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PubMed2004

Cytoplasmic asters are required for progression past the first cell cycle in cloned mouse embryos.

Unlike the oocytes of most other animal species, unfertilized murine oocytes contain cytoplasmic asters, which act as microtubule-organizing centers following fertilization. This study examined the role of asters during the first cell cycle of mouse nuclear transfer (NT) embryos. NT was performed by intracytoplasmic injection of cumulus cells. Cytoplasmic asters were localized by staining with an anti-alpha-tubulin antibody. Enucleation of MII oocytes caused no significant change in the number of cytoplasmic asters. The number of asters decreased after transfer of the donor nuclei into these enucleated oocytes, probably because some of the asters participated in the formation of the spindle that anchors the donor chromosomes. The cytoplasmic asters became undetectable within 2 h of oocyte activation, irrespective of the presence or absence of the donor chromosomes. After the standard NT protocol, a spindle-like structure persisted between the pseudopronuclei of these oocytes throughout the pronuclear stage. The asters reappeared shortly before the first mitosis and formed the mitotic spindle. When the donor nucleus was transferred into preactivated oocytes (delayed NT) that were devoid of free asters, the microtubules and microfilaments were distributed irregularly in the ooplasm and formed dense bundles within the cytoplasm. Thereafter, all of the delayed NT oocytes underwent fragmentation and arrested development. Treatment of these delayed NT oocytes with Taxol, which is a microtubule-assembling agent, resulted in the formation of several aster-like structures and reduced fragmentation. Some Taxol-treated oocytes completed the first cell cycle and developed further. This study demonstrates that cytoplasmic asters play a crucial role during the first cell cycle of murine NT embryos. Therefore, in mouse NT, the use of MII oocytes as recipients is essential, not only for chromatin reprogramming as previously reported, but also for normal cytoskeletal organization in reconstructed oocytes.

Animals↗
PubMed1999

Apoptosis is physiologically restricted to a specialized cytoplasmic compartment in rat spermatids.

Cytoplasmic caudal tags of maturing spermatids condense and are detached from the spermatidal cells just before the spermatids are released as spermatozoa. The detached cytoplasmic masses are termed "residual bodies." Features of residual bodies seem to be compatible with those of apoptosis and, just as occurs with apoptotic bodies, residual bodies are phagocytosed by Sertoli cells. Since in vitro studies have demonstrated that nucleus and cytoplasm apoptosis events can be independent phenomena, we reasoned that apoptosis pathways might be restricted to the caudal tag of the maturing spermatids in order to originate residual bodies. Consistent with this idea, here we showed that annexin V specifically bound the membranes of isolated residual bodies and that expression levels of caspase-1, c-jun, p53, and p21 were specifically increased in these cytoplasmic compartments. Electron microscopy of cytoplasmic lobes and residual bodies confirmed that their ultrastructural features were those of apoptosis. These data indicate that the mechanism responsible for the formation of residual bodies is similar to that for apoptotic bodies; and the study presents evidence, for the first time, that apoptotic signaling molecules can be restricted to a cytoplasmic compartment and proceed in the presence of a healthy nucleus.

Animals↗
PubMed1991

Cyclosporine's dual mechanism of action in inhibiting generation of the cytoplasmic activation signal and the nuclear response thereto.

The mechanism of cyclosporine's action was dissected using protocols admixing cytoplasmic extracts to trigger isolated nuclei prepared after phytohemagglutinin, OKT3, or mixed lymphocyte culture stimulation of normal volunteer and renal transplant recipient lymphocytes. CsA at 100-1000 ng/ml doses prevented cytoplasmic extracts of cells activated in vitro by OKT3 or MLC from triggering incorporation of radiolabeled nucleotides by resting isolated nuclei. In addition, nuclei isolated from cells previously treated with 200-1000 ng/ml of CsA were refractory to the potent cytoplasmic signal extracted from OKT3- or MLC-stimulated cells. CsA less effectively inhibited phytohemagglutinin-stimulated responses. CsA, but not its inactive analog cyclosporin H, inhibited 3H-thymidine incorporation into in situ nuclei. The inhibited cytoplasmic signal generation and nuclear responsiveness observed with normal lymphocytes exposed to CsA in vitro also was demonstrated with cells from renal transplant recipients who had been treated with CsA for less than 6 months. The impaired response of isolated patient lymphocyte nuclei to in vitro triggering with activated cytoplasmic extracts correlated with intranuclear CsA concentrations, but not with serum levels assessed with monoclonal antibodies selective for CsA or nonselective for CsA plus metabolites. Furthermore, patients displaying drug-induced nephrotoxicity had elevated intranuclear CsA, but not CsA plus metabolite, concentrations compared with stable transplant recipients. Thus, CsA addition in vitro or administration in vivo causes stereospecific inhibition, particularly for CD3 stimuli, of the cytoplasmic activation signal, and, to a lesser extent, of the subsequent nuclear response.

