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PubMed1990

Anti-neutrophil cytoplasm antibodies (ANCA).

Circulating antibodies directed against cytoplasmic constituents of granulocytes and monocytes (anti-neutrophil cytoplasm antibodies, ANCA) are found in about 80% of active cases of Wegener's granulomatosis and microscopic polyarteritis. Antibodies are detected by indirect immunofluorescence on normal peripheral blood leucocytes, or by ELISA or radio-immunoassay using a neutrophil cytoplasm extract. There are 2 patterns of staining seen on indirect immunofluorescent examination of leucocytes and a number of different molecules recognised on Western blots. In Wegener's granulomatosis diffusely granular cytoplasmic staining (cANCA) is associated with a 29KD molecule that has recently been identified as neutrophil proteinase 3. A finely granular pattern may also be seen in microscopic polyarteritis. A perinuclear pattern (pANCA) is present in some other cases of segmental necrotising glomerulonephritis and occasionally in rheumatoid arthritis but almost never in Wegener's granulomatosis. This pattern is often associated with the presence of anti-myeloperoxidase and anti-elastase antibodies. Levels of anti-neutrophil cytoplasm antibodies usually reflect disease activity but a pathogenetic role for these antibodies in the development of small vessel vasculitides is, as yet, unproven.

Autoantibodies↗

PubMed2001

Channel cytoplasmic loops alter voltage-dependent sodium channel activation in an isoform-specific manner.

1. The isoform-specific functional role of cytoplasmic structures of two voltage-gated sodium channel isoforms, the human cardiac channel (hH1) and the adult human skeletal muscle channel (hSkM1) was investigated through functional comparison of chimeras. 2. The voltage of half-activation (V(a)) for hH1 was shifted by > 20 mV in the hyperpolarised direction following internal papain treatment ('papain sensitive'), while V(a) for hSkM1 was unaffected ('papain insensitive'). 3. The hH1 region(s) responsible for this papain sensitivity was localised by testing a series of hH1/hSkM1 chimeras in which combinations of the large hH1 cytoplasmic loops joining the four transmembrane domains replaced analogous hSkM1 loops. Various chimeras were used to determine the smallest subset of loops that converted fully the papain-insensitive hSkM1 into a papain-sensitive channel. Then three converse chimeras were tested in which hSkM1 loops replaced hH1 loops to determine the smallest subset of loops necessary and sufficient to convert the papain-sensitive hH1 into a papain-insensitive channel. 4. Functional studies of this inclusive set of chimeras indicate that the first two cytoplasmic loops of the cardiac sodium channel that join domain I to II (loop A), and domain II to III (loop B), are both necessary, and together are sufficient to produce a papain-induced hyperpolarising shift in the voltage at which channels activate. When both loops are present (wild-type hH1 and the chimera hSkM1AB), V(a) for the channel shifts in the hyperpolarised direction by > 20 mV with papain treatment. When the analogous hSkM1 loops are present (wild-type hSkM1 and the chimera hH1AB), V(a) for the channel is not sensitive to treatment with papain. For channels that contain only one of the two hH1 loops, the effect of papain on V(a) is intermediary. 5. Experiments performed in the absence of papain showed that the activation voltages of the double loop chimeras, hSkM1AB and hH1AB, were shifted significantly from V(a) for hSkM1 and V(a) for hH1, respectively, indicating that these loops directly alter channel activation voltage. The resulting shifts in V(a) were in opposing directions, suggesting that cytoplasmic control of activation voltage is isoform specific. V(a) for hSkM1AB was about 20 mV more depolarised than V(a) for hSkM1, and V(a) for hH1AB was about 9 mV more negative than V(a) for hH1. 6. These data are the first to indicate isoform-specific cytoplasmic regions of the voltage-gated sodium channel that directly and differently alter the voltage of channel activation.

