Cell suspension culture and plant regeneration of a Brazilian plantain, cultivar Terra

The objective of this study was to establish cell suspension culture and plant regeneration via somatic embryogenesis of a Brazilian plantain, cultivar Terra Maranhão, AAB. Immature male flowers were used as explant source for generating highly embryogenic cultures 45 days after inoculation, which were used for establishment of cell suspension culture and multiplication of secondary somatic embryos. Five semisolid culture media were tested for differentiation, maturation, somatic embryos germination and for plant regeneration. An average of 558 plants per one milliliter of 5% SCV (settled cell volume) were regenerated in the MS medium, with 11.4 µM indolacetic acid and 2.2 µM 6-benzylaminopurine. Regenerated plants showed a normal development, and no visible somaclonal variation was observed in vitro. It is possible to regenerate plants from cell suspensions of plantain banana cultivar Terra using MS medium supplemented with 11.4 µM of IAA and 2.2 µM of BAP.

UV irradiation of human keratinocytes activates the inflammasome

Human keratinocytes represent a potent source of the pro-inflammatory cytokines pro-interleukin(IL)-1α and -β. ProIL-1β requires processing by caspase-1 (IL-1β-converting enzyme, ICE) for activation and receptor binding. ProIL-1α and -β lack a signal peptide and leave the cell via the alternative secretion pathway, which is independent of the classical ER/Golgi pathway. Both cytokines are stored in the cytoplasm and can be activated and released upon UV irradiation. In macrophages maturation of proIL-1β requires the activation of inflammasomes, innate multiprotein immune complexes, which are essential for the activation of caspase-1 and thereby for processing of proIL-1β. However, the intracellular pathways, which are responsible for activation of proIL-1β and secretion of IL-1β in keratinocytes, are unknown. We show that human keratinocytes express inflammasome proteins in vitro and in vivo. UVB irradiation of keratinocytes results in an increase of cytoplasmic Ca2+ from intracellular stores. This shift is required for inflammasome-dependent activation of caspase-1 and subsequent processing of proIL-1β and secretion of IL-1β. In contrast to macrophages, caspase-1 cannot activate proIL-18 in keratinocytes, although secretion of this cytokine is also induced by UVB irradiation. In vivo, caspase-1 is also essential for UVB-induced inflammation in the skin, since caspase-1 knockout mice showed a strongly reduced inflammatory response after UVB irradiation. Our results suggest that keratinocytes are important immuno-competent cells under physiological and pathological conditions.

Processing of metacaspase into a cytoplasmic catalytic domain mediating cell death in Leishmania major.

Metacaspases are cysteine peptidases that could play a role similar to caspases in the cell death programme of plants, fungi and protozoa. The human protozoan parasite Leishmania major expresses a single metacaspase (LmjMCA) harbouring a central domain with the catalytic dyad histidine and cysteine as found in caspases. In this study, we investigated the processing sites important for the maturation of LmjMCA catalytic domain, the cellular localization of LmjMCA polypeptides, and the functional role of the catalytic domain in the cell death pathway of Leishmania parasites. Although LmjMCA polypeptide precursor form harbours a functional mitochondrial localization signal (MLS), we determined that LmjMCA polypeptides are mainly localized in the cytoplasm. In stress conditions, LmjMCA precursor forms were extensively processed into soluble forms containing the catalytic domain. This domain was sufficient to enhance sensitivity of parasites to hydrogen peroxide by impairing the mitochondrion. These data provide experimental evidences of the importance of LmjMCA processing into an active catalytic domain and of its role in disrupting mitochondria, which could be relevant in the design of new drugs to fight leishmaniasis and likely other protozoan parasitic diseases.

The epidthelial sodium channel ENaC and its regulators in the epidermal permeability barrier function

The highly amiloride-sensitive epithelial sodium channel ENaC is well known to be involved in controlling whole body sodium homeostasis and lung liquid clearance. ENaC expression has also been detected in the skin of amphibians and mammals. Mice lacking ENaC expression lose rapidly weight associated with an epidermal barrier defect that develops following birth. This dehydration is accompanied with a highly abnormal lipid matrix composition and an impaired skin surface acidification. This strongly suggests a role of ENaC in the maturation of barrier function rather than in the prenatal generation of the barrier, and may be as such an important modulator for skin hydration. In parallel, gene targeting experiments of regulators of ENaC activity, membrane serine proteases, also termed channel activating proteases, like CAP1/Prss8 and matriptase/MT-SP1 by themselves have been shown to be crucial for the epidermal barrier function. In our review, we mainly focus on the role of ENaC and its regulators in the skin and discuss their importance in the epidermal permeability barrier function.

Ultrastructure of vitellogenesis and vitellocytes in the trypanorhynch cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax

This is the first TEM examination of vitellogenesis in the cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax and a member of a little-studied trypanorhynch family, the Aporhynchidae. The synthetic activity of vitellocytes plays two important functions in the developmental biology of cestodes: (1) their shell-globules serve in eggshell formation; and (2) their accumulated reserves of glycogen and lipids represent a food source for the developing embryo. In A. menezesi, vitelline follicles consist of cells at various stages of development, from peripheral, immature cells of the gonial type to mature cells towards the centre of the follicle. These stages are: (I) immature; (II) early differentiation; (III) advanced maturation; and (IV) mature. Gradual changes involved in this process occur within each stage. Vitellogenesis involves: (1) an increase in cell volume; (2) the development of a smooth endoplasmic reticulum and an accelerated formation and accumulation of both unsaturated and saturated lipid droplets, along with their continuous enlargement and fusion; (3) the formation of individual β-glycogen particles and their accumulation in the form of glycogen islands scattered among lipid droplets in the cytoplasm of maturing and mature vitellocytes; (4) the rapid accumulation of large, moderately saturated lipid droplets accompanied by dense accumulations of β-glycogen along with proteinaceous shell-globules or shell-globule clusters in the peripheral layer during the advanced stage of maturation; (5) the development of cisternae of granular endoplasmic reticulum that produce dense, proteinaceous shell-globules; (6) the development of Golgi complexes engaged in the packaging of this material; and (7) the progressive and continuous enlargement of shell-globules into very large clusters in the peripheral layer during the advanced stage of maturation. Vitellogenesis in A. menezesi, only to some extent, resembles that previously described for four other trypanorhynchs. It differs in: (i) the reversed order of secretory activities in the differentiating vitellocytes, namely the accumulation of large lipid droplets accompanied by glycogenesis or β-glycogen formation during early differentiation (stage II), i.e. before the secretory activity, which is predominantly protein synthesis for shell-globule formation (stage III); (ii) the very heavy accumulation of large lipid droplets during the final stage of cytodifferentiation (stage IV); and (iii) the small number of β-glycogen particles present in mature vitellocytes. Ultracytochemical staining with PA-TCH-SP for glycogen proved positive for a small number of β-glycogen particles in differentiating and mature vitellocytes. Hypotheses, concerning the interrelationships of patterns of vitellogenesis, possible modes of egg formation, embryonic development and life-cycles, are commented upon.