Proteome analyses of the growth inhibitory effects of NCH-51, a novel histone deacetylase inhibitor, on lymphoid malignant cells.

Recent reports showing successful inhibition of cancer and leukemia cell growth using histone deacetylase inhibitor (HDACi) compounds have highlighted the potential use of HDACi as anti-cancer agents. However, high incidence of toxicity and low stability in vivo were observed with hydroxamic acid-based HDACi such as suberoylanilide hydroxamic acid (SAHA), thus limiting its clinical applicability. In this study, we found that a novel non-hydroxamate HDACi NCH-51 could inhibit the cell growth of a variety of lymphoid malignant cells through apoptosis induction, more effectively than SAHA. Activation of caspase-3, -8 and -9, but not -7 was detected after the treatment with NCH-51. Gene expression profiles showed that NCH-51 and SAHA similarly upregulated p21 and downregulated anti-apoptotic molecules including survivin, bcl-w and c-FLIP. Proteome analysis using two-dimensional electrophoresis revealed that NCH-51 upregulated anti-oxidant molecules including peroxiredoxin 1 and 2 and glutathione S-transferase at the protein level. Interestingly, NCH-51 induced reactive oxygen species (ROS) after 8 h whereas SAHA continuously declined ROS. Pretreatment with an antioxidant, N-acetyl-L-cysteine, abolished the cytotoxicity of NCH-51. These findings suggest that NCH-51 exhibits cytotoxicity by sustaining ROS at the higher level greater than SAHA. This study indicates the therapeutic efficacy of NCH-51 and novel insights for anti-HDAC therapy.

Reversal of delayed vasospasm by TS-011 in the dual hemorrhage dog model of subarachnoid hemorrhage.

PURPOSE: Arachidonic acid is avidly metabolized to a potent vasoconstrictor, 20-hydroxyeicosatetraenoic acid (20-HETE), in the cerebral circulation. 20-HETE has been reported to contribute to the acute fall in cerebral blood flow following subarachnoid hemorrhage (SAH), but its role in the development of delayed vasospasm is unknown. The present study examined whether delayed vasospasm is associated with elevations in 20-HETE in CSF in the dual hemorrhage model of SAH in dogs and if blockade of the synthesis of 20-HETE with N-(3-chloro-4-morpholin-4-yl)phenyl-N'-hydroxyimido formamide (TS-011) can reverse delayed vasospasm in this model. MATERIALS AND METHODS: Delayed vasospasm was induced in 22 adult beagle dogs by dual injection of blood (0.5 mL/kg) into the cisterna magna on days 1 and 4. Sequential samples of CSF were collected before intracisternal injections of blood on days 1 and 4 and after the development of delayed vasospasm on day 7. Sequential angiograms were obtained before and after intracisternal injection of blood on days 1 and 4 and before and 1 hour after administration of TS-011 (1 mg/kg IV) on day 7. RESULTS: The dogs consistently developed delayed vasospasm, and the diameter of the basilar artery fell to 68 +/- 3% (n = 15), 3 days after the second intracisternal injection of blood. The levels of 20-HETE in CSF increased from 4 +/- 2 to 39 +/- 16 pg/mL. In 9 dogs with delayed vasospasm, acute blockade of the synthesis of 20-HETE with TS011 (1 mg/kg IV) significantly increased the diameter of the basilar artery by 39%. Chronic administration of TS-011 (1 mg/kg per day) attenuated the development of delayed vasospasm, and the diameter of the basilar artery fell by 17 +/- 1% versus the 33 +/- 3% decrease in diameter seen in control animals 3 days following the second injection of blood into the cisterna magna. CONCLUSIONS: These results indicate that the development of delayed vasospasm in dogs is associated with an increase in 20-HETE levels in CSF, and acute blockade of the synthesis of 20-HETE with TS-011 reverses delayed vasospasm in this model.

Mechanical behavior of bioactive composite cements consisting of resin and glass-ceramic powder in a simulated body fluid: effect of silane coupling agent.