Antibodies, Monoclonal↗
PubMed2005

New insights that link microbes with the generation of antineutrophil cytoplasmic autoantibodies: the theory of autoantigen complementarity.

PURPOSE OF REVIEW: Reviewed are recent discoveries that provide insights into novel mechanisms involved in the aetiology and pathology of anti-neutrophil cytoplasmic autoantibodies (ANCA) disease. RECENT FINDINGS: Gene expression profiles of circulating leukocytes from anti-neutrophil cytoplasmic autoantibody immunogenesis patients revealed high levels of proteinase 3 (PR3) and myeloperoxidase (MPO) mRNA. Combined with reports of increased expression of these proteins, it appears that increased antigen availability is a pathologic component of anti-neutrophil cytoplasmic autoantibody immunogenesis disease, which might be equally as important as the presence of anti-MPO or anti-PR3 autoantibodies. Genetic predisposition to develop anti-neutrophil cytoplasmic autoantibody immunogenesis disease may include a polymorphism in the promoter region of the PR3 gene. Signalling pathways affected by anti-neutrophil cytoplasmic autoantibody immunogenesis binding to neutrophils involve the p21 pathway. Lastly, a topic discussed at length in this review is the seminal observation that PR3-ANCA patients harbour antibodies reactive with a protein produced from PR3-antisense RNA, whose amino acid sequence has homologies with proteins from many microbes and viruses. Delineated in the Theory of Autoantigen Complementarity, it is proposed that the initiator of an autoimmune response is not the autoantigen, but instead is a protein that is 'antisense' or complementary to the autoantigen (e.g. from bacteria or PR3). SUMMARY: The progress in research efforts in the past year, including the identification of complementary proteins as a potential cause of anti-neutrophil cytoplasmic autoantibody immunogenesis, should highly impact future approaches therapeutic intervention.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1992

Intracellular symbionts and the evolution of uniparental cytoplasmic inheritance.

Uniparental inheritance of cytoplasmic elements is widespread among eukaryotic organisms and is achieved by a diverse range of mechanisms. This paper shows that the cytoplasmic genetic system would be expected to evolve towards uniparental inheritance, given the existence of deleterious symbionts capable of invading the host cytoplasm together with nuclear genes that lead to the elimination of cytoplasmic elements from one of the gamete types. The reason for this is that, under biparental inheritance, foreign symbionts with strong deleterious effects are able to spread through host populations. A nuclear modifier gene which leads to the loss of cytoplasmic elements from one gamete type gains a net advantage as a symbiont spreads, because the modifier sometimes gives rise to a symbiont-free zygote. Insofar as small gametes reduce the rate of symbiont transmission to the zygote, modifier genes causing small gamete size would tend to accumulate, so that cytoplasmic inheritance would become associated with maternal rather than paternal gametes. Once uniparental inheritance predominates in the host population, the population is protected from invasions by a large class of harmful symbionts, but at the same time those symbionts that benefit their hosts are still able to increase in frequency.

Animals↗
PubMed1979

Cytoplasmic and nuclear input virus RNPs in influenza virus-infected cells.

