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PubMed1990

Elemental composition of polyphosphate-containing vacuoles and cytoplasm of Leishmania major.

Leishmania major promastigotes contain electron-dense vacuoles. The elemental composition of these vacuoles and of the cytoplasm was measured by electron probe X-ray microanalysis, using rapid cryopreservation techniques to prevent alterations in composition due to diffusion. The electron-dense vacuoles are rich in P, presumably present as polyphosphate (poly P). Mg is present at about 9 times its cytoplasmic level. There is sufficient Mg to largely neutralize most of the negative charge of the Poly P. The electron-dense vacuoles also contain appreciable amounts of Ca and Zn, which are not detectable in the cytoplasm, as well as Na, K, and Cl, the latter two at concentrations below that of the cytoplasm. These results suggest that the vacuolar membranes have at least one cation transport system. Incubation of the promastigotes for 1 h in the absence of phosphate in the presence or absence of glucose did not cause significant changes in the vacuolar contents of P, Mg, or Zn, but changes in K and Cl content were observed in both the electron-dense vacuoles and in the cytoplasm.

Animals↗

PubMed1987

Rapid and selective effects of thyrotropin on [35S]methionine incorporation into cytoplasmic and nuclear proteins in calf thyroid tissue slices.

Previous studies have indicated that thyrotropin may induce general increases in RNA and protein synthesis in calf thyroid tissue slices. In this report, we show that thyrotropin selectively stimulates [35S]methionine incorporation into small numbers of specific cytoplasmic and nuclear proteins. We provide data on the time course of stimulation and on relative molecular masses and isoelectric points of hormone-response proteins. Calf thyroid tissue slices were incubated for 3 h, 6 h or 9 h in the presence or absence of thyrotropin (50 mU/ml); [35S]methionine (50-75 microCi/ml) was added for the final 3 h of incubation. Cytoplasmic and nuclear fractions were then prepared, and analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Thyrotropin increased [35S]methionine incorporation into two cytoplasmic and four nuclear proteins within 3 h; hormonal effects on the labeling of five of these six proteins were transient, and no longer evident after 6-9 h. In contrast, a second group of two cytoplasmic and four nuclear proteins exhibited increased labeling after a delay of 6-9 h. Our results suggest that thyrotropin selectively stimulates the synthesis of specific cytoplasmic and nuclear proteins in calf thyroid tissue slices, and that stimulation involves at least two mechanisms (one rapid, the other delayed).

Animals↗

PubMed1993

Neuronal death, cytoplasmic calcium and internucleosomal DNA fragmentation: evidence for DNA fragments being released from cells.

Neuronal death, secondary to endogenous agents such as glutamate, may involve changes in cytoplasmic calcium. Besides its well recognized role as a second messenger mediating cellular response, calcium is necessary for the activation of endonuclease(s), resulting in DNA fragmentation and cell death. Therefore, we investigated the relationship between changes in cytoplasmic calcium, DNA fragmentation and neuronal death, using PC12 and NCB-20 cell lines. The calcium ionophore, A23187, caused a dose-dependent increase in cytoplasmic calcium, loss of cell viability, increased lactate dehydrogenase (LDH)-release, and DNA fragmentation. DNA fragments, typical of internucleosomal digestion of genomic DNA, characteristic of endonuclease(s) activation, were consistently detected in the incubating medium. Release of DNA fragments into the medium was seen with A23187 in concentrations as low as 10 nM, and within an hour of treatment. Furthermore, calcium added to preparations of PC12 nuclei also produced DNA fragmentation, although, less pronounced than when intact cells were treated with A23187. The findings indicate that A23187-induced neuronal death involves the activation of endonuclease(s). The role of cytoplasmic calcium in this process is supported by evidence that A23187 selectively mobilizes cytoplasmic calcium, and that calcium can directly activate endonuclease(s) in nuclear preparations.

