HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A(2)α dependent PGE(2)via both PKA and PKB pathways

Cytosolic phospholipase A(2)α (cPLA(2)α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA(2)α which coincided with a significant increase in cell proliferation. The inhibition of cPLA(2)α activity by pyrrophenone or by antisense oligonucleotide against cPLA(2)α (AS) or inhibition of prostaglandin E(2) (PGE(2)) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE(2). The secreted PGE(2) activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE(2). But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE(2). AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA(2)α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA(2)α-dependent PGE(2) production. PGE(2)via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

The synergistic anti-inflammatory effects of lycopene, lutein, β-carotene, and carnosic acid combinations via redox-based inhibition of NF-κB signaling

Inflammatory mediators and cytokines play important roles in the pathogenesis of a vast number of human diseases; therefore much attention is focused on blunting their proinflammatory modes of action. The aims of the present research were to assess the effectiveness of combinations of carotenoids and phenolics, at concentrations that can be achieved in blood, to inhibit the release of inflammatory mediators from macrophages exposed to lipopolysaccharide (LPS) and to determine what the anti-inflammatory effect of the phytonutrient combinations was in an in vivo mouse model of peritonitis. Preincubation of mouse peritoneal macrophages with lycopene (1μM) or Lyc-O-Mato (1μM) and carnosic acid (2μM), lutein (1μM), and/or β-carotene (2μM) 1h before addition of LPS for 24h caused a synergistic inhibition of NO, prostaglandin E(2), and superoxide production derived from downregulation of iNOS, COX-2, and NADPH oxidase protein and mRNA expression and synergistic inhibition of TNFα secretion. We surmise that the anti-inflammatory action of the phytonutrient combinations used probably resides in their antioxidant properties, because they caused an immediate, efficient, and synergistic inhibition of LPS-induced internal superoxide production leading to a marked decrease in ERK and NF-κB activation. The anti-inflammatory effects of the selected phytonutrient combinations were also demonstrated in a mouse model of peritonitis: their supplementation in drinking water resulted in attenuation of neutrophil recruitment to the peritoneal cavity and in inhibition of inflammatory mediator production by peritoneal neutrophils and macrophages.     

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh ‘Charentais’ type melon line that accumulates β-carotene. One mutagenized M₂ family segregated for a novel recessive trait, a yellow–orange fruit flesh (‘yofI’). HPLC analysis revealed that ‘yofI’ accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of ‘yofI’ is associated with a significant change of the fruit aroma since cleavage of β-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to β-carotene in melon fruit. Cloning and sequencing of ‘yofI’ CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A–T transversion in ‘yofI’ which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.     

Effects of carbanilates on the growth and development of cell suspension cultures of Acer pseudoplatanus

The effects of three isopropyl carbanilate derivatives (propham, chlorpropham, 3,4-diCl isopropyl carbanilate) were studied on Acer pseudoplatanus L. cell suspension cultures (Lamport's strain). At 0.1 m M , three types of effects were observed: the 3,4-diCl derivative rapidly killed the cells; chlorpropham inhibited mitosis, but the cells remained alive without any significant increase in fresh or dry matter; and propham strongly inhibited mitosis, but not cell growth.
Ultrastructural analysis indicated that no observable changes in mitochondrial or plastid density occurred during the cytoplasmic expansion in the presence of propham. Size increase was also accompanied by vacuolar expansion and morphological changes of the nuclei. In Acer cell cultures, isopropyl carbanilates clearly show selective inhibition of mitosis only when the phenyl ring is not substituted by chlorine.     

Donor-Derived Brain Tumor Following Neural Stem Cell Transplantation in an Ataxia Telangiectasia Patient

Background

Neural stem cells are currently being investigated as potential therapies for neurodegenerative diseases, stroke, and trauma. However, concerns have been raised over the safety of this experimental therapeutic approach, including, for example, whether there is the potential for tumors to develop from transplanted stem cells.

Methods and Findings

A boy with ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human fetal neural stem cells. Four years after the first treatment he was diagnosed with a multifocal brain tumor. The biopsied tumor was diagnosed as a glioneuronal neoplasm. We compared the tumor cells and the patient''s peripheral blood cells by fluorescent in situ hybridization using X and Y chromosome probes, by PCR for the amelogenin gene X- and Y-specific alleles, by MassArray for the ATM patient specific mutation and for several SNPs, by PCR for polymorphic microsatellites, and by human leukocyte antigen (HLA) typing. Molecular and cytogenetic studies showed that the tumor was of nonhost origin suggesting it was derived from the transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor is derived from at least two donors.

