Glutathione plays different roles in the induction of the cytotoxic effects of inorganic and organic arsenic compounds in cultured BALB/c 3T3 cells

The cytotoxicity of arsenic compounds towards BALB/c 3T3 cells in culture was investigated, together with the role of glutathione (GSH) in the induction of the cytotoxic effects. The rank order of cytotoxicity was as follows: arsenite (As³⁺)>arsenate (As⁵⁺)>dimethylarsinic acid (DMAA)>methylarsonic acid (MAA)>trimethylarsine oxide (TMAO). Arsenobetaine, arsenocholine and the tetramethylarsonium ion were less toxic. Depletion of GSH enhanced the cytotoxic effects of As³⁺, As⁵⁺, MAA and TMAO, while the cytotoxicity of DMAA was markedly reduced by depletion of GSH. These results suggest that GSH plays a role in protecting the cells against the toxic effects of As³⁺, As⁵⁺, MAA and TMAO while it is involved in the induction of the cytotoxic effects of DMAA.     

Subpopulations of T-lymphocytes in Peyer's patches: Sensitivity to antilymphocyte serum and adult thymectomy

Summary Anti-lymphocyte (ALS) treatment or adult thymectomy of the donor have been shown to depress respectively the cell proliferation and the cytotoxicity in the graft-versus-host (GVH) reaction. A quantitative assay and the histological criteria of the GVH reaction have been used to demonstrate that all the known subpopulations of T-lymphocytes involved in the GVH reaction are present in the Peyer's patches as well as in the spleen and mesenteric lymph nodes in the rat.This work was supported by a grant of the Délégation Générale à la Recherche Scientifique et Technique (No. 74-7-0619).     

Intestinal epithelial barrier and mucosal immunity

The mucosal immune system maintains a delicate balance between providing robust defense against infectious pathogens and, at the same time, regulating responses toward innocuous environmental and food antigens and commensal microbes. The Peyer's patch (PP) has been studied in detail as a major inductive site for mucosal immunity within the small intestine. While the mechanisms responsible for the induction of mucosal immunity versus tolerance are not yet fully understood, recent studies have highlighted mucosal dendritic cells (DCs) as regulators of the immune responses to orally administered antigens. Here we discuss recent studies that describe the role of PP DCs in immune induction and speculate on the mechanism by which the resident DCs regulate T cell and immunoglobulin A (IgA) responses in the gastrointestinal mucosa.     

Subpopulations of T-lymphocytes in Peyer's patches: sensitivity to antilymphocyte serum and adult thymectomy.

Anti-lymphocyte (ALS) treatment or adult thymectomy of the donor have been shown to depress respectively the cell proliferation and the cytotoxicity in the graft-versus-host (GVH) reaction. A quantitative assay and the histological criteria of the GVH reaction have been used to demonstrate that all the known subpopulations of T-lymphocytes involved in the GVH reaction are present in the Peyer's patches as well as in the spleen and mesenteric lymph nodes in the rat.     

Contrasting effects of RNA and protein synthesis blocking on natural and lectin-dependent cell-mediated cytotoxicity against adherent HEp-2 cells

Summary In this study, earlier observations^2,9 concerning the independence of both natural (NCMC) and lectin-dependent cell-mediated cytotoxicity (LDCC) from DNA synthesis have been confirmed. In addition, blocking of RNA synthesis by actinomycin D and of protein synthesis, reversibly by puromycin (PM) and irreversibly by emetine (EM) had different effects on NCMC and LDCC against³H-thymidine-prelabeled HEp-2 target cells. Similarly to the Con A-induced proliferation of lymphocytes, LDCC activity was also inhibited by blocking of RNA and protein synthesis. NCMC to HEp-2 target cells was not affected by blocking of RNA synthesis, while both PM and EM strongly enhanced NCMC activity.     

