Elicitor-like effects of low-energy ultrasound on plant (Panax ginseng) cells: induction of plant defense responses and secondary metabolite production

In this work we examined the elicitor-like effects of low-energy ultrasound (US) on plant cells with respect to the induction of plant defense responses and secondary metabolite production. Panax ginseng cells in suspension culture were exposed to US (power 相似文献   

Involvement of nitric oxide in oxidative burst, phenylalanine ammonia-lyase activation and Taxol production induced by low-energy ultrasound in Taxus yunnanensis cell suspension cultures

This work was to characterize the generation of nitric oxide (NO) in Taxus yunnanensis cells exposed to low-energy ultrasound (US) and the signal role of NO in elicitation of plant defense responses and secondary metabolite accumulation. The US sonication (3.5-55.6 mW/cm(3) at 40 kHz fixed frequency) for 2 min induced a rapid and dose-dependent NO production in the Taxus cell culture, which exhibited a biphasic time course, reaching the first plateau within 1.5 h and the second within 7 h after US sonication. The NO donor sodium nitroprusside (SNP) potentiated US-induced H(2)O(2) production and cell death. Inhibition of nitric oxide synthase (NOS) activity by N(omega)-nitro-L-arginine (L-NNA) or scavenging NO by 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxyde (PTIO) partially blocked the US-induced H(2)O(2) production and cell death. Moreover, the NO inhibitors suppressed US-induced activation of phenylalanine ammonium-lyase (PAL) and accumulation of diterpenoid taxanes (Taxol and baccatin III). These results suggest that NO plays a signal role in the US-induced responses and secondary metabolism activities in the Taxus cells.     

Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures

This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low-energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism. Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel). The culture was exposed to low-frequency US for a short period of time (2 min). At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability. The US exposure induced transient production of O(2)*- and H(2)O(2) and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS). Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US-stimulated Taxol production. Inhibition of the ROS production also suppressed the US-induced JA accumulation. These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T. chinensis cells by low-energy US.     

Enhancement of shikonin production in single- and two-phase suspension cultures of Lithospermum erythrorhizon cells using low-energy ultrasound

This work demonstrates the use of low-energy ultrasound (US) to enhance secondary metabolite production in plant cell cultures. Suspension culture of Lithospermum erythrorhizon cells was exposed to low-power US (power density < or = 113.9 mW/cm(3)) for short periods (1-8 min). The US exposure significantly stimulated the shikonin biosynthesis of the cells, and at certain US doses, increased the volumetric shikonin yield by about 60%-70%. Meanwhile, the shikonin excreted from the cells was increased from 20% to 65%-70%, due partially to an increase in the cell membrane permeability by sonication. With combined use of US treatment and in situ product extraction by an organic solvent, or the two-phase culture, the volumetric shikonin yield was increased more than two- to threefold. Increasing in the number of US exposures during the culture process usually resulted in negative effects on shikonin yield but slight stimulation of shikonin excretion. US at relatively high energy levels caused slight cell growth depression (maximum 9% decrease in dry cell weight). Two key enzymes for the secondary metabolite biosynthesis of cells, phenylalanine ammonia lyase and p-hydroxybenzoic acid geranyltransferase, were found to be stimulated by the US. The US stimulation of secondary metabolite biosynthesis was attributed to the metabolic activity of cells activated by US, and more specifically, the defense responses of plant cells to the mechanical stress of US irradiation.     

质膜NAD(P)H氧化酶参予金瓜炭疽(Colletotrichum lagerarium)细胞壁激发子诱导人参细胞产生激发反应(英文)