Time-dependent strength behavior was investigated for bisphenol-a-glycidyl methacrylate/triethylene glycol dimethacrylate (Bis-GMA/TEGDMA) resin cements combined with glass-ceramic A-W filler treated with various kinds of silane coupling agents. The fracture strength of the composite resin cements was measured by three-point bending as a function of stressing rate in a simulated body fluid (SBF), and thereby the stress-corrosion susceptibility constant was evaluated. The fracture strength was found to depend on the kind of coupling agent used. For the present Bis-GMA/TEGDMA resin, the silane coupling agents without hydrophilic amine groups can be used to obtain good adhesion between resin and A-W filler owing to their nature of co-polymerizing with the resin. On the other hand, all the composite resin cements showed nearly the same degree of stress-corrosion susceptibility whether the A-W fillers were treated or untreated with silane coupling agents. This means that the stress-corrosion susceptibility of the present composite cements is predominantly affected by that of the matrix resin. Thus, the microcrack formation and growth at the resin matrix near particle - resin interface were thought to determine overall time-dependent strength behavior of the composite cements.

Mechanical properties of glass-ceramic A-W-polyethylene composites: effect of filler content and particle size.

Composites which comprise a bioactive filler and ductile polymer matrix are desirable as implant materials since both their biological and mechanical properties can be tailored for a given application. In the present study three-point bending was used to characterise biomedical materials composed of glass-ceramic apatite-wollastonite (A-W) particulate reinforced polyethylene (PE) (denoted as AWPEX). The effects of filler volume fraction, varied from 10 to 50 vol%, and average particle size, 4.4 and 6.7 microm, on the bending strength, yield strength, mode of fracture, Young's modulus and strain to failure were investigated. HAPEX, a commercially used composite of hydroxyapatite and polyethylene, with a 40 vol% filler content, was used for comparison. Increasing the filler content caused an increase in Young's modulus, yield strength and bending strength, and a decreased strain to failure. When filler particle size was increased, the Young's modulus, yield and bending strengths were found to be slightly reduced. A transition in fracture behaviour from ductile to brittle behaviour was observed in samples containing between 30 and 40 vol% filler.

Bonding strength of the apatite layer formed on glass-ceramic apatite-wollastonite-polyethylene composites.

Bioactive glass-ceramic apatite-wollastonite (A-W) has been incorporated into polyethylene in particulate form to create new bioactive composites for potential maxillofacial applications. The effects of varying the volume fraction of glass-ceramic A-W filler and the glass-ceramic A-W particle size were investigated by measuring the bonding strength of the bonelike apatite layer formed on the surface of glass-ceramic A-W-polyethylene composites. The bonding strength was evaluated via a modified ASTM C-333 standard in which a tensile stress was applied to the substrate and the strength of the bioactive layer was compared with that formed on commercially available hydroxyapatite-polyethylene composite samples, HAPEX. The composites demonstrated greater bonding strength with increased filler content and reduced filler particle size (maximum 6.9 +/- 0.5 MPa) and a marginally greater bonding strength as compared with HAPEX (2.8 +/- 0.5 MPa), when glass-ceramic A-W-polyethylene composite samples with the same filler content were tested. The higher bonding strength of the apatite layer formed on the A-W-polyethylene composite samples suggests that, in addition to maxillofacial applications, these composites might also be utilized in applications involving higher levels of load bearing.

Apatite-forming ability of glass-ceramic apatite-wollastonite - polyethylene composites: effect of filler content.

The bioactivity of a range of glass-ceramic apatite-wollastonite (A-W) - polyethylene composites (AWPEXs) with glass-ceramic A-W volume percentages ranging from 10 to 50, has been investigated in an acellular simulated body fluid (SBF) with ion concentrations similar to those of human blood plasma. The formation of a biologically active apatite layer on the composite surface after immersion in SBF was demonstrated by thin-film X-ray diffraction (TF-XRD) and field-emission scanning electron microscopy (FE-SEM). An apatite layer was formed on all the composites, with the rate of formation increasing with an increase in glass-ceramic A-W percentage. For composites with glass-ceramic A-W filler contents >or=30 vol %, the apatite layer was formed within 12 h of immersion, which is a comparable time for apatite formation on monolithic glass-ceramic A-W. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) demonstrated that the apatite formation on AWPEX samples with 50 vol % filler content occurred in a manner similar to that seen on pure glass-ceramic A-W, in that the calcium, silicon, and magnesium ion concentrations increased and, conversely, a decrease was observed in the phosphate ion concentration. These results indicate that a suitable in vitro response was achieved on a composite incorporating particulate glass-ceramic A-W with a particularly favorable response being observed on the AWPEX sample with 50 vol % filler content.