Animals↗

PubMed1995

Human chorionic gonadotropin protects Leydig cell function against 2,3,7,8-tetrachlorodibenzo-p-dioxin in adult rats: role of Leydig cell cytoplasmic volume.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters testicular steroidogenesis and reduces Leydig cell volume and number; however, human chorionic gonadotropin (hCG) stimulates testosterone production, increases the number and volume of Leydig cells and prevents TCDD's inhibition of testosterone production. The objective of this study was to determine if hCG protects Leydig cell function by maintaining sufficient Leydig cell cytoplasmic volume in TCDD-treated rats. Adult, male Sprague Dawley rats were divided into six treatment groups. Half of the animals received TCDD in corn oil (50 micrograms/kg body wt) and half received corn oil alone on Day 7 only. Additionally the rats received daily treatment of saline for 14 days, saline for 7 days and then hCG for 7 days, or hCG for 14 days. Rats were sacrificed on Day 14 and tissues collected. The decapsulated left testes were incubated in Eagles MEM for 2 h to determine basal production of testosterone and for 2 additional hours after the addition of hCG (100 IU) to the culture media. The right testis was evaluated by stereology to determine the volume of Leydig cells. Body weight was reduced (P < 0.01) in each TCDD-treated group; whereas, testicular weight was not affected by TCDD or hCG treatment. hCG prevented the TCDD-induced reduction in prostate and seminal vesicles weights. TCDD reduced the total volume of Leydig cell cytoplasm in saline-treated rats, but hCG eliminated the TCDD-induced reduction in Leydig cell cytoplasmic volume. hCG prevented the TCDD-induced reduction in Leydig cell function for both the 7- and 14-day treatments. Variation in the total volume of Leydig cell cytoplasm induced by the various treatment combinations was positively correlated with stimulated testosterone production in vitro (r = 0.486; P < 0.01) and the weight of the androgen sensitive organs (seminal vesicles, r = 0.562, P < 0.01; prostate, r = 0.380, P < 0.05). These data support the hypothesis that hCG prevents the TCDD-reduced Leydig cell function by maintaining sufficient Leydig cell cytoplasm and organelle content.

Animals↗

PubMed1988

Construction and quality of cDNA libraries prepared from cytoplasmic RNA not enriched in poly(A)+RNA.

Poly(A)+RNA and cytoplasmic RNA of Ehrlich ascites tumor cells grown in vivo were used to study the quality and efficiency of cDNA synthesis. It was found that the rates of oligo(dT)-primed and unprimed reverse transcription were very similar in both cases. The size distributions of the cDNA strands prepared from unfractionated RNA reflected the size of cytoplasmic mRNA populations including a significant fraction of long molecules up to 6 kb. The fraction of cDNAs primed on rRNAs by oligo(dT) was found to be as low as 2-3%. Following second-strand synthesis by means of RNase H-induced nick translation by DNA polymerase I the overall yields in double-stranded cDNA were slightly higher when unfractionated cytoplasmic RNA was used as starting template. In repeated experiments we obtained an average yield of 2.2 micrograms of double-stranded cDNA when 70 micrograms of unfractionated cytoplasmic RNA was used as starting material. This amount of cDNA synthesized in one assay was sufficient to construct representative cDNA libraries in different vectors. Southern hybridizations of DNA isolated from cDNA libraries with various radiolabelled probes show that the libraries constructed from cDNA synthesized from cytoplasmic RNA not enriched in poly(A)+RNA contain a high ratio of full-length cDNA clones. The results suggest that representative cDNA libraries of high quality can be constructed without pre-isolation of poly(A)+RNA fractions.

Animals↗

PubMed1988

Stimulation-dependent development of neuronal cytoplasm in mouse cochlear nucleus.

The extent of neuronal development in the mouse ventral cochlear nucleus was examined in normal and developmentally auditory deprived mice. Mice were unilaterally deprived on postnatal day three by external auditory meatus removal and sacrificed with controls on day 45, which is after the developmental period. Light microscopic morphometry of neuronal nucleus and cytoplasm areas demonstrated that the normal spherical and globular cells were larger in their low frequency regions than in their high frequency regions. This size difference occurred mainly in the cytoplasm. Developmental deprivation reduced neuronal cytoplasm areas similarly in the high and low frequency regions of both neuronal types, but had no effect on the size of the neuronal nucleus. It was interpreted that cytoplasmic metabolic events are dependent on developmental levels of stimulation and high and low frequency regions normally are differentially stimulated. Furthermore, these stimulation-dependent cytoplasmic events are impaired by developmental hypostimulation, which retards neuronal metabolism and growth. No changes were observed in the cochlear nucleus contralateral to the deprived side, suggesting that compensatory changes, such as hypertrophy, did not occur.