The impact of cyclin-dependent kinase 5 depletion on poly(ADP-ribose) polymerase activity and responses to radiation

Cyclin-dependent kinase 5 (Cdk5) has been identified as a determinant of sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Here, the consequences of its depletion on cell survival, PARP activity, the recruitment of base excision repair (BER) proteins to DNA damage sites, and overall DNA single-strand break (SSB) repair were investigated using isogenic HeLa stably depleted (KD) and Control cell lines. Synthetic lethality achieved by disrupting PARP activity in Cdk5-deficient cells was confirmed, and the Cdk5^KD cells were also found to be sensitive to the killing effects of ionizing radiation (IR) but not methyl methanesulfonate or neocarzinostatin. The recruitment profiles of GFP-PARP-1 and XRCC1-YFP to sites of micro-irradiated Cdk5^KD cells were slower and reached lower maximum values, while the profile of GFP-PCNA recruitment was faster and attained higher maximum values compared to Control cells. Higher basal, IR, and hydrogen peroxide-induced polymer levels were observed in Cdk5^KD compared to Control cells. Recruitment of GFP-PARP-1 in which serines 782, 785, and 786, potential Cdk5 phosphorylation targets, were mutated to alanines in micro-irradiated Control cells was also reduced. We hypothesize that Cdk5-dependent PARP-1 phosphorylation on one or more of these serines results in an attenuation of its ribosylating activity facilitating persistence at DNA damage sites. Despite these deficiencies, Cdk5^KD cells are able to effectively repair SSBs probably via the long patch BER pathway, suggesting that the enhanced radiation sensitivity of Cdk5^KD cells is due to a role of Cdk5 in other pathways or the altered polymer levels.     

Cortisone sensitive T-cells in Peyer's patches

Summary Pretreatment of donor lymphoid cells with cortisone has been shown to depress the T-cell subpopulation responsible for cellular proliferation in the GVH reaction. A quantitative assay as well as the histological criteria of the GVH reaction have been used in this study to demonstrate the presence of cortisone-sensitive T-cells within the Peyer's patches as well as in the spleen and mesenteric lymph nodes in the rat.This work was supported by a grant of the Délégation Générale à la Recherche Scientifique et Technique (No. 74-7-0619).     

Accumulation of 2-micron latex particles in mouse Peyer's patches during chronic latex feeding

2-micron latex particles accumulated in macrophages in intestinal Peyer's patches of mice given latex suspensions as drinking fluid for 2 months. The number of particles accumulating was a direct (but nonlinear) function of the number ingested. Some of the latex particles were still present in Peyer's patches 6 weeks after the cessation of latex feeding.     

Calcium-induced cleavage of DNA topoisomerase I involves the cytoplasmic-nuclear shuttling of calpain 2

Important to the function of calpains is temporal and spatial regulation of their proteolytic activity. Here, we demonstrate that cytoplasm-resident calpain 2 cleaves human nuclear topoisomerase I (hTOP1) via Ca²⁺-activated proteolysis and nucleoplasmic shuttling of proteases. This proteolysis of hTOP1 was induced by either ionomycin-caused Ca²⁺ influx or addition of Ca²⁺ in cellular extracts. Ca²⁺ failed to induce hTOP1 proteolysis in calpain 2-knockdown cells. Moreover, calpain 2 cleaved hTOP1 in vitro. Furthermore, calpain 2 entered the nucleus upon Ca²⁺ influx, and calpastatin interfered with this process. Calpain 2 cleavage sites were mapped at K¹⁵⁸ and K¹⁸³ of hTOP1. Calpain 2-truncated hTOP1 exhibited greater relaxation activity but remained able to interact with nucleolin and to form cleavable complexes. Interestingly, calpain 2 appears to be involved in ionomycin-induced protection from camptothecin-induced cytotoxicity. Thus, our data suggest that nucleocytoplasmic shuttling may serve as a novel type of regulation for calpain 2-mediated nuclear proteolysis.     

Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway

The cytotoxicity of cadmium (Cd) induced autophagy and apoptosis in MES-13 cells was determined by flow cytometry. Autophagy was also assessed by formation of autophagosomes and processing of LC3. Pharmacological inhibition of autophagy resulted in increased of cell viability, suggesting autophagy plays a role in cell death in Cd-treated mesangial cells. Cd also induced a rapid elevation in cytosolic calcium ([Ca²⁺]_i ), and modulation of [Ca²⁺]_i via treatment with IP ₃R inhibitor or knockdown of calcineurin resulted in a change in the proportion of cell death, suggesting that the release of calcium from the ER plays a crucial role in Cd-induced cell death. Inhibition of Cd-induced ERK activation by PD 98059 suppressed Cd-induced autophagy, and BAPTA-AM eliminated activation of ERK. BAPTA-AM also inhibited Cd-induced mitochondrial depolarization and activation of caspases. These findings demonstrated that Cd induces both autophagy and apoptosis through elevation of [Ca²⁺]_i, followed by Ca²⁺-ERK and Ca²⁺-mitochondria-caspase signaling pathways. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 05 July 2008; received after revision 25 August 2008; accepted 17 September 2008     

