Mitochondria-mediated Caspase-dependent and Caspase-independent apoptosis induced by aqueous extract from Moringa oleifera leaves in human melanoma cells

Molecular Biology Reports - Malignant melanoma is a very aggressive and serious type of cutaneous cancer. Previous studies indicated the anti-cancer activity of aqueous extract of Moringa oleifera...     

Myeloperoxidase generates 5-chlorouracil in human atherosclerotic tissue: a potential pathway for somatic mutagenesis by macrophages

Somatic mutations induced by oxidative damage of DNA might play important roles in atherogenesis. However, the underlying mechanisms remain poorly understood. Myeloperoxidase, a heme protein expressed by select populations of artery wall macrophages, initiates one potentially mutagenic pathway by generating hypochlorous acid. This potent chlorinating agent reacts rapidly with primary amines to yield long-lived, selectively reactive N-chloramines. In the current studies, we demonstrate that myeloperoxidase produced by human macrophages differentiated in the presence of granulocyte macrophage colony-stimulating factor generates 5-chlorouracil, a mutagenic thymine analog. The primary amine taurine fails to block the reaction, suggesting that N-haloamines produced by macrophages might oxidize uracil. Model system studies demonstrated that N-chloramines convert uracil to 5-chlorouracil. Interestingly, the tertiary amine nicotine dramatically enhances uracil chlorination, suggesting that cigarette smoke might promote nucleobase oxidation by N-chloramines. To look for evidence that myeloperoxidase promotes uracil oxidation in vivo, we measured 5-chlorouracil levels in human aortic tissue, using isotope dilution gas chromatography-mass spectrometry. The level of 5-chlorouracil was 10-fold higher in atherosclerotic aortic tissue obtained during vascular surgery than in normal aortic tissue, suggesting that halogenated nucleobases produced by macrophages might contribute to atherogenesis. Because 5-chlorouracil can be incorporated into nuclear DNA, our observations raise the possibility that halogenation reactions initiated by phagocytes provide one pathway for mutagenesis, phenotypic modulation, and cytotoxicity during atherogenesis.     

NEU1 sialidase expressed in human airway epithelia regulates epidermal growth factor receptor (EGFR) and MUC1 protein signaling

Epithelial cells (ECs) lining the airways provide a protective barrier between the external environment and the internal host milieu. These same airway epithelia express receptors that respond to danger signals and initiate repair programs. Because the sialylation state of a receptor can influence its function and is dictated in part by sialidase activity, we asked whether airway epithelia express catalytically active sialidase(s). Human primary small airway and A549 ECs expressed NEU1 sialidase at the mRNA and protein levels, and NEU1 accounted for >70% of EC sialidase activity. Blotting with Maackia amurensis and peanut agglutinin lectins established epidermal growth factor receptor (EGFR) and MUC1 as in vivo substrates for NEU1. NEU1 associated with EGFR and MUC1, and NEU1-EGFR association was regulated by EGF stimulation. NEU1 overexpression diminished EGF-stimulated EGFR Tyr-1068 autophosphorylation by up to 44% but enhanced MUC1-dependent Pseudomonas aeruginosa adhesion by 1.6-1.7-fold and flagellin-stimulated ERK1/2 activation by 1.7-1.9-fold. In contrast, NEU1 depletion increased EGFR activation (1.5-fold) and diminished MUC1-mediated bacterial adhesion (38-56%) and signaling (73%). These data indicate for the first time that human airway epithelia express catalytically active NEU1 sialidase that regulates EGFR- and MUC1-dependent signaling and bacterial adhesion. NEU1 catalytic activity may offer an additional level of regulation over the airway epithelial response to ligands, pathogens, and injurious stimuli.     

Novel HCV NS5B polymerase inhibitors derived from 4-(1',1'-dioxo-1',4'-dihydro-1'lambda6-benzo[1',2',4']thiadiazin-3'-yl)-5-hydroxy-2H-pyridazin-3-ones. Part 1: exploration of 7'-substitution of benzothiadiazine

5-Hydroxy-3(2H)-pyridazinone derivatives were investigated as inhibitors of genotype 1 HCV NS5B polymerase. The synthesis, structure-activity relationships (SAR), metabolic stability, and structure-based design approach for this new class of compounds are discussed.     