Aging↗

PubMed1986

Experimental diabetes causes mitochondrial loss and cytoplasmic enrichment of pyridoxal phosphate and aspartate aminotransferase activity.

The streptozotocin diabetic rat was selected as a model to study how insulin deficiency alters vitamin B6 utilization by focusing on pyridoxal phosphate levels and aspartate aminotransferase activities in liver tissues. Diabetes of 15 weeks' duration lowered plasma pyridoxal phosphate levels by 84%. Normal plasma pyridoxal phosphate was 480 pmole/ml. Fractionation of liver into mitochondrial and extramitochondrial compartments demonstrated that diabetes caused a 43% diminution in mitochondrial pyridoxal phosphate per gram of liver. There was no cytoplasmic change in these diabetic rats. Mitochondrial aspartate aminotransferase activity was decreased 53% per gram of diabetic liver and cytoplasmic aspartate aminotransferase activity was elevated 3.4-fold. Damage to diabetic mitochondria during preparation procedures could not account for the rise in cytoplasmic aspartate aminotransferase activity. Electrophoresis showed that in the diabetic cytoplasm both cathodal and anodal forms of the enzyme were elevated. Speculations concerning mitochondrial loss and cytoplasmic gain of enzyme activity as well as those on the reduction of plasma pyridoxal phosphate in the diabetic rat are presented.

Animals↗

PubMed1989

Activity of maturation promoting factor in pig oocytes after microinjection and serial transfer of maturing cytoplasm.

Microinjection of 120 pl homologous or heterologous (mouse) maturing cytoplasm into immature pig oocytes resulted in germinal vesicle breakdown (GVBD) and chromosome condensation (CC) after 8 h of culture. In contrast, nearly all control oocytes remained at the germinal vesicle stage. Immature cytoplasm did not induce nuclear maturation of recipient oocytes when injected. The cytoplasm of the oocytes injected with maturing cytoplasm preserved the capacity to cause GVBD in the second recipients. These results suggest that the level of injected maturation promoting factor (MPF) must rise before GVBD can occur. When the pig oocytes injected with 120 pl of mouse maturing cytoplasm were cultured in cycloheximide for 8 h, the presence of GVBD was dramatically reduced. Thus, in pig oocytes the increase in active MPF up to the effective concentration requires protein synthesis.

Animals↗

PubMed1995

Synthetic competition between cytoplasmic folding and translocation of a soluble membrane protein domain.

In wild-type strains of Escherichia coli, alkaline phosphatase (AP), either when present as a soluble protein or when fused to a membrane protein, is only active after translocation to the periplasm. In thioredoxin reductase (trxB) mutants, however, cytoplasmically localized AP can form disulphide bonds and can reach an active conformation. Once it has folded in the cytoplasm, it can no longer be translocated. On the other hand, when AP is fused to periplasmic domains of a membrane protein, translocation can be more rapid than folding. Thus, expressing hybrids of AP and integral membrane proteins in a trxB mutant generates competition between folding of AP in the cytoplasm and its translocation to the periplasm. The cellular localization of AP can be monitored in phosphoserine phosphatase (serB) mutants causing auxotrophy for L-serine. Cytoplasmically but not periplasmically localized AP can compensate for the lack of SerB, leading to growth on indicator plates. As expected, when AP was fused to cytoplasmic domains of membrane proteins, serB-mediated auxotrophy was abolished. Surprisingly, AP fusions to periplasmic domains exhibited a non-uniform response pattern. Fusions that translocate AP rapidly did not complement the SerB defect, while those that export AP only slowly could do so. The usefulness of these strains for studying a variety of aspects related to membrane protein biogenesis is discussed.

Alkaline Phosphatase↗

PubMed2006

Cytoplasmic remodelling and the acquisition of developmental competence in pig oocytes.

