BBA - General Subjects (v.1800, #7)

Activity of recombinant cysteine-rich domain proteins derived from the membrane-bound MUC17/Muc3 family mucins by Samuel B. Ho; Ying Luu; Laurie L. Shekels; Surinder K. Batra; Brandon Kandarian; David B. Evans; Phillip G. Zaworski; Cindy L. Wolfe; Robert L. Heinrikson (629-638).
The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and have cytoprotective properties. Their extracellular regions contain two EGF-like Cys-rich domains (CRD1 and CRD2) connected by an intervening linker segment with SEA module (L), and may function to stimulate intestinal cell restitution. The purpose of this study was to determine the effect of size, recombinant host source, and external tags on mucin CRD1-L-CRD2 protein activity.Four recombinant Muc3-CRD proteins and three MUC17-CRD proteins were generated using E scherichia coli or baculovirus-insect cell systems and tested in colonic cell cultures for activity related to cell migration and apoptosis.N-terminal glutathione-S-transferase (GST) or C-terminal His8 tags had no effect on either the cell migration or anti-apoptosis activity of Muc3-CRD1-L-CRD2. His-tagged Muc3-CRD1-L-CRD2 proteins with truncated linker regions, or the linker region alone, did not demonstrate biologic activity. The human recombinant MUC17-CRD1-L-CRD2-His8 was shown to have anti-apoptotic and pro-migratory activity, but did not stimulate cell proliferation. This protein showed similar in vitro biologic activity, whether produced in E. coli or a baculovirus-insect cell system.Recombinant mucin proteins containing a bivalent display of Cys-rich domains accelerate colon cell migration and inhibit apoptosis, require a full-length intervening Linker-SEA segment for optimal biologic activity, and are functional when synthesized in either E. coli and insect cell systems.These results indicate that an E scherichia coli-derived full-length His8-tagged human MUC17 CRD1-L-CRD2 recombinant protein is a biologically active candidate for further development as a therapeutic agent.
Keywords: Mucin; Cell migration; Inflammatory bowel disease; MUC17; Epidermal growth factor; Recombinant protein;

The classical NFκB pathway is required for phloroglucinol-induced activation of murine lymphocytes by Ginnae Ahn; Eunjin Park; Hyun Jeong Park; You-Jin Jeon; Jehee Lee; Jae Woo Park; Youngheun Jee (639-645).
Phloroglucinol (PG), a polyphenolic compound, has been proposed to show free radical scavenging, anti-tumor, and immunomodulation effects on immune cells. In this study, we investigated PG for its immunological activity and their molecular mechanisms, specifically focusing on the functional activation of nuclear factor-κB (NFκB), an important transcription factor involved in the activation and differentiation of lymphocytes, and subsequent recruitment of murine lymphocytes.We tested whether PG may contribute to the activation of immune response through the NFκB pathway by 3H-thymidine incorporation assay, Western blot, intracellular cytokine assays and Electrophoretic mobility shift assay (EMSA) in murine lymphocytes.PG markedly enhanced the proliferation of lymphocytes by inducing the degradation and phosphorylation of IκB, which leads to the activation of NFκB p65. Also, PG induced the activation of mitogen-activated protein kinases (MAPKs) such as ERK1/2, JNK, and p38, upstream molecules of NFκB pathway. In addition, PG augmented the secretion of interleukin (IL)-2 in mature T lymphocytes and their production of IL-2. Furthermore, the application of TPCK significantly reduced the activation of lymphocytes by PG via inhibiting the NFκB activation.These results suggest that PG induces the activation of lymphocytes by inducing the proliferative activity and secretion of IL-2 through the classical NFκB.The effect of PG on lymphocyte activation was assayed for the first time. These results would also elucidate its underlying mechanism.
Keywords: Phloroglucinol (PG); Lymphocytes; NFκB p65; IL-2;

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells by Arti Parihar; Mordhwaj S. Parihar; Rafal Nazarewicz; Pedram Ghafourifar (646-654).
Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.
Keywords: Ceramide; Cytochrome c; Mitochondria; Membrane potential; Respiration; Oxidative stress; Mammary adenocarcinoma cells;

AFM observation of single, functioning ionotropic glutamate receptors reconstituted in lipid bilayers by Nahoko Kasai; Chandra S. Ramanujan; Ichiro Fujimoto; Akiyoshi Shimada; John F. Ryan; Keiichi Torimitsu (655-661).
Ionotropic glutamate receptors (iGluRs) are responsible for extracellular signaling in the central nervous system. However, the relationship between the overall structure of the protein and its function has yet to be resolved. Atomic force microscopy (AFM) is an important technique that allows nano-scale imaging in liquid. In the present work we have succeeded in imaging by AFM of the external features of the most common iGluR, AMPA-R (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor), in a physiological environment.Homomeric GluR3 receptors were over-expressed in insect cells, purified and reconstituted into lipid membranes. AFM images were obtained in a buffer from membranes immobilized on a mica substrate.Using Au nanoparticle-conjugated antibodies, we show that proteins reconstitute predominantly with the N-terminal domain uppermost on the membrane. A tetrameric receptor structure is clearly observed, but it displays considerable heterogeneity, and the dimensions differ considerably from cryo-electron microscopy measurements.Our results indicate that the extracellular domains of AMPA-R are highly flexible in a physiological environment.AFM allows us to observe the protein surface structure, suggesting the possibility of visualizing real time conformational changes of a functioning protein. This knowledge may be useful for neuroscience as well as in pharmaceutical applications.
Keywords: AMPA receptor; Atomic force microscopy; Membrane protein; Neuroreceptor;