Critical role of the FERM domain in Pyk2 stimulated glioma cell migration

The strong tendency of malignant glioma cells to invade locally into surrounding normal brain precludes effective surgical resection, reduces the efficacy of radiotherapy, and is associated with increased resistance to chemotherapy regimens. We report that the N-terminal FERM domain of Pyk2 regulates its promigratory function. A 3-dimensional model of the Pyk2 FERM domain was generated and mutagenesis studies identified residues essential for Pyk2 promigratory function. Model-based targeted mutations within the FERM domain decreased Pyk2 phosphorylation and reduced the capacity of Pyk2 to stimulate glioma cell migration but did not significantly alter the intracellular distribution of Pyk2. Expression of autonomous Pyk2 FERM domain fragments containing analogous mutations exhibited reduced capacity to inhibit glioma cell migration and Pyk2 phosphorylation relative to expression of an autonomous wild type FERM domain fragment. These results indicate that the FERM domain plays an important role in regulating the functional competency of Pyk2 as a promigratory factor in glioma.     

Phytochemical and cytotoxic studies on the leaves of Calotropis gigantea

A new lignan, 9′-methoxypinoresinol (1), and two new glycosylated 5-hydroxymethylfurfurals, calofurfuralside A (2), and calofurfuralside B (3), together with nine known compounds (4–12) have been isolated from the active fractions, CHCl₃ (IC₅₀, 0.32 μg mL^?1) and EtOAc (IC₅₀, 0.55 μg mL^?1) fractions of the leaves of Calotropis gigantea. Their structures were elucidated based on NMR and MS data. Among the isolated compounds, compounds 1 and 9 exhibited potent cytotoxicity against PANC-1 human pancreatic cancer cell line under the normoglycemic condition with IC₅₀ values of 3.7 and 3.3 μM, respectively. 9′-Methoxypinoresinol (1) significantly inhibited the colony formation of PANC-1 cells in a concentration-dependent manner.     

Evidence calcium pump binds magnesium before inorganic phosphate

Calcium pump-catalyzed (18)O exchange between inorganic phosphate and water was studied to test the hypothesis that all P-type pumps bind Mg(2+) before P(i) and validate utilization of the rate equation for ordered binding to interpret differences between site-directed mutants and wild-type enzyme. The results were remarkably similar to those obtained earlier with sodium pump (Kasho, V. N., Stengelin, M., Smirnova, I. N., and Faller, L. D. (1997) Biochemistry 36, 8045- 8052). The equation for ordered binding of Mg(2+) before P(i) fit the data best with only a slight chance (0.6%) of P(i) binding to apoenzyme. Therefore, P(i) is the substrate, and Mg(2+) is an obligatory cofactor. The intrinsic Mg(2+) dissociation constant from metalloenzyme (K(M) = 3.5 +/- 0.3 mm) was experimentally indistinguishable from the sodium pump value. However, the half-maximal concentration for P(i) binding to metalloenzyme ((K(p)(')=6.3+/-0.6 mM)) was significantly higher ( approximately 6-fold), and the probability of calcium pump forming phosphoenzyme from bound P(i) (P(c) = 0.04 +/- 0.03) was significantly lower ( approximately 6-fold) than for the sodium pump. From estimates of the rate constants for phosphorylation and dephosphorylation, the calcium pump appears to catalyze phosphoryl group transfer less efficiently than the sodium pump. Ordered binding of Mg(2+) before P(i) implies that both calcium pump and sodium pump form a ternary enzyme.metal.phosphate complex, consistent with molecular structures of other haloacid dehalogenase superfamily members that were crystallized with Mg(2+) and phosphate, or a phosphate analogue, bound.     

Toxicity and bio-effects of CuO nanoparticles on transgenic Ipt-cotton

This study investigated the effects of copper oxide nanoparticles (CuO NPs) on the growth and development of transgenic cotton harboring the Ipt gene, which encodes isopentenyl transferase (Ipt). Three concentrations of CuO NPs were evaluated: 10, 200, and 1000 mg·L^-1, each with three replicates. The height and the root length were 26.91% and 42.80% decreased after 10-day exposure with 1000 mg·L^-1 CuO NPs, respectively.In addition, less abundant on root hairs and lower in shoot biomass of Ipt-cotton when compared with the control group. The growth of Ipt-cotton was not affected by 10 mg·L^-1 CuO NPs, but a high concentration of CuO NPs promoted the absorption of Fe and Na into roots, and inhibited the production of phytohormones in Ipt-cotton. The CuO NPs increased the concentration of iPA in shoots, which can delay senescence. The extent of the increase in iPA in response to CuO NPs should be relative to the amount of Ipt immobilized onto the NPs in the plant tissue. To our knowledge, this is the first study to evaluate the phytotoxicity of CuO NPs to Ipt-transgenic cotton. These results establish a baseline for further research on the effects of nanoparticles on transgenic crops harboring the Ipt gene.