Apatite formation on CaO-free polydimethylsiloxane (PDMS)-TiO2 hybrids.

Polydimethylsiloxane (PDMS)-TiO(2) hybrids with PDMS (M=550)/tetraethylorthotitanate molar ratios at 0.27, 0.68 and 1.35, i.e. Si/Ti atomic ratios at 2, 5 and 10 (hybrids PD2, PD5 and PD10, respectively) were prepared by a sol-gel method. Hybrid PD2 formed many cracks. Hybrids PD5 and PD10 were subjected to hot-water treatment 80 degrees C for 7 d. Hybrid PD5 produced cracks, whereas hybrid PD10 was crack-free after the hot-water treatment. Hybrid PD10 took a homogeneous amorphous structure before the hot-water treatment, and precipitated anatase particles 10-20 nm in size after the hot-water treatment. Hybrid PD10 did not form apatite on its surface in a simulated body fluid before the hot-water treatment, but formed it after the hot-water treatment. The obtained hybrid showed elastic deformation as large as 200% after the hot-water treatment. This kind of hybrid could be useful as a new type of bone-repairing material.

Immunomagnetic separation of scum-forming bacteria using polyclonal antibody that recognizes mycolic acids.

Mycolic acid-containing bacteria (mycolata) are thought to be involved in scum formation in aeration basins of activated sludge plants due to their ability to produce biosurfactants and their cell surface hydrophobicity. To isolate these bacteria, immunomagnetic separation (IMS) using an anti-mycolic acid polyclonal antibody was investigated. IMS that targeted Gordonia amarae SC1 exhibited a 100% recovery at 5x10(3) CFU ml(-1). At cell concentration of 7.8x10(6) CFU ml(-1), the recovery was lowered, but 80% of cells were still captured. Effect of bead concentrations on the recovery of SC1 at 10(6) CFU ml(-1) was examined. The results showed that addition of more than 6-7x10(6) beads for 1x10(6) CFU reached a maximum recovery (83%). Furthermore, the IMS procedure optimized with SC1 cells was tested with another mycolata. The results suggested that variation of the recovery for each mycolata is dependent on the specificity of the polyclonal antibody and that mycolata which are recognized by the antibody can be recovered by this procedure.

Bioactivity and mechanical properties of polydimethylsiloxane (PDMS)-CaO-SiO2 hybrids with different calcium contents.

Polydimethylsiloxane (PDMS)-CaO-SiO(2) hybrids with starting compositions containing PDMS/(Si(OC(2)H(5))(4)+PDMS) weight ratio=0.30, H(2)O/Si(OC(2)H(5))(4) molar ratio=2, and Ca(NO(3))(2)/Si(OC(2)H(5))(4) molar ratios=0-0.2, were prepared by the sol-gel method. The apatite-forming ability of the hybrids increased with increasing calcium content in the Ca(NO(3))(2)/Si(OC(2)H(5))(4) molar ratio range 0-0.1. The hybrids with a Ca(NO(3))(2)/Si(OC(2)H(5))(4) molar ratio range 0.1-0.2 formed apatite on their surfaces in a simulated body fluid (SBF) within 12 h. The hybrid with a Ca(NO(3))(2)/Si(OC(2)H(5))(4) molar ratio of 0.10 showed an excellent apatite-forming ability in SBF with a low release of silicon into SBF. It also showed mechanical properties analogous to those of human cancellous bones. This hybrid is expected to be useful as a new type of bioactive material.

