In-plane stress development in mesoporous thin films.

Ordered mesoporous thin films of TiO2 and CexZr1-xO2 (x = 0, 0.5, 1) were prepared via an evaporation-induced self-assembly (EISA) process and subsequently investigated in terms of the developing intrinsic and residual in-plane stress. These mechanical properties were determined by the curvature method, which is based on the determination of the deflection of light due to concave or convex bending of the films on a substrate. The films were investigated with regard to the intrinsic stress during heat treatment up to 500 °C and to the residual stress at room temperature for several annealing temperatures. Following this strategy, the influence of the decomposition of a block copolymer template on the intrinsic stress as well as the pore collapsing on the residual stress was analyzed. Nanoporous TiO2 thin films were prepared using two different block copolymers (PIB50-b-PEO45 and Pluronic® F127). A comparison between the templated and non-templated TiO2 films showed the lowest intrinsic and residual stress for the ordered mesoporous material prepared with PIB50-b-PEO45 indicating that the distributed polymer and the corresponding mesopores act as relaxing agents for the system. This was verified by mesoporous CexZr1-xO2 (x = 0, 0.5, 1) thin films showing a comparable behavior in terms of the experienced intrinsic stress. This work reveals an increase in the residual in-plane stress during pore collapse, which lays the foundation for further understanding of the stress-related mechanical properties of mesoporous thin films.

Antibacterial silver-containing silica glass prepared by sol-gel method.

Recently, various inorganic antibacterial materials containing silver have been developed and some of them are in commercial use. Colorless and more chemically durable materials which slowly release the silver ion for a long period are, however, desirable to be developed for medical applications such as composite resin for dental restoration. In the present study, Si(OC2H5)4, Al(NO3)3 x 9H2O, AgNO3, HNO3, C2H5OH and H2O solutions with various Al/Ag atomic ratios under a constant Si/Ag atomic ratio of 1/0.023 were kept at 40 degrees C for gelation and drying. Thus obtained gels were pulverized into fine powders with average particle size of approximately 10 microm and then heat-treated at 900-1000 degrees C for 2 h. For the composition Al/Ag = 0, a yellow-colored glass was formed, since the silver existed in the form of metallic colloids in the glass. However, for the compositions Al/Ag > or = 1, colorless glasses were successfully obtained, since the silver existed in the form of Ag+ ions in the glasses. For the composition Al/Ag = 0, the silver ions got released rapidly into the water, whereas, for the compositions Al/Ag > or = 1, they gradually got released into the water at a controlled rate. A composite of the obtained powders with Al/Ag atomic ratio of 1 with Bis-GMA/TEGDMA in 70:30 weight ratio showed excellent antibacterial property. The sol-gel derived silica glass powders containing silver with compositions Al/Ag > or = 1 are believed to be useful as an antibacterial material for medical applications such as filler of composite resin for dental restoration.

Comparison of 26,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3 on the resorption of bone explants ex vivo.

26,27-Hexafluoro-1 alpha,25-dihydroxyvitamin D3 [F6-1,25-(OH)2D3] is more potent than 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] in stimulating bone resorption in vitro and in vivo. The reason why F6-1,25(OH)2D3 is more active remains unclear. To clarify the relationship between the bone-resorbing activity of each vitamin D3 analogue and the metabolism of each analogue, in the present study, we used an ex vivo method that was established by Reynolds et al (Calcif Tissue Res, 1974, 15, 333-339). The effect of F6-1,25(OH)2D3 or 1,25(OH)2D3 on 45Ca release from parietal bones, prepared at 3, 14 and 24 h after injection of 1.9, 3.8, 7.6 or 15.2 pmol vitamin D analog/g body weight, was examined. F6-1,25(OH)2D3 was more potent than 1,25(OH)2D3 during each in vivo time period. 1,25(OH)2D3 at 3 h after the injection was more active compared to the control (no injection of 1,25(OH)2D3) but not at 14 and 24 h. The radioactivity of the bones after the injection of [3H]-F6-1,25(OH)2D3 was retained even at 24 h. In the case of [3H]-1,25(OH)2D3, the radioactivity of bones decreased with an increase in the in vivo period. In a HPLC analysis of the lipid extract of bone homogenate, [3H]-F6-1,25(OH)2D3 alone was detected at 3 h after the injection and both [3H]-F6-1,25(OH)2D3 and [3H]-26,27-hexafluoro-1 alpha, 23S,25-trihydroxyvitamin D3 [F6-1,23,25(OH)3D3] were detected at 14 and 24 h after the injection. [3H]-1,25(OH)2D3 was highly detected at 3 h after the injection, but it decreased with an increase in the in vivo period. In the ex vivo test, the activity of F6-1,23,25(OH)3D3 was less than that of F6-1,25(OH)2D3 but similar to that of 1,25(OH)2D3. The present study indicates that F6-1,25(OH)2D3 is more active and more long-lasting than 1,25(OH)2D3 in the ex vivo method. A higher potency of F6-1,25(OH)2D3 is explained, at least partly, by the results that the amounts of both F6-1,25(OH)2D3 and its active metabolite, F6-1,23,25(OH)3D3, in the bones are higher than that of 1,25(OH)2D3, and that F6-1,25(OH)2D3 and its metabolite are retained in bones longer than 1,25(OH)2D3.