在人参(Panax ginseng C.A.Meyer)悬浮细胞质膜上测出了NAD(P)H氧化酶活性。这类NAD(P)H氧化酶活性可以被金瓜炭疽细胞壁激发子(Cle)诱导。Cle处理还能诱导人参悬浮细胞的氧进发、促进人参悬浮细胞的皂苷合成、提高苯丙氨酸解氨酶(PAL)的活力、以及诱导查尔式酮酶(CHS)的累积和细胞壁上抗性相关蛋白基因脯氨酸富裕蛋白基因hrgp(Hydroxyprolin-rich glycoproleins)的表达。当用哺乳动物白细胞质膜NADPH氧化酶的特异性抑制剂二亚苯基碘(Diphenylene iodonium,DPI)与奎吖因(quinacrine)预处理人参悬浮细胞30 min 后,Cle诱导的H2O2释放与Cle激活的质膜NAD(P)H氧化酶活性被抑制,同时Cle诱导的PAL活性及CHS的积累下降,皂苷合成与hrgp的表达被抑制。由此推测:人参细胞质膜NAD(P)H氧化酶与哺乳动物白细胞质膜NADPH氧化酶有很大的相似性。在Cle激发人参悬浮细胞产生氧进发的过程中,NAD(P)H氧化酶活性被诱导从而导致H2O2的产生,H2O2作为第二信使,激活苯丙氨酸途径,诱发人参皂苷的合成及hrgp防御基因的表达。这一过程中还涉及到Ca2+内流,胞内Ca2+浓度的升高,蛋白磷酸化与去磷酸化。人参细胞质膜NAD(P)H氧化酶在人参细胞对Cle的反应过程中起一种介导作用。因此可能存在由Cle刺激,NAD(P)H氧化酶被诱导,H2O2释放,到人     

Lysophosphatidylcholine-induced cellular injury in cultured fibroblasts involves oxidative events

Lysophosphatidylcholine (lysoPC), formed during LDL oxidation and located within atherosclerotic plaques, induces numerous cellular responses, but via unknown mechanisms. Cellular events involved in sublethal lysoPC-induced injury were examined because these are relevant to mechanisms by which lysoPC alters cell behavior. LysoPC evoked transient membrane permeabilization in fibroblasts within 10 min. Cells underwent reversible rounding within 2 h, returning 3 h later to grossly normal appearance and a normal response to growth stimulation. We asked whether this sublethal permeabilization resulted from physical perturbation of the plasma membrane or if it required cellular events. LysoPC induced leakage of fluorescent dye from unilamellar phospholipid vesicles, suggesting physical membrane perturbation was a significant contributor. To characterize this further we increased the cholesterol content of cells and vesicles to stabilize membranes, and found decreased lysoPC-induced permeabilization in both cell and cell-free systems as cholesterol levels increased. Interestingly, vitamin E, a known antioxidant, blunted lysoPC-induced permeabilization and morphological changes in cells. Thus, lysoPC appeared to cause an unexpected oxidant stress-dependent enhancement of cell injury. To confirm this, several structurally distinct antioxidants, including N, N'-diphenyl-1,4-phenylenediamine, Desferal, Tiron, and 4-hydroxy TEMPO, were applied and these also were inhibitory. Oxidant stress was observed by a lysoPC-induced increase in fluorescence of 5- and 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, an intracellular marker of reactive oxygen species. Lysophosphatidylethanolamine (lysoPE) caused qualitatively similar morphological changes to cells and induced permeabilization, but injury by lysoPE was not inhibited by antioxidants. These data suggest that generation of intracellular reactive oxygen species follows lysoPC-induced plasma membrane destabilization and that this lysoPC-specific oxidant stress enhances cell injury. This intracellular oxidant stress in response to lysoPC may be an integral part of the multiple influences lysoPC has on gene expression and cell function.     

Immobilization of Escherichia coli JM103[pUC8] in kappa-carrageenan coupled with recombinant protein release by in situ cell membrane permeabilization.

Immobilization of Escherichia coli JM103[pUC8] was carried out with kappa-carrageenan as the support matrix. Substantial natural excretion of beta-lactamase, attributable to the less intact membrane of plasmid-harboring cells, was observed in immobilized cell cultures. Nevertheless, a significant portion of the beta-lactamase produced was retained in the cells. As compared to suspension cultures, much higher beta-lactamase activities, especially in the extracellular liquid, and much longer retention of plasmid-bearing cells (improved plasmid stability) were observed in immobilized cell cultures. Further enhancement in excretion of the recombinant protein (beta-lactamase) was achieved by permeabilization of cell membrane by periodic exposure of the immobilized cell cultures to ethylenediaminetetraacetic acid (EDTA). While the presence of EDTA led to some suppression of cell growth in suspension cultures, cell growth in gel beads was not affected by EDTA to the same extent, possibly due to lesser exposure of immobilized cells to EDTA. Exposure of immobilized cell cultures to EDTA presumably inhibited plasmid replication and led in turn to diversion of cellular resources for the support of expression of plasmid genes. Indeed, treatment of the immobilized cell cultures with EDTA resulted in increased production of beta-lactamase when compared to the enzyme production in EDTA-free cultures. More frequent addition of EDTA increased the period of retention of plasmid-bearing cells in these cultures but did not have any noticeable adverse effect on synthesis of beta-lactamase. Improvement in plasmid stability in EDTA-treated immobilized cell cultures was ascribed to the reduction in the growth rate differential between plasmid-free and plasmid-bearing cells, since plasmid-free cells were subject to more reduction in specific growth rate than were plasmid-bearing cells.     

