Notch3 signaling in vascular smooth muscle cells induces c-FLIP expression via ERK/MAPK activation. Resistance to Fas ligand-induced apoptosis

Mutations in the Notch3 receptor result in the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephelopathy (CADASIL) syndrome, a heritable arteriopathy predisposing to early onset stroke. Based upon clinical evidence that CADASIL arteriopathy results in degeneration and loss of vascular smooth muscle cells (VSMC) from the arterial wall, we postulated that Notch3 signaling is a critical determinant of VSMC survival. We initially established that both transient and constitutive Notch3 signaling promoted VSMC survival in response to the proapoptotic Fas ligand (FasL). Resistance to FasL-induced apoptosis was associated with the induction of c-FLIP, a primary inhibitor of the FasL signaling pathway. We determined that Notch3's regulation of c-FLIP was independent of the activity of the classical DNA-binding protein, RBP-Jk, but dependent upon cross-talk activation of the ERK/MAPK pathway. We extended our observations to the in vivo context by determining a coordinate regulation of Notch3 and c-FLIP within the arterial wall in response to injury. Furthermore, we defined that expression levels of Notch3 and c-FLIP are coordinately up-regulated within the neointima of remodeled arteries. Taken together, these findings provide initial evidence that Notch3 signaling may be a critical determinant of VSMC survival and vascular structure by modulating the expression of downstream mediators of apoptosis via signaling cross-talk with the ERK/MAPK pathway.     

Pharmacological characterisation of 5-hydroxytryptamine-induced contractile effects in the isolated gut of the lepidopteran caterpillar Spodoptera frugiperda

The indolealkylamine 5-hydroxytryptamine (5-HT, 0.1 nM-1 μM) caused dose-dependent increases in the number of contractions observed in guts isolated from the caterpillar Spodoptera frugiperda. Of the 5-HT analogues tested for agonist action, 2-methyl-5-HT (0.1-10 μM) was a full agonist with reduced potency while α-methyl-5-HT (0.1-100 μM), 5-carboxamidotryptamine (0.1-100 μM), 5-methoxytryptamine (5-MeOT) (10 nM-10 μM), and tryptamine (1-100 μM) were partial agonists. Incubation of isolated guts with proven mammalian 5-HT receptor antagonists showed that cyproheptadine (10 nM-1 μM), MDL 72222 (1-10 μM), tropisetron (1-10 μM) and 5-benzoyloxygramine (1-10 μM) were potent non-competitive antagonists of 5-HT-induced tissue contraction. In comparison, ketanserin (0.1-1 μM) was a competitive antagonist. The mammalian selective serotonin reuptake inhibitors, clomipramine (10 nM-10 μM) and fluoxetine (10 nM-10 μM) also caused non-competitive inhibition of 5-HT-induced contraction while fluvoxamine (10 nM-10 μM) was a weak competitive antagonist. Low doses of clomipramine (0.1 μM) caused potentiation of 5-HT-induced gut contraction thereby suggesting the presence of 5-HT reuptake systems in this tissue. The contractile effects of 5-HT were inhibited by verapamil, Li⁺ and H7 and potentiated by theophylline thereby indicating that L-type Ca²⁺ channels, phosphatidylinositol second messengers and cAMP, respectively, are involved in 5-HT-induced tissue contraction. The 5-HT receptors mediating contractility in the gut of S. frugiperda have properties in common with mammalian 5-HT₂ and Drosophila 5-HT_dro2A/2B receptors. In addition, these data suggest that the tissue also contains receptors that are similar to mammalian 5-ht₆ and 5-HT₇ as well as Drosophila_dro1 receptors. However, the primary amino acid sequence of these lepidopteran 5-HT receptors will have to be elucidated before full comparisons can be made.     

Diversifying animal models: the use of hispid cotton rats (Sigmodon hispidus) in infectious diseases

The hispid cotton rat (Sigmodon hispidus) has been a longstanding laboratory animal model of infectious diseases. In this review, the most common usage of hispid cotton rats as models of infectious diseases is discussed in detail and all organisms, which have been shown to infect cotton rats, are listed. A state of the art overview is given on handling and maintenance of hispid cotton rats as well as experimental techniques such as narcosis and blood withdrawal. Most importantly, through the development of new reagents, the hispid cotton rat can be used to study immune responses against the respective pathogen. Hispid cotton rat cytokine and chemokine genes have been sequenced and cotton rat specific antibodies and cell lines have been produced which in connection with the establishment of immunological methods should facilitate the use of hispid cotton rats as animal models in the pathogenesis of infectious diseases.     

Dietary influence of selenium on the incidence of N-nitrosodiethylamine-induced hepatoma with reference to drug and glutathione metabolizing enzymes

The dietary administration of selenium (sodium selenite; 4 p.p.m.) daily has been found to be highly effective in reducing the incidence of cancer induced by N-nitrosodiethylamine (DEN) in Wistar strain rats. Selenium treatment either before initiation, during initiation and selection/phenobarbital promotion phases of hepatocarcinogenesis has been found to be effective in elevating hepatic microsomal cytochrome b(5), NADPH-cytochrome C reductase and cytosolic aryl hydrocarbon hydroxylase activities to a statistically significant level measured either in the hyperplastic nodule or in the surrounding liver tissues compared to control animals. Moreover, selenium treatment throughout the study, decreases the cytosolic glutathione S-transferase and microsomal UDP-glucuronyl transferase activities by a significant degree when compared to control rats. Alterations in glutathione metabolizing enzyme activities (glutathione reductase, gamma-glutamyl transpeptidase, gamma-glutamylcysteine synthetase and glucose-6-phosphate dehydrogenase) were also observed in selenium-treated groups. Our results confirm the fact that selenium is particularly protective in limiting the action of DEN during the initiation phase of hepatocarcinogenesis.     