Animals↗

PubMed1990

Alpha-latrotoxin releases both vesicular and cytoplasmic glutamate from isolated nerve terminals.

alpha-Latrotoxin causes a massive release of endogenous glutamate from guinea-pig cerebrocortical synaptosomes. There appear to be two components to the release. In the first 2 min following addition of 1.3 nM alpha-latrotoxin, glutamate release is largely energy dependent. Superimposed upon this release is a more slowly developing but ultimately much more extensive release of cytoplasmic glutamate together with gamma-aminobutyric acid and nonvesicular amino acids such as aspartate and alpha-aminoisobutyrate. In parallel with this cytoplasmic release there is an extensive depletion of ATP, a massive rise in cytoplasmic free Ca2+ concentration, and a severe restriction of synaptosomal respiratory capacity. The cytoplasmic release is only partially Na+ dependent, eliminating a simple reversal of the plasma membrane acidic amino acid carrier. It is concluded that alpha-latrotoxin releases both transmitter and cytoplasmic pools of amino acids in synaptosomes and causes a major disruption of terminal integrity.

Amino Acids↗

PubMedDate not supplied

Entamoeba motility: dynamics of cytoplasmic streaming, locomotion and translocation of surface-bound particles, and organization of the actin cytoskeleton in Entamoeba invadens.

The dynamics of cytoplasmic streaming, retrograde translocation of externally bound particles and locomotion by Entamoeba invadens were compared. Locomoting amoebae were monopodial, exhibited fountain flow cytoplasmic streaming and translocated externally bound erythrocytes to the rear of cells. The rates of rearward flow of peripheral cytoplasmic vacuoles and of the externally bound particles were equal to the rate of cell forward locomotion. Rhodamine-phalloidin staining revealed a distinct cortical polymerized actin cytoskelton. This was least evident about the periphery of the advancing pseudopod, increased in density toward the rear of the cell and was most concentrated in the uroid. A monoclonal anti-eucaryotic actin antibody, which recognized monomeric Entamoeba actin on immunoblots, stained trophozoites by indirect immunofluorescence throughout the cytoplasm, but not in the cortical regions stained by rhodamine-phalloidin. This and other evidence implied that the antibody recognized only unpolymerized actin in Entamoeba. We propose that locomotion, cytoplasmic streaming and translocation of externally bound particles are driven by a common actin-based mechanism in Entamoeba, possibly involving retrograde cortical actin flow and recycling.

Actins↗

PubMed2005

Translocation biosensors to study signal-specific nucleo-cytoplasmic transport, protease activity and protein-protein interactions.

Regulated nucleo-cytoplasmic transport is crucial for cellular homeostasis and relies on protein interaction networks. In addition, the spatial division into the nucleus and the cytoplasm marks two intracellular compartments that can easily be distinguished by microscopy. Consequently, combining the rules for regulated nucleo-cytoplasmic transport with autofluorescent proteins, we developed novel cellular biosensors composed of glutathione S-transferase, mutants of green fluorescent protein and rational combinations of nuclear import and export signals. Addition of regulatory sequences resulted in three classes of biosensors applicable for the identification of signal-specific nuclear export and import inhibitors, small molecules that interfere with protease activity and compounds that prevent specific protein-protein interactions in living cells. As a unique feature, our system exploits nuclear accumulation of the cytoplasmic biosensors as the reliable readout for all assays. Efficacy of the biosensors was systematically investigated and also demonstrated by using a fully automated platform for high throughput screening (HTS) microscopy and assay analysis. The introduced modular biosensors not only have the potential to further dissect nucleo-cytoplasmic transport pathways but also to be employed in numerous screening applications for the early stage evaluation of potential drug candidates.

Biological Transport↗

PubMed1989

Genetics of inbred Drosophila melanogaster. XXII. Cytoplasmic influence on recombination.

To examine the possible influence of various sources of cytoplasms on recombination in the major part of the X-chromosome in Drosophila melanogaster, tests were performed in intra- and inter-line crosses. Two to six sister lines of various types were included. It was shown that cytoplasms of various sources could have different effects in the distal y-cx region but not in the mid (cx-v) or the sub-proximal (v-f) regions in both intra- and inter-line crosses. There were no diversities between sister-lines that had been separated for over 100 generations. Tests of two sets of sister-lines with the same source of cytoplasm in which different genomes had been introduced confirmed the earlier finding that there is an interaction of chromosomal and cytoplasmic influences on recombination. It is shown that changes in one extrachromosomal element (R-elements) had no demonstrable consequences on the effect on recombination. The necessity to consider cytoplasmic-bound variability is stressed.