Tissue transglutaminase inhibits the TRPV5-dependent calcium transport in an N-glycosylation-dependent manner

Tissue transglutaminase (tTG) is a multifunctional Ca²⁺-dependent enzyme, catalyzing protein crosslinking. The transient receptor potential vanilloid (TRPV) family of cation channels was recently shown to contribute to the regulation of TG activities in keratinocytes and hence skin barrier formation. In kidney, where active transcellular Ca²⁺ transport via TRPV5 predominates, the potential effect of tTG remains unknown. A multitude of factors regulate TRPV5, many secreted into the pro-urine and acting from the extracellular side. We detected tTG in mouse urine and in the apical medium of polarized cultures of rabbit connecting tubule and cortical collecting duct (CNT/CCD) cells. Extracellular application of tTG significantly reduced TRPV5 activity in human embryonic kidney cells transiently expressing the channel. Similarly, a strong inhibition of transepithelial Ca²⁺ transport was observed after apical application of purified tTG to polarized rabbit CNT/CCD cells. Furthermore, tTG promoted the aggregation of the plasma membrane-associated fraction of TRPV5. Using patch clamp analysis, we observed a reduction in the pore diameter after tTG treatment, suggesting distinct structural changes in TRPV5 upon crosslinking by tTG. As N-linked glycosylation of TRPV5 is a key step in regulating channel function, we determined the effect of tTG in the N-glycosylation-deficient TRPV5 mutant. In the absence of N-linked glycosylation, TRPV5 was insensitive to tTG. Taken together, these observations imply that tTG is a novel extracellular enzyme inhibiting the activity of TRPV5. The inhibition of TRPV5 occurs in an N-glycosylation-dependent manner, signifying a common final pathway by which distinct extracellular factors regulate channel activity.     

Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors

Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca²⁺ and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca²⁺], derived from intracellular stores, followed, only in myotubes, by a large Ca²⁺ influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca²⁺ permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca²⁺ mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death. Received 21 January 2009; received after revision 05 March 2009; accepted 11 March 2009     

Biochemical effects and growth inhibition in MCF-7 cells caused by novel sulphonamido oxa-polyamine derivatives

The novel polyamine derivatives sulphonamido oxa-spermine (oxa-Spm) and sulphonamido oxa-spermidine (oxa-Spd) exhibited rapid cytotoxic action towards MCF-7 human breast cancer cells with IC₅₀ values of 4.35 and 6.47 μM, respectively, after 24-h drug exposure. Neither compound is a substrate of serum amine oxidase. Both oxa-Spm and oxa-Spd caused cell shrinkage, as determined by phase-contrast microscopy. After incubation with 10 μM of either compound for 8 h, the cells underwent chromatin condensation and nuclear fragmentation. However, no clear DNA ladder was obtained by electrophoresis. The sulphonamido oxa-polyamine derivatives and especially oxa-Spd enhanced the activity of polyamine oxidase (PAO), an enzyme capable of oxidising N¹-acetylated spermine and spermidine to spermidine and putrescine, respectively, generating cytotoxic H₂O₂ and 3-acetamidopropanal as by-products. The intracellular polyamine content was only marginally reduced in response to drug treatment. In conclusion, our data show that these novel sulphonamido oxa-polyamine derivatives possess high cytotoxic activity against MCF-7 cells and indicate that induction of PAO may mediate their cytotoxicity via apoptosis. Received 17 January 2002; received after revision 22 February 2002; accepted 22 February 2002     

Cortisone sensitive T-cells in Peyers' patches.

Pretreatment of donor lymphoid cells with cortisone has been shown to depress the T-cell subpopulation responsible for cellular proliferation in the GVH reaction. A quantitative assay as well as the histological criteria of the GVH reaction have been used in this study to demonstrate the presence of cortisone-sensitive T-cells within the Peyer's patches as well as in the spleen and mesenteric lymph nodes in the rat.     

