Zinc homeostasis in the secretory pathway in yeast

It is estimated that up to 10% of proteins in eukaryotes require zinc for their function. Although the majority of these proteins are located in the nucleus and cytosol, a small subset is secreted from cells or is located within an intracellular compartment. As many of these compartmentalized metalloproteins fold to their native state and bind their zinc cofactor inside an organelle, cells require mechanisms to maintain supply of zinc to these compartments even under conditions of zinc deficiency. At the same time, intracellular compartments can also be the site for storing zinc ions, which then can be mobilized when needed. In this review, we highlight insight that has been obtained from yeast models about how zinc homeostasis is maintained in the secretory pathway and vacuole.     

Zinc homeostasis and functions of zinc in the brain

The brain barrier system, i.e., the blood-brain and blood-cerebrospinal fluid barriers, is important for zinc homeostasis in the brain. Zinc is supplied to the brain via both barriers. A large portion of zinc serves as zinc metalloproteins in neurons and glial cells. Approximately 10% of the total zinc in the brain, probably ionic zinc, exists in the synaptic vesicles, and may serve as an endogenous neuromodulator in synaptic neurotransmission. The turnover of zinc in the brain is much slower than in peripheral tissues such as the liver. However, dietary zinc deprivation affects zinc homeostasis in the brain. Vesicular zinc-enriched regions, e.g., the hippocampus, are responsive to dietary zinc deprivation, which causes brain dysfunctions such as learning impairment and olfactory dysfunction. Olfactory recognition is reversibly disturbed by the chelation of zinc released from amygdalar neuron terminals. On the other hand, the susceptibility to epileptic seizures, which may decrease vesicular zinc, is also enhanced by zinc deficiency. Therefore, zinc homeostasis in the brain is closely related to neuronal activity. Even in adult animals and probably adult humans, adequate zinc supply is important for brain functions and prevention of neurological diseases.     

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: Further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDa protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells. we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 μM), on pepsinogen secretion and phosphorylation of the 72-kDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 μM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 μM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with ³²P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 μM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS “phosphorylation/calmodulin binding domain peptide” indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion. J. Cell. Biochem. 64:514–523. © 1997 Wiley-Liss, Inc.     

Nutrients Induce an Increase in Inositol 1,4,5-Trisphosphate in Soybean Cells: Implication for the Involvement of Phosphoinositide-Specific Phospholipase C in DNA Synthesis

Abstract: Phosphoinositide-specific phospholipase C (PI-PLC) hydrolyzes the membrane lipid phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) to generate 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP₃). Both molecules serve as second messengers to carry out various cellular functions in mammals. In the present study, we demonstrate that many organic and inorganic nutrients cause the elevation of InsP₃ concentrations in cultured soybean cells. This elevation of InsP₃ content is sustained for several hours following treatment with Murashige-Skoog (MS) inorganic nutrients. Phosphate and calcium are the major components in MS salts responsible for the increase in InsP₃ levels. DNA synthesis, a measure of cell growth, was significantly suppressed by the PI-PLC-specific inhibitor 1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122), whereas its near-identical analogue 1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-2,5-pyrrolidinedione did not cause any suppression. Activation of PI-PLC by MS salts increased DNA synthesis and abolished the suppression of DNA synthesis caused by U-73122. Thus, we conclude that the higher cellular concentration of InsP₃ induced by MS treatment is involved in DNA synthesis.     

Molecular determinants for the differential coupling of Galpha(16) to the melatonin MT1, MT2 and Xenopus Mel1c receptors

The pineal neurohormone melatonin modulates a variety of physiological processes through different receptors. It has recently been reported that the cloned melatonin receptors (MT1, MT2 and Mel1c) exhibit differential abilities to stimulate phospholipase C (PLC) via G(16). Here we examined the molecular basis of such differences in melatonin receptor signaling. Coexpression of MT1 or MT2 with the alpha subunit of G(16) (Galpha(16) ) allowed COS-7 cells to accumulate inositol phosphates in response to 2-iodomelatonin. In contrast, Mel1c did not activate Galpha(16) even though its expression was demonstrated by radioligand binding and agonist-induced inhibition of adenylyl cyclase. As Mel1c possesses an exceptionally large C-terminal tail, we further asked if this structural feature prevented productive coupling to Galpha(16). Eleven chimeric melatonin or mutant receptors were constructed by swapping all or part of the C-terminal tail between MT1, MT2 and Mel1c. All chimeras were fully capable of binding 2-[(125) I]iodomelatonin and inhibiting adenylyl cyclase. Chimeras containing the full-length Mel1c tail were incapable of activating Galpha(16), while those that contained the complete C-terminal region of either MT1 or MT2 stimulated PLC. Incorporation of the extra portion of the C-terminal tail of Mel1c to either MT1 or MT2 completely abolished the chimeras' ability to stimulate PLC via Galpha(16). In contrast, truncation of the C-terminal tail of Mel1c allowed interaction with Galpha(16). Our results suggest that Galpha(16) can discern structural differences amid the three melatonin receptors and provide evidence for functional distinction of Mel1c from MT1 and MT2 receptors.     