Animals↗

PubMed1997

Cytoplasmic calcium buffers in intact human red cells.

1. Precise knowledge of the cytoplasmic Ca2+ buffering behaviour in intact human red cells is essential for the characterization of their [Ca2+]i-dependent functions. This was investigated by using a refined method and experimental protocols which allowed continuity in the estimates of [Ca2+]i, from nanomolar to millimolar concentrations, in the presence and absence of external Ca2+ chelators. 2. The study was carried out in human red cells whose plasma membrane Ca2+ pump was inhibited either by depleting the cells of ATP or by adding vanadate to the cell suspension. Cytoplasmic Ca2+ buffering was analysed from plots of total cell calcium content vs. ionized cytoplasmic Ca2+ concentration ([CaT]i vs. [Ca2+]i) obtained from measurements of the equilibrium distribution of 45Ca2+ at different external Ca2+ concentrations ([Ca2+]o), in conditions known to clamp cell volume and pH. The equilibrium distribution of 45Ca2+ was induced by the divalent cation ionophore A23187. 3. The results showed the following. (i) The known red cell Ca2+ buffer represented by alpha, with a large capacity and low Ca2+ affinity, was the main cytoplasmic Ca2+ binding agent. (ii) The value of alpha was remarkably constant; the means for each of four donors ranged from 0.33 to 0.35, with a combined value of all independent measurements of 0.34 +/- 0.01 (mean +/- S.E.M., n = 16). This contrasts with the variability previously reported. (iii) There was an additional Ca2+ buffering complex with a low capacity (approximately 80 micromol (340 g Hb)(-1)) and intermediate Ca2+ affinity (apparent dissociation constant, K(D,app) approximately 4-50 microM) whose possible identity is discussed. (iv) The cell content of putative Ca2+ buffers with submicromolar Ca2+ dissociation constants was below the detection limit of the methods used here (less than 2 micromol (340 g Hb)(-1)). 4. Vanadate (1 mM) inhibited the Vmax of the Ca2+ pump in inosine-fed cells by 99.7%. The cytoplasmic Ca2+ buffering behaviour in these cells was similar to that found in ATP-depleted cells.

Adenosine Triphosphate↗

PubMed1999

N-methyl-D-aspartate excitotoxicity: relationships among plasma membrane potential, Na(+)/Ca(2+) exchange, mitochondrial Ca(2+) overload, and cytoplasmic concentrations of Ca(2+), H(+), and K(+).

A high cytoplasmic Na(+) concentration may contribute to N-methyl-D-aspartate (NMDA)-induced excitotoxicity by promoting Ca(2+) influx via reverse operation of the Na(+)/Ca(2+) exchanger (NaCaX), but may simultaneously decrease the electrochemical Ca(2+) driving force by depolarizing the plasma membrane (PM). Digital fluorescence microscopy was used to compare the effects of Na(+) versus ions that do not support the NaCaX operation, i.e., N-methyl-D-glucamine(+) or Li(+), on: PM potential; cytoplasmic concentrations of Ca(2+), H(+), and K(+); mitochondrial Ca(2+) storage; and viability of primary cultures of cerebellar granule cells exposed to NMDA receptor agonists. In the presence of Na(+) or Li(+), NMDA depolarized the PM and decreased cytoplasmic pH (pH(C)); in the presence of Li(+), Ca(2+) influx was reduced, mitochondrial Ca(2+) overload did not occur, and the cytoplasm became more acidified than in the presence of Na(+). In the presence of N-methyl-D-glucamine(+), NMDA instantly hyperpolarized the PM, but further changes in PM potential and pH(C) were Ca-dependent. In the absence of Ca(2+), hyperpolarization persisted, pH(C) was decreasing very slowly, K(+) was retained in the cytoplasm, and cerebellar granule cells survived the challenge; in the presence of Ca(2+), pH(C) dropped rapidly, the K(+) concentration gradient across the PM began to collapse as the PM began to depolarize, and Ca(2+) influx and excitotoxicity greatly increased. These results indicate that the dominant, very likely excitotoxic, component of NMDA-induced Ca(2+) influx is mediated by reverse NaCaX and that direct Ca(2+) influx via NMDA channels is curtailed by Na-dependent PM depolarization.