Fungal elicitor induces singlet oxygen generation, ethylene release and saponin synthesis in cultured cells of Panax ginseng C. A. Meyer

Singlet oxygen is a high-energy molecular oxygen species. As one of the most active intermediates involved in chemical and biochemical reactions, singlet oxygen plays essential roles in plant responses to UV and strong light. Here, we report that Cle, an elicitor derived from fungal cell walls, induces the generation of singlet oxygen in cell cultures of ginseng, Panax ginseng. Cle treatment also triggers the activation of plasma membrane NADPH oxidase and 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), subsequently leading to ethylene release and increased saponin synthesis, as shown by increased mRNA expression of squalene synthase (SQS) and squalene epoxidase (SQE), and accumulation of beta-amyrin synthase (beta-AS). Suppression of Cle-induced singlet oxygen generation or inhibition of ethylene production blocks saponin synthesis, whereas treatment of ginseng cells with ethylene or singlet oxygen induces the synthesis of saponin. Together, these results indicate that Cle-induced production of both singlet oxygen and ethylene is required for saponin synthesis, and that singlet oxygen may function upstream of ethylene during Cle-induced saponin synthesis.     

Low intensity pulsed ultrasound accelerates macrophage phagocytosis by a pathway that requires actin polymerization, Rho, and Src/MAPKs activity

Phagocytosis is an essential event in the complex process of tissue repair. Here we examined the effect of low intensity pulsed ultrasound (US), which promotes fracture and wound healing, on phagocytosis by mouse macrophage cell line J774A.1 and human monocyte-derived macrophages. First, 10 to 40 min low intensity pulsed US increased uptake of serum opsonized E. coli by J774A.1 cells during a 50 min phagocytosis period. In addition, when the E. coli exposure time was varied between 35 to 80 min, the maximum increase in phagocytosis was observed in the first 35 min upon US exposure. In parallel, US induced robust actin polymerization in a time dependent manner in J774A.1 cells, showing the peak effect 30 min after stimulation. Interestingly, a low concentration of cytochalasin D (0.25-0.5 microM) prevented US-induced phagocytosis of E. coli. Furthermore, we demonstrated US enhanced activation of RhoA. Blocking its downstream effector Rho associated kinase (ROCK) with Y27632 abrogated US-induced phagocytosis. We also show that US induced activation of ERK and p38 MAPK. Pretreatment of the cells with the corresponding inhibitors PD98059 and SB203580 reduced US-induced phagocytosis. In addition, activity of tyrosine kinase Src was required for US-induced phagocytosis. Here Src represents an upstream activator of ERK and p38 MAPK. Depolymerization of actin by cytochalasin D prevented US-induced Src, ERK, and p38 activation. Our data provide a new insight into the cellular and molecular mechanisms by which low intensity pulsed US promotes tissue repair.     

纤维素酶降解人参细胞胞壁产生的激发子诱导人参培养细胞的反应

用纤维素酶降解人参细胞壁制备的激发子刺激人参细胞后,电子显微镜的观察结果显示激发子处理诱导了细胞内淀粉粒的降解,出现了细胞内动员能量物质的应急反应;同时还出现了大量拟脂类圆球体。进一步的研究表明,涉及降解淀粉的淀粉酶活力是升高了,而降解脂类物质的酯酶活力是降低的;同时细胞中可溶性糖和总类脂含量是增加的。这些都说明了人参细胞经激发子处理后能量物质的动员以及脂类代谢途径的调整。另外,适当浓度激发子的处理会诱导人参细胞内人参皂苷含量和苯丙氨酸解氨酶活力的升高,而过氧化物酶和多酚氧化酶活力是下降的。     

