Cellular Cytosol Research Articles

Scavenger Enzyme Activities in Subcellular Fractions of White Clover (Trifolium repens L.) under PEG-induced Water Stress

Scavenger enzyme activities in subcellular fractions under polyethylene glycol (PEG)-induced water stress in white clover (Trifolium repens L.) were studied. Water stress decreased ascorbic acid (AA) content and catalase (CAT) activity and increased the contents of hydrogen peroxide (H2O2), thiobarbituric acid reactive substances (TBARS) (measure of lipid peroxidation), and activities of superoxide dismutase (SOD), its various isozymes, ascorbate peroxidase (APOX), and glutathione reductase (GR) in cellular cytosol, chloroplasts, mitochondria, and peroxisomes of Trifolium repens leaves. In both the PEG-treated plants and the control, chloroplastic fractions showed the highest total SOD, APOX, and GR activities, followed by mitochondrial fractions in the case of total SOD and GR activities, whereas cytosolic fractions had the second greatest APOX activity. However, CAT activity was the highest in peroxisomes, followed by the cytosol, mitochondria, and chloroplasts in decreasing order. Although Mn-SOD activity was highest in mitochondrial fractions, residual activity was also observed in cytosolic fractions. Cu/Zn-SOD and Fe-SOD were observed in all subcellular fractions; however, the activities were the highest in chloroplastic fractions for both isoforms. Total Cu/Zn-SOD activity, the sum of activities observed in all fractions, was higher than other SOD isoforms. These results suggest that cytosolic and chloroplastic APOX, chloroplastic and mitochondrial GR, mitochondrial Mn-SOD, cytosolic and chloroplastic Cu/Zn-SOD, and chloroplastic Fe-SOD are the major scavenger enzymes, whereas cellular CAT may play a minor role in scavenging of O2− and H2O2 produced under PEG-induced water stress in Trifolium repens.

Niemann-Pick C1 Functions in Regulating Lysosomal Amine Content

Mutations in the late endosomal/lysosomal membrane protein Niemann-Pick C1 (NPC1) are known to cause a generalized block in retrograde vesicle-mediated transport, resulting in the hyper-accumulation of multiple lysosomal cargos. An important, yet often overlooked, category of lysosomal cargo includes the vast array of small molecular weight amine-containing molecules that are substrates for ion trapping in the highly acidic organelle lumen. We show here that the introduction of amine-containing molecules in lysosomes can significantly stimulate NPC1-mediated late endosome/lysosome fusion, and subsequently the secretion of lysosomal cargo. To illustrate the physiological importance of this NPC1-mediated transport pathway, we show that NPC1-deficient cells are more susceptible to the toxic effects of a lysosomotropic polyamine metabolite 3-aminopropanal. Moreover, NPC fibroblasts are shown to have higher levels of polyamine oxidase, an enzyme involved in the formation of 3-aminopropanal. Collectively, these findings provide strong support for a novel functional role for NPC1 and may also provide clues toward understanding NPC disease progression.

Un dithiol endogène aux propriétés antioxydantes : l’acide alpha-lipoïque, utilisation potentielle dans les pathologies cardiovasculaires

Alpha-Lipoic acid (ALA) is a natural compound, chemically named 1,2-dithiolane-3-pentanoic acid, also referred to as thioctic acid. In humans, ALA is synthetized by the liver and other tissues with high metabolic activity: heart, kidney. ALA is both water and fat soluble and therefore, is widely distributed in both cellular membranes and cytosol. Recently, a greater deal of attention has been given to antioxidant function for ALA and its reduced formed: dihydrolipoic acid (DHLA). ALA scavenges hydroxyl radicals, hypochlorous acid and singlet oxygen. It may also exert antioxidant effects in biological systems through transitional metal chelation. Dihydrolipoic acid has been shown to have antioxidant but also pro-oxidant properties in systems in which hydroxyl radical was generated. ALA/DHLA ratio has the capacity to recycle endogenous antioxidants such as vitamin E. A number of experimental as well as clinical studies point to the usefulness of ALA as a therapeutic agent for such diverse conditions as diabetes, atherosclerosis, insulin resistance, neuropathy, neurodegenerative diseases and ischemia-reperfusion injury. ALA represents a potential agent on the vascular endothelium, recording to ALA/DHLA redox couple is one of the most powerful biological antioxidant systems.