Animals↗

PubMed2002

Reconstitution of the cytoplasmic interaction between phospholamban and Ca(2+)-ATPase of cardiac sarcoplasmic reticulum.

Phospholamban (PLN) reversibly inhibits the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum (SERCA2a) through a direct protein-protein interaction, playing a pivotal role in the regulation of intracellular Ca(2+) in heart muscle cells. The interaction between PLN and SERCA2a occurs at multiple sites within the cytoplasmic and membrane domains. Here, we have reconstituted the cytoplasmic protein-protein interaction using bacterially expressed fusion proteins of the cytoplasmic domain of PLN and the long cytoplasmic loop of SERCA2a. We have developed two methods to evaluate the binding of the fusion proteins, one with glutathione-Sepharose beads and the other with a 96-well plate. Essentially the same results were obtained by the two methods. The affinity of the binding (K(D)) was 0.70 microM. The association was inhibited by cAMP-dependent phosphorylation of the PLN fusion protein and by usage of anti-PLN monoclonal antibody. It was also diminished by substitution at the phosphorylation site of PLN of Ser(16) to Asp. These results suggest that PLN can bind SERCA2a in the absence of the membrane domains and that the modifications of the cytoplasmic domain of PLN that activate SERCA2a parallel the disruption of the association between the two fusion proteins. It has been shown that the removal of PLN inhibition of SERCA2a rescues cardiac function and morphology in the mouse dilated cardiomyopathy model. Our assay system can be applied to the screening of novel inotropic agents that remove the inhibition of SERCA2a by PLN, improving the relaxation as well as the contractility of the failing heart.

Adenosine Triphosphate↗

PubMed2005

Chaperone-mediated folding and maturation of the penicillin acylase precursor in the cytoplasm of Escherichia coli.

Expression of the leaderless pac gene (LL pac), which lacks the coding region for the signal peptide of penicillin acylase (PAC), in Escherichia coli was conducted. It was demonstrated that the PAC precursor, proPAC, can be produced and even processed to form mature PAC in the cytoplasm, indicating that the posttranslational processing steps for PAC maturation can occur in both the periplasm and the cytoplasm of E. coli. The outcome of proPAC folding and PAC maturation could be affected by several factors, such as inducer type, proPAC formation rate, and chaperone availability. Misfolding of proPAC in the cytoplasm could be partially resolved through the coexpression of cytoplasmic chaperones, such as trigger factor, GroEL/ES, or DnaK/J-GrpE. The three chaperones tested showed different extents of the effect on proPAC solublization and PAC maturation, and trigger factor had the most prominent one. However, the chaperone-mediated solublization of proPAC did not guarantee its maturation, which is usually limited by the first autoproteolytic step. It was observed that arabinose could act as an effective inducer for the induction of LL pac expression regulated by the lac-derived promoter system of trc. In addition, PAC maturation could be highly facilitated by arabinose supplementation and coexpression of trigger factor, suggesting that the coordination of chaperone systems with proper culture conditions could dramatically impact recombinant protein production. This study suggests that folding/misfolding of proPAC could be a major step limiting the overproduction of PAC in E. coli and that the problem could be resolved through the search for appropriate chaperones for coexpression. It also demonstrates the analogy in the issues of proPAC misfolding as well as the expression bottleneck occurring in the cytoplasm (i.e., LL pac expression) and those occurring in the periplasm (i.e., wild-type pac expression).

Base Sequence↗

PubMed2005

Characterization of Wolbachia transfection efficiency by using microinjection of embryonic cytoplasm and embryo homogenate.

