Vongthip Souvannavong

Inhibition of caspase activity induces a switch from apoptosis to necrosis

The role of caspases in B lymphocyte cell death was investigated by using two broad spectrum inhibitors of the caspase family, Z‐Asp‐cmk and Z‐VAD‐fmk. They totally prevented spontaneous and drug‐induced apoptosis and inhibited the CPP32/caspase‐3‐like activity exhibited by apoptotic cells. However, the suppression of apoptosis was not associated with a long‐term increase of cell survival, but conversely, with a switch from apoptotic death to the necrotic form. These results strongly suggest that apoptosis and necrosis share common initiation pathways, the final issue being determined by the presence of an active caspase.

Silica induces apoptosis in macrophages and the release of interleukin-1 alpha and interleukin-1 beta.

Resident adherent peritoneal cells selectively released high amounts of interleukin‐1 (IL‐1) activity when treated with silica. The use of anti‐IL‐1 antisera showed that both IL‐Ια and IL‐1β were present in supernatants of silica‐treated macrophages. In contrast, intracellular IL‐1 activity was totally neutralized by anti‐ IL‐Ια antibodies and was easily converted into the mature IL‐Ια form by autolysis in cytoplasmic extracts. Anion exchange chromatography clearly separated the two IL‐1 species present in supernatants of silica‐ stimulated macrophages. Natural IL‐1β was further characterized by chromatofocalization; it had an apparent isoelectric point, pI, in the range 8.3‐8.6. In agreement with previous findings showing that IL‐1/3 was released only by apoptotic cells, we have found that silica‐treated macrophages underwent apoptosis. This was demonstrated by the characteristic laddering electrophoretic pattern of DNA extracted from silica‐treated cells and by the morphology of macrophage nuclei stained with the DNA‐specific dye DAPI. In addition, quantification of apoptotic cells was performed by a flow cytometric analysis based on the reduction of cellular DNA content exhibited by apoptotic cells. Treatment of macrophages with silica, therefore, results in an active process that promotes the processing and liberation of IL‐1β.

Resistance to bacterial wilt in somatic hybrids between Solanum tuberosum and Solanum phureja

Somatic hybrid plants were produced after protoplast electrofusion between a dihaploid potato, cv. BF15, and a wild tuber-bearing relative, Solanum phureja, with a view to transferring bacterial wilt resistance into potato lines. A total of ten putative hybrids were selected. DNA analysis using flow cytometry revealed that six were tetraploids, two mixoploids, one amphiploid and one octoploid. In the greenhouse, the putative hybrids exhibited strong vigor and were morphologically intermediate, including leaf form, flowers and tuber characteristics. The hybrid nature of the ten selected plants was confirmed by examining isoenzyme patterns for esterases and peroxidases, and analysis of RAPD and SSR markers. Analysis of chloroplast genome revealed that eight hybrids possessed chloroplast (ct) DNA of the wild species, S. phureja, and only two contained Solanum tuberosum ct type. Six hybrid clones, including five tetraploids and one amphiploid, were evaluated for resistance to bacterial wilt by using race 1 and race 3 strains of Ralstonia solanacearum, originating from Reunion Island. Inoculations were performed by an in vitro root dipping method. The cultivated potato was susceptible to both bacterial strains tested. All somatic hybrids except two were tolerant to race 1 strain, and susceptible to race 3 strain. Interestingly, the amphiploid hybrid clone showed a good tolerance to both strains.

Induction of apoptosis by dexamethasone in the B cell lineage

The susceptibility to induction of apoptosis by the synthetic glucocorticoid, dexamethasone (Dex), was analysed at different stages of B cell maturation. Cells of the 70Z/3 pre-B cell line, expressing cytoplasmic mu chains, and LPS-stimulated 70Z/3 cells, expressing surface IgM, were used as a model of differentiation of pre-B cells into immature B cells. Cell proliferation and cell cycle progression were similarly inhibited by Dex (100 nM) in both naive 70Z/3 pre-B cells and in LPS-stimulated 70Z/3 cells. In contrast, Dex failed to affect apoptosis of naive 70Z/3 cells while it increased that of LPS-stimulated 70Z/3 cells. Splenic mature B lymphocytes were highly susceptible to Dex-induced apoptosis since subphysiological doses (5 nM) increased the frequency of apoptotic cells to more than 80%. On the other hand, the treatment of B lymphocytes with LPS, which led to proliferation and differentiation into immunoblasts, decreased the susceptibility to Dex-induced apoptosis. These effects were mediated by the glucocorticoid receptor since they were abrogated by the RU 486 antagonist. The response of B cells to glucocorticoids is thus dependent on their stage of differentiation.

