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Dehydrins are well-known components of plant responses to different stresses that cause dehydration, including drought, freezing, salinity, etc. In conifers, the dehydrin gene family is very large, implying that the members of this family have important physiological functions in conifer stress tolerance. However, dehydrin gene expression displays a wide range of responses to stress, from thousand-fold increased expression to decreased expression, and it is generally unknown how regulatory systems are connected at the mRNA and protein levels. Therefore, we studied these aspects of dehydrin regulation in Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) H. Karst) seedlings under polyethylene glycol 6000-induced osmotic stress ranging from relatively low (culture medium water potential of −0.15 MPa) to very high (−1.0 MPa) intensities. In pine, the major dehydrin protein was Dhn1 in both the roots and needles, and in spruce, two isoforms of the Dhn4 protein were the major dehydrins; both of these proteins are AESK-type dehydrins. The genes encoding these major proteins were highly expressed even under control conditions; surprisingly, we also observed several highly expressed dehydrin genes that were not abundantly translated. Under osmotic stress, the most prominent expression changes were observed for the dehydrin genes with low basal expression levels, whereas highly expressed genes generally demonstrated rather modest changes in expression. We report proposed constitutive physiological functions of the AESK-type dehydrins in Pinaceae plants. • In pine and spruce seedlings, AESK-type proteins are the major DHNs. • DHNs are more responsive to stress at the mRNA level than at the protein level. • AESK-type DHNs probably function constitutively in both pine and spruce seedlings. [ABSTRACT FROM AUTHOR]

Two polypyridyl ruthenium(II) complexes [Ru(DIP) 2 (BIP)](PF 6) 2 (DIP = 4,7-diphenyl-1,10-phenanthrolie, BIP = 2-(1,1′-biphenyl-4-yl)-1 H -imidazo[4,5-f][1,10]phenanthroline, Ru1) and [Ru(DIP) 2 (CBIP)](PF 6) 2 (CBIP = 2-(4′-chloro-1,1′-biphenyl-4-yl)-1 H -imidazo[4,5-f][1,10]phenanthroline, Ru2) were synthesized. The cytotoxic activities in vitro of Ru1, Ru2 toward B16, A549, HepG2, SGC-7901, HeLa, BEL-7402, non-cancer LO2 were investigated using MTT method (3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide). Unexpectedly, Ru1, Ru2 can't prevent these cancer cells proliferation. To improve the anti-cancer effect, we used liposomes to entrap the complexes Ru1, Ru2 to form Ru1lipo, Ru2lipo. As expectation, Ru1lipo and Ru2lipo exhibit high anti-cancer efficacy, especially, Ru1lipo (IC 50 3.4 ± 0.1 μM), Ru2lipo (IC 50 3.5 ± 0.1 μM) display strong ability to block the cell proliferation in SGC-7901. The cell colony, wound healing, and cell cycle distribution show that the complexes can validly inhibit the cell growth at G2/M phase. Apoptotic studied with Annex V/PI doubling method showed that Ru1lipo and Ru2lipo can effectively induce apoptosis. Reactive oxygen species (ROS), malondialdehyde, glutathione and GPX4 demonstrate that Ru1lipo and Ru2lipo improve ROS and malondialdehyde levels, inhibit generation of glutathione, and finally result in a ferroptosis. Ru1lipo and Ru2lipo interact on the lysosomes and mitochondria and damage mitochondrial dysfunction. Additionally, Ru1lipo and Ru2lipo increase intracellular Ca2+ concentration and induce autophagy. The RNA-sequence and molecular docking were performed, the expression of Bcl-2 family was investigated by Western blot analysis. Antitumor in vivo experiments confirm that 1.23 mg/kg, 2.46 mg/kg of Ru1lipo possesses a high inhibitory rate of 53.53% and 72.90% to prevent tumor growth, hematoxylin-eosin (H&E) results show that Ru1lipo doesn't cause chronic organ damage and strongly promotes the necrosis of solid tumor. Taken together, we conclude that Ru1lipo and Ru2lipo cause cell death through the following pathways: autophagy, ferroptosis, ROS-regulated mitochondrial dysfunction, and blocking the PI3K/AKT/mTOR. Two new Ru(II) polypyridyl complexes [Ru(dip) 2 (BIP)](PF 6) 2 (dip = 4,7-diphenyl-1,10-phenanthrolie, BIP = 2-(1,1′-biphenyl-4-yl)-1 H -imidazo[4,5-f] [1,10]phenanthroline, Ru1) and [Ru(dip) 2 (CBIP)](PF 6) 2 (CBIP = 2-(4′-chloro-1,1′-biphenyl-4-yl)-1 H -imidazo[4,5-f] [1,10]phenanthroline, Ru2) and complexes-loaded liposomes Ru1lipo and Ru2lipo were synthesized and characterized. The cytotoxicity of the complexes Ru1, Ru2, Ru1lipo and Ru2lipo against B16, BEL-7402, A549, HepG2, SGC-7901 cancer cells and non-cancer cell LO2 was evaluated using 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide (MTT) method. The high inhibitory rate in vivo of Ru1lipo inhibiting SGC-7901 tumor growth reaches 72.90%. [Display omitted] • Two new Ru(II) polypyridyl complexes were synthesized and characterized. • The cytotoxicity in vitro and in vivo of the complexes and liposomes was investigated. • Apoptosis, reactive oxygen species and cell cycle arrest were performed. • RNA-sequence assay was investigated. • The antitumor activity in vivo was investigated. [ABSTRACT FROM AUTHOR]

Three new iridium (III) complexes [Ir (ppy) 2 (ipbc)](PF 6 ) ( 1 ), [Ir (bzq) 2 (ipbc)](PF 6 ) ( 2 ) and [Ir (piq) 2 (ipbc)](PF 6 ) ( 3 ) were designed and synthesized. All the complexes were tested for anticancer activity using 3-(4,5-dimethylthiazole)-2,5-diphenyltetraazolium bromide (MTT) method. The complexes show no cytotoxic activity toward cancer BEL-7402, SGC-7901, Eca-109, A549, HeLa and HepG2 cells. However, upon irradiation with white light, the complexes display high cytotoxicity against BEL-7402 cells with an IC 50 value of 5.5 ± 0.8, 7.3 ± 1.3 and 11.5 ± 1.6 μM for 1 , 2 and 3 , respectively. AO/EB staining and comet assay show that the complexes can induce apoptosis in BEL-7402 cells. The complexes can increase intracellular ROS and Ca 2+ levels and cause a decrease in the mitochondrial membrane potential. Autophagic assays exhibit that the complexes can induce autophagy and regulate the expression of Beclin-1 and LC3 proteins. The cell cycle distribution in BEL-7402 cells was carried out by flow cytometry. The expression of Bcl-2 family proteins was studied by western blot. Additionally, the complexes can release cytochrome c and inhibit the polymerization of α-tubulin. Our study reveals that the complexes inhibit the cell growth in BEL-7402 cells through an ROS-mediated mitochondria dysfunction and targeting tubules pathways. These complexes are a promising new entity for the development of multi-target anticancer drugs. [ABSTRACT FROM AUTHOR]

