NMR solution structure of viscotoxin C1 from Viscum album species Coloratum ohwi: toward a structure-function analysis of viscotoxins

The high resolution three-dimensional structure of the newly discovered plant viscotoxin C1, from the Asiatic Viscum album ssp. Coloratum ohwi, has been determined in solution by (1)H NMR spectroscopy at pH 3.6 and 285 K. The viscotoxin C1-fold, consisting of a helix-turn-helix motif and a short stretch of an antiparralel beta-sheet is very similar to that found for the highly similar viscotoxins A2 and A3 and for other related thionins. Different functional properties of members of the thionin family are discussed here in light of the structural and electrostatic properties. Among the very homologous family of alpha- and beta-thionins, known for their antimicrobial activity, the viscotoxin subfamily differs from the other members because of its high toxicity against tumoral cells. Key residues for the modulation of viscotoxin cytotoxicity have been identified on the basis of sequence and structural alignment.     

High levels of exogenous C2-ceramide promote morphological and biochemical evidences of necrotic features in thyroid follicular cells

CD95 and ceramide are known to be involved in the apoptotic mechanism. The triggering of CD95 induces a cascade of metabolic events that progressively and dramatically modifies the cell shape by intense membrane blebbing, leading to apoptotic bodies production. Although the CD95 pathway has been abundantly described in normal thyrocytes, the effects of cell permeable synthetic ceramide at morphological and biochemical levels are not fully known. In the present study, we show that thyroid follicular cells (TFC) exposed to 20 microM of C(2)-ceramide for 4 h are characterized by morphological features of necrosis, such as electron-lucent cytoplasm, mitochondrial swelling, and loss of plasma membrane integrity without drastic morphological changes in the nuclei. By contrast, TFC treated with 2 microM of C(2)-ceramide for 4 h are able to accumulate GD3, activate caspases cascade, and induce apoptosis. Furthermore, we provide evidence that 20 microM of C(2)-ceramide determine the destruction of mitochondria and are not able to induce PARP cleavage and internucleosomal DNA fragmentation, suggesting that the apoptotic program is not activated during the death process and nuclear DNA is randomly cleaved as the consequence of cellular degeneration. Pretreatment with 30 microM of zVAD-fmk rescued TFC from 2 microM of C(2)-ceramide-induced apoptosis, whereas, 20 microM of C(2)-ceramide exposure induced necrotic features. Deltapsi(m) was obviously altered in cells treated with 20 microM of C(2)-ceramide for 4 h (75% +/- 3.5%) compared with the low percentage (12.5% +/- 0.4%) of cells with altered Deltapsi(m) exposed to 2 microM of C(2)-ceramide. Whereas, only 20% +/- 1.1% of cells treated with anti-CD95 for 1 h showed altered Deltapsi(m). Additionally, Bax and Bak, two pro-apoptotic members, seem to be not oligomerized in the mitochondrial membrane following ceramide exposure. These results imply that high levels of exogenous ceramide contribute to the necrotic process in TFC, and may provide key molecular basis to the understanding of thyroid signaling pathways that might promote the apoptotic mechanism in thyroid tumoral cells.     

Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation

The amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG) contains a single high affinity steroid-binding site. Crystal structures of this domain in complex with several different steroid ligands have revealed that estradiol occupies the SHBG steroid-binding site in an opposite orientation when compared with 5 alpha-dihydrotestosterone or C19 androgen metabolites (5 alpha-androstan-3 beta,17 beta-diol and 5 alpha-androstan-3 beta,17 alpha-diol) or the synthetic progestin levonorgestrel. Substitution of specific residues within the SHBG steroid-binding site confirmed that Ser(42) plays a key role in determining high affinity interactions by hydrogen bonding to functional groups at C3 of the androstanediols and levonorgestrel and the hydroxyl at C17 of estradiol. Among residues participating in the hydrogen bond network with hydroxy groups at C17 of C19 steroids or C3 of estradiol, Asp(65) appears to be the most important. The different binding mode of estradiol is associated with a difference in the position/orientation of residues (Leu(131) and Lys(134)) in the loop segment (Leu(131)-His(136)) that covers the steroid-binding site as well as others (Leu(171)-Lys(173) and Trp(84)) on the surface of human SHBG and may provide a basis for ligand-dependent interactions between SHBG and other macromolecules. These new crystal structures have also enabled us to construct a simple space-filling model that can be used to predict the characteristics of novel SHBG ligands.     