The progression of oocyte meiosis is accompanied by major changes in the ooplasm that play a key role in the completion of a coordinate nuclear and cytoplasmic maturation. We review evidence from the literature and present data obtained in our laboratory on different aspects of pig oocyte cytoplasm compartmentalization during maturation and early embryo development. In particular, we will discuss the changes in adenosine triphosphate (ATP) concentration and distribution taking place during the maturation process and their possible significance for oocyte developmental competence. We describe two important aspects of cytoplasmic streaming: mitochondrial distribution patterns in oocytes and early embryos and the complex rearrangements of cytoplasmic microtubule networks, while discussing their possible correlations with ooplasm compartmentalization. Recent evidence indicates that the cytoskeleton is used to shuttle not only organelles but also mRNAs to specific sites within the oocyte cytoplasm. Localization is driven by specific molecular motors belonging to the kinesin superfamily and requires the involvement of the RNA targeting molecule Staufen. We present recent experimental evidence, obtained in our laboratory, on the pig orthologues for kinesin KIF5B and Staufen, describe their expression patterns and discuss their possible role in oocyte maturation.

Animals↗

PubMed2006

Genome-wide analyses show that nuclear and cytoplasmic RNA levels are differentially affected by dioxin.

The aryl hydrocarbon receptor (AHR) mounts the body's main molecular defense against environmental toxicants by inducing a battery of genes encoding xenobiotic metabolizing proteins. The AHR is activated by polycyclic aromatic hydrocarbon toxicants, including the pervasive teratogen and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). The TCDD-activated AHR significantly changes the cytoplasmic mRNA levels of hundreds of genes, but little is known of the mechanism by which the activated AHR causes such a strong effect on global gene expression. We used high-density microarrays to compare nuclear and cytoplasmic RNA levels from untreated and TCDD-treated mouse embryonic fibroblasts (MEF) to test the hypotheses that (1) TCDD has a large impact on nuclear RNA levels and (2) that cytoplasmic RNA levels are dependent on nuclear RNA levels. We found that nuclear RNA levels are strongly affected by TCDD, and that nuclear and cytoplasmic RNA levels are only weakly correlated, indicating that other regulatory mechanisms are controlling cytoplasmic RNA levels. The nuclear RNAs most affected by TCDD encode proteins involved in nuclear RNA processing and transcription. We conclude that although the AHR regulates key xenobiotic metabolizing genes at the transcriptional level, a larger impact of the TCDD-activated AHR may be at post-transcriptional levels.

Animals↗

PubMed2006

Cytoplasmic pH-dependent spreading of polymorphonuclear leukocytes: regulation by pH of PKC subcellular distribution and F-actin assembly.

Cytoplasmic pH (pHi) plays an important role in the regulation of polymorphonuclear leukocyte (PMN) spreading, but the molecular mechanisms involved have long been obscure. In the present study, we investigated the pH-dependence of phorbol myristate acetate (PMA)-induced PMN spreading. A change in pHi alone did not induce spreading, but cytoplasmic alkalinization promoted the spreading induced by PMA, whereas acidification inhibited it. To further investigate the mechanism by which pHi affects cell spreading, we employed subcellular fractionation and immunoblot analyses to evaluate the effect of pH on the subcellular distribution of protein kinase C (PKC) and assembly of the actin cytoskeleton. We found that cytoplasmic alkalinization enhanced PKC membrane distribution and quantitatively up-regulated the actin cytoskeleton. On the other hand, cytoplasmic acidification was found to have effects on these signaling molecules that were opposite to those of cytoplasmic alkalinization. These results may provide a potential explanation for the pH-regulation of the PMA-induced PMN spreading.

Actins↗

PubMed2005

Cytoplasmic-to-nuclear volume ratio affects AP-1 complex formation as an indicator of cell cycle responsiveness.

Cytoplasmic volume undergoes a series of changes during mitosis in eukaryotes; in turn, signaling events such as osmotic stress can regulate the cytoplasmic volume in cells. In some organisms, increase in cytoplasmic-to-nuclear volume ratio was seen to affect the growth potential in cells, however, the mechanistics of such a regulation, if at all present, was unclear. In a computational model, we have constructed a growth factor-induced signaling pathway leading to AP-1 heterodimer formation through transcriptional regulation, and analyzed the effects of increasing the cytoplasmic-to-nuclear ratio on c-jun transcription and AP-1 complex. We have observed that larger cytoplasmic volumes caused both an increase in the final AP-1 product and a delay in the time of AP-1 accumulation.