As one of the major cell regulated center, mitochondria are closely associated with cell proliferation, apoptosis of tumor cell. In this work, four new ruthenium (II) polypyridyl complexes [Ru(bpy) 2 (FTTP)](ClO 4 ) 2 ( 1 ) (FTTP = 11-(3-fluoro-naphthalen-2-yloxy)-4,5,9,14-tetraaza-benzo[ b ]triphenylene, bpy = 2,2′-bipyridine), [Ru(phen) 2 (FTTP)](ClO 4 ) 2 ( 2 ) (phen = 1,10-phenanthroline), [Ru(bpy) 2 (PTTP)](ClO 4 ) 2 ( 3 ) (PTTP = 2-phenoxy-1,4,8,9-tetraazatriphenylene) and [Ru(phen) 2 (PTTP)](ClO 4 ) 2 ( 4 ) were synthesized and characterized by elemental analysis, ESI-MS, 1 H NMR and 13 C NMR. The cytotoxic activity, ability of inhibiting cell invasion, cell cycle arrest and apoptosis-inducing mechanism of these Ru(II) complexes have been investigated in detail by MTT (3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide) method, invasion assay, comet assay as well as western blotting techniques. Notably, complexes 1 – 4 displayed high cytotoxic activity against liver carcinoma HepG2 cells and the IC 50 values of complexes 1 – 4 against HepG2 cells are 10.4 ± 1.2, 9.3 ± 0.6, 29.1 ± 1.5 and 5.6 ± 1.2 μM, respectively. The comet assay showed that the complexes can induce DNA damage. The acridine orange (AO) and ethidium bromide (EB) staining method indicated that the complexes can cause apoptosis in HepG2 cells. Further studies showed that complexes 1 – 4 caused cell cycle arrest at G0/G1 phase and induced HepG2 cells apoptosis through a ROS-mediated mitochondrial dysfunction pathway, which involved an increase in the levels of reactive oxygen species (ROS), a decrease in the mitochondrial membrane potential, activation of caspases and Bcl-2 family proteins. [ABSTRACT FROM AUTHOR]

Ligand HMSPIP (2-(4-(methylsulfonyl)phenyl)-1 H -imidazo[4,5-f][1,10]phenanthroline) and its iridium(III) complexes [Ir(ppy) 2 (HMSPIP)]PF 6 (ppy = 2-phenylpyridine, Ir1) and [Ir(bzq) 2 (HMSPIP)]PF 6 (bzq = benzo[ h ]quinoline, Ir2) were synthesized. The complexes were characterized by 1H NMR, 13C NMR, and UV/Vis spectra. The cytotoxicity of the complexes toward cancer cells were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, the scratch wound healing and colony-forming were also investigated. MTT assay certificated that the complexes show high toxic effect on the HeLa cells. The cell cycle assay illustrated that the complexes blocked cell growth at G 0 /G 1 phase in HeLa cells. A series of subsequent experiments showed that the complexes first enter the endoplasmic reticulum (ER) and then enter the mitochondria, leading to an increase in intracellular Ca2+ and reactive oxygen species (ROS) content, depolarizing mitochondrial membrane potential (MMP), and ultimately resulting in apoptosis. In addition, the experimental results revealed that the complexes not only increase the level of ROS but also inhibit the production of GSH and eventually produce large amounts of MDA and further leading to cell death. Taken together, we consider that the complexes can be used as potential candidate drugs for HeLa cancer treatment. [Display omitted] • Two new iridium(III) complex was synthesized and characterized. • The cytotoxicity in vitro of the complexes was studied. • Apoptosis, reactive oxygen species and cell cycle arrest were carried out. • The location and mitochondrial membrane potential were investigated. • The expression of B-cell lymphoma-2 family proteins was examined. [ABSTRACT FROM AUTHOR]

Four new ruthenium(II) polypyridyl complexes [Ru(N–N) 2 (dhbn)](ClO 4 ) 2 (N–N = dmb: 4,4′-dimethyl-2,2′-bipyridine 1 ; bpy = 2,2′-bipyridine 2 ; phen = 1,10-phenanthroline 3 ; dmp = 2,9-dimethyl-1,10-phenanthroline 4 ) were synthesized and characterized. The cytotoxicity in vitro of the ligand and complexes toward HepG-2, HeLa, MG-63 and A549 were assayed by MTT method. The IC 50 values of the complexes against the above cells range from 17.7 ± 1.1 to 45.1 ± 2.8 μM. The cytotoxic activity of the complexes against HepG-2 cells follows the order of 4 > 2 > 3 > 1 . Ligand shows no cytotoxic activity against the selected cell lines. Cellular uptake, apoptosis, comet assay, reactive oxygen species, mitochondrial membrane potential, cell cycle arrest, and the expression of proteins involved in apoptosis pathway induced by the complexes were investigated. The results indicate that complexes 1–4 induce apoptosis in HepG-2 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway. [ABSTRACT FROM AUTHOR]

In this work, four phenolic compounds, N 1 ,N 5 -di-[(E)- p -coumaroyl]-spermidine ( 1 ), p -coumaric acid ( 2 ), ferulic acid ( 3 ), and rutin ( 4 ) were isolated from n-butanol fraction of acetone extract of defatted adlay ( Coix lachryma-jobi L . var. ma-yuen Stapf) seed meal. Compound 1 was identified from adlay for the first time. The structures of these compounds were characterised by ESI-MS, 1 H NMR, 13 C NMR and UV–vis techniques. These compounds were evaluated for their anti-tumour activities against HepG-2, MCF-7 and CaCo-2 cancer cell lines by MTT assay. Of all purified compounds, compound 1 possessed the highest anti-tumour activity than other compounds with IC 50 values between 46.34 ± 3.99 and 92.69 ± 5.63 µg/ml. Furthermore, activation of the caspase cascade in HepG-2 cells for intrinsic pathways caused by compound 1 were observed using western blot analysis. The result also showed that the expression of caspase-3, caspase-9, cytochrome c and Bax protein increased and the protein level of Bcl-2 decreased. [ABSTRACT FROM AUTHOR]

A new ligand TCPI and its three ruthenium(II) polypyridyl complexes [Ru(N-N) 2 (TCPI)](PF 6 ) 2 (N-N = bpy: 2,2′-bipyridine 1 ; phen: phenanthroline 2 ; dmp: 2,9-dimethyl-1,10-phenanthroline 3 ) were synthesized and characterized by elemental analysis, ESI-MS, 1 H NMR, IR, absorption and emission spectra. The cytotoxic activity in vitro of the ligand and complexes against cancer cells SGC-7901, PC-12, HepG-2, SiHa, Eca-109, HeLa and normal cell LO2 was evaluated by MTT method. Complex 3 shows the highest cytotoxic activity toward SGC-7901 cell among the complexes. Interestingly, the complexes show low or no cytotoxic activity against normal cell LO2. The apoptosis in SGC-7901 cell was investigated with AO/EB staining method. The ROS levels and the changes of mitochondrial membrane potential were studied under fluorescent microscope and flow cytometry. The cell invasion, cell cycle arrest and the expression of Bcl-2 family proteins were studied in detail. The results demonstrate that the complexes induce apoptosis in SGC-7901 cell through a ROS-mediated mitochondrial dysfunction pathway, which was accompanied by the regulation of Bcl-2 family proteins. [ABSTRACT FROM AUTHOR]

Four new ruthenium(II) polypyridyl complexes [Ru( N - N ) 2 (bddp)](ClO 4 ) 2 1–4 (N-N = dmb: 4,4′-dimethyl-2,2′-bipyridine 1 , bpy: 2,2′-bipyridine 2 , phen: 1,10-phenanthroline 3 and dmp: 2,9-dimethyl-1,10-phenanthroline 4 , bddp = benzilo[2,3- b ]-1,4-diazabenzo[ i ]dipyrido[3,2- a :2′,3′- c ]phenazine) were synthesized and characterized by elemental analysis, ESI-MS and 1 H NMR. The cytotoxicity in vitro of the complexes against BEL-7402, HeLa, MG-63 and A549 cell lines was investigated by MTT method. The complexes show high cytotoxic activity toward the selected cell lines with an IC 50 value ranging from 5.3 ± 0.6 to 15.7 ± 3.6 μM. The apoptosis was studied with acridine orange (AO)/ethdium bromide (EB) and Hoechst 33258 staining methods. The cellular uptake was investigated with DAPI staining method. The reactive oxygen species (ROS) and mitochondrial membrane potential were performed under fluorescent microscope and flow cytometry. The complexes can induce an increase in the ROS levels and a decrease in the mitochondrial membrane potential. The comet assay was studied with fluorescent microscope. The percentage in apoptotic and necrotic cells and cell cycle arrest were assayed by flow cytometry. The effects of the complexes on the expression of caspases and Bcl-2 family proteins were studied by western blot analysis. The results show that the complexes induce apoptosis in A549 cells through an ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulating the expression of Bcl-2 family proteins. [ABSTRACT FROM AUTHOR]