Synthesis and biological evaluation of all A-ring stereoisomers of 5,6-trans-2-methyl-1,25-dihydroxyvitamin D(3) and their 20-epimers: possible binding modes of potent A-ring analogues to vitamin D receptor.

BACKGROUND: The secosteroid 1 alpha,25-dihydroxyvitamin D(3) (1) has a wide variety of biological activities, which makes it a promising therapeutic agent for the treatment of cancer, psoriasis and osteoporosis. Insight into the structure-activity relationships of the A-ring of 1 is still needed to assist the development of more potent and selective analogues as candidate chemotherapeutic agents, as well as to define the molecular mode of action. RESULTS: All possible A-ring stereoisomers of 5,6-trans-2-methyl-1,25-dihydroxyvitamin D(3) (6a-h) and their 20-epimers (7a-h) were designed and efficiently synthesized. The dependence of the affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP), as well as the HL-60 cell differentiation-inducing activity, upon the stereochemistry of the A-ring and at C20 in the side chain was evaluated. CONCLUSIONS: The binding affinities and potency of the 5,6-trans and 5,6-cis analogues were enhanced by a 2-methyl substituent in a certain orientation. Molecular docking studies based upon the X-ray crystal structure of VDR suggested that the axial 2-methyl group would be accommodated in a pocket surrounded by hydrophobic amino acid residues in the ligand binding domain, resulting in enhanced interaction.

Efficient and versatile synthesis of novel 2alpha-substituted 1alpha,25-dihydroxyvitamin D(3) analogues and their docking to vitamin D receptors.

Novel 2alpha-substituted 1alpha,25-dihydroxyvitamin D(3) analogues with 2alpha-alkyl and 2alpha-hydroxyalkyl groups were systematically synthesized from D-xylose. Their conformation on binding to the ligand binding domain (LBD) of the vitamin D receptor was analyzed. It has been found that the 2alpha-hydroxypropyl group best fits the cavity of the LBD, and the binding activity is three times higher than that for the natural hormone.

Discovery of a N'-hydroxyphenylformamidine derivative HET0016 as a potent and selective 20-HETE synthase inhibitor.

N-(4-Butyl-2-methylphenyl)-N'-hydroxyformamidine (HET0016) was evaluated as the first potent and selective inhibitor of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) synthase. The IC(50) value of HET0016 for the production of 20-HETE from arachidonic acid (AA) by human renal microsomes was 8.9+/-2.7 nM, with over 200 times the selectivity of xenobiotic-metabolizing cytochrome P450 enzymes. An examination of the structure-activity relationship revealed that the unsubstituted hydroxyformamidine moiety and the substituent at the para-position of the N-hydroxyformamidine moiety are necessary for the potent activity of HET0016.

Syntheses, structures, stability, and insulin-like activities of peroxovanadium(V) complexes with a heteroligand.

Several peroxovanadium(V) complexes were prepared with a tripodal or a quasi-tripodal tetradentate ligand. The structures of K(2)[VO(O(2))(nta)].2H(2)O and K[VO(O(2))(DL-cmhist)].H(2)O have been determined by X-ray crystallography (nta, nitrilotriacetate; cmhist, N-carboxymethylhistidinate). The structure of Cs[VO(O(2))(pda)].2H(2)O (pda, N-pyridylmethyliminodiacetate) has been estimated to be similar to that of K[VO(O(2))(DL-cmhist)].H(2)O. Each complex anion in these compounds adopts a distorted pentagonal bipyramidal structure, which is typical for heptacoordinate oxoperoxovanadium(V) complexes. The peroxide ion binds in a side-on fashion to the vanadium(V) center in the pentagonal plane. The peroxide anion in the cmhist complex dissociates rather easily in an acidic solution (pH approximately 3), while that in the other complexes stays intact under similar conditions. The in vitro insulin mimetic effect of the peroxovanadium(V) complexes has been evaluated by the inhibitory effect on free fatty acid (FFA) release in isolated rat adipocytes treated with epinephrine. The cmhist complex is effective, while the others are almost totally ineffective.