Wolbachia spp. are intracellular alpha proteobacteria closely related to Rickettsia. The maternally inherited infections occur in a wide range of invertebrates, causing several reproductive abnormalities, including cytoplasmic incompatibility. The artificial transfer of Wolbachia between hosts (transfection) is used both for basic research examining the Wolbachia-host interaction and for applied strategies that use Wolbachia infections to affect harmful insect populations. Commonly employed transfection techniques use embryonic microinjection to transfer Wolbachia-infected embryo cytoplasm or embryo homogenate. Although microinjections of both embryonic cytoplasm and homogenate have been used successfully, their respective transfection efficiencies (rates of establishing stable germ line infections) have not been directly compared. Transfection efficiency may be affected by variation in Wolbachia quantity or quality within the donor embryos and/or the buffer types used in embryo homogenization. Here we have compared Wolbachia bacteria that originate from different embryonic regions for their competencies in establishing stable germ line infections. The following three buffers were compared for their abilities to maintain an appropriate in vitro environment for Wolbachia during homogenization and injection: phosphate-buffered saline, Drosophila Ringer's buffer, and a sucrose-phosphate-glutamate solution (SPG buffer). The results demonstrate that Wolbachia bacteria from both anterior and posterior embryo cytoplasms are competent for establishing infection, although differing survivorships of injected hosts were observed. Buffer comparison shows that embryos homogenized in SPG buffer yielded the highest transfection success. No difference was observed in transfection efficiencies when the posterior cytoplasm transfer and SPG-homogenized embryo techniques were compared. We discuss the results in relation to intra- and interspecific Wolbachia transfection and the future adaptation of the microinjection technique for additional insects.

Animals↗

PubMed2005

Overlapping roles of the cytoplasmic and mitochondrial redox regulatory systems in the yeast Saccharomyces cerevisiae.

Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.

Cytoplasm↗

PubMed1980

Analysis of cytoplasmic antigens of the yeast and mycelial phases of Candida albicans by two-dimensional electrophoresis.

The extent of the macromolecular change accompanying yeast to mycelium morphogenesis of Candida albicans was analyzed by two-dimensional gel electrophoresis of the cytoplasmic proteins of the two growth forms after antibody cross-absorption experiments. Pure cultures of yeasts and true hyphae (i.e., without concomitant production of pseudohyphae) were grown in a synthetic low-sulfate medium (LSM). The two strains selected for this study were strain 4918, which produces pure mycelial (M) cultures in LSM at 37 degrees C (designated 4918-37M) and yeasts (Y) at 24 degrees C (4918-24Y), and strain 2252, which produces yeasts exclusively at both 24 and 37 degrees C in LSM (2252-24Y and 2252-37Y). The proteins of both strains were labeled at both temperatures with [35S]sulfate, and cytoplasmic fractions were prepared by mechanical disruption and ultracentrifugation. Rabbits were immunized with the 4918-24Y and 4918-37M cytoplasmic fractions to produce anti-yeast-phase and anti-mycelial-phase hyperimmune sera. Each radiolabeled cytoplasmic fraction was absorbed with anti-mycelial-phase immunoglobulin, anti-yeast immunoglobulin, and immunoglobulin from normal rabbit serum. Staphyloccal protein A was used to remove immune complexes. The labeled, nonabsorbed proteins were also analyzed by two-dimensional electrophoresis. Highly reproducible protein spot patterns were obtained which defined hundreds of proteins in each extract. The specificity of the immunoglobulin hundreds of proteins in each extract. The specificity of the immunoglobulin preparations was extremely broad, and as many as 168 cytoplasmic antigens were detected. Eighty-three antigens were recognized in the mycelial-phase extract only by the anti-mycelial-phase immunoglobulin. However, comparative analysis revealed that all of these proteins were present in at least one other extract. Therefore, none of them was unique to the mycelial morphology. Eleven antigens were detected in the 2252-37Y extract that were not present in the extracts from strain 4918, which indicates that proteins obtained from different strains may express similar antigenic determinants, but differ in their physiochemical properties.