Hatchlings of the painted turtle, Chrysemys picta marginata can endure long-term freezing of their extracellular body fluids. We hypothesized that freezing survival would include adaptive up-regulation of antioxidant defenses to deal with ischemia–reperfusion injuries associated with the freeze–thaw cycle. A number of antioxidant enzymes are under the control of the NF-E2 related factor 2 (Nrf2) transcription factor including members of the glutathione S-transferase (GST) and aldo–keto reductase (AKR) families.RT–PCR and Western immunoblotting were used to measure changes in transcript and protein levels in response to 5-h freezing exposure of hatchlings.Transcript levels of Nrf2 increased in turtle brain, liver, and muscle by 1.5- to 2-fold, and protein levels increased in the brain and muscle by 1.6- to 2.3-fold in response to freezing. GSTs responded strongly to freezing in turtle brain with amounts of GSTP1, M1, K1, and A3 elevated by 1.5- to 2.4-fold. GSTM3 and T1 rose by 1.8- to 2.3-fold in gut, whereas reduced levels of GSTP1, M1, M3, and K1 were found in livers of frozen animals. Levels of the AKR1B4 isozyme rose 2.1-fold during freezing in brain.Freezing triggered tissue-specific changes in the antioxidant defenses in C. picta marginata organs.These data indicate that activation of an antioxidant response is an important aspect of natural freeze tolerance in turtles.
Keywords: Chrysemys picta marginata; Midland painted turtle; Freeze tolerance; Ischemia–reperfusion; Reactive oxygen species; Oxidative stress; Nrf2; Glutathione S-transferase; Aldo–keto reductase;

Agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells by Masahiro Endo; Hidehiko Beppu; Hidehiko Akiyama; Kazumasa Wakamatsu; Shosuke Ito; Yasuko Kawamoto; Kan Shimpo; Toshimitu Sumiya; Takaaki Koike; Taei Matsui (669-673).
Mushrooms of the genus Agaricus are a common folk remedy against carcinoma. The active ingredients, polysaccharides and protein-polysaccharide complexes containing β-glucan, have been isolated and shown to have indirect tumor-suppressing activity via an immunological activation.The diffusible fraction of a hot-water extract of Agaricus blazei Murrill (ABM) powder was fractionated by HPLC based on the anti-tumor activity against leukemic cells in vitro. The structure of the anti-tumor substance was determined by NMR and MS analyses.We purified a tumorcidal substance from the diffusible fraction of ABM and identified it as agaritine, β-N-(γ-l(+)-glutamyl)-4-(hydroxymethyl) phenylhydrazine, having a molecular mass of 267 Da. This compound inhibited the proliferation of leukemic cell lines such as U937, MOLT4, HL60 and K562 with IC50 values of 2.7, 9.4, 13.0, and 16.0 μg/mL, respectively, but showed no significant effect on normal lymphatic cells at concentrations up to 40 μg/mL. Although agaritine has been suspected of having genotoxic or carcinogenic properties, agaritine did not activate the umu gene of Salmonella, which reacts to carcinogens.The results indicate that agaritine from ABM has direct anti-tumor activity against leukemic tumor cells in vitro. This is in contrast to the carcinogenic activity previously ascribed to this compound. Our results also show that this activity is distinct from that of β-glucan, which indirectly suppresses proliferation of tumor cells.
Keywords: Agaricus blazei; Agaritine; Anti-tumor activity; Leukemic cell; Non-carcinogenicity;

Xyloglucan xyloglucosyl transferases (EC, known as xyloglucan endotransglycosylases (XETs) use a disproportionation reaction mechanism and modulate molecular masses of xyloglucans. However, it is not known precisely how these size modulations and transfer reactions occur with polymeric acceptor substrates.cDNAs encoding three barley HvXETs were expressed in Pichia pastoris and reaction mechanism and molecular properties of HvXETs were investigated.Significant differences in catalytic efficiencies (k cat·K m 1) were observed and these values were 0.01, 0.02 and 0.2 s 1·mg 1·ml for HvXET3, HvXET4 and HvXET6, respectively, using tamarind xyloglucan as a donor substrate. HPLC analyses of the reaction mixtures showed that HvXET6 followed a stochastic reaction mechanism with fluorescently or radioactively labelled tamarind xyloglucans and xyloglucan-derived oligosaccharides. The analyses from two successive reaction cycles revealed that HvXET6 could increase or decrease molecular masses of xyloglucans. In the first reaction cycle equilibrium was reached under limiting donor substrate concentrations, while xyloglucan mass modulations occurred during the second reaction cycle and depended on the molecular masses of incoming acceptors. Deglycosylation experiments indicated that occupancy of a singular N-glycosylation site was required for activity of HvXET6. Experiments with organic solvents demonstrated that HvXET6 tolerated DMSO, glycerol, methanol and 1,4-butanediol in 20% (v/v) concentrations.The two-phase experiments demonstrated that large xyloglucan molecules can bind in the acceptor sites of HvXETs.The results characterise donor and acceptor binding sites in plant XET, report that HvXETs act on xyloglucan donor substrates adsorbed onto nanocrystals and that HvXETs tolerate the presence of organic solvents.
Keywords: Catalysis; Cell wall; Family GH16 glycoside hydrolases; Organic solvents; Transglycosylation; Water content;