Chicken fibroblasts and MDCK cells were infected with influenza virus labelled with either 3H-uridine or 14C-amino acids, and the location in infected cells and properties of input virus-labelled structures were studied. Input virus RNA and protein were found in the cytoplasm of nuclei 1 h p.i. A part of the intranuclear parental structures was associated with chromatin while the other part could be extracted from nucleoplasm by 0.16 M-NaCl and represented free ribonucleoprotein (RNP) particles. These RNPs sedimented in glycerol velocity gradients at 40 to 70S, very similar to cytoplasmic RNPs, but differed distinctly from them in buoyant density. The bulk of cytoplasmic RNPs after fixation with formaldehyde banded in CsCl at 1.34 g/ml while nucleoplasmic RNPs banded at 1.39 or 1.41 g/ml. RNPs isolated from virions and infected cells contained the NP polypeptide which was revealed by SDS-PAGE analysis as a double band. The ratio of the two bands varied in cytoplasmic and nucleoplasmic RNPs, the lower band being dominant in cytoplasmic but not in nucleoplasmic RNPs. In addition, cytoplasmic RNPs were phosphorylated. The possible significance of intracellular RNP modifications for virus replication is discussed.

Animals↗
PubMed1989

Identification of a mitochondrial protein associated with cytoplasmic male sterility in petunia.

The petunia fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS. To determine whether the pcf gene is expressed at the protein level, we produced antibodies to synthetic peptides specified by the coxII and urfS portions of the pcf gene. Anti-COXII peptide antibodies recognized petunia COXII but no other mitochondrial proteins. Anti-URF-S peptide antibodies recognized a 20-kilodalton protein present in both cytoplasmic male sterile and fertile lines and a protein with an apparent molecular mass of 25 kilodaltons present only in cytoplasmic male sterile lines. The 25-kilodalton protein was found to be synthesized by isolated mitochondria and to fractionate into both the soluble and membrane portions of disrupted mitochondria, whereas the 20-kilodalton protein was found only in the membrane fraction. The abundance of the 25-kilodalton protein was much lower in fertile plants carrying the cytoplasmic male sterile cytoplasm and a single dominant nuclear fertility restorer gene, Rf. Thus, the pcf gene is correlated with cytoplasmic male sterility not only by its co-segregation with the phenotype in somatic hybrids, but also by the modification of its expression at the protein level through the action of a nuclear gene that confers fertility.

Amino Acid Sequence↗
PubMed1982

Antibodies to human epidermal cytoplasmic antigens: incidence, patterns, and titers.

Serum or plasma specimens were assayed in indirect immunfluorescence tests on cryostat sections of normal human skin for the presence and titer of antibodies reactive with human epidermal cytoplasmic antigens. A polyvalent fluorescein-labeled goat anti-human immunoglobulin antiserum was used in all tests. Three distinct staining patterns were noted: upper epidermal cytoplasmic fluorescence, U-CYT, produced by antibodies reactive with antigen present in cells of the upper and middle layers of the epidermis; general cytoplasmic fluorescence, G-CYT, produced by antibodies reactive with antigens present in cells throughout the epidermis; and basal cell cytoplasmic fluorescence, BCL, produced by antibodies reactive with components present only in basal cells. Sera from 8% of 52 normal blood donors produced the U-CYT pattern at dilutions greater than 1:10. The incidence of antibodies reactive with epidermal cytoplasmic antigens in patients with a clinical history of not more than 2 basal cell carcinomas of the skin was 5%, compared to an incidence of 89% in those individuals with 3 or more separate instances of skin neoplasms. There was no difference in the frequency with which cryosurgery was used in the treatment of skin neoplasms in either of these 2 groups. Antibodies to epidermal cytoplasmic antigens were also detected in 10% of patients with nondermatologic, nonpulmonary neoplasms, in 43% of patients with pulmonary neoplasms and in 1 of 11 patient with nonneoplastic diseases. Positive sera yielded titers ranging from 1:16 to 1:1024. The most common staining patterns noted in all of these cases were the U-CYT and G-CYT patterns; the BCL staining pattern was noted in only one instance.

