Regulation of Fas (CD95)-induced apoptosis by nuclear factor-kappaB and tumor necrosis factor-alpha in macrophages

The APO-1/Fasligand (FasL) and tumor necrosis factor- (TNF-) are twofunctionally related molecules that induce apoptosis ofsusceptible cells. Although the two molecules have been reported toinduce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-, raising thepossibility that TNF- may be involved in FasL-inducedapoptosis. Because TNF- gene expression is under the controlof nuclear factor-B (NF-B), we investigated whether FasL caninduce NF-B activation and whether such activation plays a role inFasL-mediated cell death in macrophages. Gene transfection studiesusing NF-B-dependent reporter plasmid showed that FasL did activateNF-B promoter activity. Gel shift studies also revealed that FasLmobilized the p50/p65 heterodimeric form of NF-B. Inhibition ofNF-B by a specific NF-B inhibitor, caffeic acid phenylethylester, or by dominant expression of the NF-B inhibitory subunitIB caused an increase in FasL-induced apoptosis and areduction in TNF- expression. However, neutralization of TNF- byspecific anti-TNF- antibody had no effect on FasL-inducedapoptosis. These results indicate that FasL-mediated cell deathin macrophages is regulated through NF-B and is independent ofTNF- activation, suggesting the antiapoptotic role of NF-Band a separate death signaling pathway mediated by FasL.

     

Synthesis of potential thrombin inhibitors. Incorporation of tartaric acid templates as P2 proline mimetics

With the objective to prepare novel non-peptidic thrombin inhibitors, bioisosteres of the inhibitory tripeptide D-Phe-Pro-Arg chain have been examined. Thus, the P1 Arg was replaced with p-amidinobenzylamine, an elongated homologue of the same and with 2,5-dichloro benzylamine. The P2-P3, D-Phe-Pro, was replaced with a novel tartaric acid template coupled to a series of readily available, mainly lipophilic, amines. Some of these compounds exhibit promising thrombin inhibition activity in vitro, IC(50 ) approximately 5.9 microM.     

XPORT-dependent transport of TRP and rhodopsin

TRP channels have emerged as key biological sensors in vision, taste, olfaction, hearing, and touch. Despite their importance, virtually nothing is known about the folding and transport of TRP channels during biosynthesis. Here, we identify XPORT (exit protein of rhodopsin and TRP) as a critical chaperone for TRP and its G protein-coupled receptor (GPCR), rhodopsin (Rh1). XPORT is a resident ER and secretory pathway protein that interacts with TRP and Rh1, as well as with Hsp27 and Hsp90. XPORT promotes the targeting of TRP to the membrane in Drosophila S2 cells, a finding that provides a critical first step toward solving a longstanding problem in?the successful heterologous expression of TRP. Mutations in xport result in defective transport of TRP and Rh1, leading to retinal degeneration. Our results identify XPORT as a molecular chaperone and provide a mechanistic link between TRP channels and their GPCRs during biosynthesis and transport.     

Targeting MAGE-C1/CT7 expression increases cell sensitivity to the proteasome inhibitor bortezomib in multiple myeloma cell lines

The MAGE-C1/CT7 encodes a cancer/testis antigen (CTA), is located on the chromosomal region Xq26-27 and is highly polymorphic in humans. MAGE-C1/CT7 is frequently expressed in multiple myeloma (MM) that may be a potential target for immunotherapy in this still incurable disease. MAGEC1/CT7 expression is restricted to malignant plasma cells and it has been suggested that MAGE-C1/CT7 might play a pathogenic role in MM; however, the exact function this protein in the pathophysiology of MM is not yet understood. Our objectives were (1) to clarify the role of MAGE-C1/CT7 in the control of cellular proliferation and cell cycle in myeloma and (2) to evaluate the impact of silencing MAGE-C1/CT7 on myeloma cells treated with bortezomib. Myeloma cell line SKO-007 was transduced for stable expression of shRNA-MAGE-C1/CT7. Downregulation of MAGE-C1/CT7 was confirmed by real time quantitative PCR and western blot. Functional assays included cell proliferation, cell invasion, cell cycle analysis and apoptosis. Western blot showed a 70-80% decrease in MAGE-C1/CT7 protein expression in inhibited cells (shRNA-MAGE-C1/CT7) when compared with controls. Functional assays did not indicate a difference in cell proliferation and DNA synthesis when inhibited cells were compared with controls. However, we found a decreased percentage of cells in the G2/M phase of the cell cycle among inhibited cells, but not in the controls (p<0.05). When myeloma cells were treated with bortezomib, we observed a 48% reduction of cells in the G2/M phase among inhibited cells while controls showed 13% (empty vector) and 9% (ineffective shRNA) reduction, respectively (p<0.01). Furthermore, inhibited cells treated with bortezomib showed an increased percentage of apoptotic cells (Annexin V+/PI-) in comparison with bortezomib-treated controls (p<0.001). We found that MAGE-C1/CT7 protects SKO-007 cells against bortezomib-induced apoptosis. Therefore, we could speculate that MAGE-C1/CT7 gene therapy could be a strategy for future therapies in MM, in particular in combination with proteasome inhibitors.     