Source of resistance against Ralstonia solanacearum in fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L

Solanum aethiopicum is reported to carry resistance to bacterial wilt disease caused by Ralstonia solanacearum, which is one of the most important diseases of eggplant (Solanum melongena). These two species can sexually be crossed but the fertility of their progeny is very low. In order to transfer the resistance and improve the fertility, somatic hybrids between S. melongena cv. Dourga and two groups of S. aethiopicum were produced by electrical fusion of mesophyll protoplasts. Thirty hybrid plants were regenerated. When transferred to the greenhouse and transplanted in the field, they were vigorous and showed intermediate morphological traits. Their ploidy level was determined by DNA analysis through flow cytometry, and their hybrid nature was confirmed by examining isozymes and RAPDs patterns. Chloroplast DNA microsatellite analysis revealed that 18 hybrids had the chloroplasts of the eggplant and 12 those of the wild species. The parents and 16 hybrids were evaluated in the field for their fertility and resistance to bacterial wilt using a race 1, biovar 3 strain of R. solanacearum. All hybrids were fertile and set fruit with viable seeds. Their yield was either intermediate or as high as that of the cultivated eggplant. Both groups of S. aethiopicum were found tolerant to R. solanacearum, as about 50% of plants wilted after 8 weeks. The cultivated eggplant was susceptible with 100% of wilted plants 2 weeks after inoculation. All somatic hybrids tested were as tolerant as the wild species, except six hybrids showing a better level of resistance.

Specific dual effect of cycloheximide on B lymphocyte apoptosis: involvement of CPP32/caspase-3

Cycloheximide (CHX) is known to stimulate or to prevent apoptosis, according to the cell type used. We found that CHX, in a dose-dependent way, exerted the two opposite effects in B lymphocytes. CHXhigh (2.5 microg/mL) inhibited protein synthesis (>90%) and greatly increased B cell apoptosis but failed to prevent apoptosis induction by dexamethasone (DEX) or dibutyryl-cAMP (dbcAMP), which is in opposition with CHX activity in thymocytes. On the contrary, CHXlow (0.05 microg/mL), modestly inhibited protein synthesis (<15%) and reduced spontaneous as well as drug-induced apoptosis and further augmented the protection conferred by interleukin-4 or lipopolysaccharide. To examine the role of caspases in CHX effects, we used the broad spectrum peptide caspase inhibitor Z-VAD-fmk: it totally abrogated spontaneous as well as drug- and CHXhigh-induced cell death. Apoptosis was associated with CPP32/caspase-3 activation, since cleavage of CPP32/caspase-3 and caspase-3 activity were increased by DEX, dbcAMP as well as by CHXhigh treatment. Meanwhile, caspase-3 activity was reduced by CHXlow treatment. Therefore, CHX exerts opposite effects on B lymphocyte apoptosis which are associated with a dual action on caspase-3 activation.

Macrophages from C3H/HeJ mice require an additional step to produce monokines: synergistic effects of silica and poly(I:C) in the release of interleukin 1.