Antibodies, Neoplasm↗
PubMed2006

Nuclear and cytoplasmic c-Ski differently modulate cellular functions.

c-Ski is a proto-oncogene product that induces morphologic transformation, anchorage independence, and myogenic differentiation when it is over-expressed in mesenchymal cells. c-Ski also inhibits signaling of transforming growth factor-beta (TGF-beta) superfamily members through interaction with Smad proteins. Although c-Ski is predominantly localized in the nucleus, aberrant cytoplasmic localization of it has also been reported in some tumor tissues and cell lines. In the present study, we identified the nuclear localization signal (NLS) in c-Ski. By introducing a mutation to abolish NLS activity, we examined the function of cytoplasmic c-Ski. Although cytoplasmic c-Ski suppressed TGF-beta superfamily-induced Smad signaling through sequestration of activated Smad complex to the cytoplasm, it failed to exhibit some of the activities that require nuclear localization of c-Ski, including suppression of basal transcription of the Smad7 gene. These findings indicate that subcellular localization of c-Ski affects its biologic activities. We also found that c-Ski accumulated in the cytoplasm when proteasome activity was inhibited. Mapping of the regions required for cytoplasmic accumulation by proteasome inhibitors suggests that subcellular localization of c-Ski may be regulated by proteasome-sensitive processes through amino acid residues 94-210 and 491-548.

Amino Acid Motifs↗
PubMed2005

Integrin CD11a cytoplasmic tail interacts with the CD45 membrane-proximal protein tyrosine phosphatase domain 1.

Leucocyte adhesion receptor integrin CD11aCD18 and the transmembrane receptor-like protein tyrosine phosphatase (RPTP) CD45 mediate immune synapse formation and signalling during antigen presentation. Previous cocapping studies on human naïve T cells demonstrate an interaction between CD11aCD18 and CD45. CD45 cross-linking also has an effect on the ligand-binding activity of CD11aCD18. However, the mode of interaction between CD11aCD18 and CD45 remains unclear. Herein, yeast two-hybrid analysis identified a partial CD45 cytoplasmic tail interacting with that of CD11a. The CD45 cytoplasmic tail comprises a membrane proximal (Mp) region, protein tyrosine phosphatase domain 1 (D1), spacer, D2, and carboxyl terminus. CD45 Mp-D1 was found to be the main interacting region for the CD11a cytoplasmic tail. In contrast, the full-length CD45 cytoplasmic tail interacted weakly with that of CD11a. It has been reported that CD45 Mp-D1 but not the full-length cytoplasmic tail forms a homodimer whose enzymatic activity is inhibited. Our in vitro binding and enzymatic assays showed that the homodimeric CD45 cytoplasmic tail interacts with that of CD11a. The biological function of CD45 dimerization and its association with CD11a remains to be investigated.

Amino Acid Sequence↗
PubMed1976

Quantitative estimates of cytoplasmic and nuclear oestrogen receptors in chick oviduct. Effect of oestrogen on receptor concentration and subcellular distribution.

(3H)Oestradiol exchange techniques were developed for the determination of specific oestrogen receptor site concentrations in the cytoplasm and nuclei of chick oviduct cells. Non-labelled, receptor-bound oestrogens were exchanged with (3H)oestradiol during a 24-h incubation at 20 degrees C, 2 h at 30 degrees C or 45 min at 3 degrees C. Both "soluble" and "insoluble" nuclear receptors were stable for at least 6 h at 30 degrees C and 3 degrees C but a proportion (approx. 30%) of cytoplasmic sites from withdrawn chickens were inactivated after 2 h at 20 degrees C. The magnum of 4-week-old immature chickens (weight = 15 mg) contained 0.20 pmol of oestrogen receptor which corresponds to 4275 receptor sites/cell, when it is assumed that all magnum cells have equal concentrations of receptor. In primarily stimulated chickens of similar age which had received 10x1 mg of oestradiol benzoate/day, the magnum weighed approximately 800 mg and contained 8.65 pmol of oestrogen receptor (4610 sites/cell). Withdrawal from primary oestrogenic stimulation for 3-6 weeks resulted in a 110 mg magnum which contained 1.20 pmol of receptor (2225 sites/cell). Oviducts from immature and withdrawn chickens had the majority (73-77%) of their oestrogen receptors sites in the receptor sites in the cytoplasmic fraction, while in primary stimulated chicken oviducts the majority (82%) of receptor sites were located in the nucleus. A single secondary injection of oestradiol, to oestrogen-withdrawn chickens, resulted in apparent translocation of cytoplasmic receptors to the nucleus during the first hour after injection. The magnitude of the decline in cytoplasmic receptor, and the concurrent increase in nuclear receptor concentration, was dose-dependent between 2 and 100 mug oestradiol/kg body weight. Larger doses of oestradiol up to 1 mg/kg did not increase the concentration of nuclear receptor above the maximum level seen at 100 mug oestradiol/kg. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. The initial rapid accumulation of nuclear receptor sites was followed by a period of progressive decline. By 15 h after an injection of 100 mug oestradiol/kg, the concentration of nuclear sites had reached pre-injection levels. During the same time period, the depleted cytoplasmic receptor levels were replenished such that they reached control values by 12 h and were about 150% of the pre-injection level at 24 h.

