Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on beta-amyloid-induced toxicity in PC12 cells

Abstract Alzheimer's disease is widely held to be associated with oxidative stress due, in part, to the membrane action of beta-amyloid peptide aggregates. Here, we studied the effect of cannabidiol, a major non-psychoactive component of the marijuana plant (Cannabis sativa) on beta-amyloid peptide-induced toxicity in cultured rat pheocromocytoma PC12 cells. Following exposure of cells to beta-amyloid peptide (1 micro g/mL), a marked reduction in cell survival was observed. This effect was associated with increased reactive oxygen species (ROS) production and lipid peroxidation, as well as caspase 3 (a key enzyme in the apoptosis cell-signalling cascade) appearance, DNA fragmentation and increased intracellular calcium. Treatment of the cells with cannabidiol (10(-7)-10(-4)m) prior to beta-amyloid peptide exposure significantly elevated cell survival while it decreased ROS production, lipid peroxidation, caspase 3 levels, DNA fragmentation and intracellular calcium. Our results indicate that cannabidiol exerts a combination of neuroprotective, anti-oxidative and anti-apoptotic effects against beta-amyloid peptide toxicity, and that inhibition of caspase 3 appearance from its inactive precursor, pro-caspase 3, by cannabidiol is involved in the signalling pathway for this neuroprotection.     

Connexin43 levels are increased in mouse neural crest cells exposed to homocysteine

BACKGROUND: Elevated homocysteine levels during embryonic development can result in neural tube and cardiovascular defects. The mechanisms that underlie the toxic effect of homocysteine are largely unknown. METHODS: We cultured mouse neural tube explants to study the effects of homocysteine on the migratory behavior of neural crest cells and on the levels of the gap junction protein Connexin43 (C x 43) and the actin- and C x 43-interacting protein ZO-1. RESULTS: Homocysteine exposure resulted in a significantly augmented maximal migration distance (MMD). The level of C x 43 immunolabeling was 2 times higher in the cytoplasm and cell protrusions of neural crest cells in homocysteine-treated cultures than in control cultures. Furthermore, colocalization of C x 43 and ZO-1 was increased in neural crest cell protrusions by this treatment. CONCLUSION: Increased C x 43 levels were previously shown to result in abnormal embryonic development. Our data raises the hypothesis that the embryotoxic effects of homocysteine may be mediated in part by its effects on C x 43 expression level and gap junction function in neural crest cells.     

Random amplification of polymorphic DNA (RAPD) typing of carnobacteria isolated from hindgut chamber and large intestine of Atlantic cod (Gadus morhua l.)

Autochthonous and allochthonous bacteria were isolated from hindgut chamber and large intestine of fed and starved Atlantic cod (Gadus morhua L.). All bacterial strains isolated from hindgut chamber belong to carnobacteria. However, only 10.2% of the bacteria strains from the large intestine belong to carnobacteria. Random amplification of polymorphic DNA (RAPD) analysis using three selective primers, was performed to further identify the carnobacteria strains. Nine of these were isolated from hindgut chamber contents, ten associated with epithelial cells of the hindgut chamber, and six isolated from the large intestines of fed and starved fish. The 25 isolates segregated into eight clusters. The major cluster comprised nine strains isolated from the hindgut chamber of both fed and starved fish showing low similarity with the reference strains. The other strains isolated from the hindgut were located in clusters showing high similarity with Carnobacterium gallinarum or Carnobacterium piscicola. Strains isolated from large intestine appeared more divergent and were located in five different clusters. Autochthonous (indigenous) bacteria were clearly demonstrated in the hindgut chamber as transmission electron microscopy revealed rod-shaped bacteria between adjacent microvilli. Endocytosis of bacteria by epithelial cells was observed in the hindgut chamber.     

Efficient immunization and cross-priming by vaccine adjuvants containing TLR3 or TLR9 agonists complexed to cationic liposomes

Complexing TLR9 agonists such as plasmid DNA to cationic liposomes markedly potentiates their ability to activate innate immunity. We therefore reasoned that liposomes complexed with DNA or other TLR agonists could be used as effective vaccine adjuvants. To test this hypothesis, the vaccine adjuvant effects of liposomes complexed to TLR agonists were assessed in mice. We found that liposomes complexed to nucleic acids (liposome-Ag-nucleic acid complexes; LANAC) were particularly effective adjuvants for eliciting CD4(+) and CD8(+) T cell responses against peptide and protein Ags. Notably, LANAC containing TLR3 or TLR9 agonists effectively cross-primed CD8(+) T cell responses against even low doses of protein Ags, and this effect was independent of CD4(+) T cell help. Ag-specific CD8(+) T cells elicited by LANAC adjuvants were functionally active and persisted for long periods of time in tissues. In a therapeutic tumor vaccine model, immunization with the melanoma peptide trp2 and LANAC adjuvant controlled the growth of established B16 melanoma tumors. In a prophylactic vaccine model, immunization with the Mycobacterium tuberculosis protein ESAT-6 with LANAC adjuvant elicited significant protective immunity against aerosol challenge with virulent M. tuberculosis. These results suggest that certain TLR agonists can be combined with cationic liposomes to produce uniquely effective vaccine adjuvants capable of eliciting strong T cell responses against protein and peptide Ags.     

