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An atisane diterpene was isolated from Xylopia langsdorfi ana St. Hilaire & Tulasne, Annonaceae, leaves,. Preliminary study showed that xylodiol was cytotoxic and induced differentiation on human leukemia cell lines. However, the molecular mechanisms of xylodiol-mediated cytotoxicity have not been fully defined. Thus, we investigated the anti-tumor effect of xylodiol in human leukemia HL60 cell line. Xylodiol induced apoptosis and necrosis. HL60 cells treated with xylodiol showed biochemical changes characteristic of apoptosis, including caspases-8, -9 and -3 activation and loss of mitochondrial transmembrane potential (∆ψ m ). However, there was a condensation rather than swelling of mitochondria. Moreover, the formation of condensed mitochondria and the loss of ∆ψ m occurred downstream of caspase activation. Cyclosporine A did not protect HL60 cells from the cytotoxic effects of xylodiol, suggesting that the loss of ∆ψ m is a late event in xylodiol-induced apoptosis. Oxidative stress was involved in xylodiol-induced apoptosis. Thus, we conclude that activated caspases cleave cellular proteins resulting in mitochondrial damage leading to mitochondrial condensation, loss of ∆ψ m and ROS release from the mitochondria. ROS can further induce and maintain a collapse of ∆ψ m leading to cellular damage through oxidation of lipids and proteins resulting in apoptotic cell death.

Two new diterpenes were isolated from stems and leaves of Xylopia langsdorffi ana, entatisane- 7α,16α-diol (xylodiol) and ent-7α-acetoxytrachyloban-18-oic acid (trachylobane), along with the known 8(17),12E,14-labdatrien-18-oic acid (labdane). We investigated their antitumour effects on HL60, U937 and K562 human leukemia cell lines. We found that xylodiol was the most potent diterpene in inhibiting cell proliferation of HL60, U937 and K562 cells, with mean IC50 values of 90, 80 and 50 μM, respectively. Based on the nitroblue tetrazolium (NBT) reduction assay, all the diterpenes were found to induce terminal differentiation in HL60 and K562 cells, with xylodiol being the most effective. NBT reduction was increased by almost 120% after 12 h exposure of HL60 cells to xylodiol at a concentration lower than the IC50 (50 μM). Thus, xylodiol inhibited human leukemia cell growth in vitro partly by inducing cell differentiation, and merits further studies to examine its mechanism of action as a potential antitumoural agent

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Misfolded proteins, aggregates, and inclusion bodies are hallmarks of the cytopathology of neurodegenerative disorders including Huntington's disease, Amyotropic lateral sclerosis, Parkinson's disease, Prion diseases, and Alzheimer's disease. The appearance of proteins with altered folded states is regulated by the protein folding quality control machinery and agedependent. We have identified an unexpected molecular link between metabolic state, accumulation of damaged proteins, the heat-shock response and chaperones, and longevity. Mutations (age-1, daf-2) in the insulin-like signaling (ILS) pathway in C. elegans leading to longevity results in the suppression of polyglutamine toxicity and aggregate formation. Because overexpression of HSF-1, a known regulator of chaperone networks and quality control, was also shown to suppress polyglutamine aggregation, we examined whether HSF-1-regulated lifespan. Downregulation of hsf-1 by RNAi in neurons and muscle cells suppressed longevity, which reveals a new molecular link between longevity and stress resistance. To identify other modifiers of protein quality control, we screened transgenic polyglutamine-expressing strains using genome-wide RNAi to identify genes that regulate polyglutamine aggregation. Nearly 200 genes were identified defining a ''protein quality control proteome'' corresponding to five principal classes of polyglutamine regulators: genes involved in RNA metabolism, protein synthesis, protein folding, protein degradation, and those involved in protein trafficking. We propose that each of these classes represents a molecular machine that collectively comprises the protein homeostatic buffer that responds to the expression of damaged proteins to prevent their misfolding and aggregation.Small heat-shock proteins are low-molecular mass proteins, abundant in all kingdoms of life. Their role is to maintain substrate proteins in folding-competent state. These small heat-shock proteins generally form large multimeric complexes; the oligomers usually have a dynamic quaternary structure. The oligomerization is a prerequisite for the chaperone function, but the Escherichia coli chaperone GroEL (hsp60) is known to prevent non-specific association of non-native proteins by the interaction with them and to promote their native structure formation in a ligand-dependent manner. In the present work, the complexes of GroEL with non-native proteins (lactalbumin, ribonuclease, lysozyme, casein, apocytochrome C and pepsin) were studied using size-exclusion and affinity chromatographies, small angle X-ray scattering, scanning microcalorimetry, limited proteolysis and fluorescence spectroscopy. The results demonstrate the following: (i) GroEL binds more then one protein target; (ii) the interaction with non-native protein increases GroEL thermostability and affects the orientation of its apical and intermediate domains as well as inter-ring distance; (iii) GroEL affinity to negatively charged non-native proteins is determinated by Mg 2+ or Ca 2+ ions; (iv) the interaction of...

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The present work analyzed the tetrameric stability of the hemoglobins from the rattlesnake C. durissus terrificus using analytical gel filtration chromatography, SAXS and osmotic stress. We show that the dissociation mechanism proposed for L. miliaris hemoglobin does not apply for these hemoglobins, which constitute stable tetramers even at low concentrations.