In our previous studies, benzimidazole–oxindole conjugates were synthesized and evaluated by National Cancer Institute (NCI) for their cytotoxic activity and the new molecules like 5c and 5p were considered as potential leads. These conjugates arrested the cell cycle at G2/M phase and inhibited tubulin polymerization. These observations prompted us to investigate the apoptotic mechanism induced by these lead molecules against human breast cancer cells (MCF-7). Studies like measurement of mitochondrial membrane potential (Δ Ψ m ), generation of reactive oxygen species (ROS) and Annexin V-FITC assay revealed that these compounds induced mitochondrial mediated (intrinsic apoptotic pathway) apoptosis in human breast cancer cells. It was further confirmed by western blot analysis of pro apoptotic protein Bax, anti apoptotic protein Bcl-2, cytochrome c release, caspase-9 activity and cleavage of PARP. [ABSTRACT FROM AUTHOR]

Four new ruthenium(II) polypyridyl complexes [Ru(dmb) 2 (dqtbt)](ClO 4 ) 2 ( 1 ) (dqtbt = 12-(2,3-diphenyl-quinoxalin-6-yl)-4,5,10,13-tetraazabenzo[ b ]triphenylene, dmb = 4,4′-dimethyl-2,2′-bipyridine), [Ru(bpy) 2 (dqtbt)](ClO 4 ) 2 ( 2 ) (bpy = 2,2′-bipyridine), [Ru(phen) 2 (dqtbt)](ClO 4 ) 2 ( 3 ) (phen = 1,10-phenanthroline) and [Ru(dmp) 2 (dqtbt)](ClO 4 ) 2 ( 4 ) (dmp = 2,9-dimethyl-1,10-phenanthroline) were synthesized and characterized. The cytotoxicity in vitro of the complexes was evaluated against human BEL-7402, A549, HeLa, HepG-2 and MG-63 cancer cell lines. These complexes are sensitive to BEL-7402 cells, the IC 50 values are 4.9 ± 0.5, 4.6 ± 0.4, 7.7 ± 1.8 and 1.9 ± 0.3 μM toward BEL-7402 cells. The complexes can increase the levels of reactive oxygen species and induce the decrease of mitochondrial membrane potential. Morphological and comet assay studies show that the complexes can effectively induce apoptosis in BEL-7402 cells. Complexes 1 – 4 inhibit the cell growth at G0/G1 phase in BEL-7402 cell line. The complexes can downregulate the expression of Bcl-2 and Bcl-x proteins and upregulate the levels of Bid protein in BEL-7402 cells. The results show that the complexes induce BEL-7402 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway. In addition, the complexes show strong protein-binding affinities. [ABSTRACT FROM AUTHOR]

Four new ruthenium(II) polypyridyl complexes [Ru(dmb) 2 (DQTT)](ClO 4 ) 2 ( 1 ) (DQTT = 12-(1,4-dihydroquinoxalin-6-yl)-4,5,9,14-tetraazabenzo[b]triphenylene, dmb = 4,4′-dimethyl-2,2′-bipyridine), [Ru(bpy) 2 (DQTT)](ClO 4 ) 2 ( 2 ) (bpy = 2,2′-bipyridine), [Ru(phen) 2 (DQTT)](ClO 4 ) 2 ( 3 ) (phen = 1,10-phenanthroline) and [Ru(dmp) 2 (DQTT)](ClO 4 ) 2 ( 4 ) (dmp = 2,9-dimethyl-1,10-phenanthroline) were synthesized and characterized by elemental analysis, ESI-MS, 1 H NMR and 13 C NMR. The cytotoxic activity in vitro of the complexes was evaluated against human BEL-7402, A549, HeLa, HepG-2 and MG-63 cancer cell lines by MTT (3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide) method. The IC 50 values of complexes 1 – 4 against BEL-7402 cells are 31.8 ± 1.0, 35.8 ± 1.6, 29.0 ± 0.8 and 25.0 ± 0.9 μM, respectively. The morphological apoptosis was investigated with AO/EB (acridine orange/ethidium bromide) and Hoechst 33258 staining methods. The DNA damage was assayed by comet assay. The inhibition of cell migration was evaluated by the wound healing assay. The levels of ROS (reactive oxygen species) and the changes of mitochondrial membrane potential were studied under fluorescent microscope. The percentages in the cells of apoptotic and necrotic cells and the cell cycle arrest were determined by flow cytometry. The expression of Bcl-2 family proteins was investigated by western blot analysis. The results show that the complexes induce BEL-7402 cells apoptosis through a ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulation of the expression of Bcl-2 family proteins. [ABSTRACT FROM AUTHOR]

A new ligand Adbdp and its four ruthenium(II) polypyridyl complexes [Ru(N-N) 2 (Adbdp)](ClO 4 ) 2 [N-N = dmb: 4,4′-dimethyl-2,2′-bipyridine 1 , bpy: 2,2′-bipyridine 2 , phen: 1,10-phenanthroline 3 and dmp: 2,9-dimethyl-1,10-phenanthroline 4 , Adbdp = acenaphtheno[2,3-b]-1,4-diazabenzo[i]dipyrido[3,2- a :2′,3′- c ]phenazine] were synthesized and characterized by elemental analysis, ESI-MS and 1 H NMR. The cytotoxicity in vitro of the complexes against BEL-7402, HeLa, MG-63 and A549 cells was studied by MTT method. The IC 50 values are 24.2 ± 1.9, 11.5 ± 3.6, 3.4 ± 0.3 and 9.7 ± 0.5 μM for complexes 1 , 2 , 3 and 4 toward A549 cells, respectively. The apoptosis was assayed with AO/EB and Hoechst 33258 staining methods. The cellular uptake was investigated with DAPI-stained cell nuclei. The reactive oxygen species and mitochondrial membrane potential were performed under fluorescent microscope. The cell cycle distribution was studied by flow cytometry and the protein expression of Bcl-2 family proteins was analyzed by western blot. The results show that the complexes induce A549 cell apoptosis through a ROS-mediated mitochondrial dysfunction pathway. [ABSTRACT FROM AUTHOR]

A Ru(II) polypyridyl complex [Ru(bpy) 2 (HMSPIP)](ClO 4 ) 2 ( 1 ) (bpy = 2,2′-bipyridine, HMSPIP = 2-(4-methylsulfonyl)phenyl-1H-imidazo[4,5- f ][1,10] phenanthroline) was synthesized. The IC 50 value of the complex against human hepatocellular cell BEL-7402 is 21.6 ± 2.7 μM. The complex shows no cytotoxic activity toward human lung adenocarcinoma cell A549, human osteosarcoma cell MG-63 and human breast cancer cell SK-BR-3 cells. It is easily for complex 1 to be taken up by BEL-7402 cells. The complex can enhance the reactive oxygen species (ROS) levels and induce the decrease in the mitochondrial membrane potential. The complex inhibits the cell growth in BEL-7402 cells at G2/M phase. Complex 1 can regulate the expression of Bcl-2 family proteins. The results show that the complex induces apoptosis of BEL-7402 cells through a ROS-mediated mitochondrial dysfunction pathway. [ABSTRACT FROM AUTHOR]

A new ruthenium(II) polypyridyl complex [Ru(dmp) 2 (pddppn)](ClO 4 ) 2 Ru1 was synthesized and characterized. The cytotoxic activity in vitro of the complex was evaluated by MTT method. Ru1 shows high effect on the inhibition of the cell growth against BEL-7402, HeLa, MG-63 and A549 cells with low IC 50 values of 1.6 ± 0.4, 9.0 ± 0.8, 1.5 ± 0.2 and 1.5 ± 0.3 μM, respectively. The cellular uptake indicates that Ru1 can enter into the cytoplasm and accumulate in the cell nuclei. Ru1 can induce apoptosis in A549 cells and enhance the levels of reactive oxygen species (ROS) and induce the decrease of mitochondrial membrane potential. In addition, Ru1 can down-regulate the levels of Bcl-2, Bcl-x, Bak, and Bim expression and up-regulate the expression of Bag-1 and Bad. The complex induces apoptosis of A549 cells through an intrinsic ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulating the expression of caspases and Bcl-2 family proteins. [ABSTRACT FROM AUTHOR]