The lytic cycle of Epstein-Barr virus is associated with decreased expression of cell surface major histocompatibility complex class I and class II molecules

Human herpesviruses utilize an impressive range of strategies to evade the immune system during their lytic replicative cycle, including reducing the expression of cell surface major histocompatibility complex (MHC) and immunostimulatory molecules required for recognition and lysis by virus-specific cytotoxic T cells. Study of possible immune evasion strategies by Epstein-Barr virus (EBV) in lytically infected cells has been hampered by the lack of an appropriate permissive culture model. Using two-color immunofluorescence staining of cell surface antigens and EBV-encoded lytic cycle antigens, we examined EBV-transformed B-cell lines in which a small subpopulation of cells had spontaneously entered the lytic cycle. Cells in the lytic cycle showed a four- to fivefold decrease in cell surface expression of MHC class I molecules relative to that in latently infected cells. Expression of MHC class II molecules, CD40, and CD54 was reduced by 40 to 50% on cells in the lytic cycle, while no decrease was observed in cell surface expression of CD19, CD80, and CD86. Downregulation of MHC class I expression was found to be an early-lytic-cycle event, since it was observed when progress through late lytic cycle was blocked by treatment with acyclovir. The immediate-early transactivator of the EBV lytic cycle, BZLF1, did not directly affect expression of MHC class I molecules. However, BZLF1 completely inhibited the upregulation of MHC class I expression mediated by the EBV cell-transforming protein, LMP1. This novel function of BZLF1 elucidates the paradox of how MHC class I expression can be downregulated when LMP1, which upregulates MHC class I expression in latent infection, remains expressed in the lytic cycle.     

Lamellar and tubular associations of the mitochondrial cristae: unique forms of the cristae present in steroid-producing cells

This report provides a survey of mitochondrial structure in numerous cell types from all basic tissue types (epithelial, connective, muscular and nervous) with the main purpose being to determine the presence or absence of a form of the cristae previously termed the lamellar association (LA) (Anat. Rec. , 254 (1999) 534). The LA is a complex of closely apposed lamellar cristae within the inner membrane system of the mitochondria of Leydig cells, the steroid-producing cells of the testis. These lamellae are separated by a gap of approximately 4 nm. This survey has found no evidence of the LA in non-steroid-producing cells. The LA is a common cristae morphology in human Leydig cells, but is rare in marmoset Leydig cells, where instead, tubular associations (TA) are found. Cells of other steroid-producing cells are not included in this survey. Published micrographs from the literature do, however, have evidence of the existence of the LA and TA in other steroid-producing cells. It is concluded that these membrane substructures of the inner mitochondrial membrane are unique to steroid-producing cells. It is suggested that the LA is a region of the cristae, which is not involved with adenosine triphosphate (ATP) production, since the dimensions do not allow for F1 complexes on the matrix side of the cristae. The significance of this remains elusive.     

Design of a targeted peptide nucleic acid prodrug to inhibit hepatic human microsomal triglyceride transfer protein expression in hepatocytes

In this study, we present the design and synthesis of an antisense peptide nucleic acid (asPNA) prodrug, which displays an improved biodistribution profile and an equally improved capacity to reduce the levels of target mRNA. The prodrug, K(GalNAc)(2)-asPNA, comprised of a 14-mer sequence complementary to the human microsomal triglyceride transfer protein (huMTP) gene, conjugated to a high-affinity tag for the hepatic asialoglycoprotein receptor (K(GalNAc)(2)). The prodrug was avidly bound and rapidly internalized by HepG2s. After iv injection into mice, K(GalNAc)(2)-asPNA accumulated in the parenchymal liver cells to a much greater extent than nonconjugated PNA (46% +/- 1% vs 3.1% +/- 0.5% of the injected dose, respectively). The prodrug was able to reduce MTP mRNA levels in HepG2 cells by 35-40% (P < 0.02) at 100 nM in an asialoglycoprotein receptor- and sequence-dependent fashion. In conclusion, hepatocyte-targeted PNA prodrugs combine a greatly improved tropism with an enhanced local intracellular availability and activity, making them attractive therapeutics to lower the expression level of hepatic target genes such as MTP.     

Membrane-anchored cytochrome c as an electron carrier in photosynthesis and respiration: past,present and future of an unexpected discovery

In the mid 1980s, it was observed that photosynthesis could still occur in the absence of the diffusible electron carrier cytochrome c ₂ in the purple non-sulfur facultative phototrophic bacterium Rhodobacter capsulatus. This serendipic finding led to the discovery of a novel class of membrane-anchored electron carrier cytochromes and their associated electron transfer pathways. Studies of cytochrome c _y of R. capsulatus (and its homologues in other species) have modified the previous dogma of electron transfer between photosynthetic and respiratory membrane protein complexes with a new paradigm, in which these proteins and their electron carriers can form `hard-wired' structural super-complexes. Here, we reminisce on the early days of this discovery, its impacts on our understanding of cellular energy transduction pathways and the physiological roles played by the electron carrier cytochromes c, and discuss the current knowledge and emerging future challenges of this field. This revised version was published online in August 2006 with corrections to the Cover Date.