Qualitative and quantitative discrimination of fake and true alkene rotation processes in pd(η-olefin) complexes. A new bimolecular mechanism

The fluxional behaviour of [Pd(η²-fn)(N-SMe)] (2) (fn = fumaronitrile, N-SMe = 2-methylthiomethylpyridine) and of [Pd(η²-tmetc)(N-N′-4-anisyl)] (3) (tmetc = tetramethylethylenetetracarboxylate, N-N′-4-anisyl = 2-(4-methoxyphenyliminomethane)pyridine) were monitored by ¹H NMR spectroscopy and quantitatively determined by line-shape analyses (for 2) and selective inversion recovery experiments (for 3). The coalescence of the AB multiplet of fn hydrogens of 2 is concentration dependent and presents a strongly negative ΔS^≠, suggesting the intermediacy of a dimeric complex and ruling out the hypothesis of olefin rotation. The accurate evaluation of all spectral features also allows determination of the approaching mode of the monomeric units. The inversion transfer between the tmetc methyls of 3 reveals a true propeller-like olefin rotation. The presence of a nucleophilic electron pair at sulfur in 2 triggers the formation of the dimeric intermediate.     

Characterization of the MMP/TIMP Imbalance and Collagen Production Induced by IL-1β or TNF-α Release from Human Hepatic Stellate Cells

Inflammation has an important role in the development of liver fibrosis in general and the activation of hepatic stellate cells (HSCs) in particular. It is known that HSCs are themselves able to produce cytokines and chemokines, and that this production may be a key event in the initiation of fibrogenesis. However, the direct involvement of cytokines and chemokines in HSC (self-)activation remains uncertain. In this study, the effects of pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8 on the activation state of HSCs were examined, in comparison to the pro-fibrogenic mediator TGF-β1. LX-2 cells were stimulated for 24 or 48 hours with recombinant human form of the pro-inflammatory cytokines IL-1α and β, TNF-α, and IL-8, and also the pro-fibrogenic mediator TGF-β1. Two drugs were also evaluated, the anti-TNF-α monoclonal antibody infliximab and the IL-1 receptor antagonist anakinra, regarding their inhibitory effects. In LX-2 human HSC, treatment with TGF-β1 are associated with downregulation of the metalloproteinase (MMP)-1 and MMP-3, with upregulation of tissue inhibitor of metalloproteinase (TIMP)-1, collagen type I α1, collagen type IV α1, α-SMA, endothelin-1 and PDGF-BB. Cytokines and chemokines expression were found to be downregulated, excepting IL-6. In contrast, we observed that LX-2 exposure to IL-1, TNF-α and IL-8 can reverse the phenotype of pro-fibrogenic activated cells. Indeed, MMP-1, MMP-3 and MMP-9 were found elevated, associated with downregulation of α-SMA and/or PDGF-BB, and a greater expression of IL-1β, IL-6, IL-8, CXCL1 and CCL2. Lastly, we found that infliximab and anakinra successfully inhibits effects of TNF-α and IL-1 respectively in LX-2 cells. Infliximab and anakinra may be of value in preclinical trials in chronic liver disease. Overall, our results suggest that (i) pro-inflammatory mediators exert complex effects in HSCs via an MMP/TIMP imbalance, and (ii) targeting IL-1 signaling may be a potentially valuable therapeutic strategy in chronic liver diseases.     

PML-RARA-targeted DNA vaccine induces protective immunity in a mouse model of leukemia

Despite improved molecular characterization of malignancies and development of targeted therapies, acute leukemia is not curable and few patients survive more than 10 years after diagnosis. Recently, combinations of different therapeutic strategies (based on mechanisms of apoptosis, differentiation and cytotoxicity) have significantly increased survival. To further improve outcome, we studied the potential efficacy of boosting the patient's immune response using specific immunotherapy. In an animal model of acute promyelocytic leukemia, we developed a DNA-based vaccine by fusing the human promyelocytic leukemia-retinoic acid receptor-alpha (PML-RARA) oncogene to tetanus fragment C (FrC) sequences. We show for the first time that a DNA vaccine specifically targeted to an oncoprotein can have a pronounced effect on survival, both alone and when combined with all-trans retinoic acid (ATRA). The survival advantage is concomitant with time-dependent antibody production and an increase in interferon-gamma (IFN-gamma). We also show that ATRA therapy on its own triggers an immune response in this model. When DNA vaccination and conventional ATRA therapy are combined, they induce protective immune responses against leukemia progression in mice and may provide a new approach to improve clinical outcome in human leukemia.     