Role of guanine nucleotide regulatory protein in polyphosphoinositide degradation and activation of phagocytic leukocytes by chemoattractants

Leukocyte activation by chemoattractants provides an important model to study the biochemical mechanisms of stimulus-response coupling in these cells. Well-defined chemotactic factors induce readily quantifiable responses in phagocytic leukocytes. These include directed migration and the production and release of toxic substances including oxygen radicals and lysosomal enzymes. The development of radiolabeled synthetic oligopeptides with potent chemotactic activity allowed the demonstration of chemoattractant receptors on polymorphonuclear leukocytes (PMNs) as well as macrophages. In membrane preparations from these cells, these receptors exist in high- and low-affinity states which are regulated by guanosine di- and triphosphates. This suggested that chemoattractant receptors interact with guanine nucleotide regulatory proteins (N or G proteins). Although chemoattractants elicit a rapid but transient increase in intracellular cAMP levels, they neither stimulate nor inhibit membrane-bound adenylate cyclase, suggesting a novel role for N proteins in certain receptor-transduction mechanisms. Stimulation of phagocytes by chemoattractants is also associated with a rapid increase in cytosolic Ca2+ concentrations ([ Ca2+]i) which appears to result from the production of inositol 1,4,5-triphosphate (IP3) as a consequence of the diesteric cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2). Treatment of phagocytes with pertussis toxin (PT), which ADP-ribosylates and thereby inactivates certain N proteins, abolishes the cells' responsiveness to chemoattractants. More direct evidence for a role of a PT-sensitive N protein in leukocyte activation was provided by the demonstration that chemoattractants stimulate the hydrolysis of PIP2 in PMN membranes only in the presence of GTP. This receptor-mediated hydrolysis of PIP2 is not observed in plasma membranes prepared from PT-treated PMNs. Therefore, these studies suggest that occupancy of chemoattractant receptors activates a PT-sensitive N protein. The activated N protein shifts the Ca2+ requirement for phospholipase C activity from supraphysiological levels to ambient cytosolic Ca2+ concentrations. Cleavage of PIP2 results in the formation of the second messenger molecules, IP3 and 1,2-diacylglycerol, which can initiate cellular activation. These messengers also seem to activate responses which feed back to attenuate receptor stimulation of phospholipase.     

Expression pattern and sub‐cellular distribution of phosphoinositide specific phospholipase C enzymes after treatment with U‐73122 in rat astrocytoma cells

Phosphoinositide specific phospholipase C (PI‐PLC) enzymes interfere with the metabolism of inositol phospholipids (PI), molecules involved in signal transduction, a complex process depending on various components. Many evidences support the hypothesis that, in the glia, isoforms of PI‐PLC family display different expression and/or sub cellular distribution under non‐physiological conditions such as the rat astrocytes activation during neurodegeneration, the tumoural progression of some neoplasms and the inflammatory cascade activation after lipopolysaccharide administration, even if their role remains not completely elucidated. Treatment of a cultured established glioma cell line (C6 rat astrocytoma cell line) induces a modification in the pattern of expression and of sub cellular distribution of PI‐PLCs compared to untreated cells. Special attention require PI‐PLC beta3 and PI‐PLC gamma2 isoforms, whose expression and sub cellular localization significantly differ after U‐73122 treatment. The meaning of these modifications is unclear, also because the use of this N‐aminosteroid compound remains controversial, inasmuch it has further actions which might contribute to the global effect recorded on the treated cells. J. Cell. Biochem. 110: 1005–1012, 2010. © 2010 Wiley‐Liss, Inc.     