CD1d Degradation in Chlamydia trachomatis-infected Epithelial Cells Is the Result of Both Cellular and Chlamydial Proteasomal Activity

Chlamydia trachomatis is an obligate intracellular pathogen that can persist in the urogenital tract. Mechanisms by which C. trachomatis evades clearance by host innate immune responses are poorly described. CD1d is MHC-like, is expressed by epithelial cells, and can signal innate immune responses by NK and NKT cells. Here we demonstrate that C. trachomatis infection down-regulates surface-expressed CD1d in human penile urethral epithelial cells through proteasomal degradation. A chlamydial proteasome-like activity factor (CPAF) interacts with the CD1d heavy chain, and CPAF-associated CD1d heavy chain is then ubiquitinated and directed along two distinct proteolytic pathways. The degradation of immature glycosylated CD1d was blocked by the proteasome inhibitor lactacystin but not by MG132, indicating that degradation was not via the conventional proteasome. In contrast, the degradation of non-glycosylated CD1d was blocked by lactacystin and MG132, consistent with conventional cellular cytosolic degradation of N-linked glycoproteins. Immunofluorescent microscopy confirmed the interruption of CD1d trafficking to the cell surface, and the dislocation of CD1d heavy chains into both the cellular cytosol and the chlamydial inclusion along with cytosolic CPAF. C. trachomatis targeted CD1d toward two distinct proteolytic pathways. Decreased CD1d surface expression may help C. trachomatis evade detection by innate immune cells and may promote C. trachomatis persistence.

Subcellular Distribution of Heavy Metals in Organs of BivalveModiolus Modiolus Living Along a Metal Contamination Gradient

Concentration and distribution of Fe, Zn, Cu, Cd, Mn, Pb, Ni among subcellular fractions (cellular membrane structures and cytosol) and Zn, Cu, Cd among cytoplasmic proteins in the kidney and digestive gland of musselModiolus modiolus living along a polymetallic concentration gradient were studied. It was found in the kidney ofM. modiolus from contaminated sites that the Fe percent increased in the “membrane” fraction, whereas Zn, Pb, Ni and Mn percent increased in the cytosol compared to the kidney of the control mussel. Note kidney cytosol ofM. modiolus from clean and contaminated sites sequestered major parts of Cu and Cd. In the digestive gland ofM. modiolus from contaminated sites Fe, Zn, Cd, Mn, Ni percent increased in the “membrane” fraction, whereas Cu, Pb percent increased in the cytosol compared to digestive gland of control mussel. Gel-filtration chromatography shows kidney ofM. modiolus contains increased metallothionein-like protein levels irrespective of ambient dissolved metal concentrations. It was shown that the metal detoxification system in the kidney and digestive gland ofM. modiolus was efficient under extremely high ambient metal levels. However, under complex environmental contamination in the kidney ofM. modiolus, the metal detoxification capacity of metallothionein-like proteins was damaged.

Multiple oxidative stress-response members of the Adapt78 family

Adapt78 is an oxidative and calcium stress-response gene. Its protein product is a potent natural inhibitor of the intracellular calcium signaling protein calcineurin. Much of what is known about Adapt78 protein is based on cell-transfection studies. Toward understanding natural endogenous Adapt78, we used an antibody raised against cellular Adapt78 and recently determined that endogenous Adapt78 protein, like its mRNA, is oxidative and calcium stress responsive. Here we report the identification of a second endogenous form of this protein family of 41 kDa. Subcellular fractionation of human HeLa cells revealed that in contrast to results of previous transfection studies, most endogenous Adapt78, characterized as 29 and 41 kDa electrophoretic doublets, resides in the cellular cytosol. The 41 kDa form of Adapt78 was abundant and found to exhibit many characteristics in common with the previously reported oxidative stress-responsive 29 kDa form, including hypo- and hyperphosphorylation variants, rapid loss of the hypophosphorylated form following oxidative stress, response to various kinase and phosphatase inhibitors, and localization. However, it also exhibited some unique characteristics, most notably the lack of calcium inducibility. Finally, the 29 kDa form exhibited a much shorter half-life and strong stabilization following oxidant exposure compared with the 41 kDa Adapt78 form. These data reveal the presence of a novel oxidative stress-responsive 41 kDa Adapt78 species, lend further insight into the Adapt78 family of proteins and their distribution, and challenge previous conclusions obtained using transfection protocols.