The reactions in ginseng cells induced by elicitor derived from digesting ginseng cell walls by cellulase]

After treatment of the elicitor derived from digesting ginseng cell walls by cellulase, the starch grains were degraded, and a large number of spherosomes were produced in Ginseng cells. In the mean time, the activity of amylase in the cells was increased and the activities of esterases in the cells were reduced; in addition, the content of soluble sugar and the content of lipoid were increased. These indicate that the emergency reactions and the adjustment of metabolism of lipoid were taken placed in the cells elicited. Besides, after the treatment with certain concentrations of elicitor, the content of ginseng saponin and the activity of phenylalanine ammonia lyase (PAL) were increased in ginseng cells. On the contrary, the activities of peroxidase (POD) and polyphenoloxidase (PPO) were reduced.     

Molecular mechanisms of low intensity pulsed ultrasound in human skin fibroblasts

Soluble factors such as polypeptide growth factors, mitogenic lipids, inflammatory cytokines, and hormones are known regulators of cell proliferation. However, the effect of mechanical stimuli on cell proliferation is less well understood. Here we examined the effect of low intensity pulsed ultrasound (US), which is used to promote wound healing, on the proliferation of primary human foreskin fibroblasts and the underlying signaling mechanisms. We show that a single 6-11-min US stimulation increases bromodeoxyuridine incorporation. In addition, an increase in the total cell number is observed after sequential US stimulation. US induced stress fiber and focal adhesion formation via activation of Rho. We further observed that US selectively induced activation of extracellular signal-regulated kinase (ERK) 1/2. Inhibition of Rho-associated coiled-coil-containing protein kinase (ROCK) prevented US-induced ERK1/2 activation, demonstrating that the Rho/ROCK pathway is an upstream regulator of ERK activation in response to US. Consequently, activation of ROCK and MEK-1 was required for US-induced DNA synthesis. Finally, an integrin beta(1) blocking antibody as well as a RGD peptide prevented US-induced DNA synthesis. In addition, US slightly increased phosphorylation of Src at Tyr(416), and Src activity was found to be required for ERK1/2 activation in response to US. In conclusion, our data demonstrate for the first time that US promotes cell proliferation via activation of integrin receptors and a Rho/ROCK/Src/ERK signaling pathway.     

IgA responses in xid mice: oral antigen primes Peyer's patch cells for in vitro immune responses and secretory antibody production

Because the gut-associated lymphoreticular tissue (GALT), e.g., Peyer's patches (PP), of X-linked immunodeficient (xid) mice possesses a subpopulation of mature B cells, we have characterized the ability of xid mice to respond to the thymic-dependent antigen sheep erythrocytes (SRBC) given by the oral route. Gastric intubation of SRBC to xid (CBA/N X DBA/2) F1 male or CBA/N mice, followed by the in vitro culture of dissociated PP cells with SRBC, resulted in IgM, IgG1, IgG2, and high IgA anti-SRBC plaque-forming cell (PFC) responses. The addition of unprimed PP but not splenic T cells to splenic xid B cell cultures resulted in IgM anti-SRBC PFC responses, suggesting the importance of GALT T cells for support of the immune responses to SRBC by splenic B cells from xid mice. Furthermore, purified PP T cells from SRBC orally primed xid mice supported in vitro IgA anti-SRBC PFC responses in B cell cultures from either the PP or the spleens of nonprimed xid mice. Higher IgA responses, however, occurred in PP, when compared with splenic B cell cultures. Additional evidence that the GALT of xid mice contains functional IgA precursor cells was provided by the finding that cloned H-2k PP T helper cells (PP Th A) supported IgA responses in PP B cell cultures derived from (CBA/N X C3H/HeN) F1 male (xid) mice. On the other hand, splenic B cells from these xid mice, in the presence of PP Th A cells, did not support in vitro responses. These results suggest that unique subpopulations of T cells occur in the GALT of xid and normal mice; one T cell subpopulation may induce immature B cells to become precursor IgA cells in the PP. A separate GALT T cell subpopulation, e.g., isotype-specific helper T cells, effectively collaborates with mature IgA B cells for the induction of IgA responses to orally administered antigen. When xid mice were gastric intubated with SRBC, followed by i.p. injection of SRBC, good splenic IgA anti-SRBC PFC responses were seen. Salivary and serum IgA antibodies were also detected in these xid mice. Nevertheless, the magnitude of the anti-SRBC response in xid mice was lower than that seen in similarly treated normal mice. These studies indicate that the GALT of both xid and normal mice possess unique populations of T cells that support in vitro responses in xid B cell cultures from either the spleen or the PP, which direct the mature B cell populations present toward IgA isotype-specific responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of the epithelial permeation enhancing effect of basic butylated methacrylate copolymer--in vitro studies