Photochemical generation of nitric oxide from 6-nitrobenzo[a]pyrene.

Photolabile 6-nitrobenzo[a]pyrene (6-nitroBaP) released nitric oxide (NO) under visible-light irradiation. The generation of NO and the concomitant formation of the 6-oxyBaP radical were confirmed by ESR. BaP quinones were also detected as further oxidized products of the 6-oxyBaP radical. No such photodegradation was observed with other nitrated BaPs, such as 1-nitroBaP and 3-nitroBaP. DNA-strand breakage, caused by photoexcited 6-nitroBaP, was closely related to its NO-releasing activity. MO calculations of nitrated BaP suggest that the perpendicular conformation of the nitro substituent to the aromatic ring is important for the release of NO with light. These findings may be useful for the development of a new type of NO donor.

Optical anisotropy of oxidized Si(001) surfaces and its oscillation in the layer-by-layer oxidation process.

Reflectance-difference (RD) measurements for the oxidation of single-domain (2x1)-reconstructed Si(001) surfaces show that the polarity of the interface-induced optical anisotropy is reversed repeatedly with increasing oxide thickness. The oscillation of the RD amplitude, which we show is due to layer-by-layer progression of the oxidation, has allowed us to count the number of oxidized Si layers in situ during oxidation. The origins of the observed spectral line shape are discussed.

Resveratrol, a naturally occurring polyphenol, induces sister chromatid exchanges in a Chinese hamster lung (CHL) cell line.

We tested the genotoxicity of 3,5,4'-trihydroxystilbene (resveratrol), a polyphenolic phytoalexin found in grapes, in a bacterial reverse mutation assay, in vitro chromosome aberration (CA) test, in vitro micronucleus (MN) test, and sister chromatid exchange (SCE) test. Resveratrol was negative in the strains we used in the bacterial reverse mutation assay (S. typhimurium TA98 and TA100 and E. coli WP2uvrA) in the absence and presence of a microsomal metabolizing system. It induced structural CAs at 2.5-20 microg/ml and showed weak aneuploidy induction in a Chinese hamster lung (CHL) cell line. It induced MN cells and polynuclear and karyorrhectic cells after 48h treatments in the in vitro MN test. In the SCE test, resveratrol caused a clear cell-cycle delay; at 10 microg/ml, the cell cycle took twice as long as it did in the control. Resveratrol induced SCEs dose-dependently at up to 10 microg/ml, at which it increased SCE six-fold, and the number was almost as large as mitomycin C, a strong SCE inducer. No second mitoses were observed at 20 microg/ml even after 54h. Cell cycle analysis by FACScan indicated that resveratrol caused S phase arrest, and 48h treatment induced apoptosis. Our results suggest that resveratrol may preferentially induce SCE but not CA, that is, it may cause S phase arrest only when SCEs are induced.

HET0016, a potent and selective inhibitor of 20-HETE synthesizing enzyme.

The present study examined the inhibitory effects of N-hydroxy-N'-(4-butyl-2-methylphenyl)-formamidine (HET0016) on the renal metabolism of arachidonic acid by cytochrome P450 (CYP) enzymes. HET0016 exhibited a high degree of selectivity in inhibiting the formation of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) in rat renal microsomes. The IC(50) value averaged 35+/-4 nM, whereas the IC(50) value for inhibition of the formation of epoxyeicosatrienoic acids by HET0016 averaged 2800+/-300 nM. In human renal microsomes, HET0016 potently inhibited the formation of 20-HETE with an IC(50) value of 8.9+/-2.7 nM. Higher concentrations of HET0016 also inhibited the CYP2C9, CYP2D6 and CYP3A4-catalysed substrates oxidation with IC(50) values of 3300, 83,900 and 71,000 nM. The IC(50) value for HET0016 on cyclo-oxygenase activity was 2300 nM. These results indicate that HET0016 is a potent and selective inhibitor of CYP enzymes responsible for the formation of 20-HETE in man and rat.

Treatment of Vogt-Koyanagi- Harada's disease during pregnancy.