A series of twenty seven oxa/azaspiro[4,5]trienone derivatives were synthesized and their anticancer properties have been explored. GI 50 values of all these compounds were evaluated against four types of human cancer cell lines, i.e. MCF-7 (breast), DU-145 (prostate), A549 (lung) and HepG2 (liver). Five compounds of the series exhibited good anticancer potential against MCF-7 with GI 50 values less than 2 μM. Detailed biological studies of the two representative compounds 9b and 9e revealed that they arrest cell cycle in G0/G1 phase and induce mitochondria mediated apoptosis, that was further confirmed by measurement of mitochondrial membrane potential (ΔΨm), intracellular ROS generation, caspase 9 activity and Annexin V-FITC assay. Furthermore, western blot analysis suggested that these compounds up-regulate the levels of p53, p21, p27 and Bax, and down-regulate the level of Bcl-2 confirming the apoptosis inducing properties. [ABSTRACT FROM AUTHOR]

A new ligand dmdppz and its four ruthenium(II) polypyridyl complexes [Ru(dmb) 2 (dmdppz)](ClO 4 ) 2 ( 1 ), [Ru(bpy) 2 (dmdppz)](ClO 4 ) 2 ( 2 ), [Ru(phen) 2 (dmdppz)](ClO 4 ) 2 ( 3 ) and [Ru(dmp) 2 (dmdppz)](ClO 4 ) 2 ( 4 ) (where dmb, bpy, phen, dmp and dmdppz stand for 4,4′-dimethyl-2,2′-bipyridine, 2,2′-bipyridine, 1,10-phenanthroline, 2,9-dimethyl-1,10-phenanthroline and 5,8-dimethoxylpyrido[3,2- a :2′,3′- c ]phenazine, respectively) have been synthesized and characterized. Their DNA binding behaviors show that the complexes bind to calf thymus DNA by intercalation. The complexes exhibit efficient photocleavage of pBR322 DNA on irradiation. The cytotoxicity of the ligand and the complexes toward HepG-2, HeLa, MG-63, A549 and BEL-7402 were assayed by MTT ((3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide)) method. The IC 50 values of the complexes 1 , 2 , 3 and 4 toward BEL-7402 cells are 14.6, 16.8, 18.0 and 16.7 μM, respectively. Dmdppz shows no cytotoxic activity against selected cell lines. The cellular uptake, apoptosis, comet assay, reactive oxygen species (ROS), mitochondrial membrane potential and western blot analysis were investigated. These results indicate that complexes 1 – 4 exert their toxicity through the intrinsic ROS-mediated mitochondrial pathway, which is accompanied by the regulation of Bcl-2 family proteins. [ABSTRACT FROM AUTHOR]

The aim of our study was to investigate DNA-binding and cytotoxic activity of the four new Ru(II) polypyridyl complexes [Ru(dmb) 2 (HMHPIP)](ClO 4 ) 2 ( 1 ), [Ru(bpy) 2 (HMHPIP)](ClO 4 ) 2 ( 2 ), [Ru(phen) 2 (HMHPIP)](ClO 4 ) 2 ( 3 ) and [Ru(dmp) 2 (HMHPIP)](ClO 4 ) 2 ( 4 ). The complexes interact with DNA through intercalative mode and show relatively high cytotoxic activity against A549 cells, no cytotoxicity toward MG-63 cells. Complexes 1 – 4 can enhance the levels of ROS in A549 cells and induce the decrease of the mitochondrial membrane potential. These complexes inhibit the cell growth in A549 cells at G0/G1 or S phase. Complex 3 activated caspase 7, and down-regulated the expression of the anti-apoptotic protein Bcl-2. Complexes 1 – 4 induce apoptosis in A549 cells through ROS-mediated mitochondrial dysfunction pathway. [ABSTRACT FROM AUTHOR]

Abstract: The major epitope region of the glycoprotein B (gB) gene of the porcine cytomegalovirus (PCMV), with a length of 270bp, was cloned and expressed in Escherichia coli Rosetta (DE3). The major gB epitope was detected using an agar gel precipitation and Western blot analysis with the polyclonal antibodies specific for the major epitope. An indirect-blocking enzyme-linked immunosorbent assay (ELISA) was developed using the expressed major gB epitope as a coating antigen for the detection of PCMV antibodies. The results of the tests show that the indirect-blocking ELISA has 98% specificity and 97.8% sensitivity. No cross-reactions were observed between the major gB epitope and the antibodies against other virus, which indicates that the gB epitope is specific for PCMV antibodies. The indirect-blocking ELISA is a highly specific, sensitive method for detecting anti-PCMV gB antibodies. [Copyright &y& Elsevier]

Abstract: Although the risk factor for harboring the apolipoprotein E4 (apoE4) allele in late-onset Alzheimer''s disease (AD) is well known, the mechanism by which apoE4 contributes to AD pathogenesis has yet to be clarified. Preferential cleavage of the ApoE4 isoform relative to other polymorphic forms appears to be significant, as the resulting fragments are associated with hallmarks of AD. To examine the possible role of apoE4 proteolysis in AD, we designed a site-directed antibody directed at position D172, which would yield a predicted amino-terminal fragment previously identified in AD brain extracts. Western blot analysis utilizing this novel antibody, termed the amino-terminal apoE4 cleavage fragment (nApoE4CF) Ab, consistently identified the predicted amino-terminal fragment (∼18kDa) in several commercially available forms of human recombinant apoE4 purified from E. coli. Mass spectrometry confirmed the identity of this 18kDa fragment as being an amino-terminal fragment of apoE4. Immunohistochemical experiments indicated the nApoE4CF Ab specifically labeled neurofibrillary tangles (NFTs) in AD frontal cortex sections that colocalized with the mature tangle marker PHF-1. Taken together, these results suggest a novel cleavage event of apoE4, generating an amino-terminal fragment that localizes within NFTs of the AD brain. [Copyright &y& Elsevier]

Abstract: Duck circovirus (DuCV) is classified in the genus Circovirus of the Circoviridae family. Two major open reading frames (ORFs), encoding the replicase (ORF1/rep) and the capsid protein (ORF2/cap), have been recognized for DuCV. Sequence analysis show that another major conserved ORF (named ORF3) is located in the complementary strand of ORF1/rep of DuCV, and its function remains to be investigated. In this study, the ORF3 of DuCV was expressed in recombinant baculovirus-infected Sf9 cells. By IFA and Western blot analysis, the ORF3 protein was positive for the sera from ducks infected with DuCV. The percentages of apoptotic cells of the Sf9 cells infected with the recombinant baculovirus encoding ORF3 of DuCV were significantly higher than (P <0.05) that of the Sf9 cells infected with wild-type baculovirus at 24, 48 and 72h postinfection. Based on our knowledge, we deduced that the ORF3 protein of DuCV might play an important role in viral pathogenesis via its apoptotic activity. [Copyright &y& Elsevier]

Abstract: The natural p300-specific histone acetyltransferase (HAT) inhibitor, curcumin (CUR), has been widely investigated for its potential therapeutic effect as an anticancer and anti-inflammatory agent. Notwithstanding this interesting pharmacological profile, CUR shows some drawbacks, such as poor absorption and a very fast metabolism and elimination, that limit its clinical use. Aim of the present study was to formulate CUR loaded nanostructured lipid carriers (NLC-CUR) in order to improve the bioavailability and stability of this compound after systemic administration with increased effects in the central nervous system (CNS). NLC-CUR were prepared and characterized on their physicochemical properties by PCS and DSC analyses. Thus, NLC-CUR were systemically injected and the effects in the CNS were compared with a CUR control formulation containing 0.05% DMSO (DMSO-CUR). Our results demonstrate that CUR is able to decrease histone acetylation in the CNS when included in NLCs. Western blot analysis shows that intraperitoneal injection of NLC-CUR (100mg/kg) in mice induces a marked hypoacetylation of histone 4 (H4) at lysine 12 (K12) in the spinal cord compared with control group. Notably, DMSO-CUR (100mg/kg) did not change the H4K12 acetylation level in the CNS. Our study suggests a novel approach to ameliorate the pharmacokinetics of CUR that allows a better permeation in the CNS. [Copyright &y& Elsevier]