Synthesis of pyridinium dinitrobenzyl sulfates and potassium (dinitrobenzyl beta-D-glucopyranosid)uronates.

Sulfates and glucuronides of 2,4-dinitrobenzyl alcohol 1a and 2,6-dinitrobenzyl alcohol 1b, which are major or putative metabolites of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT), were synthesized from 1a and 1b by reaction with pyridinium sulfonate and methyl (2,3,4-tri-O-acetyl-alpha-D-glucopyranosyl)uronate bromide 3, respectively, as their pyridinium salts (2a, 2b) and potassium salts (6a, 6b). These conjugates are important for the study of the carcinogenicity of 2,4-DNT and 2,6-DNT.

Lead perturbs the regulation of spontaneous release of tissue plasminogen activator and plasminogen activator inhibitor-1 from vascular smooth muscle cells and fibroblasts in culture.

To evaluate the toxicity of lead on the blood fibrinolytic system during hemostasis, human aortic smooth muscle cells and human fetal lung fibroblasts were cultured in the presence of lead chloride. Tissue plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor-1 antigen (PAI-1:Ag) released were determined by enzyme immunoassay. It was found that lead decreased the release of both t-PA:Ag and PAI-1:Ag from vascular smooth muscle cells. On the other hand, in fibroblasts, the release of t-PA:Ag was markedly decreased whereas that of PAI-1:Ag was markedly increased by the metal. Fibrin zymography showed that lead reduced the plasminogen activator activity in the conditioned medium of both cell types. However, lead did not cause a nonspecific cell damage and an alteration of protein synthesis when evaluated by lactate dehydrogenase leakage and [14C]leucine incorporation, respectively. Lead accumulated within either vascular smooth muscle cells or fibroblasts in a dose-dependent manner; intracellular accumulation of calcium could be increased by lead. However, the effects of lead on the release of t-PA:Ag and PAI-1:Ag were different from those of calcium ionophore A23187. It was therefore suggested that regulation of spontaneous release of fibrinolytic proteins from subendothelial cells is disturbed by lead through intracellular calcium-independent pathway.

Inhibitory effect of lead on the repair of wounded monolayers of cultured vascular endothelial cells.

We investigated the effect of lead nitrate (0.5-5.0 microM) on the repair of wounded monolayer of cultured bovine aortic endothelial cells. It was morphologically found that lead decreases the appearance of the cells in the wounded area in a concentration-dependent manner without degenerative changes after a 48-h incubation. Although mercury weakly inhibited the repair with nonspecific cell damage, the other cations including bismuth, cobalt, manganese and nickel failed to affect the repair. The inhibition of endothelial repair caused by lead was observed even when stimulated by exogenous either basic or fibroblast growth factor. These results indicated that inhibition of the repair process of damaged endothelial cell layer is a component of lead-induced vascular lesions such as atherosclerosis.

Sensitive response of cultured vascular smooth-muscle cells to cadmium cytotoxicity: comparison with cultured vascular endothelial cells and kidney epithelial LLC-PK1 cells.