Magnetic bead isolation of neutrophil plasma membranes and quantification of membrane-associated guanine nucleotide binding proteins

A protocol for isolation of neutrophil plasma membranes utilizing a plasma membrane marker antibody, anti-CD15, attached to superparamagnetic beads was developed. Cells were initially disrupted by nitrogen cavitation and then incubated with anti-CD15 antibody-conjugated superparamagnetic beads. The beads were then washed to remove unbound cellular debris and cytosol. Recovered plasma membranes were quantified by immunodetection of G β2 in Western blots. This membrane marker-based separation yielded highly pure plasma membranes. This protocol has advantages over standard density sedimentation protocols for isolating plasma membrane in that it is faster and easily accommodates cell numbers as low as 10 6. These methods were coupled with immunodetection methods and an adenosine 5 ′-diphosphate-ribosylation assay to measure the amount of membrane-associated G iα proteins available for receptor coupling in neutrophils either stimulated with N-formyl peptides or treated to differing degrees with pertussis toxin. As expected, pertussis toxin treatment decreased the amount of membrane G protein available for signaling although total membrane G protein was not affected. In addition, activation of neutrophils with N-formyl peptides resulted in an approximately 50% decrease in G protein associated with the plasma membrane.

Study of interaction between the polyoxidonium immunomodulator and the human immune system cells

Polyoxidonium (PO) is a high-molecular weight physiologically active compound with pronounced immunomodulating activity, an N-oxidized polyethylene–piperazine derivative. The aim of our work was to study cellular and molecular mechanisms of the action of PO on the human peripheral blood leukocytes. By means of flow cytometry it was established that the binding of fluorescein-isothiocyanate-labeled PO (FITC-labeled PO) occurs more rapidly with monocytes and neutrophils than with lymphocytes (7- to 8-fold weaker as compared with monocytes). Using colloidal gold-labeled PO and electron microscopy it was shown with that the preparation penetrates into leukocytes by endocytosis. PO is localized in endoplasmic vesicles of cellular cytosol. Analysis of one of the crucial signal transducer, the intracellular Ca 2+, performed with the Fluo-3 fluorescent dye, showed that PO does not induce Ca 2+ mobilization from the intracellular calcium stores and influx of extracellular Ca 2+. The study of the intracellular hydrogen peroxide (H 2O 2) production with the 2′,7′-dichlorfluorescein indicator demonstrated that PO significantly increases the level of intracellular H 2O 2 in monocytes and neutrophils, however, this increase is much less as compared with phorbol myristate acetate stimulation. The analysis of immunomodulating effect produced by PO proved its stimulating activity on some cytokines production in vitro, e.g. interleukin 1β (IL-1β), tumor necrosis factor (TNF)-α and IL-6. A dose-dependent increase in the intracellular killing by blood phagocytes was established under the action of PO.

Localization of α-, β-, and γ-synuclein during neuronal development and alterations associated with the neuronal response to axonal trauma

Genetic and protein studies have indicated abnormalities in α-synuclein in neurodegenerative diseases. However, the developmental localization and cellular role of synuclein isoforms is contentious. We investigated the cellular localization of α-, β-, and γ-synuclein in developing cultured rat neurons and following axonal transection of relatively mature neurons, a model that disrupts the axonal cytoskeleton and results in regenerative sprouting. Cortical neurons were grown up to 21 days in vitro (DIV). Axon bundles at 21 DIV were transected and cellular changes examined at 4 and 24 h post-injury. Immunohistochemistry demonstrated that α- and β-synuclein were localized to cellular cytosol and growth cones at 3DIV, with accumulating puncta-like labeling within axons and growth cones by 10-21DIV. In contrast, γ-synuclein immunoreactivity was limited at all time points. By 21DIV, α- and β-synuclein were present in the same neurons but largely in separate subregions, only 26% of puncta contained both α- and β-synuclein immunoreactivity. Less than 20% of α-, β-, and pan-synuclein immunoreactive puncta directly colocalized to synaptophysin profiles at 10DIV, decreasing to 10% at 21DIV. Both α- and β-synuclein accumulated substantially within damaged axons at 21DIV and were localized to cytoskeletal abnormalities. At latter time points post-injury, α- and β-synuclein immunoreactive puncta were localized to growth cone-like structures in regenerating neurites. This study shows that α- and β-synuclein have a precise localization within cortical neurons and are generally nonoverlapping in their distribution within individual neurons. In addition, synuclein proteins accumulate rapidly in damaged axons and may have a role in regenerative sprouting.