Conclusions

This is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor-derived brain tumor. Further work is urgently needed to assess the safety of these therapies.     

Concepts and schemes for the re-engineering of physical protein modules: generating nanodevices via targeted replacements with constrained amino acids

Physically building complex multi-molecular structures from naturally occurring biological macromolecules has aroused a great deal of interest. Here we focus on nanostructures composed of re-engineered, natural 'foldamer' building blocks. Our aim is to provide some of the underlying concepts and schemes for crafting structures utilizing such conformationally relatively stable molecular components. We describe how, via chemical biology strategies, it is further possible to chemically manipulate the foldamer building blocks toward specific shape-driven structures, which in turn could be used toward potential-designed functions. We outline the criteria in choosing candidate foldamers from the vast biological repertoire, and how to enhance their stability through selected targeted replacements by non-proteinogenic conformationally constrained amino acids. These approaches combine bioinformatics, high performance computations and mathematics with synthetic organic chemistry. The resulting artificially engineered self-organizing molecular scale structures take advantage of nature's nanobiology toolkit and at the same time improve on it, since their new targeted function differs from that optimized by evolution. The major challenge facing nanobiology is to be able to exercise fine control over the performance of these target-specific molecular machines.     

Specific cysteines in beta3 are involved in disulfide bond exchange-dependent and -independent activation of alphaIIbbeta3

Disulfide bond exchange among cysteine residues in epidermal growth factor (EGF)-like domains of beta3 was suggested to be involved in activation of alphaIIbbeta3. To investigate the role of specific beta3 cysteines in alphaIIbbeta3 expression and activation, we expressed in baby hamster kidney cells normal alphaIIb with normal beta3 or beta3 with single or double cysteine substitutions of nine disulfide bonds in EGF-3, EGF-4, and beta-tail domains and assessed alphaIIbbeta3 surface expression and activation state by flow cytometry using P2 or PAC-1 antibodies, respectively. Most mutants displayed reduced surface expression of alphaIIbbeta3. Disruptions of disulfide bonds in EGF-3 yielded constitutively active alphaIIbbeta3, implying that these bonds stabilize the inactive alphaIIbbeta3 conformer. Mutants of the Cys-567-Cys-581 bond in EGF-4 were inactive even after exposure to alphaIIbbeta3-activating antibodies, indicating that this bond is necessary for activating alphaIIbbeta3. Disrupting Cys-560-Cys-583 in the EGF-3/EGF-4 or Cys-608-Cys-655 in beta-tail domain resulted in alphaIIbbeta3 activation only when Cys-560 or Cys-655 of each pair was mutated but not when their partners (Cys-583, Cys-608) or both cysteines were mutated, suggesting that free sulfhydryls of Cys-583 and Cys-608 participate in alphaIIbbeta3 activation by a disulfide bond exchange-dependent mechanism. The free sulfhydryl blocker dithiobisnitrobenzoic acid inhibited 70% of anti-LIBS6 antibody-induced activation of wild-type alphaIIbbeta3 and had a smaller effect on mutants, implicating disulfide bond exchange-dependent and -independent mechanisms in alphaIIbbeta3 activation. These data suggest that different disulfide bonds in beta3 EGF and beta-tail domains play variable structural and regulatory roles in alphaIIbbeta3.     

Lipid transport in the developing bovine follicle: messenger RNA expression increases for selective uptake receptors and decreases for endocytosis receptors

Differences in rates of steroid production and secretion will, eventually, determine the developmental rates of ovarian follicles. The major supply of cholesterol, the precursor for steroid and androgen biosynthesis, to ovarian cells is from circulating lipoproteins via membrane receptors from the low density lipoprotein receptor (LDL) superfamily. This occurs by either endocytosis, which has been described for very low density lipoprotein receptors (VLDLr), for LDL receptors (LDLr), and by the selective uptake pathway described for the scavenger receptor class B type 1 receptor (SRB1) and the recently described ovarian receptor, lipoprotein receptor-related protein 8 (LRP8). In this study, the mRNA expression of these four cholesterol receptors in bovine ovarian cells was determined at different stages of follicular development. In small antral follicles, mRNA expression of the endocytosis receptors was higher than in large antral follicles. Expression of LRP8 mRNA increased linearly with follicular size together with an increase in LDL, VLDL, and cholesterol concentrations in the follicular fluid. SRB1 mRNA expression tended to increase with follicular diameter. Because different mRNA expression patterns were found for the two types of receptor, this may imply different regulation of cholesterol supply at different stages of follicular development. Accumulation of LDL and VLDL particles in the follicular fluid of large antral follicles may enhance cholesterol availability for the intense steroidogenic activity that is essential at these stages.