Response to cadmium cytotoxicity of cultured bovine aortic smooth-muscle cells was compared with that of cultured bovine aortic endothelial cells and porcine kidney epithelial LLC-PK1 cells. The cell damage was evaluated by morphology and the lactate dehydrogenase leakage assay. It was found that vascular smooth-muscle cells are markedly sensitive to cadmium cytotoxicity. The accumulation of intracellular cadmium was much higher but that of metallothionein was much less in vascular smooth-muscle cells than in LLC-PK1 cells; vascular endothelial cells were in between vascular smooth-muscle cells and LLC-PK1 cells. The content of reduced glutathione was slightly increased by cadmium in all three cell types. The present data suggest that a much lower inducibility of metallothionein with a high accumulation of intracellular cadmium in vascular smooth-muscle cells resulted in a marked sensitivity of the cells to cadmium cytotoxicity. Vascular smooth-muscle cells may be one of the critical target of cadmium toxicity.

S-methylmethionine sulfonium in fruits of citrus hybrids.

The S-methylmethionine sulfonium (MMS) concentrations in fruits of citrus hybrids were measured, and found to increase during ripening of the fruit. However, there of eleven hybrids of 'Seto unshiu' crossed with 'Morita ponkan' and four of 9 hybrids of 'Murcott' tangor crossed with 'Seto unshiu' had low MMS concentrations even at late harvest stage. Crossbreeding is useful in producing new citrus fruits that have juices with the desirable characteristics of their parents without formation of dimethyl sulfide which is an off-flavor.

Interaction between cadmium and zinc in the production and sulfation of glycosaminoglycans in cultured bovine vascular endothelial cells.

Previously, we showed that cadmium stimulates the production of glycosaminoglycans (GAGs) but inhibits their sulfation in cultured bovine aortic endothelial cells. The effect of zinc on such alterations of GAGs induced by cadmium was investigated in the present study. The incorporation of [3H]glucosamine and [35S]sulfate into GAGs was determined by the cetylpyridinium chloride precipitation method as a marker of GAG production and GAG sulfation, respectively. The incorporation of both [3H]glucosamine and [35S]sulfate was not changed in GAGs accumulated in the endothelial cell layer and the conditioned medium after exposure to zinc at 20 micrometers or less alone. A simultaneous exposure of the endothelial cell layer to zinc at 20 micrometers or less and cadmium at 2 micrometers resulted in prevention of the cadmium-induced decrease in [35S]sulfate incorporation; however, the cadmium-induced increase in [3H]glucosamine incorporation was not affected by zinc. Characterization of GAGs in the cell layer revealed that such an interaction between zinc and cadmium occurred in both heparan sulfate and the other GAGs. Zinc significantly prevented the inhibition of either [3H]thymidine or [3H]leucine incorporation caused by cadmium with less accumulation of intracellular cadmium suggesting that zinc decreased intracellular cadmium and protected endothelial cells from cadmium-induced inhibition of DNA and protein synthesis. The present data showed that a simultaneous exposure to cadmium and zinc resulted in an increase in heparan sulfate without a reduction of sulfation in the endothelial cell layer. The alteration may potentiate the antihrombogenic property of vascular endothelium.

Further studies on the urinary metabolites of 2,4-dinitrotoluene and 2,6-dinitrotoluene in the male Wistar rat.

1. Conjugates of 2,4-dinitrobenzyl alcohol (2,4-DNB) and 2,6-dinitrobenzyl alcohol (2,6-DNB), which were major urinary metabolites of the male Wistar rat dosed orally with 2,4-dinitrotoluene (2,4-DNT) or 2,6-dinitrotoluene (2,6-DNT), were examined by hplc using potassium 2,4-dinitrobenzyl glucuronide (2,4-DNB-G), potassium 2,6-dinitrobenzyl glucuronide (2,6-DNB-G), pyridinium 2,4-dinitrobenzyl sulphate (2,4-DNB-S), and pyridinium 2,6-dinitrobenzyl sulphate (2,6-DNB-S) as authentic compounds. Other metabolites were also examined by hplc. 2. Conjugates detected from urine following administration of 2,4-DNT and 2,6-DNT were 2,4-DNB-G and 2,6-DNB-G, which accounted for about 10.7 and 17.4% of the administered dose respectively. No peaks corresponding to pyridinium 2,4-DNB-S and pyridinium 2,6-DNB-S were detected in urine samples. 3. 2-Amino-4-nitrobenzoic acid (0.71%), 4-amino-2-nitrobenzoic acid (0.52%) and 4-acetylamino-2-nitrobenzoic acid (3.9%), in addition to known metabolites 4-amino-2-nitrotoluene (0.04%), 2,4-DNB (0.25%), 2,4-dinitrobenzoic acid (6.9%) and 4-acetylamino-2-aminobenzoic acid (3.4%), were detected in ether extracts of urine of rat given 2,4-DNT. 2,6-Dinitrobenzoic acid (0.17%) and two known metabolites, 2-amino-6-nitrotoluene (0.44%) and 2,6-DNB (0.53%), were detected in ether extracts of urine of rat given 2,6-DNT.