Effects of the Ca ionophore A23187 on zinc-induced apoptosis in C6 glioma cells

Zinc ions are essential, but at elevated concentrations, they also have toxic effects on mammalian cells. Zinc plays a crucial role in cell proliferation and differentiation and it even protects cells against apoptosis caused by various reagents. On the other hand, zinc at high concentrations causes cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, and breakdown of the mitochondrial membrane potential. In the present work, a clone of rat C6 glioma cells that was resistant to toxic effects of ZnCl₂ up to 250 μM was employed to study the effect of the ionophore A23187 on zinc-induced apoptosis. Neither 150 μM Zn²⁺ nor 100 nM A23187 alone caused apoptosis as measured by internucleosomal DNA fragmentation. However, combined exposure of C6 cells to 100 nM A23187 and 150 μM Zn²⁺ for 48 h was effective in inducing apoptosis. Because the so-called calcium ionophore A23187 is not specific for Ca²⁺ ions but also transports Zn²⁺ with high selectivity over Ca²⁺, we investigated whether this substance promoted the uptake of Zn²⁺ ions into C6 cells. Employing the zinc-specific fluorescence probe Zinquin, we observed that the very low concentration of 1.9 nM A23187 significantly and rapidly raised the intracellular mobile Zn²⁺ content. Analysis by atomic absorption spectroscopy revealed that incubation with 1.9 nM A23187 caused a doubling of the total intracellular zinc level within 60 min. We conclude that the apoptosis evoked by the combined action of Zn²⁺ and A23187 was the result of enhanced Zn²⁺ influx evoked by the ionophore, resulting in higher intracellular zinc levels.     

Cadmium-induced apoptosis in C6 glioma cells: Mediation by caspase 9-activation

The induction of apoptotic cell death by cadmium was investigated in eight mammalian cell lines. Great differences in the cytotoxicity of cadmium were found with different cell lines: Rat C6 glioma cells turned out to be most sensitive with an IC₅₀-value of 0.7 M, while human A549 adenocarcinoma cells were relatively resistant with an IC₅₀-value of 164 M CdCl₂. The mode of cadmium-induced cellular death was identified to involve apoptotic DNA fragmentation in three cell lines, i.e., in C6 glioma cells, E367 neuroblastoma cells and NIH3T3 fibroblasts. In C6 glioma cells, this process was investigated in detail. Internucleosomal DNA-fragmentation occurred 40 h after application of CdCl₂ and was concentration-dependent between 1–100 M CdCl₂, followed by a decrease at higher concentrations due to necrotic processes. Apoptotic chromatin-condensation and nuclear fragmentation was observed 48 h after application of 2.5 M CdCl₂. Furthermore, cadmium (1 M, 48 h) caused a breakdown of the mitochondrial membrane potential as shown by the decline in mitochondrial uptake of rhodamine 123. Also, we found an activation of caspase 9, a protease known to be activated in apoptotic processes following mitochondrial damage. Besides Cd²⁺, other toxic heavy metal ions (Hg²⁺, Pb²⁺, Ni²⁺, Fe²⁺, CrO₄ ^2–, Cu²⁺ or Co²⁺) did not induce apoptotic DNA fragmentation in C6 cells. The only exception was Zn²⁺ which caused apotosis at high concentrations (>150 M) whereas it protected against cadmium-induced apoptosis at low concentrations (10–50 M).     

Zinc metabolism and homeostasis: The application of tracer techniques to human zinc physiology

Tracer kinetic techniques based on zinc stable isotopes have a vital role in advancing knowledge of human zinc physiology and homeostasis. These techniques have demonstrated the complexity of zinc metabolism, and have been critical to estimating the size and interrelationships of those pools of zinc that exchange rapidly with zinc in plasma and which are likely to be especially important for zinc dependent biology. This paper presents findings from recent research linking a steady state compartmental model with non-steady state post-prandial sampling from the intestine, utilizing a combination of intestinal intubation/perfusion and stable isotope tracer kinetic techniques. The gastrointestinal tract has a central role in maintaining whole body zinc homeostasis. While the fractional absorption of zinc from a meal depends on the quantity of exogenous zinc and on such dietary factors as phytic acid, the fractional absorption does not appear to be dependent on the size of the rapidly exchanging pool of the host. In contrast, the quantity of endogenous zinc excreted via the intestine is positively correlated with both the amount of absorbed zinc and the zinc `status' of the host, and thus this process has an equally critical role in maintaining zinc homeostasis. The observed alterations in zinc metabolism in some disease states can be understood in the context of known homeostatic processes. In other conditions, however, such alterations as inflammation-associated hyperzincuria and zinc redistribution, the links between homeostatic perturbation and cellular biology are yet to be explained. Thus the challenge remains for research at the whole body level to carefully characterize zinc distribution and exchange under diverse circumstances, while research at the cellular level must elucidate the regulatory processes and the factors to which they respond.     