Abstract: The outbreak of severe acute respiratory syndrome (SARS) in 2002 affected thousands of people and an efficient diagnostic system is needed for accurate detection of SARS coronavirus (SARS CoV) to prevent or limit future outbreaks. Of the several SARS CoV structural proteins, the nucleocapsid protein has been shown to be a good diagnostic marker. In this study, an ssDNA aptamer that specifically binds to SARS CoV nucleocapsid protein was isolated from a DNA library containing 45-nuceotide random sequences in the middle of an 88mer single-stranded DNA. After twelve cycles of systematic evolution of ligands by exponential enrichment (SELEX) procedure, 15 ssDNA aptamers were identified. Enzyme-linked immunosorbent assay (ELISA) analysis was then used to identify the aptamer with the highest binding affinity to the SARS CoV nucleocapsid protein. Using this approach, an ssDNA aptamer that binds to the nucleocapsid protein with a K d of 4.93±0.30nM was identified. Western blot analysis further demonstrated that this ssDNA aptamer could be used to efficiently detect the SARS CoV nucleocapsid protein when compared with a nucleocapsid antibody. Therefore, we believe that the selected ssDNA aptamer may be a good alternative detection probe for the rapid and sensitive detection of SARS. [Copyright &y& Elsevier]

Abstract: Stratifin (14-3-3σ or SFN) is a member of the 14-3-3 family of proteins which play critical roles in different cellular signaling processes. Stratifin as a potential tumor suppressor gene plays an important role in carcinogenesis and metastasis. The aim of this study was to investigate the expression of Stratifin in human gliomas and to analyze its expression profile with respect to tumor development. The expression pattern of Stratifin was analyzed by immunohistochemistry and/or Western blotting in tumor samples from 186 patients with different grades of gliomas. Prognostic significance was assessed using Kaplan–Meier survival estimates and Cox regression analyses. The expression pattern of Stratifin was correlated with the pathological and clinical characteristics of the patients with gliomas. Western blot analysis indicated that the average optical densitometry (OD) ratio of Stratifin in high-grade gliomas (World Health Organization [WHO] grade III/IV) was lower than in low-grade tumors (WHO grade I/II, p =0.026). In addition, statistical analysis showed that patients expressing a high level of Stratifin have favorable overall survival rates relative to those expressing a low level of this protein. Cox multi-factor analysis showed that Stratifin (p =0.02) was an independent prognosis factor for human gliomas. Our results provide convincing evidence that the expression of Stratifin is down-regulated in human gliomas. Its expression level is correlated with the clinicopathological parameters and prognosis in patients with gliomas. Pending validation targeting, Stratifin might also be a novel opportunity to improve the therapy of this tumor. [Copyright &y& Elsevier]

Abstract: Puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP) is a glycoprotein (200 kDa as a dimer) that we previously isolated from the plasma of Fugu pardalis (). For the study on functions of PSTBP, here we examined distribution of homologous proteins to PSTBP in the plasma of seven species of puffer fish, and among the tissues of F. pardalis by Western blot analysis probed with a polyclonal IgG against unglycosylated PSTBP1 expressed in Echelichia coli. One or two major positive broad bands were detected at 105–140 kDa molecular weight range in the plasma (0.5 μg protein) of all species of puffer fish tested, while no band was detected in the plasma (5 μg protein) of fish other than puffer fish. Glycopeptidase F treated plasma of all species of puffer fish tested commonly showed the bands at approximately 42 kDa that was consistent to the molecular weight of unglycosylated PSTBP. These data suggest that puffer fish commonly possess glycoproteins homologous to PSTBP, but the sizes of N-glycan are specific to the species. Among soluble protein extracts (5 μg protein) from the tissues of F. pardalis, PSTBP was detected in all tissues examined, most prominently in heart, skin, and gall. [Copyright &y& Elsevier]

Abstract: Sprouting of uninjured nociceptive axons was examined in young adult, middle aged and aged rats. Axon sprouting from the spared sural nerve, both into adjacent denervated skin and into end-to-side coapted nerve graft, was significantly higher in young rats than in aged rats. Cross-transplantations of the end-to-side coapted nerve grafts between young and aged rats demonstrated that axon sprouting from young recipient nerves into aged donor nerve grafts was significantly deteriorated, whereas the axon sprouting from aged recipient nerves into young donor nerve grafts was not statistically significantly affected. The levels of laminin polypeptides in peripheral nerves were 50–100% higher in young adult than in aged rats. However, the levels of peripherin, NGF isoforms and TrkA in skin, peripheral nerves and DRG, respectively, were not significantly reduced in aged rats. Therefore, impaired sprouting of nociceptive axons in aged rats is due rather to the alterations in peripheral neural pathways, than to the limited sprouting capacity of aged sensory neurons. Decreased levels of extracellular matrix components might be important in this respect. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate the impact of gender difference in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated animal model of Parkinson''s disease (PD). In the present study, we investigated the time-dependent alterations of dopamine and its metabolites, striatal tyrosine hydroxylase (TH) protein, dopamine transporter (DAT) protein, glial fibrillary acidic protein (GFAP) protein and midbrain TH protein and motor function in male and female mice 5h and 1, 3 and 7 days after four administrations of MPTP (20mg/kg) at 2-h intervals. The present study showed that the decrease of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and HVA (homovanillic acid) content in female mice was more pronounced than that in male animals 1, 3 and 7 days after MPTP treatment. Our Western blot analysis study also demonstrated that the decrease of both striatal and midbrain TH protein levels in female mice was more pronounced than that in male animals from 1 to 7 days after MPTP treatment. As compared to male mice, in contrast, the increase of striatal GFAP protein levels in female mice was observed from 5h to 7 days after MPTP treatment. Furthermore, the present study showed that motor deficits were found in both male and female mice 1 and 7 days after MPTP treatment. In the present study, moreover, the decrease of striatal DAT protein levels in female mice was more pronounced than that in male animals 1, 3 and 7 days after MPTP treatment. These results demonstrate that our administrations of MPTP at 2-h intervals can cause more severe damage in female mice as compared with male animals. The gender difference may be due to the decrease of DAT expression caused by MPTP. Thus our findings provide further valuable information for the pathogenesis of PD. [Copyright &y& Elsevier]

Abstract: Nematode parasites of the genus Trichinella are intracellular and distinct life cycle stages invade intestinal epithelial and skeletal muscle cells. Within the genus, Trichinella spiralis and Trichinella pseudospiralis exhibit species-specific differences with respect to host-parasite complex formation and host immune modulation. Parasite excretory-secretory (ES) proteins play important roles at the host-parasite interface and are thought to underpin these differences in biology. Serine proteases are among the most abundant group of T. spiralis ES proteins and multiple isoforms of the muscle larvae-specific TspSP-1 serine protease have been identified. Recently, a similar protein (TppSP-1) in T. pseudospiralis muscle larvae was identified. Here we report the cloning and characterisation of the full-length transcript of TppSP-1 and present comparative data between TspSP-1 and TppSP-1. [Copyright &y& Elsevier]

Abstract: The localization of dopamine stores and the expression and localization of vesicular monoamine transporter (VMAT) type-1 and 2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat thymus and spleen by immunohistochemical, immunochemical techniques and by RT-PCR. In the thymus dopamine immunoreactivity was developed in the cortico-medullary junction and in the medulla, but not in the thymic cortex. In the spleen, dopamine stores were found in reticular structures in the white pulp border and in the white pulp, but not in the red one. Both thymus and spleen expressed VMAT-1 and VMAT-2 immunoreactivity as well as dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity. Immunohistochemistry revealed VMAT-1, VMAT-2 and dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity primarily in the thymic cortical-medulla transitional zone and to a lesser extent in the medulla but not in the cortex. In the spleen, VMAT-1, VMAT-2 and dopamine D1, D2, D3, D4 and D5 receptor immunoreactivity was located primarily in the white pulp border and to a lesser extent in the white pulp. These findings indicate that both thymus and spleen express a dopaminergic system characterized by the presence of dopamine, vesicular monoamine transporters and the five subtypes of dopamine receptors. The presence of these dopaminergic markers suggests that dopamine likely originating from immune cells and/or from sympathetic neuroeffector plexus is released in the lymphoid microenvironment. Based on the microanatomical localization of dopaminergic markers investigated, a role of dopamine in maturation and selection of lymphocytes and activation of immune responses is suggested. [Copyright &y& Elsevier]