Actions of ionomycin, 4-BrA23187 and a novel electrogenic Ca 2+ ionophore on mitochondria in intact cells

We have used fluorescence digital imaging techniques to explore the actions of two groups of Ca 2+ ionophores: (i) ferutinin, an electrogenic naturally occurring ionophore, and (ii) the neutral ionophores 4-BrA23187 and ionomycin, on cytosolic [Ca 2+] ([Ca 2+] c), mitochondrial [Ca 2+] ([Ca 2+] m) and mitochondrial membrane potential (Δ ψ m) in HepG2 cells and primary hippocampal neurones in culture. 4-BrA23187 and ionomycin promoted the equilibration of [Ca 2+] gradients between cellular compartments, including ER, mitochondria and cytosol. Thus, [Ca 2+] c and [Ca 2+] m increased together and then recovered in parallel on removal of the ionophore. In contrast, following a rise in [Ca 2+] c in response to ferutinin, [Ca 2+] m remained elevated for prolonged periods after the recovery of [Ca 2+] c levels despite washout of the compound. Both groups of Ca 2+ ionophores caused some mitochondrial depolarisation, although this was highly variable in degree. Mitochondrial depolarisation induced by ionomycin and 4-BrA23187 was often modest, independent of cyclosporin A (CsA), was suppressed in the absence of extracellular Ca 2+ and was enhanced by pre-incubation of cells with the inhibitor of the mitochondrial Ca 2+/2Na +-exchanger, CGP37157, suggesting that the change in potential reflects the prior state of mitochondrial calcium loading. The mitochondrial depolarisation induced by ferutinin was not influenced by CGP37157 but was completely blocked by CsA, suggesting that it reflects opening of the mitochondrial permeability transition pore (mPTP). We suggest that ferutinin may provide a very valuable tool to promote mitochondrial calcium overload experimentally and to promote calcium-dependent opening of the mPTP.

Effects of garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity and 2-aminofluorene-DNA adducts in human promyelocytic leukemia cells.

Two components of garlic, diallyl sulfide (DAS) and diallyl disulfide (DADS), inhibited arylamine N-acetyltransferase (NAT) activity and 2-aminofluorene-DNA adduct in human promyelocytic leukemia cells (HL-60). The NAT activity was measured by high performance liquid chromatography assaying for amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF). Cellular cytosols and intact cell suspensions were assayed. The inhibition of NAT activity and 2-AF-DNA adduct formation in human leukemia cells by DAS and DADS were dose-dependent and were directly proportional. The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax from human leukemia cells in both assays. This is the first report of garlic components affecting human leukemia cell NAT activity and 2-AF-DNA adduct formation.

Ellagica acid inhibits arylamine N-acetyltransferase activity and DNA adduct formation in human bladder tumor cell lines (T24 and TSGH 8301)

The fact that vitamin C (ascorbic acid) exhibits a protective effect in certain types of cancer is well documented. Our previous studies demonstrated that human bladder tumor cell line (T24) has N-acetyltransferase (NAT) activity in cytosols and intact cells. The present studies examined the inhibition of arylamine NAT activity and carcinogen (2-aminofluorene)-DNA adduct formation by ellagic acid (EA) in human bladder tumor cell lines (T24 and TSGH 8301). Two assay systems were performed, one with cellular cytosols (9,000g supernatant), the other with intact bladder tumor cell suspensions. NAT activity and 2-aminofluorene-DNA adduct formation in T24 and TSGH 8301 cells was inhibited by EA in a dose-dependent manner in both systems, i.e., the greater the concentration of EA in the reaction the greater the inhibition of NAT activity (dose-and time-course dependent effects). The data also indicated that EA decreased the apparent Km and Vmax of NAT enzymes from T24 and TSGH 8301 cells in cytosols. NAT activity and 2-aminofluorene-DNA adducts in T24 is higher than in TSGH 8301. This report is the first to demonstrate that EA affects human bladder tumor cell NAT activity.