Dual coupling of the cloned 5-HT1A receptor to both adenylyl cyclase and phospholipase C is mediated via the same Gi protein.

The coloned 5-HT_1A receptor, stably expressed in HeLa cells, has been shown to mediate the effects of 5-hydroxytryptamine (5-HT) to inhibit cAMP formation and to stimulate the hydrolysis of phosphatidylinositol. Both responses were found to be pertussis toxin sensitive. We have examined these two responses in membranes derived from these cells and show that the 5-HT_1A receptor can directly regulate the activity of adenylyl cyclase and phospholipase C in response to agonist. In order to examine whether the same or distinct guanine nucleotide-binding regulatory protein(s) (G protein) are involved in these two signal transduction pathways, we used anti-peptide antibodies recognizing the -subunits of G_i1, G_i2, G_i3 as specific tools, since these pertussis toxin substrates are expressed in HeLa cells. These antibodies have previously been shown to prevent receptor-G protein coupling by binding to the regions of G proteins which are putatively involved in interaction with receptors. Our results indicate that the G_i proteins, but preferentially G₃, mediate the effects of 5-HT both to inhibit adenylyl cyclase and to stimulate phospholipase C. These findings demonstrate that the same receptor interacting with the same C protein can regulate several distinct effector molecules.     

Evidence that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of gene expression from human cytomegalovirus promoter in CHO cells

The signal transduction from insulin to its receptors and Ras has been extensively studied, while little has been reported beyond these steps. We found that the expression of human interleukin 6 gene under the control of immediate early gene promoter of human cytomegalovirus was enhanced by insulin sitmulation in Chinese hamster ovary cells. The induction effect of insulin was not significantly affected by inhibitors or activators of conventional protein kinase C, cAMP dependent protein kinase and phosphoinositide -3 kinase, however, pre-incubation of the cells with D609, a specific inhibitors of phosphatidylcholine-specific phospholipase C completely abolished the induction effect. These results clearly demonstrate that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of hIL-6 expression from the human cytomegalovirus promoter in Chinese hamster ovary cells and strongly suggest that it plays an important role in the insulin signaling pathways.Abbreviations CHO – Chinese hamster ovary; hCMV promoter – immediate early gene promoter of human cytomegalovirus; hIL-6 – human interleukin 6; PC-PLC-phosphatidylcholine-specific phospholipase C; PI-3 kinase – phosphoinositide 3 kinase; PKA – cAMP dependent protein kinase; PKC – protein kinase C.     

Expression of phosphoinositide-specific phospholipase C isoenzymes in cultured astrocytes

Signal transduction from plasma membrane to cell nucleus is a complex process depending on various components including lipid signaling molecules, in particular phosphoinositides and their related enzymes, which act at cell periphery and/or plasma membrane as well as at nuclear level. As far as the nervous system may concern the inositol lipid cycle has been hypothesized to be involved in numerous neural as well as glial functions. In this context, however, a precise panel of glial PLC isoforms has not been determined yet. In the present experiments we investigated astrocytic PLC isoforms in astrocytes obtained from foetal primary cultures of rat brain and from an established cultured (C6) rat astrocytoma cell line, two well known cell models for experimental studies on glia. Identification of PLC isoforms was achieved by using a combination of RT-PCR and immunocytochemistry experiments. While in both cell models the most represented PI-PLC isoforms were beta4, gamma1, delta4, and epsilon, isoforms PI-PLC beta2 and delta3 were not detected. Moreover, in primary astrocyte cultures PI-PLC delta3 resulted well expressed in C6 cells but was absent in astrocytes. Immunocytochemistry performed with antibodies against specific PLC isoforms substantially confirmed this pattern of expression both in astrocytes and C6 glioma cells. In particular while some isoenzymes (namely isoforms beta3 and beta4) resulted mainly nuclear, others (isoforms delta4 and epsilon) were preferentially localized at cytoplasmic and plasma membrane level.     