Animals↗

PubMed2005

Phosphagen kinase of the giant tubeworm Riftia pachyptila. Cloning and expression of cytoplasmic and mitochondrial isoforms of taurocyamine kinase.

The giant tubeworm Riftia pachyptila lives at deep-sea hydrothermal vents along the East Pacific Rise and the Galapagos Rift. The large size and high growth rate of R. pachyptila is supported by an endosymbiotic relationship with a chemosynthetic bacterium. Elucidation of the regulation of energy metabolism of the giant tubeworm remains an interesting problem. The purpose of this study is to determine the cDNA sequence of phosphagen kinase, one of the most important enzymes in energy metabolism, and to characterize its function. Two phosphagen kinase cDNA sequences amplified from the cDNA library of R. pachyptila showed high derived amino acid sequence identity (74%) with those of cytoplasmic taurocyamine kinase (TK) and mitochondrial TK from an annelid Arenicola brasiliensis. The cytoplasmic form of the Riftia recombinant enzyme showed stronger activity for the substrates taurocyamine and also considerable activity for lombricine (21% that of taurocyamine). The mitochondrial form, which was structurally similar to mitochondrial creatine kinase, showed stronger activity for taurocyamine, and a broader activity for various guanidine compounds: glycocyamine (35% that of taurocyamine), lombricine (31%) and arginine (3%). Both forms showed no activity for creatine. The difference in substrate specificities between the cytoplasmic and mitochondrial forms might be attributable to the large difference in the amino acid sequence of the GS region and/or several key amino acid residues for establishing guanidine substrate specificity. Based on these results, we conclude that Riftia contains at least two forms of TK as phosphagen kinase. We also report the kinetic parameters, Km and kcat, of Arenicola and Riftia TKs for the first time. The Km values for taurocyamine of Arenicola and Riftia TKs ranged from 0.9 to 4.0 mM and appear to be comparable to those of other annelid-specific enzymes, lombricine kinase and glycocyamine kinase, but are significantly lower than those of Neanthes cytoplasmic and mitochondrial creatine kinases. Comparison of kcat/Km value in various annelid phosphagen kinases indicates that Arenicola mitochondrial TK has the highest catalytic efficiency (16.2 s-1 mM-1). In Arenicola TKs, the mitochondrial form has seven-fold higher efficiency than the cytoplasmic form.

Amino Acid Sequence↗

PubMed2005

Lamp-2a facilitates MHC class II presentation of cytoplasmic antigens.

Extracellular antigens are internalized and processed before binding MHC class II molecules within endosomal and lysosomal compartments of professional antigen presenting cells (APC) for subsequent presentation to T cells. Yet select cytoplasmic peptides derived from autoantigens also intersect and bind class II molecules via an unknown mechanism. In human B lymphoblasts, inhibition of the peptide transporter associated with antigen processing (TAP) failed to alter class II-restricted cytoplasmic epitope presentation. By contrast, decreased display of cytoplasmic epitopes via class II molecules was observed in cells with diminished expression of the lysosome-associated membrane protein-2 (Lamp-2). Overexpression of Lamp-2 isoform A (Lamp-2a), an established component of chaperone-mediated autophagy, enhanced cytoplasmic autoantigen presentation. Manipulating APC expression of heat shock cognate protein 70 (hsc70), a cofactor for Lamp-2a, also altered cytoplasmic class II peptide presentation. These results demonstrate a novel role for the lysosomal Lamp-2a-hsc70 complex in promoting immunological recognition and antigen presentation.

ATP-Binding Cassette Transporters↗

PubMed2006

Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of Escherichia coli: evidence that chaperone activity is the prime effect of thioredoxin.