Abstract: Emerging evidence shows a beneficial effect of estrogens for Parkinson''s disease, yet the exact potency of these compounds implicated remain obscured. In this study, we investigated the neuroprotective effect of 17β-estradiol and estrone against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced striatal toxicity in mice. The neuroprotective effects of both compounds were evaluated by HPLC and Western blot analyses 5 days after the last of 4 consecutive injections of MPTP at 1-h intervals to mice. Subacute treatment (10 days) with estrone or 17β-estradiol at low doses (0.05 and 0.2mg/kg) showed no significant changes against MPTP-induced damage of striatal dopamine terminals in mice. Furthermore, acute treatment with estrone at high doses (0.5 and 2.0mg/kg) showed no significant alterations against MPTP-induced damage of striatal dopamine terminals in mice. In contrast, acute treatment with 17β-estradiol at high doses exhibited a neuroprotective effect against the damage of striatal dopamine terminals in both male and female mice after MPTP treatments. The results demonstrate that estrogen therapy with high doses may have a neuroprotective effect on the damage of striatal dopamine terminals in the MPTP-induced mice. These findings may lead to be development of estrogen therapy for the prevention and treatment of Parkinson''s disease. [Copyright &y& Elsevier]

Abstract: Cochlin (encoded by COCH) constitutes 70% of non-collagenous protein in the inner ear, and the expression of cochlin is highly specific to the inner ear. Eleven missense mutation and one in-frame deletion have been reported in the COCH gene, causing hereditary progressive sensorineural hearing loss and vestibular dysfunction, DFNA9. These data imply that cochlin should bear an essential and crucial role in the inner ear function. However, the role of cochlin has not been fully clarified. We have investigated the spatiotemporal expression of cochlin in the inner ear of rats during postnatal development to better understand the functional role of cochlin. By immunohistochemistry, cochlin expression was faint in the cochlea and vestibule on the 6th day after birth (DAB6). At DAB70, strong expression of cochlin was detected in the spiral limbus and spiral ligament within the cochlea, and in the stromata of the maculae of otolithic organs and crista ampullaris within the vestibule. Immunoreactivity for cochlin increased during the postnatal development. Western blot analysis also showed an increase in the expression of cochlin isoforms. Furthermore, the dominant isoform of cochlin expressed changed from p63s to p40s between DAB24 and DAB70. These results suggest that the expression of cochlin may be related to the maturation of inner ear function, and the change in isoforms of cochlin expressed will provide important insight into the understanding of both cochlin function and formation of cochlin isoforms. This is the first to report about the spatiotemporal expression of cochlin in the developing rat inner ear. [Copyright &y& Elsevier]

The purpose of this study was to test the effect of modulating the expression of the human low-density lipoprotein receptor (LDLr) in human embryonic kidney (293T) cells on Cyclosporin A (CsA) cellular uptake and CsA-mediated cytotoxicity. LDLr expression was modulated using RNA interference (RNAi) and an LDLr overexpression plasmid. One of the small-interfering RNA (siRNA) constructs, LDLr-792, showed a 60% decrease in LDLr protein expression. The downregulation effect was specific as transfection with an annexin V (AxV) siRNA construct did not decrease LDLr expression levels. AxV and ABCA1 expression levels were not affected in the cells transfected with LDLr-792 (LDLrLOW cells) compared to the controls. At a functional level, fluorescent low-density lipoprotein (LDL) (DiI-LDL) internalization in the LDLrLOW cells was decreased (30%) compared to control cells. We tested the dose-dependent cytotoxicity induced by CsA using a respiration assay. We found a decrease in CsA-mediated cytotoxicity in the range of CsA doses studied (1–10 µg/mL) in the LDLrLOW cells compared to the pSHAG-transfected cells, reaching a statistical significance at 10 µg/mL CsA. At higher CsA doses we found a significant decrease in LDLr expression. When the control and LDLrLOW cells were treated with another cytotoxic drug, gentamycin, there was no difference in the cell viability, suggesting that this effect is specific for CsA. We confirmed the association of LDLr expression levels with CsA uptake by overexpressing the LDLr. The LDLr overexpressing cells showed an enhanced uptake of radiolabelled CsA. Taken together these results suggest that CsA internalization and cytotoxicity are affected by the LDL receptor expression levels. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:2349–2361, 2008 [ABSTRACT FROM AUTHOR]

Abstract: Nardilysin is a metalloendopeptidase that in vitro cleaves peptides such as dynorphin-A, somatostatin-28, α-neoendorphin and glucagon at the N-terminus of arginine and lysine residues in dibasic moieties. The enzyme is highly expressed in many endocrine tissues. Nardilysin has also been found in the brain. Previously, we have detected that nardilysin interacts with brain-specific proteins, i.e. p42IP4/centaurin-α1 [Stricker R, Chow KM, Walther D, Hanck T, Hersh LB, Reiser G (2006) Interaction of the brain specific protein p42IP4/centaurin-α1 with the peptidase nardilysin is regulated by the cognate ligands of p42IP4, PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4, with stereospecificity. J Neurochem 98:343–354]. However, very little is known about the distribution of nardilysin in the brain. The aim of the present study was to reveal its regional distribution and cellular localization in developing and adult human brain. Using immunohistochemistry and Western blot analysis we demonstrate that the enzyme is widely, but unevenly, expressed in the human brain. We found high staining intensity in the hypothalamus, neocortex and brain stem nuclei. The cellular localization is almost exclusively confined to neurons. In pre- and perinatal human brain cortex, most neurons express the enzyme. In cortical neurons nardilysin protein was found to be partially co-localized with parvalbumin but not calretinin. No co-expression was seen with somatostatin-28 immunoreactivity. A considerable overlap was revealed between p42IP4 and nardilysin. Our data support the hypothesis that nardilysin might possibly play a role in brain development, whereas its putative function in brain peptide metabolism remains to be clarified further. [Copyright &y& Elsevier]

Abstract: The proteome of the Thai cobra, Naja kaouthia, venom, revealed by two-dimensional liquid chromatography/tandem mass spectrometry, was found to consist of peptides which could be matched with 61 proteins in the database. These proteins were classified into 12 groups according to the differences in their biological activities: cardiotoxins, cobra venom factors, a cysteine-rich toxin, cytotoxins, kaouthiagin, mocarhagin, muscarinic toxin-like proteins, neurotoxins, an oxoglutarate dehydrogenase, phospholipases, serum albumin, and a weak toxin. Horse derived- anti-N. kaouthia venom hyperimmune serum currently used for the treatment of cobra ophitoxaemia reacted only to the cobra venom factors and phospholipases in the cobra holovenom by two-dimensional gel electrophoresis based-immunoblotting. The venom proteomic insight of this study should pave the way for preparing a therapeutic anti-venom of improved quality, i.e. also containing antibodies to the newly revealed toxic, but poorly immunogenic, minor venom components. It is expected that such a preparation should have a higher effectiveness than the currently used anti-venom in resuscitating cobra-bite victims. [Copyright &y& Elsevier]

Summary: We analysed the biologic properties of a small cell lung carcinoma cell line (designated KK0206) established from a patient with SCLC who had cancer-associated retinopathy (CAR). Morphological and immunohistochemical studies showed that KK0206 cells have features of the classic type of SCLC. KK0206 cells grew in suspension, forming relatively small clumps of cells with a doubling time of 72h. On light microscopy, the cells were relatively small with little cytoplasm. On immunohistochemistry using anti-bovine recoverin rabbit antibody, the cells were intensely positive for recoverin. In addition, they were positive for NSE, Ki-67, and TP53. They also expressed human recoverin, a photoreceptor protein, whose presence was confirmed by RT-PCR analysis with cDNA sequencing and Western blot analysis. The point mutation of their TP53 gene (exon 156) was detected as well. The present study demonstrates that human recoverin is expressed in SCLC cells cultured from an anti-recoverin antibody-negative patient with CAR. KK0206 might be important for further research on SCLC related retinopathy. [Copyright &y& Elsevier]