Levodopa modulating effects of inducible nitric oxide synthase and reactive oxygen species in glioma cells

Neurological injury and Parkinson disease (PD) are often associated with the increase of nitric oxide (NO) and free radicals from resident glial cells in the brain. In vitro, exposure to L-3-4-dihydroxyphenylalanine (L-DOPA), one of the main therapeutic agents for the treatment of PD, can lead to neurotoxicity. In this study, lipopolysaccharide (LPS) and interferon-gamma (IFN-g) were used to stimulate C6 glioma cells in the presence of varying concentrations of L-DOPA (1 microM-1 mM). The results indicated a slight augmentation of NO(2)(-) production at low concentrations of L-DOPA (<100 microM) and complete inhibition of NO(2)(-) at higher concentrations (500 microM, 1 mM), (p < 0.001). Western blot analysis corroborated that L-DOPA effects on iNOS was at the level of its protein expression. Total reactive oxygen species (ROS) were detected using 2', 7'-dichlorofluorescein diacetate fluorescence dye (2', 7'-DCFC) and there was an increase of intensity with the increasing concentrations of L-DOPA. Furthermore, large amounts of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) were generated from the autoxidation of L-DOPA. C6 cells contain high levels of catalase, with inadequate levels of superoxide dismutase (SOD); therefore, there was an accumulation of O(2)(-), tantamount to elevation in 2'7'-DCFC intensity. Simultaneous accumulation of O(2)(-) and NO(2)(-) would propel formation of peroxynitrite (ONOO-). SOD completely attenuated the autoxidation of L-DOPA and significantly reversed the inhibitory effects on iNOS at high concentrations. The data obtained confirmed that the observed effects on iNOS were not due to the activation of the D(1) or beta1 adrenergic receptors by L-DOPA. It was concluded from this study that L-DOPA contributed to the modulation of iNOS and to the increase of O(2)(-) production in the stimulated glioma cells in vitro.     

Tubulin stimulates adenylyl cyclase activity in C6 glioma cells by bypassing the beta-adrenergic receptor: a potential mechanism of G protein activation

While the cytoskeleton is known to play several roles in the biology of the cell, one role, which has been revealed only recently, is that of a participant in the signal transduction process. Tubulin binds specifically to the alpha subunits of Gs (stimulatory GTP-binding regulatory protein of adenylyl cyclase), Gi1 (inhibitory protein of adenylyl cyclase), and Gq and transactivates those molecules through direct transfer of GTP. The relevance of this transactivation process to G proteins which are normally activated by a neurotransmitter-occupied receptor is the subject of this study. C6 glioma cells, made permeable with saponin, retained tight coupling between Gs and the beta-adrenergic receptor. Although 5-guanylylimidodiphosphate (GppNHp) was incapable of activating Gs (and subsequently, adenylyl cyclase) in the absence of agonist, tubulin with GppNHp bound (tubulin-GppNHp) activated adenylyl cyclase with an EC(50) of 30 nM. Desensitization of beta-adrenergic receptors by isoproterenol exposure had no effect on the ability of tubulin-GppNHp to activate Gs and adenylyl cyclase. When the photoaffinity GTP analog, azidoanilido GTP (AAGTP; P3(4-azidoanilido)-P1-5'-GTP), was added to C6 membranes or permeable C6 cells, it was only weakly incorporated by G alpha s in the absence of isoproterenol. When the same concentration of dimeric tubulin with AAGTP bound was introduced, AAGTP was transferred from tubulin to G alpha s, activating the latter species. Similar 'preferential' activation of G alpha s by tubulin-AAGTP versus the free nucleotide was seen using purified components. Thus, membrane-associated tubulin may serve to activate G alpha s, independent of signals not normally coupled to that protein. Tubulin may act as an agent to link a variety of membrane-associated signalling systems.     