Effect of norcantharidin on N-acetyltransferase activity in HepG2 cells.

The inhibition ofarylamine N-acetyltransferase (NAT) activity by norcantharidin (NCTD), the demethylated form of cantharidin, in human hepatocellular carcinoma HepG2 cells was investigated. By using high performance liquid chromatography, NAT activity on acetylation of 2-aminofluorene (AF) and p-aminobenzoic acid (PABA) were examined. Two assay systems were performed, one with cellular cytosols, the other with intact HepG2 cell suspensions. The NAT activity in HepG2 cell line was inhibited by norcantharidin in a dose-dependent manner in both types of examined systems: i.e. the greater the concentration of norcantharidin in the reaction, the greater the inhibition of NAT activities. This report is the first to show that norcantharidin has an inhibitory effect on NAT activity in HepG2 cell.

Modifications in Brain CaM Kinase II after Long-Term Treatment with Desmethylimipramine

The present study investigated the effect of long-term (15 mg/kg for 15 days) and acute (15 mg/kg, single administration) treatment with desmethylimipramine, a tricyclic antidepressant drug, on calcium/calmodulin-dependent protein kinase II (CaMKII), a kinase implicated in the mechanism of antidepressant drug action. Similar to selective and non-selective serotonin reuptake inhibitors, long-term, but not acute, treatment with desmethylimipramine markedly increased the activity of CaMKII in the hippocampal synaptic vesicle fraction (+51.9%). The kinase activity was also increased in the same fraction of frontal cortex (+24.2%) and in the striatum (+45.9%), although in this last area the mechanism appeared to be different because the protein level of the kinase was also markedly increased (+43.7%). However, the effect of treatment was not restricted to the presynaptic kinase, because CaMKII activity was also increased in the total cellular cytosol in cortical areas. The autonomous (calcium-independent) activity of CaMKII was assayed for the first time after antidepressant treatment, and found to be increased in synaptic vesicles of all three areas. These results confirmed the involvement of CaMKII in antidepressant drug action and suggested that modulation of transmitter release is a primary component in the action of psychotropic drugs.

Inhibition of N-Acetyltransferase activity and DNA–2-Aminofluorene adducts by glycyrrhizic acid in human colon tumour cells

Glycyrrhizic acid (GA) was tested for inhibition of arylamine N-acetyltransferase (NAT) activity in a human colon tumour (adenocarcinoma) cell line (colo 205). Two assay systems were performed, one with cellular cytosols (9000 g supernatant), the other with intact colon tumour cell cultures. The NAT activity in a human colon tumour cell line was inhibited by GA in a dose-dependent manner in both types of systems examined. The data also indicated that GA decreased the apparent values of K m and V max of NAT enzymes from human colon tumour cells in both examined systems. The DNA-2-aminofluorene adduct formation in human colon tumour cells were inhibited by GA. This report is the first to demonstrate that GA does inhibit human colon tumour cell NAT activity and DNA adduct formation.

Aplysia gut epithelial cells: Luminal membrane chloride channel activity

The Cl(-) energy gradient across the luminal membrane of Aplysia foregut epithelial cells is directed downhill from the lumen to the cellular cytosol. No primary or secondary active transporters had been shown to be involved in Cl(-) translocation across the luminal membrane. Cl(-) channel blockers impeded the movement of Cl(-) from the lumen into the foregut cellular cytosol. It was concluded that the primary means of Cl(-) transport across the luminal membrane was via Cl(-) conductance.

Effects of ellagic acid on arylamine N‐acetyltransferase activity in human colon tumor cells

The inhibition of arylamine N‐acetyltransferase (NAT) activity by ellagic acid was determined in a human colon tumor (adenocarcinoma) cell line. Two assay systems were performed, one with cellular cytosols (9000g supernatant), the other with intact colon tumor cell suspensions. The NAT activity in a human colon tumor cell line was inhibited by ellagic acid in a dose‐dependent manner in both types of examined systems: i.e. the greater the concentration of ellagic acid in the reaction, the greater the inhibition of NAT activities in both systems. The data also indicated that ellagic acid decreased the apparent K m and V max of NAT enzymes from human colon tumor cells in both the systems examined. This report is the first demonstration which showed ellagic acid affect human colon tumor cell NAT activity.