Resident macrophages from C3H/HeJ mice, in contrast with those of other strains of mice such as BDF1 mice, did not release a 35 kD m.w. factor having macrophage replacing activity (FRM) or interleukin 1 (IL‐1) when, respectively, cultured alone or in the presence of silica. C3H/HeJ macrophages were nevertheless capable of producing an intracellular IL‐1‐like activity. In addition, after a two‐step activation process, macrophages from BDF1 mice spontaneously released IL‐1, whereas silica was required to induce the release of IL‐1 from similarly treated C3H/HeJ macrophages. Such in vivo primed and in vitro stimulated macrophages failed to release FRM. In contrast, poly(l:C) was able to induce the release of FRM by C3H/HeJ macrophages but not that of IL‐1; moreover, the addition of silica to poly(l:C)‐stimulated cells led to an IL‐1 release similar to that obtained with normal mice treated with silica alone. Since poly(l:C) is able to elicit the production of interferons (IFN), the involvement of IFNs was investigated in poly(l:C) activity. Neither IFN‐α/β nor IFN‐γ, when used alone or in the presence of silica, could induce the release of IL‐1 by C3H/HeJ macrophages. In addition, antibodies to IFN‐α/β and IFN‐γ were unable to affect the poly(l:C) and silica induced release of IL‐1. Thus, the signal provided by poly(l:C) does not appear to be mediated by IFN(s).

Muramyl dipeptide (MDP) synergizes with interleukin 2 and interleukin 4 to stimulate, respectively, the differentiation and proliferation of B cells☆

The synthetic immunomodulator muramyldipeptide (MDP) can stimulate B cells. MDP, when used alone, was apparently unable to induce the differentiation or proliferation of resting B cells. In contrast, MDP appeared to synergize with a single recombinant interleukin (IL) to stimulate either their differentiation or proliferation. We used single interleukins to avoid synergistic and antagonistic effects inherent in the use of several factors. IL-2 was found to be sufficient to restore the specific immune response of resting B cells to sheep erythrocytes; MDP greatly increased the number of plaque-forming cells of such IL-2-stimulated B cells. In contrast, IL-4 and interferon-gamma (IFN-gamma), either alone or in the presence of MDP, had no effect in this differentiation assay. MDP was also able to stimulate polyclonally activated B cells. IL-4 increased the proliferation of anti-IgM-stimulated B cells, leading to enlargement and driving more cells into the cell cycle; these effects were further enhanced by MDP, more cells being induced to proliferate, to enlarge, and to progress into the cycle with a higher frequency of cells in the G1B, S, and G2/M compartments. Intracellular free calcium levels were not increased by IL-4 and/or MDP, and the two compounds did not modify the anti-IgM-induced calcium mobilization. Therefore, MDP appears to amplify cytokine effects in B cell activation, by a mechanism which does not appear to involve free calcium mobilization.

Photoaffinity labelling of macrophages and B-lymphocytes using 125I-labelled aryl-azide derivatives of muramyldipeptide

Two 125I-labelled aryl-azide derivatives of MDP have been synthesized. Photoaffinity labelling experiments demonstrated cell surface binding sites for muramylpeptides on activated B-lymphocytes and intracellular muramylpeptides binding protein(s) of 40-45 kD in rat alveolar macrophages.

Effect of genotype, gelling agent, and auxin on the induction of somatic embryogenesis in sweet potato (Ipomoea batatas Lam.).

Lateral buds of six cultivars of sweet potato were induced to form embryogenic callus in a culture medium solidified with two types of gelling agents, Agar or Gelrite, and supplemented with various concentrations of auxins, 2,4-D, 2,4,5-T and Picloram. Of the six cultivars screened, only three gave an embryogenic response. Best results with an average of 3.53% embryogenic response were obtained with the medium solidified with Agar, while in Gelrite only 0.45% of lateral buds gave rise to embryogenic callus. The interaction between the genotype and auxins was highly significant; particularly the optimal response was obtained with cv. Zho and 865 yielding 10.7 and 14.7% somatic embryogenesis, respectively, in the medium containing 2,4,5-T or Picloram. The plant conversion was dramatically improved by subculture of the embryogenic callus on the medium with the combination of 1 microM 2,4-D and 1 microM Kinetin or 5 microM ABA alone before transfer of mature embryos onto hormone-free medium. The embryogenic callus of sweet potato and its sustained ability to further regenerate plants have regularly been maintained for several years by frequent subculture in 5 microM 2,4,5-T or the combination of 10 microM 2,4-D and 1 microM BAP or kinetin. The embryo-derived plants seemed apparently genetically stable and similar to the hexaploid parental plants, based on morphological analysis and their ploidy level determined by using flow cytometry.