A critical role for tetraspanin CD151 in alpha3beta1 and alpha6beta4 integrin-dependent tumor cell functions on laminin-5

The basement membrane protein laminin-5 supports tumor cell adhesion and motility and is implicated at multiple steps of the metastatic cascade. Tetraspanin CD151 engages in lateral, cell surface complexes with both of the major laminin-5 receptors, integrins alpha3beta1 and alpha6beta4. To determine the role of CD151 in tumor cell responses to laminin-5, we used retroviral RNA interference to efficiently silence CD151 expression in epidermal carcinoma cells. Near total loss of CD151 had no effect on steady state cell surface expression of alpha3beta1, alpha6beta4, or other integrins with which CD151 associates. However, CD151-silenced carcinoma cells displayed markedly impaired motility on laminin-5, accompanied by unusually persistent lateral and trailing edge adhesive contacts. CD151 silencing disrupted alpha3beta1 integrin association with tetraspanin-enriched microdomains, reduced the bulk detergent extractability of alpha3beta1, and impaired alpha3beta1 internalization in cells migrating on laminin-5. Both alpha3beta1- and alpha6beta4-dependent cell adhesion to laminin-5 were also impaired in CD151-silenced cells. Reexpressing CD151 in CD151-silenced cells reversed the adhesion and motility defects. Finally, loss of CD151 also impaired migration but not adhesion on substrates other than laminin-5. These data show that CD151 plays a critical role in tumor cell responses to laminin-5 and reveal promotion of integrin recycling as a novel potential mechanism whereby CD151 regulates tumor cell migration.     

Specific Chaperones for the Type VII Protein Secretion Pathway

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.     

Virologically suppressed HIV patients show activation of NK cells and persistent innate immune activation

FcRγ is an ITAM-containing adaptor required for CD16 signaling and function in NK cells. We have previously shown that NK cells from HIV patients receiving combination antiretroviral therapy (cART) have decreased FcRγ expression, but the factors causing this are unknown. We conducted a cross-sectional study of cART-naive viremic patients (ART(-)), virologically suppressed patients receiving cART (ART(+)), and HIV-uninfected controls. CD8(+) T cells were activated, as assessed by CD38(+)HLA-DR(+) expression, in ART(-) patients (p < 0.0001), which was significantly reduced in ART(+) patients (p = 0.0005). In contrast, CD38(+)HLA-DR(+) NK cells were elevated in ART(-) patients (p = 0.0001) but did not decrease in ART(+) patients (p = 0.88). NK cells from both ART(-) and ART(+) patients showed high levels of spontaneous degranulation in ex vivo whole blood assays as well as decreased CD16 expression (p = 0.0001 and p = 0.0025, respectively), FcRγ mRNA (p < 0.0001 for both groups), FcRγ protein expression (p = 0.0016 and p < 0.0001, respectively), and CD16-dependent Syk phosphorylation (p = 0.0001 and p = 0.003, respectively). HIV-infected subjects showed alterations in NK activation, degranulation, CD16 expression and signaling, and elevated plasma markers of inflammation and macrophage activation, that is, neopterin and sCD14, which remained elevated in ART(+) patients. Alterations in NK cell measures did not correlate with viral load or CD4 counts. These data show that in HIV patients who achieve viral suppression following cART, NK cell activation persists. This suggests that NK cells respond to factors different from those driving T cell activation, but which are associated with inflammation in HIV patients.     

Functional and structural effects of seven-residue deletions on the coiled-coil cytoplasmic domain of a chemoreceptor

Chemoreceptors transmit signals from the environment to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors is strongly conserved and consists of a long alpha-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Changes in this domain during evolution are characterized by the presence of seven-residue insertions/deletions located symmetrically with respect to the hairpin turn, suggesting that specific interactions between the helices that form the hairpin are required for function. We assessed the impact of seven-residue deletions on the signalling ability and higher-order organization of the serine chemoreceptor from Escherichia coli. Our results indicate that symmetry alterations between the two branches of the cytoplasmic hairpin seriously compromise chemoreceptor function. Shorter functional versions of Tsr with symmetrical deletions form mixed trimers of dimers when coexpressed with Tar, the aspartate receptor of E. coli. However, Tar function in those cells is impaired, suggesting that the length difference between receptors introduces non-functional distortions into the chemoreceptor cluster. This observation is reinforced by the analysis of coexpression of Tar with chemoreceptors from Rhodobacter sphaeroides that naturally belong to a shorter-length class.