taurus A and B. taurus R cytoplasm (P < 0.05 and P < 0.02). Body weight of nucleocytoplasmic hybrid fetuses was not significantly different from Brown Swiss control fetuses produced by artificial insemination (AI), but fetuses reconstructed with random cytoplasts of the same breed as the nuclear donor exhibited overgrowth (P < 0.01) and a higher coefficient of variation in weight. Furthermore, body weight, crown rump length, thorax circumference (P < 0.05), and femur length (P < 0.01) of fetuses with B. taurus A cytoplasm differed from fetuses with B. taurus R cytoplasms. Fetal skin, heart, and liver cells with B. indicus cytoplasm showed a greater increase in number per time period (P < 0.001) and oxygen consumption rate per cell (skin and liver, P < 0.001; heart, P < 0.08) in comparison with their counterparts with B. taurus A cytoplasm. These data point to complex oocyte cytoplasm-dependent epigenetic modifications and/or nuclear DNA-mitochondrial DNA interactions with relevance to nuclear transfer and other reproductive technologies such as ooplasmic transfer in human assisted reproduction.

Animals↗
PubMed1996

Restricted propagation of cytoplasmic Ca2+ oscillation into the nucleus in guinea pig cardiac myocytes as revealed by rapid scanning confocal microscopy and indo-1.

Two-dimensional images of cytoplasmic and nuclear free Ca2+ movements in cardiac myocytes were obtained at 67-msec intervals using a Ca(2+)-sensitive fluorescence probe, indo-1, and a rapid scanning confocal laser microscope, Nikon RCM8000. Isolated guinea pig ventricular cells were loaded with indo-1 and stimulated at 0.5 Hz through patch pipettes. On stimulation, nuclear Ca2+ concentration ([Ca2+]) was observed to rise and fall following cytoplasmic [Ca2+] with an obvious delay. Application of isoproterenol significantly increased the peak [Ca2+] on stimulation in both the cytoplasm and nucleus with no substantial change in the basal [Ca2+]; the increase in peak [Ca2+] produced by application of isoproterenol was larger in the cytoplasm than in the nucleus. Under a low [Na+] condition, the basal [Ca2+] was increased from the control values in both the cytoplasm and nucleus; no difference in basal [Ca2+] was observed between the two regions. The increase in peak [Ca2+] by low [Na+] in the cytoplasm was significantly larger than that in the nucleus. When the cells were voltage clamped at 0 mV for 3 sec, no difference in the steady state [Ca2+] was observed between the cytoplasm and nucleus. Nuclear [Ca2+] was also observed to increase following a Ca2+ wave, a local increase in [Ca2+] propagating within the cytoplasm, with a delay. Thus, we demonstrated in isolated myocardial cells that cytoplasmic Ca2+ movements, although hampered by the nuclear envelope, are propagated into the nucleus, a mechanism through which factors affecting cytoplasmic Ca2+ may influence intranuclear events.

Animals↗
PubMed1990

Functional analysis of the cytoplasmic domains of the human thyrotropin receptor by site-directed mutagenesis.

The thyrotropin (TSH) receptor belongs to a family of guanine nucleotide protein-coupled receptors with seven transmembrane-spanning regions joined regulatory together by extracellular and intracellular loops. The cytoplasmic domain comprises three cytoplasmic loops and a cytoplasmic tail that are likely to be important in coupling of the receptor to the guanine nucleotide proteins. To address the question of which portions of the cytoplasmic domain of the TSH receptor are important in this process, we have altered groups of amino acids in the region of the TSH receptor by site-directed mutagenesis. Because of the low affinity of TSH binding to the TSH receptor mutated in the amino terminus of the second cytoplasmic loop and the amino terminus of the cytoplasmic tail, definitive conclusions cannot be made regarding the roles of these regions in signal transduction. However, our data indicate that the first cytoplasmic loop (residues 441-450), the carboxyl-terminal region of the second cytoplasmic loop (residues 528-537), and the carboxyl-terminal (but not the amino-terminal) region of the third cytoplasmic loop (residues 617-625) are important in the ability of the TSH receptor to mediate an increase in intracellular cAMP production. Furthermore, two-thirds of the carboxyl-terminal end of the cytoplasmic tail (residues 709-764; corresponding to the region not conserved between the TSH and lutropin/chorionic gonadotropin receptors) can be removed without functional impairment of the TSH receptor.

Amino Acid Sequence↗
PubMed1979

Involvement of anti-Ig-activated serine protease in the generation of cytoplasmic factor(s) that are responsible for the transmission of Ig-receptor-mediated signals.