Oxidative stress in diabetes-induced endothelial dysfunction involvement of nitric oxide and protein kinase C

Reactive oxygen species (ROS) formation plays a major role in diabetes-induced endothelial dysfunction, though the molecular mechanism(s) involved and the contribution of nitric oxide (NO) are still unclear. This study using bovine retinal endothelial cells was aimed at assessing (i) the role of oxygen-dependent vs. NO-dependent oxidative stress in the endothelial cell permeability alterations induced by the diabetic milieu and (ii) whether protein kinase C (PKC) activation ultimately mediates these changes. Superoxide, lipid peroxide, and PKC activity were higher under high glucose (HG) vs. normal glucose throughout the 30 d period. Nitrite/nitrate and endothelial NO synthase levels increased at 1 d and decreased thereafter. Changes in monolayer permeability to 125I-BSA induced by 1 or 30 d incubation in HG or exposure to advanced glycosylation endproduct were reduced by treatment with antioxidants or PKC inhibitors, whereas NO blockade prevented only the effect of 1 d HG. HG-induced changes were mimicked by a PKC activator, a superoxide generating system, an NO and superoxide donor, or peroxynitrite (attenuated by PKC inhibition), but not a NO donor. The short-term effect of HG depends on a combined oxidative and nitrosative stress with peroxynitrite formation, whereas the long-term effect is related to ROS generation; in both cases, PKC ultimately mediates permeability changes.     

Behaviour of interleukin-2 serum levels in advanced non-small-cell lung cancer patients: relationship with response to therapy and survival

 Interleukin(IL)-2 is a T helper (Th) 1 type cytokine that has been shown to play an important role in antitumour immune responses. In this study, the prognostic significance of serum IL-2 levels was investigated in 60 advanced non-small-cell lung cancer (NSCLC) patients. IL-2 serum levels were determined before chemotherapy, at the end of chemotherapy and during follow-up, using a commercially available enzyme-linked immunoadsorbent assay kit. The results were analysed according to the response to therapy and were used to generate a model predicting overall survival and time to treatment failure. All 60 patients were shown to have higher IL-2 serum levels than controls (P < 0.0001). Stage IV patients had significantly lower IL-2 levels than stage III patients (P < 0.0001), although they were still significantly higher than controls (P < 0.0001). It is interesting that, when patients were divided into responders and non-responders according to the response to therapy, the former were shown to have significantly higher pre-chemotherapy levels than the latter (P < 0.0001). Moreover, a further significant increase in IL-2 serum levels (P=0.004) and a significant decrease (P < 0.0001) were shown in responders and non-responders, respectively at the end of the therapy. Using univariate and multivariate analyses, both overall survival and time to treatment failure were shown to be affected by the mean pathological levels of IL-2. Furthermore, the prognostic significance of the serum level of IL-2 was confirmed by the stepwise regression analysis. In conclusion, determination of pre-treatment IL-2 serum levels was shown to be of independent prognostic utility in patients with advanced NSCLC; therefore, its possible use for prediction of outcome is proposed. Received: 16 March 2000 / Accepted: 27 July 2000     

Dietary Omega-3 Fatty Acid Supplementation Reduces Inflammation in Obese Pregnant Women: A Randomized Double-Blind Controlled Clinical Trial

Objective

Long-chain omega 3 fatty acids, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) exert potent anti-inflammatory properties in humans. This study characterized the effects of omega-3 ω-3 fatty acids supplements (ω-3 FA) on the inflammatory status in the placenta and adipose tissue of overweight/obese pregnant women.