Animals↗
PubMed2005

Accumulation of cytoplasmic beta-catenin correlates with reduced expression of E-cadherin, but not with phosphorylated Akt in esophageal squamous cell carcinoma: immunohistochemical study.

Accumulation of beta-catenin in cytoplasm occurs frequently during the pathogenesis of esophageal squamous cell carcinoma (ESCC). The mechanism leading to this alteration, however, is largely unknown. In the present study, immunohistochemistry was performed for beta-catenin, E-cadherin and Ser473 phosphorylated Akt (P-Akt) in 44 tissue samples of ESCC and corresponding normal esophageal epithelium. Exon 3 of the beta-catenin gene was analyzed by using single-strand conformation polymorphism and direct sequencing. In addition to the reduced expression of E-cadherin and membranous beta-catenin observed in 65.9% and 68% of ESCC tested, respectively, cytoplasmic accumulation of beta-catenin was also detected in 68% (30/44) cases. However, only two cases were found to have the same beta-catenin gene mutation. The data showed that cytoplasmic accumulation of beta-catenin was significantly associated with reduced expression of E-cadherin (P < 0.05) and that of membranous beta-catenin (P < 0.05). Furthermore, cytoplasmic beta-catenin was correlated significantly with lymph node metastasis (P < 0.05). In contrast, although strong staining of P-Akt occurred in 14 of 44 cases (32%), there was no significant correlation between the positive staining of P-Akt and cytoplasmic beta-catenin. Taken together these results suggest that the lost membranous beta-catenin might translocate to cytoplasm depending on reduced expression of E-cadherin, while Akt seems unlikely to play a role in this process.

Adult↗
PubMed1983

The identity of hexokinase activities from mitochondrial and cytoplasmic fractions of rat brain homogenates.

Cytoplasmic hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified from the soluble fraction of a rat brain homogenate by a procedure that included a unique affinity elution of the enzyme from Blue Dextran-Sepharose. The purified enzyme was examined with respect to properties in which the impure cytoplasmic enzyme has been reported to differ from the solubilized mitochondrial enzyme. These included the ability to bind to mitochondria, inhibition by quercetin, effect of pH on activity, and kinetics. In all regards the purified mitochondrial and cytoplasmic enzymes appeared identical. In addition, comparative peptide maps after partial proteolysis showed no detectable differences. These results do not support the view that there exist distinct mitochondrial and cytoplasmic forms of hexokinase, the latter being permanently relegated to a cytoplasmic location and unable to participate in a dynamic equilibrium with the mitochondrially-bound enzyme. Alternatives are proposed to explain previous results that had been interpreted as indirect evidence for the existence of a distinct cytoplasmic hexokinase.

Animals↗
PubMed1996

Differential effects of chronic ethanol consumption on hepatic mitochondrial and cytoplasmic ribosomes.