Membrane destabilizing properties and increased efflux of doxorubicin from liposomes caused by basic butylated methacrylate copolymer (BBMC), better known under its commercial trade name EUDRAGIT E, have been described in the scientific literature. Here, we investigated the effect of BBMC on suspended and filter-grown Caco2 cells with respect to apical-to-basal transport and membrane permeabilization using transport assays, trypan blue exclusion assay, measurements of transepithelial electrical resistance (TEER), confocal laser scanning microscopy, and transmission electron microscopy. The effect of inhibiting protein phosphatase 2A (PP2A) by okadaic acid was investigated by measuring TEER, but a link between PP2A and the observed effects could not be established. Overall, membrane permeabilization of Caco2 cells by BBMC was demonstrated, which went along with increased apical-to-basal transport of the model compounds mannitol, talinolol, and trospium. The effect was concentration- and time-dependent, and reversible. Enhancement occurred at polymer concentrations as low as 20 microg/mL.     

Role of P2z purinergic receptors in ATP-mediated killing of tumor necrosis factor (TNF)-sensitive and TNF-resistant L929 fibroblasts.

Two closely related cell lines were characterized in their responses to extracellular ATP (ATPo): the fibroblast cell line L929 and a TNF-resistant variant L929/R. Both lines showed ATPo-activated increases in intracellular Ca2+, inward current, and sustained depolarization of the plasma membrane, cell responses compatible with activation of purinergic receptors of the P2y, P2x, or P2z subtype; however, only the L929/R variant was susceptible to ATPo-dependent early permeabilization of the plasma membrane to hydrophilic solutes of M(r) below 900, a response uniquely caused by the activation of P2z receptors. Both cell types were susceptible to the cytotoxic effect of ATPo, but killing of the L929/R variant required much shorter incubations in the presence of this nucleotide. Morphologic examination of ATPo-challenged L929 and L929/R cells showed that cell death occurred by two alternative mechanisms: colloido-osmotic lysis or apoptosis. Occurrence of apoptosis was confirmed by agarose gel analysis of cellular DNA. Although ATPo caused a fast mobilization of intracellular Ca2+, neither colloido-osmotic lysis nor apoptosis were Ca2+ dependent. Our results show that the L929/R variant, but not the L929 parental fibroblast cell line, expresses functional purinergic receptors of the P2z subtype. The presence of P2z receptors confers to L929/R cells enhanced susceptibility to ATPo-mediated cytotoxicity.     

Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.     

Mucosal immunoregulation: environmental lipopolysaccharide and GALT T lymphocytes regulate the IgA response