Antigens↗

PubMed1983

Effect of antibody on entry of Rickettsia tsutsugamushi into polymorphonuclear leukocyte cytoplasm.

The effects of rickettsial antibodies on the entry of Rickettsia tsutsugamushi, Gilliam strain, into guinea pig polymorphonuclear leukocyte cytoplasm were studied by electron microscopy. Immunoglobulin G fractions were obtained from four rabbit antisera against: yolk sac-propagated rickettsiae; baby hamster kidney cell (BHK-21)-propagated rickettsiae; formaldehyde-fixed, BHK-21 cell-propagated rickettsiae; and glutaraldehyde-fixed, BHK-21 cell-propagated rickettsiae. A fuzzy coating was observed on the rickettsiae after reaction with each of the antibodies. All of the antibodies increased the uptake of rickettsiae by polymorphonuclear leukocytes. The opsonization effect was higher with an antibody against BHK-21 cell-propagated rickettsiae than with an antibody against yolk sac-propagated rickettsiae, and an antibody to live rickettsiae had a higher opsonization effect than did antibodies to chemically fixed rickettsiae. Rickettsiae were released from phagosomes into the cytoplasm with the four antibodies. The highest number of rickettsiae were released into the cytoplasm with antibody against live rickettsiae propagated in BHK-21 cells. The four antibodies inhibited the translocation of the cytoplasmic rickettsiae from the filamentous area to glycogen-rich zones. Almost 100% inhibition of translocation was observed with antibodies against live rickettsiae. These results indicate that rabbit antibodies against rickettsiae, when used alone, were unable to completely prevent rickettsial entry into polymorphonuclear leukocyte cytoplasm in vitro.

Animals↗

PubMed1989

Movement of a falciparum malaria protein through the erythrocyte cytoplasm to the erythrocyte membrane is associated with lysis of the erythrocyte and release of gametes.

Erythrocytes containing mature gametocytes of Plasmodium falciparum circulate in the blood until they are ingested by a mosquito, an event that triggers gametogenesis and lysis of the infected erythrocyte. It was previously shown that a parasite protein (Pf155/RESA) accumulates in the erythrocyte cytoplasm next to the parasitophorous vacuolar membrane (S. Uni, A. Masuda, M. J. Stewart, R. Nussenzweig, and M. Aikawa, Am. J. Trop. Med. Hyg., 36:481-488, 1987). Using a monoclonal antibody to Pf155/RESA and rabbit sera to two different repeat peptides of Pf155/RESA, we have studied the location of Pf155/RESA after induction of gametogenesis. Five minutes after triggering gametogenesis, the parasitophorous membrane no longer surrounded the parasite, bringing the parasite membrane in contact with the erythrocyte cytoplasm. Clear spaces appeared throughout the hemoglobin-rich host cytoplasm; Pf155/RESA was now localized in the cytoplasm directly surrounding the spaces. No membrane existed between the spaces and the erythrocyte cytoplasm. The spaces with surrounding Pf155/RESA protein extended to the erythrocyte membrane. After lysis of the erythrocyte membrane (15 min after triggering gametogenesis), the protein was distributed along the erythrocyte membrane and throughout the space between the gamete and the erythrocyte membrane. The mechanism by which Pf155/RESA remained aggregated around the spaces and its role in erythrocyte lysis are unknown. It is of interest that the parasite appeared to use the same molecule during invasion of erythrocytes and during release of gametes from infected erythrocytes.

Animals↗

PubMed1999

Differentiation of monocytes to macrophages primes cells for lipopolysaccharide stimulation via accumulation of cytoplasmic nuclear factor kappaB.