Abstract: We report here the in vivo effects of estrogen (E2) on modulation of synaptic plasticity and the agonistic (estrogen-like) role of selective estrogen receptor modulator (SERM), tamoxifen (TAM) in the CA1 of the rat hippocampus. Effects on synaptophysin (SYP), a presynaptic vesicular protein, and phosphorylated cyclic AMP responsive element-binding (p-CREB) protein, a signal transduction pathway molecule, were studied using the ovariectomized (OVX) experimental rat model. Bilateral ovariectomy was performed on 40 rats and these were divided into 4 groups based on the treatment they received (at 2 weeks post-ovariectomy, a subcutaneous injection daily for 4 weeks) viz., OVX+E2 (0.1 mg/kg body weight), OVX+TAM (0.05 mg/kg body weight), OVX+vehicle and one group served as OVX control. An additional 10 animals served as the ovary intact control group. At the end of the treatment schedule, five animals/group were used for immunohistochemical staining of SYP and p-CREB using specific antibodies with peroxidase anti-peroxidase technique on paraformaldehyde-fixed cryostat sections. Protein estimation and Western blot analysis coupled with densitometric analysis (using gel-documentation system and image analysis software) were performed on unfixed hippocampus collected from rest of the five animals/group. Serum estradiol levels were estimated with radioimmunoassay prior to sacrifice. The results revealed that ovariectomy reduced SYP and p-CREB expression whereas E2 or TAM administration resulted in their upregulation. Serum estradiol levels of E2 administered animals were comparable with the ovary intact group whereas those of TAM administered group persisted in the range of OVX controls. To conclude, long-term estrogen therapy modulates the synaptic plasticity of hippocampal neurons and presumably, the agonist biocharacter of TAM as observed in the present investigations, may in the long run have a potential in the treatment and prevention of various estrogen-related disorders. [Copyright &y& Elsevier]

Abstract: In the present study, the formation of modified proteins by methyl docosahexaenoate (DHA) peroxidation products in the presence of a metal-catalyzed oxidation system was investigated. Metal-catalyzed oxidation of mixtures containing bovine serum albumin (BSA) and DHA led to formation of two high molecular weight derivatives of BSA. One had a mass of 71.5 kDa as determined by two-dimensional electrophoresis, matrix assisted laser desorption and ionization mass spectrometer (MALDI MS) analysis. The other was estimated to be 93 kDa by SDS-PAGE electrophoresis. The exposure of BSA to DHA also led to the generation of carbonyl groups. Oxygen radical scavengers could inhibit these modifications induced by DHA peroxidation. Furthermore, there was little difference of the peptides mass fingerprinting between the two kinds of modified high-molecular-weight proteins. These results suggest that oxygen radicals formed during lipid peroxidation are involved in the formation of protein derivatives. Our study may be important in the understanding the specific role of docosahexaenoic acid in the formation of modified proteins during aging and its related diseases. [Copyright &y& Elsevier]

Abstract: Target-derived neurotrophins such as nerve growth factor (NGF) and neurotrophin-3 (NT-3) regulate sympathetic neuron survival. Here, NGF and NT-3 protein and transcript were examined in sympathetic neurons and targets in order to determine their role in age-related neuronal atrophy. One obvious alteration was a dramatic increase (up to 50-fold) in NGF protein forms, corresponding to proNGF-B, in the superior cervical ganglion (SCG) and targets where sympathetic innervation shows atrophy. In the iris, where sympathetic innervation is protected into old age, proNGF-B was decreased. Alterations in NGF transcript paralleled changes in NGF protein, albeit to a lesser degree. Though significantly increased in aged SCG, NT-3 protein, found primarily as the ‘mature’ form, showed only minor changes in most tissues, though NT-3 mRNA generally was decreased. In contrast, both NT-3 transcript and NT-3 precursors were increased in iris. The dramatic increases in proNGF, together with minimal changes in NT-3, suggest that alterations in NGF regulation may contribute to the loss of sympathetic innervation observed in many aged peripheral targets. [Copyright &y& Elsevier]

Abstract: Parathyroid hormone (PTH) and stanniocalcin (STC) are primarily produced by the parathyroid glands and corpuscles of Stannius in tetrapods and fish, respectively. However, it is now known that both calcitropic peptides are also synthesized outside of these specialized endocrine glands. The current study employed Western blot analysis to characterize PTH and STC in neural tissues of high- (rats) and low- (hagfish, dogfish, rockfish, trout and skate) vertebrates and invertebrates (starfish, squid, cuttlefish, snails, prawns). Immunoreactive PTH-like peptides, comparable in size to PTH 1-84, were readily detectable in brains of vertebrates lacking (fish) and possessing (rat) parathyroid glands and in invertebrate (snail) ganglia. Immunoreactive STC-like peptides of varying size were similarly detected in brains of vertebrates lacking (rat) and possessing (fish) corpuscles of Stannius and in invertebrate (snail, prawn) ganglia. STC and PTH may thus have evolved as ancestral neuropeptides. [Copyright &y& Elsevier]

Abstract: The present study was conducted to investigate protein levels of inducible NO synthase (iNOS) and cayclooxygenase-2 (COX-2) in human colon cancer tissues. Significant increase of iNOS and COX-2 levels in tumors as compared to paired normal tissue was observed in 15 and 28, respectively, of 29 cases investigated. No correlation was observed between the two enzymes, and immunohistochemically demonstrated iNOS expression was mainly observed in tumor epithelial cells and was higher in well-differentiated than moderately-differentiated adenocarcinomas, while COX-2 expression was mainly detected in stromal cells and higher in Dukes'' C and D than Dukes'' A and B cases. These results suggest that iNOS and COX-2 have separate roles in colon carcinogenesis, and might both be usefully targeted by chemoprevention. [Copyright &y& Elsevier]

Abstract: The cDNA encoding eclosion hormone (EH), which plays an integral role in triggering ecdysis behavior at the end of each molt, was cloned from the cotton bollworm, Helicoverpa armigera (Har) (Lepidoptera: Noctuidae). The EH polyprotein precursor contains a 26-amino acid signal peptide and a single 62-amino acid mature EH. Compared the mature Har-EH with other known EHs, it shows 94%, 84%, and 59% identities to Manduca sexta, Bombyx mori, and Drosophila melanogaster, respectively. Har-EH mRNA is expressed only in the brain by Northern blot and RT-PCR, but not in other tissues. By in situ hybridization and immunocytochemistry, both Har-EH mRNA and protein are localized in two pairs of neurosecretory cells of the brain. Prior to a molt, expression level of Har-EH gene reaches the highest point, and then drops after molt. EH release is detected both centrally, within the ganglia, and peripherally, into the hemolymph. A peak of the EH titer in hemolymph measured by ELISA presents at ecdysis. These results are consistent with the biological function of Har-EH associated with ecdysis. Furthermore, Har-EH gene is expressed throughout all of the developmental stages examined, implicating that the EH gene may possess other biological functions in post-embryonic development other than triggering ecdysis behavior. [Copyright &y& Elsevier]

Olfactory receptors are G-protein coupled receptors and are encoded by an extremely large and diverse family of genes in mammals. There is increasing evidence that olfactory receptors are widely distributed in many organs, suggesting that olfactory receptors do not only recognize airborne odorants but also play important roles in chemotaxis or organ construction in embryo. In this study, we investigated whether olfactory receptors and their transduction molecule, Golf are expressed in the rat placenta. By RT-PCR, we identified 11 different olfactory receptor genes, which are all members of class II, in the rat placenta cDNAs, and our results suggested that particular members of the olfactory receptor gene family might be preferentially expressed in the placenta. By western blot analysis, we demonstrated that Golf protein is expressed in the placenta and its expression levels are developmentally regulated. We found that Golf immunoreactivity is exclusively localized to giant cell trophoblasts and spongiotrophoblast cells. These findings raised a possibility that a particular subset of olfactory receptors might be coupled with Golf and function in giant cell trophoblasts and spongiotrophoblast cells. [Copyright &y& Elsevier]