The effects of chronic ethanol consumption on the properties of mitochondrial and cytoplasmic ribosomes were investigated in rat liver. Sedimentation properties of purified mitochondrial (55S) and cytoplasmic (80S) ribosomes were determined by analyses on sucrose density gradients. Mitochondrial ribosomes from control animals moved further in the gradients than did those isolated from ethanol-fed rats, which suggests that ethanol ribosomes have a lower molecular weight. In addition, mitochondrial from ethanol-fed animals contained a lower percentage of ribosomes present as the intact monosome, suggesting that ethanol may have an effect on the stability of the functional mitochondrial ribosomes. This was confirmed by the presence of the larger 39S subunit in preparations from ethanol-fed animals. No such ethanol-related alterations were seen with cytoplasmic ribosomes. The protein composition of mitochondrial cytoplasmic ribosomes was investigated using two-dimensional gel electrophoresis, followed by two-dimensional densitometry. As indicated by differences in protein staining intensity, ethanol consumption seemed to alter the concentration of seven mitochondrial ribosomal proteins. In contrast, no such changes were observed in the protein pattern from cytoplasmic ribosomes. Observations in this study provide for the possibility that alterations in the amounts of selected proteins in the mitochondrial ribosome lead to impaired assembly of the ribosome. These ethanol-related structural changes may be responsible for the decreased activity of mitochondrial ribosomes that results in impaired hepatic mitochondrial protein synthesis (W.B. Coleman and C.C. Cunningham, Biochim. Biophys, Acta 1058:178-186, 1991). Furthermore, this study reemphasizes the increased susceptibility of the hepatic mitochondrial translation system, compared with the cytoplasmic system to chronic ethanol consumption.

Animals↗
PubMed2005

Origin and properties of cytoplasmic and mitochondrial isoforms of taurocyamine kinase.

Taurocyamine kinase (TK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase. TK is found only in certain marine annelids. In this study we used PCR to amplify two cDNAs coding for TKs from the polychaete Arenicola brasiliensis, cloned these cDNAs into the pMAL plasmid and expressed the TKs as fusion proteins with the maltose-binding protein. These are the first TK cDNA and deduced amino acid sequences to be reported. One of the two cDNA-derived amino acid sequences of TKs shows a high amino acid identity to lombricine kinase, another phosphagen kinase unique to annelids, and appears to be a cytoplasmic isoform. The other sequence appears to be a mitochondrial isoform; it has a long N-terminal extension that was judged to be a mitochondrial targeting peptide by several on-line programs and shows a higher similarity in amino acid sequence to mitochondrial creatine kinases from both vertebrates and invertebrates. The recombinant cytoplasmic TK showed activity for the substrates taurocyamine and lombricine (9% of that of taurocyamine). However, the mitochondrial TK showed activity for taurocyamine, lombricine (30% of that of taurocyamine) and glycocyamine (7% of that of taurocyamine). Neither TK catalyzed the phosphorylation of creatine. Comparison of the deduced amino acid sequences of mitochondrial CK and TK indicated that several key residues required for CK activity are lacking in the mitochondrial TK sequence. Homology models for both cytoplasmic and mitochondrial TK, constructed using CK templates, provided some insight into the structural correlation of differences in substrate specificity between the two TKs. A phylogenetic analysis using amino acid sequences from a broad spectrum of phosphagen kinases showed that annelid-specific phosphagen kinases (lombricine kinase, glycocyamine kinase and cytoplasmic and mitochondrial TKs) are grouped in one cluster, and form a sister-group with CK sequences from vertebrate and invertebrate groups. It appears that the annelid-specific phosphagen kinases, including cytoplasmic and mitochondrial TKs, evolved from a CK-like ancestor(s) early in the divergence of the protostome metazoans. Furthermore, our results suggest that the cytoplasmic and mitochondrial isoforms of TK evolved independently.

Amino Acid Sequence↗
PubMed1978

The distribution of sodium, potassium and chloride in the nucleus and cytoplasm of Bufo bufo oocytes measured by electron microprobe analysis.