During infection, circulating blood monocytes migrate from the vasculature to the extravascular compartments where they mature into tissue macrophages. The maturation process prepares the cell to actively participate in the inflammatory and the immune responses, and many transcription factors have been found to be involved. Here we report on a novel role for nuclear factor kappaB (NF-kappaB) in this process. Its accumulation in the cytoplasm of differentiated macrophages is responsible for the enhanced ability of the cell to respond to lipopolysaccharide (LPS) stimulation, as determined by tumor necrosis factor alpha (TNF-alpha) secretion. Differentiation of the human monocytic cell line THP-1 into macrophage-like cells was induced by exposure of the cells to phorbol myristate acetate. DNA-bindable NF-kappaB was not detected in the cytoplasm of undifferentiated THP-1 cells but accumulated in the cytoplasm of the cells following differentiation. No TNF-alpha was detected in the media of resting differentiated and nondifferentiated THP-1 cells. Stimulation with LPS of differentiated cells induced the production of higher levels of TNF-alpha than stimulation of nondifferentiated cells. This hyperresponsiveness to LPS was found in the mRNA and secreted TNF-alpha levels. Furthermore, stimulation with LPS induced the translocation of NF-kappaB from the cytoplasm into the nucleus. This translocation process was more rapid in the differentiated cells than in the nondifferentiated cells, and the resultant accumulated levels of NF-kappaB in the nucleus were higher. The DNA-bindable NF-kappaB was identified as a heterodimer of p65 and p50. The results suggest that NF-kappaB accumulation in the cytoplasm during maturation of monocytes to macrophages primes the cells for enhanced responsiveness to LPS and results in the rapid secretion of inflammatory mediators, such as TNF-alpha, by mature macrophages following LPS challenge.

Cell Differentiation↗

PubMed1980

Streptococcus faecalis mutants defective in regulation of cytoplasmic pH.

We have isolated two acid-sensitive mutants of Streptococcus faecalis (ATCC 9790), designated AS13 and AS25, which grew at pH 7.5 but not at pH below 6.0. The ionophore gramicidin D, which collapsed the pH gradient between the cytoplasm and the medium, had little effect on the growth of these mutants, indicating that growing cells maintain only a small pH gradient. In the presence of gramicidin D the growth rates of the parent and mutant strains were identical over a range of pH values. When glucose was added to a cell suspension at pH 6.4, the parent strain generated a pH gradient of 1.0 unit, interior alkaline; AS13 generated a pH gradient of only 0.5 units, and AS25 generated no measurable pH gradient. The proton permeability of the mutant strains was the same as that of the parent strain. These results suggest that a cytoplasmic pH of around 7.5 is required for the growth of the cells and that the mutant strains are unable to establish a neutral cytoplasmic pH in acidic medium because of damage to the regulatory system of the cytoplasmic pH. Mutant strains also have a reduced capacity to extrude protons and take up potassium. Therefore, it is likely that these cation transport systems are involved in the regulation of cytoplasmic pH.

Cell Membrane Permeability↗

PubMed1984

Phase separation between nucleoid and cytoplasm in Escherichia coli as defined by immersive refractometry.

The refractive indices of nucleoid and cytoplasm in Escherichia coli were derived theoretically and experimentally. For the theoretical estimates, we made use of the known macromolecular composition of E. coli B/r (G. Churchward and H. Bremer, J. Theor. Biol. 94:651-670, 1982) and of estimates of cell and nucleoid volumes. These were obtained from micrographs of living bacteria made with a confocal scanning light microscope. The theoretical values were calculated, assuming that all DNA occurred in the nucleoid and that all protein and RNA occurred in the cytoplasm. Comparison with experimental refractive index values directly obtained by immersive refractometry showed that, besides its DNA, the nucleoid must contain an additional amount of solids equivalent to 8.6% (wt/vol) protein. With the nucleoid containing 6.8% (wt/vol) DNA and 8.6% (wt/vol) protein and the cytoplasm containing 21% (wt/vol) protein and 4% (wt/vol) RNA, a mass difference is obtained, which accounts for the phase separation observed between the nucleoid and cytoplasm in living cells by phase-contrast microscopy. The decrease in the refractive index of the nucleoid relative to that of the cytoplasm observed upon, for instance, OsO4 fixation was interpreted as being indicative of the loss of protein content in the nucleoid.

Chloramphenicol↗