In this review, we have emphasized: 1) bacterial lipopolysaccharide (LPS) involvement in IgA responses to orally administered thymic-dependent (TD) antigens; 2) characterization of Peyer's patch (PP) lymphoreticular cells; and 3) gastrointestinal immunization with gram negative pathogens and anti-LPS immunity to infection. Gut LPS, which interacts with PP lymphoreticular cells, is a major determinant for host responses to orally administered TD antigens. Bacteroides species are the principal microflora present in the gastrointestinal tract and our studies with phenol-water LPS extracts from Bacteroides fragilis indicate that both polysaccharide and lipid A activate lymphoreticular cells. The B. fragilis lipid A moiety, like that derived from E. coli and Salmonella LPS, induces B cell mitogenic responses in cultures from LPS responsive mice, but does not stimulate C3H/ H3J B cells. The inability of lipid A to stimulate gut-associated lymphoreticular tissue (GALT) cells of C3H/HeJ mice results in the induction of greater T helper cell activity in this tissue in response to orally administered TD antigens and ultimately results in an elevated IgA response pattern. Murine PP contain accessory cells (approximately 1% dendritic cells and 6-8% macrophages) and lymphocytes T (35-38%) and B (40-42%). Recent studies with antigen-specific T cell clones from C3H/ H3J PP have resulted in the isolation of IgA isotype-specific T helper cells (PP Th A cells). PP Th A cells are antigen-specific, bear Fc alpha receptors, and require H-2 histocompatibility with B cells for helper activity. PP Th A cells most effectively collaborate with surface IgA (sIgA)-bearing B cells (IgA committed B cells) for IgA isotype responses. Other studies have shown that PP dendritic cells and T cells form clusters when stimulated in vitro with sodium periodate and that these clusters promote polyclonal IgA responses in B cell cultures. Polyclonal IgA responses in cultures containing PP cell clusters from C3H/ H3J mice are considerably higher than those in identical cultures from LPS responsive mice. In other studies, the environmental influence on GALT B cells and their resultant commitment to IgA isotype is under investigation. CBA/N, X-linked immunodeficient (xid) mice possess an immature splenic B cell population which cannot respond to thymic-independent class-2 (TI-2) or certain TD antigens. However, GALT B cells of xid mice possess a mature Lyb-5+ B cell subpopulation capable of both TI-2 and TD responses.(ABSTRACT TRUNCATED AT 400 WORDS)     

Real time analysis of tumor necrosis factor-related apoptosis-inducing ligand/cycloheximide-induced caspase activities during apoptosis initiation

Employing fluorescence resonance energy transfer (FRET) imaging, we previously demonstrated that effector caspase activation is often an all-or-none response independent of drug choice or dose administered. We here investigated the signaling dynamics during apoptosis initiation via the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor pathway to investigate how variability in drug exposure can be translated into largely kinetically invariant cell death execution pathways. FRET-based microscopy demonstrated dose-dependent responses of caspase-8 activation and activity within individual living HeLa cells. Caspase-8 on average was activated 45-600 min after TRAIL/cycloheximide addition. Caspase-8-like activities persisted for 15-60 min before eventually inducing mitochondrial outer membrane permeabilization. Independent of the TRAIL concentrations used or the resulting caspase-8-like activities, mitochondrial outer membrane permeabilization was induced when 10% of the FRET substrate was cleaved. In contrast, in Bid-depleted cells, caspase-8-like activity persisted for hours without causing immediate cell death. Our findings provide detailed insight into the intracellular signaling kinetics during apoptosis initiation and describe a threshold mechanism controlling the induction of apoptosis execution.     

Extracellular ATP induces cytokine expression and apoptosis through P2X7 receptor in murine mast cells

Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.     

High-yield production of functional human lactoferrin in transgenic cell cultures of siberian ginseng (Acanthopanax senticosus)

Human lactoferrin (hLf) is an iron-binding glycoprotein that has been considered to play many biological roles in the human, including the stimulation of the immune system, antimicrobial and anti-inflammatory effects, and regulation of iron absorption. We generated transgenic Siberian ginseng (Acanthopanax senticosus) cell cultures producing a functional hLf protein using the signal peptide sequence from the endoplasmic reticulum and driven by an oxidative stress-inducibleSWPA2 promoter which is highly expressed in plant cell cultures. The production of hLf increased proportionally to cell growth and showed a maximal level (up to 3.6% of total soluble protein) at the stationary phase in suspension cultures. Full-length hLf protein was identified by immunoblot analysis in transgenic cell cultures of Siberian ginseng. Recombinant hLf (rhLf) was purified from suspension cells of Siberian ginseng by ammonium sulfate precipitation, cation-exchange and gel filtration chromatography. N-terminal sequences of rhLf were identical to native hLf (nhLf). The overall monosaccharide composition of rhLf showed the presence of plant specific xylose while sialic acid is absent. Antibacterial activity of purified rhLf was higher than that of nhLf. Taken together, we anticipate that medicinal Siberian ginseng cultured cells, as demonstrated by this study, will be a biotechnologically useful source for commercial production of functional hLf not requiring further purification.