1. Measurements of cytoplasmic and nuclear Na, K and Cl have been made by electron microprobe analysis on freeze-dried sections of oocytes of Bufo bufo, using standards of bovine plasma albumin and gamma-globulin. Concentrations were obtained per kilogram of dry mass, were converted to concentrations per litre of water content using known figures for water and solid concentration of nucleus and cytoplasm, and were then compared with measurements on cells from the same animal obtained by flame photometry. 2. In fresh oocytes concentrations were (mean +/- S.E. of mean in m-mole/l. H2O) in cytoplasm Na 10.9 +/- 1.95, K 70.2 +/- 3.22, Cl 98.8 +/- 11.0, and in nucleus Na 10.4 +/- 1.79, K 266.4 +/- 22.8, Cl 91.3 +/- 9.0. 3. After treatment with Na-free Ringer (Li substituted for Na) for 5 hr, concentrations were in cytoplasm Na 11.1 +/- 2.44, K 64.4 +/- 5.7, Cl 88.7 +/- 8.8, and in nucleus Na 2.4 +/- 0.73, K 141 +/- 13.9, Cl 75.0 +/- 6.7. Na inexchangeable with Li therefore lay in the cytoplasm but not in the nucleus as previously shown by autoradiography. 4. For K electron microscopic analysis measurements agreed well with those obtained by flame photometry but the former measured only 35% of Na measured by flame photometry. This discrepancy may be due either to technical difficulties with the electron microprobe analysis or to localization of Na in the cytoplasm.

Animals↗
PubMed1984

Alteration of cytoplasmic ionized calcium levels in smooth muscle by vasodilators in the ferret.

Aequorin was used as an indicator of cytoplasmic Ca2+ levels during the action of vasodilators in potassium-depolarized strips of ferret portal vein. Moderate concentrations of isoprenaline produced either no change or an increase in cytoplasmic Ca2+ levels during smooth muscle relaxation. Only in the presence of very high concentrations of isoprenaline (greater than 10(-4) M) was a decrease in intracellular Ca2+ levels detectable. Both papaverine and forskolin also caused relaxations of the muscle while cytoplasmic Ca2+ levels were observed either not to change or to increase. When the muscles were relaxed either by decreasing the calcium concentration in the bathing medium or by the addition of sodium nitroprusside, light and force fell together. The ratio of force to light was greater when the muscle was relaxed with calcium depletion than with the addition of sodium nitroprusside, indicating that sodium nitroprusside was relaxing the muscle by more than just decreasing cytoplasmic Ca2+ levels. These results indicate that not all vasodilators cause a lowering of cytoplasmic Ca2+ levels and indicate that, in intact cells, agents associated with increases in cytoplasmic cyclic AMP levels can cause an uncoupling of calcium-force relations.

Aequorin↗
PubMed2006

Mitochondrial dynamics in chondrocytes and their connection to the mechanical properties of the cytoplasm.

BACKGROUND: The motion and redistribution of intracellular organelles is a fundamental process in cells. Organelle motion is a complex phenomenon that depends on a large number of variables including the shape of the organelle, the type of motors with which the organelles are associated, and the mechanical properties of the cytoplasm. This paper presents a study that characterizes the diffusive motion of mitochondria in chondrocytes seeded in agarose constructs and what this implies about the mechanical properties of the cytoplasm. METHOD OF APPROACH: Images showing mitochondrial motion in individual cells at 30 s intervals for 15 min were captured with a confocal microscope. Digital image correlation was used to quantify the motion of the mitochondria, and the mean square displacement (MSD) was calculated. Statistical tools for testing whether the characteristic motion of mitochondria varied throughout the cell were developed. Calculations based on statistical mechanics were used to establish connections between the measured MSDs and the mechanical nature of the cytoplasm. RESULTS: The average MSD of the mitochondria varied with time according to a power law with the power term greater than 1, indicating that mitochondrial motion can be viewed as a combination of diffusion and directional motion. Statistical analysis revealed that the motion of the mitochondria was not uniform throughout the cell, and that the diffusion coefficient may vary by over 50%, indicating intracellular heterogeneity. High correlations were found between movements of mitochondria when they were less than 2 microm apart. The correlation is probably due to viscoelastic properties of the cytoplasm. Theoretical analysis based on statistical mechanics suggests that directed diffusion can only occur in a material that behaves like a fluid on large time scales. CONCLUSIONS: The study shows that mitochondria in different regions of the cell experience different characteristic motions. This suggests that the cytoplasm is a heterogeneous viscoelastic material. The study provides new insight into the motion of mitochondria in chondrocytes and its connection with the mechanical properties of the cytoplasm.

Animals↗