Involvement of serine protease-activation in the generation of cytoplasmic factor(s) that induced NHP-specific protein kinase activity in nuclei in anti-Ig-stimulated cells was described. DFP or PMSF with anti-Ig inhibited the induction of cytoplasmic factor(s), whereas pretreatment of cells with DFP or PMSF without anti-Ig did not show any inhibitory effect on anti-Ig-induced generation of cytoplasmic factor(s). TAME or BAME with anti-Ig inhibited the generation of cytoplasmic factor(s) and the simultaneous addition of TAME or BAME with DFP protected the generation of cytoplasmic factor(s) against the inhibitory effect of DFP, showing the involvement of trypsin-like, arginine-type serine protease in anti-Ig-induced generation of cytoplasmic factor(s). Anti-Ig-stimulated membrane preparations induced cytoplasmic factor(s) in normal cytoplasm. The m.w. of precursor proteins present in resting B cells and active cytoplasmic factor(s) were approximately 150,000 and 45,000, respectively. These results showed that anti-Ig-activated membrane-bound serine protease split precursor proteins in resting B cells into active cytoplasmic factor(s) responsible for signal transmission.

Animals↗
PubMed1996

The cytoplasmic domains of E- and P-selectin do not constitutively interact with alpha-actinin and are not essential for leukocyte adhesion.

The selectins are a family of carbohydrate-binding adhesion molecules involved in the regulation of leukocyte migration. Although there is strong homology between different selectins in their extracellular regions, there is none in the cytoplasmic tails, suggesting selectin-specific functions for these domains. Our previous work showed that the cytoplasmic tail of L-selectin interacts with the actin cytoskeleton via alpha-actinin and vinculin, and that truncation of the cytoplasmic tail of L-selectin blocked both association with alpha-actinin and vinculin and leukocyte adhesion. In the present study, the effects of truncation of the cytoplasmic tails of E- or P-selectin on cell adhesion and cell surface expression were examined, and possible interactions between alpha-actinin and the E- and P-selectin cytoplasmic tails were assessed. In contrast to previous observations demonstrating a requirement for the L-selectin cytoplasmic tail, truncation of the E- or P-selectin cytoplasmic domains had no effect on cell adhesion, or on cell surface expression, when assessed in transiently transfected COS cells. This lack of effect on cell surface expression and adhesion was also observed when transfections were performed with lower amounts of cDNA, which led to submaximal levels of expression. In addition, no interaction between alpha-actinin and the cytoplasmic tails of either E- or P-selectin could be detected under conditions in which binding of alpha-actinin to the L-selectin cytoplasmic tail could be readily demonstrated. Therefore, interactions between the cytoplasmic tail of E- or P-selectin and alpha-actinin or other cytoskeletal proteins are not necessary for leukocyte adhesion per se, but may facilitate downstream biologic events.

Actinin↗
PubMed1996

Reducing the amount of cytoplasm available for early embryonic development decreases the quality but not quantity of embryos produced by in vitro fertilization and nuclear transplantation.

The effect of reducing the amount of cytoplasm available for early embryonic development was investigated in embryos produced by in vitro fertilization (IVF) and nuclear transplantation. In Experiment 1, approximately 1/2 or 1/20 of the cytoplasm was removed from bovine embryos at the pronuclear-stage of development. The percentage of embryos developing to the compact morula or blastocyst stage was significantly higher in non-manipulated controls (26%) than in embryos with 1/20 of the cytoplasm removed (16%), and those with 1/2 of the cytoplasm removed (10%; P < 0.05). There was also a significant difference in the average number of cells between blastocysts in which 1/20 of their cytoplasm was removed (67), those with 1/2 of their cytoplasm removed (55), and nonmanipulated controls (77; P < 0.05). In Experiment 2, nuclear transfer embryos were produced in which approximately 1/2 or 1/20 of the cytoplasm was removed during oocyte enucleation. The percentage of embryos developing to the blastocyst stage was 17% for both groups of nuclear transfer embryos compared to 44% for control embryos (P < 0.05). The mean number of cells in blastocysts produced by nuclear transfer in which 1/20 of the cytoplasm was removed during oocyte enucleation (61) was no different than that in control embryos (66), but significantly higher than the mean number of cells in blastocysts produced by nuclear transfer in which 1/2 of the cytoplasm was removed (42; P < 0.05). There was no indication that altering the amount of cytoplasm available for early embryonic development of IVF embryos affected the timing of differentiation events, including those of embryo compaction and blastocyst formation.

Journal Article↗
PubMed1995

The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-actinin.

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L-selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L-selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha-actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L-selectin cytoplasmic domain. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L-selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L-selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.

Actinin↗
PubMed1989

Measurement of the Cytoplasmic and Vacuolar Buffer Capacities in Chara corallina.

The cytoplasm and the vacuole were isolated from internodal cells of Chara corallina by using the intracellular perfusion technique, and their buffer capacities (beta(i)) were determined from the titration curves. The pH of the isolated vacuolar sap was 5.19 +/- 0.029 (mean +/- standard error). At this pH, beta(i) was minimal and amounted to 0.933 +/- 0.11 millimoles H(+)/pH unit/liter vacuolar sap. The pH of isolated cytoplasm was 7.22 +/- 0.028. beta(i) was minimal in this pH region and amounted to 14.2 +/- 0.80 millimoles H(+)/pH unit/liter cytoplasm. When 1% (volume/volume) Triton X-100 was added to the cytoplasmic solution to permeabilize the subcellular organelles, the cytoplasmic pH increased to 7.32 +/- 0.026, where beta(i) was 20.35 +/- 2.66 millimoles H(+)/pH unit/liter cytoplasm. This shows that alkaline subcellular compartments exist in the cytoplasm and also that the cytoplasmic pH before adding Triton X-100 may represent the cytosolic pH. These data indicate that the pH values of the cytoplasm and the vacuole are regulated at the values where the beta(i) values are minimal. This suggests that ATP- and inorganic pyrophosphate-dependent H(+) pumps in the plasma membrane and the tonoplast could efficiently regulate the pH of both cytoplasm and vacuole in Chara internodal cells.