Effects of cadmium on the release of tissue plasminogen activator and plasminogen activator inhibitor type 1 from cultured human vascular smooth muscle cells and fibroblasts.

To evaluate the toxicity of cadmium on the blood fibrinolytic system during hemostasis, human vascular smooth muscle cells and human fibroblasts were cultured in the presence of cadmium chloride. It was found that cadmium markedly decreased the release of both tissue plasminogen activator antigen (t-PA:Ag) and plasminogen activator inhibitor type-1 antigen (PAI-1:Ag) from vascular smooth muscle cells. Other heavy metals including lead, manganese, mercury and nickel also decreased the t-PA:Ag and PAI-1:Ag release, however, cadmium was the most potent inhibitor. On the other hand, the release of t-PA:Ag was significantly increased whereas that of PAI-1:Ag was unaffected in fibroblasts after exposure to cadmium. Of the tested heavy metals, only cadmium increased the t-PA:Ag release from the cells. Electrophoretic enzymography revealed that cadmium reduced the activity of plasminogen activators in the conditioned medium of both vascular smooth muscle cells and fibroblasts. Cadmium markedly decreased the incorporation of [3H]leucine accompanied with a significant increase in the leakage of lactate dehydrogenase in vascular smooth muscle cells; however, the metal did not change these markers in fibroblasts. These results suggest that the regulation of fibrinolysis mediated by vascular smooth muscle cells and fibroblasts during hemostasis may be disturbed by cadmium.

Stimulation of cultured vascular smooth muscle cell proliferation by thrombospondin is potentiated by zinc.

The interaction of zinc sulfate with thrombospondin (TSP) derived from bovine platelets, on the proliferation of bovine aortic smooth muscle cells was investigated using cell culture. Zinc alone has no effect but potentiates the stimulatory effect of TSP on the incorporation of [3H]thymidine into the acid-insoluble cell fraction. Other heavy metals including copper, lead, manganese and nickel did not exhibit a similar effect. In bovine aortic endothelial cells, [3H]thymidine incorporation was stimulated by zinc alone and suppressed by TSP alone; the stimulation by zinc was significantly reduced by TSP. In human fibroblastic IMR-90 cells, [3H]thymidine incorporation was stimulated by either zinc or TSP; a simultaneous treatment with zinc and TSP showed an additive effect. The present data suggest that zinc may be a unique heavy metal that potentiates the proliferation of vascular smooth muscle cells stimulated by TSP; in addition, such an interaction was observed selectively in this cell type. Since a large amount of TSP is released from alpha-granules of aggregated platelets, zinc may augment TSP activity which is involved in the physiology of vascular tissues.

Zinc promotes the repair of wounded monolayers of cultured vascular endothelial cells.

The effect of zinc on the repair of wounded monolayers of bovine aortic endothelial cells in a culture system was investigated. It was morphologically found that zinc promotes the appearance of the cells in the wounded area; cell number in the area was significantly increased by zinc. However, other heavy metals including copper, manganese, nickel and cobalt failed to exhibit a similar effect. The repair induced by exogenous basic fibroblast growth factor (bFGF) was potentiated by zinc but that by exogenous acidic fibroblast growth factor was unaffected by the metal. Promotion of the repair of the wounded area by zinc was completely blocked by either cycloheximide or anti-bFGF antibody. In addition, zinc-induced repair was significantly inhibited by a lipoxygenase inhibitor, nordihydroguaiaretic acid but not by a cyclooxygenase inhibitor, indomethacin. From these results, it is suggested that zinc promotes the repair process of damaged vascular endothelium through the lipoxygenase pathway that mediates the response of vascular endothelial cells to endogenous bFGF.

Regulation by basic fibroblast growth factor of glycosaminoglycan biosynthesis in cultured vascular endothelial cells.