Cdc14 Inhibition by the Spindle Assembly Checkpoint Prevents Unscheduled Centrosome Separation in Budding Yeast

The spindle assembly checkpoint (SAC) is an evolutionarily conserved surveillance mechanism that delays anaphase onset and mitotic exit in response to the lack of kinetochore attachment. The target of the SAC is the E3 ubiquitin ligase anaphase-promoting complex (APC) bound to its Cdc20 activator. The Cdc20/APC complex is in turn required for sister chromatid separation and mitotic exit through ubiquitin-mediated proteolysis of securin, thus relieving inhibition of separase that unties sister chromatids. Separase is also involved in the Cdc-fourteen early anaphase release (FEAR) pathway of nucleolar release and activation of the Cdc14 phosphatase, which regulates several microtubule-linked processes at the metaphase/anaphase transition and also drives mitotic exit. Here, we report that the SAC prevents separation of microtubule-organizing centers (spindle pole bodies [SPBs]) when spindle assembly is defective. Under these circumstances, failure of SAC activation causes unscheduled SPB separation, which requires Cdc20/APC, the FEAR pathway, cytoplasmic dynein, and the actin cytoskeleton. We propose that, besides inhibiting sister chromatid separation, the SAC preserves the accurate transmission of chromosomes also by preventing SPBs to migrate far apart until the conditions to assemble a bipolar spindle are satisfied.     

Hyperactivation of the yeast DNA damage checkpoint by TEL1 and DDC2 overexpression.

The evolutionarily conserved yeast Mec1 and Tel1 protein kinases, as well as the Mec1-interacting protein Ddc2, are involved in the DNA damage checkpoint response. We show that regulation of Tel1 and Ddc2-Mec1 activities is important to modulate both activation and termination of checkpoint-mediated cell cycle arrest. In fact, overproduction of either Tel1 or Ddc2 causes a prolonged cell cycle arrest and cell death in response to DNA damage, impairing the ability of cells to recover from checkpoint activation. This cell cycle arrest is independent of Mec1 in UV-irradiated Tel1-overproducing cells, while it is strictly Mec1 dependent in similarly treated DDC2-overexpressing cells. The Rad53 checkpoint kinase is instead required in both cases for cell cycle arrest, which correlates with its enhanced and persistent phosphorylation, suggesting that unscheduled Rad53 phosphorylation might prevent cells from re-entering the cell cycle after checkpoint activation. In addition, Tel1 overproduction results in transient nuclear division arrest and concomitant Rad53 phosphorylation in the absence of exogenous DNA damage independently of Mec1 and Ddc1.     

A DIGE analysis of developing poplar leaves subjected to ozone reveals major changes in carbon metabolism

Tropospheric ozone pollution is described as having major negative effects on plants, compromising plant survival. Carbon metabolism is especially affected. In the present work, the effects of chronic ozone exposure were evaluated at the proteomic level in developing leaves of young poplar plants exposed to 120 ppb of ozone for 35 days. Soluble proteins (excluding intrinsic membrane proteins) were extracted from leaves after 3, 14 and 35 days of ozone exposure, as well as 10 days after a recovery period. Proteins (pI 4 to 7) were analyzed by 2-D DIGE experiments, followed by MALDI-TOF-TOF identification. Additional observations were obtained on growth, lesion formation, and leaf pigments analysis. Although treated plants showed large necrotic spots and chlorosis in mature leaves, growth decreased only slightly and plant height was not affected. The number of abscised leaves was higher in treated plants, but new leaf formation was not affected. A decrease in chlorophylls and lutein contents was recorded. A large number of proteins involved in carbon metabolism were identified. In particular, proteins associated with the Calvin cycle and electron transport in the chloroplast were down-regulated. In contrast, proteins associated with glucose catabolism increased in response to ozone exposure. Other identified enzymes are associated with protein folding, nitrogen metabolism and oxidoreductase activity.