Interactome modeling

A long-term goal of the field of interactome modeling is to understand how global and local properties of complex macromolecular networks impact on observable biological properties, and how changes in such properties can lead to human diseases. The information available at this stage of development of the field provides strong evidence for the existence of such interesting global and local properties, but also demonstrates that many more datasets will be needed to provide accurate models with increasingly predictive capacity. This review focuses on an early attempt at mapping a multicellular interactome network and on the lessons learned from that attempt.     

Role of Nrf2 signaling in regulation of antioxidants and phase 2 enzymes in cardiac fibroblasts: protection against reactive oxygen and nitrogen species-induced cell injury

Understanding the molecular pathway(s) of antioxidant gene regulation is of crucial importance for developing antioxidant-inducing agents for the intervention of oxidative cardiac disorders. Accordingly, this study was undertaken to determine the role of Nrf2 signaling in the basal expression as well as the chemical inducibility of endogenous antioxidants and phase 2 enzymes in cardiac fibroblasts. The basal expression of a scope of key cellular antioxidants and phase 2 enzymes was significantly lower in cardiac fibroblasts derived from Nrf2-/- mice than those from wild type control. These include catalase, reduced glutathione (GSH), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). Incubation of Nrf2+/+ cardiac fibroblasts with 3H-1,2-dithiole-3-thione (D3T) led to a significant induction of superoxide dismutase (SOD), catalase, GSH, GR, glutathione peroxidase (GPx), GST, and NQO1. The inducibility of SOD, catalase, GSH, GR, GST, and NQO1, but not GPx by D3T was completely abolished in Nrf2-/- cells. The Nrf2-/- cardiac fibroblasts were much more sensitive to reactive oxygen and nitrogen species-mediated cytotoxicity. Upregulation of antioxidants and phase 2 enzymes by D3T in Nrf2+/+ cardiac fibroblasts resulted in a dramatically increased resistance to the above species-induced cytotoxicity. In contrast, D3T-treatment of the Nrf2-/- cells only provided a slight cytoprotection. Taken together, this study demonstrates for the first time that Nrf2 is critically involved in the regulation of the basal expression and chemical induction of a number of antioxidants and phase 2 enzymes in cardiac fibroblasts, and is an important factor in controlling cardiac cellular susceptibility to reactive oxygen and nitrogen species-induced cytotoxicity.     

Inhibitory effects of anticancer peptide from Mercenaria on the BGC-823 cells and several enzymes

An anti-cancer peptide was purified from the Mercenaria (Meretrix meretrix Linnaeus) by the method of chromatography on Sephadex G-25 and FPLC, and its molecular weight was determined to be 3147 Da by the way of MALDI-TOF mass spectrum. The effects of this peptide on human gastric gland carcinoma cells (BGC-823) and their cytoskeletal morphology were investigated. The results showed that the peptide could inhibit the proliferation of BGC-823 cells and obviously destroy the skeletal structures of the cells. When the concentration of the peptide reached 4.0 microg/ml, the inhibition percentage of the cell growth was about 60%. The effects of this anticancer peptide on the activities of superoxide dismutase (SOD), alkaline phosphatase (ALP) and tyrosinase were studied. The results showed that the peptide activated ALP and SOD, but inhibit the tyrosinase activity. When the concentration of the peptide reached to 0.5 microg/ml, the relative activities of SOD, ALP and tyrosinase were determined to be 188.5%, 122.0% and 27.5%, respectively.     

Fibroblast growth and H-7 protein kinase inhibitor response monitored in microimpedance sensor arrays

Functional genomic studies and drug candidate testing both require high throughput, parallel experimentation strategies to screen for variable cellular behaviors. In this article we describe the use of an impedance sensing electrode array that is capable of sensing cell "presence" as well as the extent of cell (focal) attachment to the substrate. The signals provided by mouse fibroblasts on a sensing structure containing four different sized electrodes are reported. In the absence of cells, each electrode's impedance was found to depend as expected on electrode size and frequency. The impedance increased by several-fold when fibroblasts attached and spread out over time. More notably, the sensors also detected the cellular response to the protein kinase C inhibitor, H-7. H-7 inhibits actomyosin contractility; thereafter, the loss of focal adhesion complexes occurs. The sensors, in turn, detected an impedance decrease after H-7 addition and an increase in impedance after H-7 removal.     

New class of quaternary ammonium salts, derivatives of methyl D-glucopyranosides

Reactions of two aromatic and two aliphatic amines with methyl 6-O-p-toluenesulfonyl-alpha-D-glucopyranoside or methyl 6-O-p-toluenesulfonyl-beta-D-glucopyranoside were performed on a micro-scale. The synthesis and preparative isolation methods have been developed for quaternary N-(methyl 2,3,4-tri-O-acetyl-6-deoxy-alpha- and -beta-D-glucopyranoside-6-yl)ammonium salts derived from three amines: trimethylamine, 2-methylpyridine, and pyridine. The reaction products were examined with 1H, 13C NMR spectroscopy. N-(Methyl 2,3,4-tri-O-acetyl-6-deoxy-beta-d-glucopyranoside-6-yl)trimethylammonium tosylate was additionally analyzed with X-ray crystallography.     