Summary: Cyclooxygenase-2 (COX-2) has been shown to be involved in multiple steps of carcinogenesis. In this study, we examined COX-2 expression in nasopharyngeal carcinoma (NPC). COX-2 mRNA analyzed by reverse-transcription polymerase chain reaction (RT-PCR) was detected in 66% (16 of 24) of tumor tissues. Western blot analysis demonstrated that COX-2 protein level was increased in tumor tissues and was correlated with the expression level of mRNA. Immunohistochemical study showed that COX-2 was predominantly detected in cancer cells, and the staining pattern was cytoplasmic. Ten histologically normal nasopharyngeal tissues obtained from nasopharyngeal hyperplasia were also investigated. We found that COX-2 mRNA was detectable in three tissues and the COX-2 protein level was very low. The frequency of COX-2 overexpression was significantly higher in patients of the N1–N3 group than in patients of the N0 group (P =0.006). Taken together, these data suggest that COX-2 is overexpressed and is associated with increased lymphatic invasion in NPC. [Copyright &y& Elsevier]

Abstract: The ability of unsaturated fatty acid methyl esters to modify bovine serum albumin (BSA) in the presence of a metal-catalyzed oxidation system [ascorbic acid/Fe (II)/O2] was investigated. The exposure of BSA to PUFA esters led to the generation of carbonyl groups and the formation of high-molecular-weight proteins, which were strongly dependent on the degree of fatty acid unsaturation. The high-molecular-weight proteins were detected by Western blot analysis and were not recognized by five antibodies. The observation that levels of conjugated dienes and malondialdehyde formed in the presence of BSA were substantially lower in the presence than in the absence of BSA indicated that radicals formed during the degradation of lipid hydroperoxides are likely involved in the formation of protein derivatives. These results may be important in understanding the specific roles of different polyunsaturated fatty acids in the pathophysiological effects associated with oxidative stress. [Copyright &y& Elsevier]

Abstract: Introduction: The purpose of this study was to identify and characterize the presence of low-density lipoprotein receptors (LDLr) in LLC-PK1 cells. Methods: LLC-PK1 cells were assessed for the presence of LDLr by conducting dose–response, LDL specific binding and competitive studies with DiI-LDL, and Western blot and RT-polymerase chain reaction (PCR) analyses. Assay conditions with IgG-C7, a monoclonal antibody (mAb) to the LDLr, were optimized, including temperature, preincubation time, and concentration in LLC-PK1 cells. Results: LLC-PK1 cells express LDL receptors as determined by LDL specific and competitive binding studies and Western blot and RT-PCR analysis (specific binding 0.5 ng DiI-LDL/μg of cellular protein). Discussion: Taken together, these findings confirm the presence of LDL receptors on LLC-PK1 cells and support the appropriateness of using these cells in studies involving renal cell cholesterol uptake and metabolism. [Copyright &y& Elsevier]

Objective: The purpose of this study was to investigate the expression of epidermal growth factor receptor and c-erbB-2 oncoprotein in trophoblast populations of placenta accreta. Study design: Paraffin sections of 43 placental specimens with (cases) and 43 without (controls) placenta accreta were studied immunohistochemically for epidermal growth factor receptor and c-erbB-2 expression in the syncytiotrophoblast, villous cytotrophoblast, and extravillous cytotrophoblast. Epidermal growth factor receptor and c-erbB-2 protein levels were also performed by Western blot analysis. Controls were matched for gestational age in this case- control study. Results: The percentage of strong/intermediate immunoreactivity of epidermal growth factor receptor in the syncytiotrophoblast was significantly higher in cases (98%) than controls (79%; odds ratio,l 1.1; 95% CI, 1.3-92.0; p = .03), although strong/intermediate immunoreactivity of c-erbB-2 in the syncytiotrophoblast was significantly lower in cases (35%) than controls (81%; odds ratio, 0.1; 95% CI, 0.1-0.3; p < .001). Epidermal growth factor receptor and c-erbB-2 expression in the villous and extravillous cytotrophoblastic cells were not significantly different between controls and cases (P > .0 5). Most immunoreactive bands on Western blots were consistent with the trends of immunohistochemical findings. Conclusion: The development of placenta accreta is related closely to the differential expression of epidermal growth factor receptor and c-erbB-2 in the syncytiotrophoblast. [ABSTRACT FROM AUTHOR]

This study investigated the expression and cellular localization of neuronal nitric oxide synthase in the rat retina following optic nerve transection (ONT). In the normal rat retina, nNOS immunoreactivity was localized to amacrine cells and displaced amacrine cells. A few bipolar cells were also labeled. In the axotomized retina, ganglion cells showed nNOS immunoreactivity from 3 days after ONT, and these cells increased in number, peaking 5 days after ONT. Quantitative evaluation using immunoblotting confirmed that nNOS expression showed a peak value (255% of control levels) 5 days after ONT and decreased to 137% of controls by 28 days. These findings suggest that axotomized ganglion cells degenerate via NO-mediated excitotoxicity. [Copyright &y& Elsevier]

Pregnancy results in attenuated baroreflex mediated sympathoexcitatory responses which may be due to potentiation of γ-aminobutyric acid (GABA) inhibition in the rostral ventrolateral medulla (RVLM). The major metabolite of progesterone, 3α-hydroxy-dihydroprogesterone (3α-OH-DHP), which is elevated in pregnancy, is a potent neurosteroid positive modulator of GABAA receptors, and sensitivity of GABAA receptors to 3α-OH-DHP is dependent on the receptor subunit composition. The purpose of this study was to evaluate the GABAA α1 and α2 receptor subunit mRNA and protein expression in the RVLM of nonpregnant and late term pregnant rats. Micropunches of RVLM were collected from nonpregnant and late term pregnant rats and the expression levels of GABAA α1 and α2 receptor subunits were analyzed using quantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot techniques. The competitive RT-PCR analysis allows comparison of expression levels between different mRNA, and the mRNA expression level of GABAA α1 was several hundred fold greater than GABAA α2 in both groups. However, this relative distribution of GABAA α1 and α2 receptor subunits protein or mRNA expression was not altered in late term pregnant compared to nonpregnant rats. These data demonstrate, that within the RVLM of both nonpregnant and late term pregnant rats, the relative expression levels of GABAA α1,2 receptor subunits favor GABAA receptors susceptible to positive modulation by progesterone metabolites. [Copyright &y& Elsevier]

We investigated c-Met expression in cultured astrocytes and their regulation by cytokines. Immunocytochemistry revealed that c-Met was expressed in cultured astrocytes. Western blotting revealed that acidic and basic fibroblast growth factor (FGF) enhanced and hepatocyte growth factor (HGF) reduced c-Met expression. Reverse transcription-polymerase chain reaction revealed that FGFs and HGF enhanced c-met expression. These findings suggest that c-Met expressed in astrocytes may have important roles during the nervous system regeneration. [Copyright &y& Elsevier]

The coding region of guinea pig peroxisome proliferator activated receptor gamma1 (gpPPARγ1) cDNA was successfully cloned from adipose tissue by reverse transcription polymerase chain reaction (RT-PCR) using the designated primers based on the conserved regions of the other mammalian PPARγ1 sequence. From RT-PCR, a combination of three cDNA fragments that comprised of the full length coding region PPARγ1 cDNA gene were amplified, with the size of 498, 550 and 557 bp, respectively. All three fragments were then successfully assembled by utilising the internal restriction sites present at the overlapping regions to give rise to the full-length coding region of gpPPARγ1 with the size of 1428 bp and consisting of 475 amino acids. Guinea pig PPARγ1 is highly conserved with those of other species at protein and nucleotide levels. Gene expression studies showed that gpPPARγ mRNA was predominantly expressed in adipose tissue followed by lung and spleen. However, at the protein level, PPARγ was also found to be expressed in skeletal muscle. [Copyright &y& Elsevier]