Journal Article↗
PubMed1971

Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100.

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.

Bacterial Proteins↗
PubMed2003

Intranuclear cytoplasmic pseudoinclusions in pituitary adenomas.

Intranuclear pseudoinclusions are well known in papillary carcinomas of the thyroid gland, hepatocellular carcinomas, meningiomas, paragangliomas, pheochromocytomas, and melanomas. Only two papers on the intranuclear inclusions of adenohypophyseal cells in humans have been reported. This study found that intranuclear cytoplasmic pseudoinclusions occur frequently in pituitary adenoma cases (70.3%, 97 of 138 pituitary adenomas) and are uncommon in normal pituitary tissue (11.1%, 1 of 9 normal pituitary tissues). In addition, the frequency of intranuclear cytoplasmic pseudoinclusions between the functional and non-functional pituitary adenomas was found to be similar. Electron microscopy and immunostaining was used to reveal the entity of the intranuclear inclusion. These intranuclear inclusions are due to cytoplasmic invagination because 1) the inclusions are continuous with the cytoplasm, 2) all cytoplasmic organelles, such as the endoplasmic reticulum, the Golgi apparatus, and the secretory granules are found in the inclusions, 3) immunoreactivity of the intranuclear inclusion is the same as that of the cytoplasm. In conclusion, intranuclear cytoplasmic pseudoinclusions in pituitary adenomas occur frequently (70.3%) and are formed by cytoplasmic invagination. This study suggests that pituitary intranuclear inclusions caused by cytoplasmic invagination be called "intranuclear cytoplasmic pseudoinclusions".

Adenoma↗
PubMed1975

Calcium regulation of the contractile state of isolated mammalian fibroblast cytoplasm.

Calcium-dependent contractions have been induced in fresh, naked cytoplasm of L-929 fibroblasts using physiological solutions (rigor, relaxing and contracting) similar to those designed to control the contractile state of vertebrate striated muscle. Free access of solutions to the cytoplasm was achieved by popping and stripping the plasma membrane from cells using 7-10 strokes of a Dounce homogenizer. Contracting solution (free Ca2+ 7 X 10(-5) M; with added MgATP) applied locally from a micropipette to cells popped in rigor (free Ca2+ less than 10(-8) M) or relaxing (free Ca2+ less than 10(-8) M; with added MgATP) solutions induced symmetrical contractions of unstretched cytoplasm and directional shortening of stretched cytoplasm. The contractions produced 12-18% shortenings and were complete in 1-3 s. The cytoplasm could be cycled repeatedly through the contracted state from the relaxed state. Exogenous MgATP was required for the Ca2+-dependent contractions. At low free Ca2+ concentrations (less than 10(-8) M), MgATP had a marked plasticizing effect on the cytoplasm. Thus cytoplasm prepared in relaxing solution was less cohesive and more easily deformed than cytoplasm prepared in rigor solution. When induced to contract, relaxed cytoplasm showed a loss of plasticity. Using this criterion, the threshold concentration of free Ca2+ for contraction was determined to lie between 7 X 10(-8) M and 5 X 10(-7) M.

Adenosine Triphosphate↗
PubMed2001

Multifunctional regulatory proteins that control gene expression in both the nucleus and the cytoplasm.

The multistep pathway of eukaryotic gene expression involves a series of highly regulated events in the nucleus and cytoplasm. In the nucleus, genes are transcribed into pre-messenger RNAs which undergo a series of nuclear processing steps. Mature mRNAs are then transported to the cytoplasm, where they are translated into protein and degraded at a rate dictated by transcript- and cell-type-specific cues. Until recently, these individual nuclear and cytoplasmic events were thought to be primarily regulated by different RNA- and DNA-binding proteins that are localized either only in the nucleus or only the cytoplasm. Here, we describe multifunctional proteins that control both nuclear and cytoplasmic steps of gene expression. One such class of multifunctional proteins (e.g., Bicoid and Y-box proteins) regulates both transcription and translation whereas another class (e.g., Sex-lethal) regulates both nuclear RNA processing and translation. Other events controlled by multifunctional proteins include assembly of spliceosome components, spliceosome recycling, RNA editing, cytoplasmic mRNA localization, and cytoplasmic RNA stability. The existence of multifunctional proteins may explain the paradoxical involvement of the nucleus in an RNA surveillance pathway (nonsense-mediated decay) that detects cytoplasmic signals (premature termination codons). We speculate that shuttling multifunctional proteins serve to efficiently link RNA metabolism in the cytoplasmic and nuclear compartments.

Animals↗