BACKGROUND: Caution should be exercised in treating patients with autoimmune diseases during pregnancy. CASES: We successfully treated three cases of Vogt-Koyanagi-Harada's disease (VKH disease) during pregnancy. OBSERVATIONS: In the second trimester (14-27 weeks) of 1 patient, inflammation was mild and could be treated by topical corticosteroid. There is the possibility that her immune response had been modified by pregnancy. Systemic corticosteroid in a high dose was administered to the two cases in the second and third trimesters of pregnancy (28-41 weeks). The severity of inflammation in these 2 patients was similar to that in nonpregnant women. Inflammation subsided immediately without recurrence in all cases. No abnormality was found during the deliveries or in the babies. CONCLUSIONS: Treatment for VKH disease during pregnancy should be chosen according to the severity of inflammation, the stage of pregnancy, and the maternal and fetal conditions.

Micronucleus induction and chromosomal aberration of 1- and 3-nitroazabenzo[a]pyrene and their N-oxides.

Nitro-azabenzo[a]pyrenes, 1- or 3-nitro-azabenzo[a]pyrene and their N-oxides are nitrated derivatives of azabenzo[a] pyrene (ABP) containing nitrogen in the 6-position of benzo[a]pyrene (B[a]P). The nitro-ABP-N-oxides (ABPOs) were formed by reaction of ABP with excess HNO(3). These derivatives were noteworthy as potent mutagens for Salmonella strains, and were present in fine particles of diesel particulates. In this study, micronucleus induction in mice and chromosomal aberrations due to means of Chinese hamster lung fibroblast (CHL) cells were investigated to determine genotoxicity in order to define the relationship with the mutagenic potency of these derivatives. The induction of micronucleus polychromatic erythrocytes (MNPCEs) was dependent on the dose response of 10-40 mg for 3-N-6-ABP, and of 10-40 mg for 1-N-6-ABP, and in addition, 1- and 3-N-6-ABPOs markedly induced MNPCEs in a dose range of 10-400 mg and from 1 to 80 mg, respectively, when the compound was intraperitoneally administrated in two mice at each dose. The results show that of the four compounds, 3-N-6-ABPO demonstrated a marked increase in MNPCES: On the other hand, chromosomal aberrations of the four compounds were investigated by the duplicate tests using CHLS: The results after a 48 h treatment induced aberrations of the chromatid type, chromatid breaks and exchanges for 1- and 3-N-6-ABP, and mainly chromatid exchanges for 1- and 3-N-6-ABPO. The frequency of chromosomal aberrations associated with nitro substitution on the ABPO structure. Chromosomal aberrations of nitro derivatives of ABPO substituted at the 3-position on the structure were more potent than those at the 1-postion. N-oxide derivatives have been found to be reduced to anion radicals much more easily than azaB[a]P and its nitro derivatives. This suggests that the electrochemical reduction of the chemicals plays an important role in the metabolic activation of nitrated B[a]P derivatives.

Oxidative DNA damage by a metabolite of carcinogenic 1-nitropyrene.

Nitropyrenes are carcinogenic pollutants. Adduct formation following nitro-reduction is considered to be a major cause of nitropyrene-mediated DNA damage. We investigated the role of 1-nitrosopyrene, a metabolite of 1-nitropyrene, in causing oxidative DNA damage, using 32P-5'-end-labeled DNA. 1-Nitrosopyrene was found to facilitate Cu(II)-mediated DNA damage in the presence of NADH. Catalase and a Cu(I)-specific chelator attenuated DNA damage, indicating the involvement of H2O2 and Cu(I). Typical *OH scavenger did not have a significant effect. These results suggest that the main reactive species is probably a DNA-copper-hydroperoxo complex. We also measured 8-oxo-7,8-dihydro-2'-deoxyguanosine formation by 1-nitrosopyrene in the presence of Cu(II) and NADH, using an electrochemical detector coupled to a high-pressure liquid chromatograph. We conclude that oxidative DNA damage, in addition to DNA adduct formation, may play an important role in the carcinogenesis of nitropyrenes.