The alteration of glycosaminoglycans (GAGs) in cultured bovine aortic endothelial cells after exposure to basic fibroblast growth factor (bFGF) was investigated. It was found that the incorporation of [3H]glucosamine into GAGs was markedly increased by bFGF in both the cell layer and the conditioned medium; however, that of [35S]sulfate was not changed by the growth factor. These results indicated that bFGF enhanced the sugar-chain formation but did not affect their sulfation in endothelial GAG production. Similar changes were observed in either bovine aortic smooth-muscle cells and human fibroblastic IMR-90 cells to greater and lesser degrees, respectively. Characterization of GAGs in the endothelial cell layer and the conditioned medium revealed that bFGF enhanced both heparan sulfate and the other GAGs to a similar degree. The present data suggest that bFGF may be involved in the regulation of the blood coagulation system via altering GAGs of the vascular tissue when the endothelium was damaged.

Characterization of hepatic responses of rat to administration of perfluorooctanoic and perfluorodecanoic acids at low levels.

Male rats were fed a diet that contained perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) at concentrations ranging from 0.0025-0.04% (w/w) and from 0.00125-0.01% (w/w), respectively, for 1 week. The hepatic responses of the rats to PFOA and PFDA were examined. Upon the administration of PFOA and PFDA, three peroxisome proliferator-responsive parameters, peroxisomal beta-oxidation, microsomal 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase and cytosolic long-chain acyl-CoA hydrolase, were induced in a dose-dependent manner. A multiple regression analysis of the three parameters revealed that the data from rats treated with PFOA and PFDA shared one common line, indicating a marked correlation among the inductions of the three parameters. The activities of glutathione (GSH) S-transferases towards 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) were depressed by PFOA and PFDA. Significant inverse correlations were found between activities of GSH S-transferases and peroxisomal beta-oxidation. The administration of PFOA and PFDA significantly increased hepatic concentration of triacylglycerol. The perfluorocarboxylic acids at relatively high doses caused accumulation of cholesterol in liver. Electron microscopic studies showed that the administration of PFOA and PFDA caused an increase in cell size and proliferations of peroxisomes, and that the treatment of rats with PFDA at dietary concentration of 0.01% caused a marked increase in small lipid droplet in hepatocytes, indicative of hepatotoxic manifestations. The present results suggest that when PFOA and PFDA are administered at low levels, there are no differences between the properties of the perfluorocarboxylic acids as peroxisome proliferators, although the administration of PFDA at the doses exceeding a certain level becomes markedly toxic to hepatocytes.

Stimulatory effect of lead on the proliferation of cultured vascular smooth-muscle cells.

The effect of lead nitrate on the DNA synthesis of cultured bovine aortic smooth-muscle cells has been studied. Lead at 0.5-10 microM stimulated the incorporation of [3H]thymidine into the acid-insoluble fraction of the cells in a dose-dependent manner. However, other heavy metals including zinc, copper, manganese and nickel did not show such a stimulatory effect. Stimulation of [3H]thymidine incorporation by basic fibroblast growth factor was additive to that by lead. However, stimulation by either platelet-derived growth factor or acidic fibroblast growth factor was reduced by the metal. The leakage of lactate dehydrogenase into the medium from smooth-muscle cells, a marker of cell death, was not changed by the metal. Calcium ionophore A23187 inhibited [3H]thymidine incorporation in vascular endothelial cells but stimulated it in vascular smooth-muscle cells. These results suggest that lead may stimulate the proliferation of cultured vascular smooth-muscle cells probably via a calcium-dependent pathway; the metal may mimic calcium within the cells.

Stimulative effects of lead on bone resorption in organ culture.

To clarify whether hypercalcemia after injection of Pb to rats is due to biological bone resorption or physicochemical mineral dissolution, the effect of lead (Pb) on release of previously incorporated 45Ca in organ culture was investigated. Pb at 50 microM and above stimulated the release of 45Ca and hydroxyproline (Hyp). Pb did not stimulate 45Ca release from the bones inactivated by freezing and thawing. Eel calcitonin (ECT), bafilomycin A1 and scopadulcic acid B (SDB) inhibited Pb-stimulated 45Ca release. These results indicate that Pb-induced 45Ca release is due to osteoclastic bone resorption. Pb-stimulated bone resorption was inhibited by indomethacin and flurbiprofen. Pb stimulated the release of prostaglandin E2 (PGE2) from the bones into the media. There was significantly high correlation between 45Ca and PGE2 release. Pb-induced bone resorption was inferred to be mediated by PGE2. From these results, it was suggested that hypercalcemia after Pb injection might be caused by biological bone resorption.