Structural features of pectic polysaccharides from the skin of Opuntia ficus-indica prickly pear fruits

After removal of the mucilage with water at room temperature, pectic polysaccharides were solubilized from Opuntia ficus-indica fruit skin, by sequential extraction with water at 60 degrees C (WSP) and EDTA solution at 60 degrees C (CSP). Polysaccharides with neutral sugar content of 0.48 and 0.36 mol/mol galacturonic acid residue were obtained, respectively, in the WSP and CSP extracts. These pectic polysaccharides were de-esterified and fractionated by anion-exchange chromatography, yielding for each extract five fractions, which were thereafter purified by size-exclusion chromatography. Two of these purified fractions were characterized by sugar analysis combined with methylation and reduction-methylation analysis. The study was then supported by (1)H and (13)C NMR spectroscopy. The results showed that the water-soluble fraction WSP3 and the EDTA soluble fraction CSP3, consisted of a disaccharide repeating unit -->2)-alpha-l-Rhap-(1-->4)-alpha-d-GalpA-(1--> backbone, with side chains attached to O-4 of the rhamnosyl residues. The side chains contained highly branched alpha-(1-->5)-linked arabinan and short linear beta-(1-->4)-linked galactan.     

Protection of tamoxifen against oxidation of mitochondrial thiols and NAD(P)H underlying the permeability transition induced by prooxidants

The effects of tamoxifen (TAM) were studied on the mitochondrial permeability transition (MPT) induced by the prooxidant tert-butyl hydroperoxide (t-BuOOH) or the thiol cross-linker phenylarsine oxide (PhAsO), in the presence of Ca2+, in order to clarify the mechanisms involved in the MPT inhibition by this drug. The combination of Ca2+ with t-BuOOH or PhAsO induces mitochondrial swelling and depolarization of membrane potential (deltapsi). These events are inhibited by cyclosporine A (CyA), suggesting the inhibition of the MPT. The pre-incubation of mitochondria with TAM also prevents those events and induces a time-dependent reversal of deltapsi depolarization following MPT induction, similarly to CyA. Moreover, TAM inhibits the Ca2+ release and the oxidation of NAD(P)H and protein thiol (-SH) groups promoted by t-BuOOH plus Ca2+. On the other hand, the MPT induced by PhAsO plus Ca2+ does not induce -SH groups oxidation, supporting the notion that MPT induction by this compound is not mediated by the oxidation of specific membrane proteins groups. However, TAM also inhibits the PhAsO induced MPT, suggesting that this drug may inhibit this phenomenon by inhibiting PhAsO binding to -SH vicinal groups, implicated in the MPT induction. These data indicate that the MPT inhibition by TAM may be related to its antioxidant capacity in preventing the oxidation of NAD(P)H and -SH groups or by blocking these groups, since the oxidation of these groups increases the sensitivity of mitochondria to the MPT induction. Additionally, they suggest an MPT-independent pathway for TAM-induced apoptosis and a potential ER-independent mechanism for the effectiveness of this drug in the cancer therapy and prevention.     

Interaction of the fusogenic peptide B18 in its amyloid-state with lipid membranes studied by solid state NMR

The interaction of the fusogenic polypeptide segment "B18" from the fertilization protein binding with lipid membranes was investigated by solid state 2H and 31P NMR, and by differential scanning calorimetry. B18 is known to adopt different conformations depending on peptide concentration, ionic conditions, pH and lipid environment. Here, the peptide was studied in its beta-stranded amyloid conformation. According to 31P NMR, the lamellar morphology of the DMPC bilayer remains intact in the presence of B18. In going from low (1:90) to high (1:10) peptide/lipid ratios, an increasing effect on several different 2H-labeled lipid segments was observed, reflecting changes in phase behavior and local dynamics. The strongest influence of B18 was detected at the acyl-chains, while no significant effect on the lipid headgroup conformation was observed. This suggests an insertion of B18 in its fibrillar state into the membrane driven by hydrophobic interactions, rather than a peripheral binding mediated by electrostatics.     

Role of programmed cell death in patterning the Drosophila antennal arista

Programmed cell death is a critical process for the patterning and sculpting of organs during development. The Drosophila arista, a feather-like structure at the tip of the antenna, is composed of a central core and several lateral branches. A homozygous viable mutation in the thread gene, which encodes an inhibitor of apoptosis protein, produces a branchless arista. We have found that mutations in the proapoptotic gene hid lead to numerous extra branches, suggesting that the level of cell death determines the number of branches in the arista. Consistent with this idea, we have found that thread mutants show excessive cell death restricted to the antennal imaginal disc during the middle third instar larval stage. These findings point to a narrow window of development in which regulation of programmed cell death is essential to the proper formation of the arista.