In this study we investigate the effect of thioredoxin (Trx1) protein fusions in the production, oxidation, and folding of single chain Fv (scFv) antibodies in the cytoplasm of Escherichia coli. We analyze the expression levels, solubility, disulfide-bond formation, and antigen-binding properties of Trx1-scFv fusions in E. coli wild-type cells and isogenic strains carrying mutations in the glutathione oxidoreductase (gor) and/or thioredoxin reductase (trxB) genes. We compare the Trx1-scFv fusions with other reported systems for production of scFv in the cytoplasm of E. coli, including protein fusions to the maltose-binding protein. In addition, we analyze the effect of co-expressing a signal-sequence-less derivative of the periplasmic chaperone and disulfide-bond isomerase DsbC (DeltassDsbC), which has been shown to act as a chaperone for scFvs in the cytoplasm. The results reported here demonstrate that Trx1 fusions produce the highest expression level and induce the correct folding of scFvs even in the absence of DeltassDsbC in the cytoplasm of E. coli trxB gor cells. The disulfide bridges of Trx1-scFv fusions were formed correctly in E. coli trxB gor cells, but not in trxB single mutants. Antigen-binding assays showed that Trx1 has only a minor influence in the affinity of the scFv, indicating that Trx1-scFv fusions can be used without removal of the Trx1 moiety. In addition, we proved that a Trx1"AGPA" variant, having its catalytic cysteine residues mutated to alanine, was fully capable of assisting the folding of the fused scFvs. Taken together, our data indicate that the Trx1 moiety acts largely as an intramolecular protein chaperone, not as a disulfide bond catalyst, inducing the correct folding of scFvs in the cytoplasm of E. coli trxB gor cells.

Cytoplasm↗

PubMed2006

Amyloid precursor protein cytoplasmic domain antagonizes reelin neurite outgrowth inhibition of hippocampal neurons.

The function of the amyloid precursor protein (APP), a key molecule in Alzheimer's disease (AD) remains unknown. Among the proteins that interact with the APP cytoplasmic domain in vitro and in heterologous systems is Disabled-1, a signaling molecule of the reelin pathway. The physiological consequence of this interaction is unknown. Here we used an in vitro model of hippocampal neurons grown on a reelin substrate that inhibits neurite outgrowth. Our results show that an excess of APP cytoplasmic domain internalized by a cell permeable peptide, is able to antagonize the neurite outgrowth inhibition of reelin. The APP cytoplasmic domain binds Disabled-1 and retains it in the cytoplasm, preventing it from reaching the plasma membrane and sequesters tyrosine phosphorylated Disabled-1, both of which disrupt reelin signaling. In the context of AD, increased formation of APP cytoplasmic domain in the cytosol released after cleavage of the A beta peptide, could then inhibit reelin signaling pathway in the hippocampus and thus influence synaptic plasticity.

Amino Acid Sequence↗

PubMed2005

Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli.

c-Met, a high affinity receptor for hepatocyte growth factor/scatter factor, shown to be overexpressed in a variety of malignant cells, is a potential biomarker as well as a therapeutic target. Thus, single-chain antibody fragment (scFv) specific for c-Met is expected to be efficiently employed in the clinical treatment or imaging of many cancer cells. Here, we constructed the expression system for anti-c-Met scFv fused with T7 tag at its N-terminus using pET vector and investigated the expression conditions to achieve a functional and soluble expression of the scFv in the cytoplasm of recombinant Escherichia coli. The redox potential of E. coli cytoplasm was the most critical factor for the functional expression of anti-c-Met scFv. The employment of a host with oxidizing cytoplasm, E. coli trxB/gor double mutant, improved the productivity of functional anti-c-Met scFv by approximately 10-fold compared to the production of anti-c-Met scFv in the reducing cytoplasm of wild type E. coli. Productivity of functional anti-c-Met scFv could be further enhanced by co-expressing molecular chaperones such as GroELS, trigger factor, and DsbC with the scFv. Coexpression of DsbC increased the yield of functional anti-c-Met scFv about 2.5-fold in the cytoplasm of E. coli trxB/gor mutant compared to the production of scFv without DsbC coexpression. Lowering the IPTG concentration from 1 to 0.05 mM led to the slight enhancement, approximately 1.6-fold, of productivity of functional scFv. Although the use of low temperature for anti-c-Met scFv expression increased the ratio of soluble scFv fraction to insoluble fraction, productivity of soluble scFv decreased owing to the significant reduction of expression rate. The addition of 0.5 M sucrose in the medium inhibited the formation of intracellular insoluble anti-c-Met scFv. To purify the anti-c-Met scFv simply, we fused hexahistidine at the C-terminus of scFv and purified the scFv showing 98% of purity through the interaction between Ni2+ and histidine.

Antibodies↗