Study Design

A randomized, double-masked controlled trial was conducted in overweight/obese pregnant women that were randomly assigned to receive DHA plus EPA (2g/day) or the equivalent of a placebo twice a day from week 10–16 to term. Inflammatory pathways were characterized in: 1) adipose tissue and placenta of treated vs. untreated women; and 2) adipose and trophoblast cells cultured with long chain FAs.

Results

The sum of plasma DHA and EPA increased by 5.8 fold and ω-3 FA/ ω-6 FA ratio was 1.5 in treated vs. untreated women (p< 0.005). Plasma CRP concentrations were reduced (p<0.001). The adipose tissue and placenta of treated women exhibited a significant decrease in TLR4 adipose and placental expression as well as IL6, IL8, and TNFα In vitro, EPA and DHA suppressed the activation of TLR4, IL6, IL8 induced by palmitate in culture of adipose and trophoblast cells.

Conclusion

Supplementation of overweight/obese pregnant women with dietary ω-3 FAs for >25 weeks reduced inflammation in maternal adipose and the placental tissue. TLR4 appears as a central target of the anti-inflammatory effects at the cellular level.

Trial Registration

ClinicalTrials.gov NCT00957476     

Phylogenetic morphometrics (I): the use of landmark data in a phylogenetic framework

A method for the direct use of aligned landmark data (2D or 3D coordinates of comparable points) in phylogenetic analysis is described. The approach is based on finding, for each of the landmark points, the ancestral positions that minimize the distance between the ancestor/descendant points along the tree. Doing so amounts to maximizing the degree to which similar positions of the landmarks in different taxa can be accounted for by common ancestry, i.e. parsimony. This method requires no transformation of the aligned data or the results: the data themselves are the x, y, z coordinates of the landmarks, and the output of mapping a character onto a given tree is the x, y, z coordinates for the hypothetical ancestors. In the special case of collinear points, the results are identical to those of optimization of (continuous) additive characters.     

Lycopene prevents 7-ketocholesterol-induced oxidative stress,cell cycle arrest and apoptosis in human macrophages

The present study was undertaken to examine whether lycopene is able to counteract 7-ketocholesterol (7-KC)-induced oxidative stress and apoptosis in human macrophages. Human THP-1 macrophages were exposed to 7-KC (10–25 μM) alone and in combination with lycopene (0.5–2 μM), and we monitored changes in cell oxidative status [reactive oxygen species (ROS) production, NOX-4, hsp70 and hsp90 expressions, 8-OHdG formation] and in cell proliferation and apoptosis. After 24 h of treatment, lycopene significantly reduced the increase in ROS production and in 8-OHdG formation induced by the oxysterol in a dose-dependent manner. Moreover, the carotenoid strongly prevented the increase of NOX-4, hsp70 and hsp90 expressions as well as the phosphorylation of the redox-sensitive p38, JNK and ERK1/2 induced by the oxysterol. The attenuation of 7-KC-induced oxidative stress by lycopene coincided with a normalization of cell growth in human macrophages. Lycopene prevented the arrest in G0/G1 phase of cell cycle induced by the oxysterol and counteracted the increased expression of p53 and p21. Concomitantly, it inhibited 7-KC-induced apoptosis, by limiting caspase-3 activation and the modulatory effects of 7-KC on AKT, Bcl-2, Bcl-xL and Bax. Comparing the effects of lycopene, β-carotene and (5Z)-lycopene on ROS production, cell growth and apoptosis show that lycopene and its isomer were more effective than β-carotene in counteracting the dangerous effects of 7-KC in human macrophages. Our study suggests that lycopene may act as a potential antiatherogenic agent by preventing 7-KC-induced oxidative stress and apoptosis in human macrophages.