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.

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.

The design and characterization of receptor-selective APRIL variants.

A proliferation-inducing ligand (APRIL), a member of the TNF ligand superfamily with an important role in humoral immunity, is also implicated in several cancers as a prosurvival factor. APRIL binds two different TNF receptors, B cell maturation antigen (BCMA) and transmembrane activator and cylclophilin ligand interactor (TACI), and also interacts independently with heparan sulfate proteoglycans. Because APRIL shares binding of the TNF receptors with B cell activation factor, separating the precise signaling pathways activated by either ligand in a given context has proven quite difficult. In this study, we have used the protein design algorithm FoldX to successfully generate a BCMA-specific variant of APRIL, APRIL-R206E, and two TACI-selective variants, D132F and D132Y. These APRIL variants show selective activity toward their receptors in several in vitro assays. Moreover, we have used these ligands to show that BCMA and TACI have a distinct role in APRIL-induced B cell stimulation. We conclude that these ligands are useful tools for studying APRIL biology in the context of individual receptor activation.

Co- and posttranslational modification of the alpha(1B)-adrenergic receptor: effects on receptor expression and function.

We have characterized the maturation, co- and posttranslational modifications, and functional properties of the alpha(1B)-adrenergic receptor (AR) expressed in different mammalian cells transfected using conventional approaches or the Semliki Forest virus system. We found that the alpha(1B)-AR undergoes N-linked glycosylation as demonstrated by its sensitivity to endoglycosidases and by the effect of tunicamycin on receptor maturation. Pulse-chase labeling experiments in BHK-21 cells demonstrate that the alpha(1B)-AR is synthesized as a 70 kDa core glycosylated precursor that is converted to the 90 kDa mature form of the receptor with a half-time of approximately 2 h. N-Linked glycosylation of the alpha(1B)-AR occurs at four asparagines on the N-terminus of the receptor. Mutations of the N-linked glycosylation sites did not have a significant effect on receptor function or expression. Surprisingly, receptor mutants lacking N-linked glycosylation migrated as heterogeneous bands in SDS-PAGE. Our findings demonstrate that N-linked glycosylation and phosphorylation, but not palmitoylation or O-linked glycosylation, contribute to the structural heterogeneity of the alpha(1B)-AR as it is observed in SDS-PAGE. The modifications found are similar in the different mammalian expression systems explored. Our findings indicate that the Semliki Forest virus system can provide large amounts of functional and fully glycosylated alpha(1B)-AR protein suitable for biochemical and structural studies. The results of this study contribute to elucidate the basic steps involved in the processing of G protein-coupled receptors as well as to optimize strategies for their overexpression.