Effects of berberine on arylamine N-acetyltransferase activity in human bladder tumour cells

Berberine was used to determine inhibition of arylamine N-acetyltransferase (NAT) activity in human bladder tumour cells. The NAT activity was measured by HPLC assaying for the amounts of N-acetyl-2-aminofluorene (AAF) and N-acetyl- p-aminobenzoic acid (N-Ac-PABA) and remaining 2-aminofluorene (AF) and p-aminobenzoic acid (PABA). Two assay systems were performed, one with cellular cytosols, the other with intact bladder tumour cell suspensions. The NAT activity in human bladder tumour cells was inhibited by berberine in a dose-dependent manner, that is, the higher the concentration of berberine, the higher the inhibition of NAT activity. The values of apparent K m and V max calculated from cytosol NAT and intact cells were also decreased by berberine. This report is the first demonstration to show berberine did affect human bladder tumour cell NAT activity.

Chronic Ethanol‐Initiated Apoptosis in Hepatocytes Is Induced by Changes in Membrane Biogenesis and Intracellular Transport

The outcome of chronic ethanol consumption recorded in liver by in situ staining of the genomic DNA in fragmented nuclei indicates the course of cellular events that has been coined as apoptosis or programmed cell death. Hence, we designed the study to determine which ethanol-induced modification of the cellular make-up is responsible for the hastening the cell damage. The in vitro assays were performed with cellular organelles and cytosol prepared from hepatocytes derived from rats subjected to 9 weeks of chronic alcohol consumption. The results were compared with the pair-fed controls receiving isocaloric liquid diet. In the initial phase of the studies, we established that the process of apoptosis was not triggered by the aberrant activity of neutral or acidic sphingomyelinase. The hepatocytes derived from alcohol and control diets manifested equal enzymatic activity. The de novo synthesis of sphingoid bases and ceramides in the alcohol-derived sample of endoplasmic reticulum was reduced, but the in situ apoptosis was up to 36-fold higher than in the control. Also, the isolated hepatocytes contained a 2- to 4-fold higher amount of nucleosomal fragments in the cytosolic extracts. The endosomes from liver hepatocytes of ethanol-consuming rats, in the presence of the cytosol and mitochondria from pair-fed controls, disclosed 2 to 3 times higher apoptotic potential than sample consisting of ethanol-derived fractions only, and 3 to 5 times higher than the control-derived fractions. The substantial increase in apoptosis, as recorded in the amount of DNA fragments released to the cytosol from the fresh nuclei, was also recorded when the microsomal membranes of endoplasmic reticulum and Golgi were incubated in the conditions with preserved intracellular transport. The maximal 20-fold increase of apoptotic activity was recorded in the incubation mixtures of ethanol-derived endoplasmic reticulum-Golgi membranes with control-derived cytosol in the presence of the ATP generating system. Results infer that the intracellular transport vesicles, generated from ethanol-modified membranes in the presence of the substrates that are available in the cytosol of the control hepatocytes, activate the apoptotic activity in the in vitro system. This interpretation is supported by the results of analysis of the clathrin-coated transport vesicles that, in contrast to nonclathrin transport vesicles, contain a sizable accumulation of ceramides that are known to induce apoptosis.

Effects of ibuprofen on arylamine N-acetyltransferase activity in human colon tumor cells.

The inhibition of arylamine N-acetyltransferase (NAT) activity by ibuprofen was determined in a human colon tumour (adenocarcinoma) cell line. Two assay systems were employed, one with cellular cytosols (9000 g supernatant) and the other with intact colon tumour cell suspensions. The NAT activity in a human colon tumour cell line was inhibited by ibuprofen in a dose-dependent manner in both systems, i.e. the greater the concentration of ibuprofen in the reaction, the greater the inhibition of NAT activities in both systems. The data also indicated that ibuprofen decreases the apparent Km and Vmax of NAT enzyme from human colon tumour cells in both systems examined. This report is the first demonstration to show that ibuprofen affects human colon tumour cell NAT activity.