Coordinated Regulation of Radioadaptive Response by Protein Kinase C and p38 Mitogen-Activated Protein Kinase

Eukaryotic cells are known to have an inducible or adaptive response that enhances radioresistance after a low priming dose of radiation. This radioadaptive response seems to present a novel cellular defense mechanism. However, its molecular processing and signaling mechanisms are largely unknown. Here, we studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the expression of radioadaptive response in cultured mouse cells. Protein immunoblot analysis using isoform-specific antibodies showed an immediate activation of PKC-alpha upon X-irradiation as indicated by a translocation from cytosol to membrane. A low priming dose caused a prolonged translocation, while a nonadaptive high dose dramatically downregulated the total PKC level. Low-dose X-rays also activated the p38 MAPK. The activation of p38 MAPK and resistance to chromosome aberration formation were blocked by SB203580, an inhibitor of p38 MAPK, and Calphostin C, an inhibitor of PKC. Furthermore, it was demonstrated that p38 MAPK was physically associated with delta1 isoform of phospholipase C (PLC-delta1), which hydrolyzed phosphatidylinositol bisphosphate into diacylglycerol, an activator of PKC, and that SB203580 also blocked the activation of PKC-alpha. These results indicate the presence of a novel mechanism for coordinated regulation of adaptive response to low-dose X-rays by a nexus of PKC-alpha/p38 MAPK/PLC-delta1 circuitry feedback signaling pathway with its breakage operated by downregulation of labile PKC-alpha at high doses or excess stimuli.     

Coordinated effects of electromagnetic field exposure on erythropoietin-induced activities of phosphatidylinositol-phospholipase C and phosphatidylinositol 3-kinase

Initial studies with the erythropoietin-sensitive human hematopoietic cell line, TF1, demonstrated both multifarious effects of pulsed electromagnetic field (EMF) exposure on lipid signal transduction and antiproliferative effects of EMF. Stimulation of TF1 cells with erythropoietin resulted in increased phosphatidylinositol 3-kinase activity within 2 min. Addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, produced a decrease in cell proliferation as measured by accumulation of cells in the G₀/G₁ phase of the cell cycle and suppression of erythropoietin-induced DNA synthesis. Similar effects on cell proliferation were seen under EMF treatment. Phosphatidylinositol 3-kinase activity in erythropoietin-stimulated TF1 cells, measured in whole-cell extracts, increased 34% within 2 min and remained above basal levels for at least 20 min. EMF decreased erythropoietin-stimulated phosphatidylinositol 3-kinase activity to lower than basal levels. Additionally, translocation of the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase to the membrane was prevented by EMF. Phosphatidylinositol-specific phospholipase C was activated, as reflected by increases in diacylglycerol and inositol trisphosphate at 15–60 s after EMF treatment. These results provide the first evidence of subtle coordinated changes by EMF associated with loss of phosphatidylinositol 3-kinase activity, inhibition of the translocation of p85 to the membrane, and activation of phosphatidylinositol-phospholipase C.     

SVCT与维生素C内稳态的保持

维生素C(又名抗坏血酸)是一种基本的微量营养素,作为辅助因子参与多个酶促反应,同时还是一种自由基清除剂。维生素C内稳态主要由两种钠离子依赖的维生素C转运蛋白(sodium-dependent vitamin C transporter,SVCT)——SVCT1和SVCT2来保持。SVCT1在内皮系统表达,介导了维生素C的肠吸收和肾脏重吸收;而SVCT2表达广泛,表达于脑、骨骼和其他组织,保护这些组织免遭氧化损伤。SVCT的遗传多态性与癌症的发生密切相关。对SVCT介导的维生素C内稳态的保持机制的研究,可使维生素C更好地应用于临床。     

Induction of apoptosis in rat C6 glioma cells by panaxydol

Panaxydol is a naturally occurring non-peptidyl small molecule isolated from the lipophilic fractions of Panax notoginseng, a well-known Chinese traditional medicine. Previous studies have shown that panaxydol inhibited the growth of various kinds of malignant cell lines. To date, there has been no report concerning the effect of panaxydol on cell growth inhibition in glioma cells. In this paper, we examined panaxydol's antiproliferation and proapoptotic effects on rat C6 glioma cells and investigated its mechanism. Cell growth inhibition of panaxydol was determined by MTT reduction assay. Apoptosis of cells was measured by both Hoechst 33258 staining and Annexin V analysis. It was found that panaxydol markedly inhibited proliferation of C6 cells in a dose-dependent manner with ID(50) of 40 microM. The cell apoptosis was observed at 48 h in the presence of panaxydol. In concert with these findings, Western blot analysis showed a decreased expression of bcl-2 and increased levels of Bax and caspase-3 in C6 cells treated by panaxydol. In conclusion, panaxydol has profound effects on growth and apoptosis of C6 cells, suggesting that panaxydol may be a potential candidate for the treatment of malignant gliomas.