Accumulation of 2-μm latex particles in mouse Peyer's patches during chronic latex feeding

Summary 2-m latex particles accumulated in macrophages in intestinal Peyer's patches of mice given latex suspensions as drinking fluid for 2 months. The number of particles accumulating was a direct (but nonlinear) function of the number ingested. Some of the latex particles were still present in Peyer's patches 6 weeks after the cessation of latex feeding.Acknowledgments. Research supported by the U. S. Energy Research and Development Administration. We thank Mr W. Marin, Division of Photography and Graphic Arts, Brookhaven National Laboratory, for figure 2.The research described in this report involved animals maintained in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Interaction of volkensin with HeLa cells: binding,uptake, intracellular localization,degradation and exocytosis

Among two-chain ribosome-inactivating proteins (RIPs), volkensin is the most toxic to cells and animals, and is retrogradely axonally transported in the rat central nervous system, being an effective suicide transport agent. Here we studied the binding, endocytosis, intracellular routeing, degradation and exocytosis of this RIP. The interaction of volkensin with HeLa cells was compared to that of nigrin b, as an example of a type 2 RIP with low toxicity, and of ricin, as a reference toxin. Nigrin b and volkensin bound to cells with comparable affinity (approx. 10^-10 M) and had a similar number of binding sites (2 × 105/cell), two-log lower than that reported for ricin. The cellular uptake of volkensin was lower than that reported for nigrin b and ricin. Confocal microscopy showed the rapid localization of volkensin in the Golgi stacks with a perinuclear localization similar to that of ricin, while nigrin b was distributed between cytoplasmic dots and the Golgi compartment. Consistently, brefeldin A, which disrupts the Golgi apparatus, protected cells from the inhibition of protein synthesis by volkensin or ricin, whereas it was ineffective in the case of nigrin b. Of the cell-released RIPs, 57% of volkensin and only 5% of ricin were active, whilst exocytosed nigrin b was totally inactive. Despite the low binding to, and uptake by, cells, the high cytotoxicity of volkensin may depend on (i) routeing to the Golgi apparatus, (ii) the low level of degradation, (iii) rapid recycling and (iv) the high percentage of active toxin remaining after exocytosis.Received 21 April 2004; received after revision 26 May 2004; accepted 9 June 2004     

Two serotonin-sensitive potassium channels in the identified heart excitatory neurone of the African giant snail,Achatina fulica Fèrussac

Summary By the patch clamp experiments, two serotonin-sensitive K⁺ channels (SL-channel and SS-channel) were demonstrated in the identified heart excitatory neurone (PON) of the African giant snail,Achatina fulica Fèrussac. The activities of both channels could be recorded in the steady state and those activities disappeared on application of 5-HT.     

Protective effect of interferon on the target cells of cytotoxic lymphocytes

Interferon treatment of sensitized T lymphocytes enhances cytotoxicity against target cells. On the contrary, the same treatment of the target cells alone protects them, whatever the cytotoxicity of effector cells might be. We suggest that the cells having once survived contact with cytotoxic T lymphocytes could become resistant to any new attack by the same cells.     

Rat pancreatic islet cells in primary culture: occurrence of giant cells amenable to patch clamping

Summary Neonatal and adult rat pancreatic islet cells were maintained in dissociated cell culture for up to three weeks. The unexpected occurrence of giant (40–50 m) cells was noted, some of which reacted positively to an insulin antiserum, indicating the presence of insulin. The giant cells were amenable to study using the extracellular patch clamp technique, which was used to demonstrate a population of membrane channels gating outwardly directed current in these cells.     

Cell-mediated cytotoxicity by natural killer and killer cells, lipid peroxidation and glutathione

In the course of spontaneous cell-mediated cytotoxicity (SCMC) and antibody-dependent cell-mediated cytotoxicity (ADCC) with human peripheral lymphocytes as effector cells, no lipid peroxidation occurred as measured by the production of ethane and thiobarbituric acid-reactive material. Furthermore, impairment of major cellular defense systems of target cells (K562 cells for SCMC, Chang liver cells for ADCC), by decreasing their glutathione content, had no effect on either lipid peroxidation or the cytotoxic response. These findings indicate that peroxidative damage is not a mechanism of NK and K cell-mediated cytotoxicity.