Construction of Saccharomyces cerevisiae strain FAV20 useful in detection of immunosuppressants produced by soil actinomycetes

The screening of microbial natural products continues to represent an important route to the discovery of novel chemicals for development of new therapeutic agents. The aim of this work was to develop an efficient method for the detection of immunosuppressive compounds produced by soil actinomycetes. Mutant strain of Saccharomyces cerevisiae, named FAV20, sensitive to FK506 was constructed by disrupting VMA22 gene using the selectable marker kanMX4 which allowed detection of integration events. Actinomycetes were isolated from different soil samples and in a newly developed test with S. cerevisiae FAV20, six strains have been identified that produce bioactive compounds with the same mechanism of action as FK506. S. cerevisiae FAV20 can be easily used as a test strain in drug screening programs based on inhibition of the calcineurin phosphatase dependent signaling pathway in the cell.     

Natural and modified (1-->3)-beta-D-glucans in health promotion and disease alleviation

A number of polysaccharides with beta-glycosidic linkage are widespread in nature in a variety of sources. All have a common structure and the (1-->3)-beta-D-glucan backbone is essential. They have attracted attention over the years because of their bioactive and medicinal properties. In many cases their functional role is a mystery, in others it is well established. Because of their insoluble chemical nature, particulate (1-->3)-beta-D-glucans are not suitable for many medical applications. Various methods of changing or modifying the beta-D-glucan chemical structure and transforming it to a soluble form have been published. The beta-D-glucan bioactive properties can be affected positively or negatively by such modifications. This review examines beta-glucan sources in nature, health effects and structure-activity relationships. It presents the current state of beta-D-glucan solubilization methods and discusses their effectiveness and application possibilities for the future.     

Regulation of Fas (CD95)-induced apoptotic and necrotic cell death by reactive oxygen species in macrophages

Although reactive oxygen species (ROS) have long been suspected to play a key role in Fas (CD95)-induced cell death, the identity of specific ROS involved in this process and the relationship between apoptotic and necrotic cell death induced by Fas are largely unknown. Using electron spin resonance (ESR) spectroscopy, we showed that activation of Fas receptor by its ligand (FasL) in macrophages resulted in a rapid and transient production of hydrogen peroxide (H2O2) and hydroxyl radicals (*OH). The response was visible as early as 5 min and peaked at approximately 45 min post-treatment. Morphological analysis of total death response (apoptosis vs. necrosis) showed dose and time dependency with apoptosis significantly increased at 6 h after the treatment, while necrosis remained at a baseline level. Only at a 35-fold increase in apoptosis did necrosis become significant. Inhibition of apoptosis by a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (zVAD-fmk), significantly inhibited cell necrosis, indicating the linkage between the two events. Catalase (H2O2 scavenger) and deferoxamine (*OH scavenger) effectively inhibited the total death response as well as the ESR signals, while superoxide dismutase (SOD) (O2*- scavenger) had minimal effects. These results established the role for H2O2 and *OH as key participants in Fas-induced cell death and indicated apoptosis as a primary mode of cell death preceding necrosis. Because the Fas death pathway is implicated in various inflammatory and immunologic disorders, utilization of antioxidants and apoptosis inhibitors as potential therapeutic agents may be advantageous.     

Role of boundary conditions in an experimental model of epithelial wound healing

Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis, but our understanding of the mechanisms that coordinate the behavior of multiple cells in these processes is far from complete. Recent experiments with Madin-Darby canine kidney epithelial monolayers revealed a wave-like pattern of injury-induced MAPK activation and showed that it is essential for collective cell migration after wounding. To investigate the effects of the different aspects of wounding on cell sheet migration, we engineered a system that allowed us to dissect the classic wound healing assay. We studied Madin-Darby canine kidney sheet migration under three different conditions: 1) the classic wound healing assay, 2) empty space induction, where a confluent monolayer is grown adjacent to a slab of polydimethylsiloxane and the monolayer is not injured but allowed to migrate upon removal of the slab, and 3) injury via polydimethylsiloxane membrane peel-off, where an injured monolayer migrates onto plain tissue culture surface, as in the case of empty space induction allowing for direct comparison. By tracking the motion of individual cells within the sheet under these three conditions, we show how the dynamics of the individual cells' motion is responsible for the coordinated migration of the sheet and is coordinated with the activation of ERK1/2 MAPK. In addition, we demonstrate that the propagation of the waves of MAPK activation depends on the generation of reactive oxygen species at the wound edge.     

Targeting polyketide synthase gene pool within actinomycetes: new degenerate primers

Natural products provide a unique element of molecular diversity and biological functionality and they are still indispensable for drug discovery. The polyketides, comprising a large and structurally diverse family of bioactive natural products, have been isolated from a group of mycelia-forming Gram-positive microorganisms, the actinomycetes. Relatively high amino acid sequence identity of the actinomycetes type I polyketide synthases (PKSs) was used to design three degenerate primer pairs for homology-based PCR detection of novel PKS genes, with particular interest into PKSs involved in biosynthesis of immunosuppressive-like metabolites. The stepdown PCR method, described here, enables fast insight into the PKS arsenal within actinomycetes. Designed primers and stepdown PCR were applied for the analysis of two natural isolates, Streptomyces sp. strains NP13 and MS405. Sequence analysis of chosen clones revealed the presence of two distinctive sequences in strain Streptomyces sp. NP13, but only one of these showed homology to PKS-related sequences. On analysing PCR amplicons derived from Streptomyces sp. strain MS405, three different PKS-related sequences were identified demonstrating a potential of designed primers to target PKS gene pool within single organism.