Isolation and characterization of a novel lectin from the wild mushroom Xerocomus spadiceus

A lectin was isolated from extracts of fruiting bodies of the mushroom Xerocomus spadiceus using a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, and gel filtration by fast protein liquid chromatography on Superdex 75. The lectin was unadsorbed on DEAE-cellulose and Affi-gel blue gel and adsorbed on CM-Sepharose. The lectin was capable of eliciting an approximately four-fold stimulation of mitogenic response in murine splenocytes. The hemagglutinating activity was stable up to 60 degrees C, halved at 70 degrees C, reduced to 25% at 75 degrees C and dwindled to an undetectable level at 80 degrees C. The activity remained unaltered in the presence of various divalent (Ca2+, Mg2+, Zn2+ and Mn2+) chlorides up to a salt concentration of 10 mM except in the case of ZnCl2. FeCl3 up to a concentration of 10 mM did not affect the hemagglutinating activity of the lectin. The hemagglutinating activity of the lectin was doubled in the presence of 5 mM AlCl3 or 10 mM ZnCl2 and quadrupled in the presence of 10 mM AlCl3. The activity was reduced in the presence of HCl and NaOH. Among the large number of carbohydrates tested, only inulin was able to inhibit the hemagglutinating activity of the lectin.     

Purification and partial characterization of manganese peroxidase from immobilized Phanerochaete chrysosporium

Solid-state culture of the white-rot fungus Phanerochaete chrysosporium BKMF-1767 (ATCC 24725) has been carried out, using an inert support, polystyrene foam. Suitable medium and culture conditions have been chosen to favor the secretion of manganese peroxidase (MnP). The enzyme was isolated and purified from immobilized P. chrysosporium and partially characterized. Partial protein precipitation in crude enzyme was affected using ammonium sulphate, polyethylene glycol, methanol, and ethanol methods. Fractionation of MnP was performed by DEAE-Sepharose ion exchange chromatography followed by Ultragel AcA 54 gel filtration chromatography. This purification attained 23.08% activity yield with a purification factor of 5.8. According to data on gel filtration chromatography and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weight of the enzyme was 45 000±1000 Da. The optimum pH and temperature of purified MnP were 4.5 and 30 °C, respectively. This enzyme was stable in the pH range 4.5–6.0, at 25 °C and also up to 35 °C at pH 4.5 for 1 h incubation period. MnP activity was inhibited by 2 mM NaN₃, ascorbic acid, β-mercaptoethanol and dithreitol. The K_m values of MnP for hydrogen peroxide and 2.6-dimetoxyphenol were 71.4 and 28.57 μM at pH 4.5, respectively. The effects of possible inhibitors and activators of enzyme activity were investigated.     

Arachidonate lipoxygenase inhibitors in guinea-pig isolated trachea. Effects on contractions to antigen and various agonists

We compared the effects of the leukotriene (LT) D₄ receptor antagonist FPL55712 and some lipoxygenase inhibitors on contractions of isolated guinea-pig trachea induced by antigen (ovalbumin, OA) and calcium ionophore A23187 in the presence of the cyclooxygenase inhibitor indomethacin (5 μM), and by arachidonic acid (AA), melittin and LTD₄. FPL55712 (0.1 and 1 μM) inhibited contractions induced by AA (100 μM) and the phospholipase A₂ activator melittin (3 μg/ml), while the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 μM) was a more effective inhibitor of the melittin response than the response. FPL55712 inhibited contractions induced by OA (100 μg/ml) more than by A23187 (1 μg/ml), and these inhibitory effects of FPL55712 were much less in the presence of l-serine-borate complex (45 mM), an inhibitor of LTC₄ conversion to LTD₄. NDGA (10 μM) had no significant effect on the OA response, whereas the lipoxygenase inhibitors 1-phenyl-3-pyrazolidone (phenidone, 10 μM) and 5,8,11,14-eicosatetraynoic acid (ETYA, 10 μM) clearly inhibited it. In contrast, NDGA and phenidone inhibited the A23187 response, but ETYA had no effect on it. FPL55712, phenidone and ETYA, but not NDGA, had a large inhibitory effect on LTD₄-induced contractions, but these inhibitors had no effect on histamine-induced contractions. These results suggest that in the guinea-pig trachea inhibitors of LTD₄-induced contractions decrease antigen-induced contractions, whereas lipoxygenase inhibitors reduce the contraction to A23187.     

Carotenoid oxidative degradation products inhibit Na+-K+-ATPase

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na⁺-K⁺-ATPase activity as an index. β-Carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gasliquid chromatography and high performance liquid chromatography. The Na⁺-K⁺-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of β-carotene oxidative breakdown products, β-Apo-10'-carotenal and retinal. Most of the β-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na⁺-K⁺-ATPase activity by 50% (IC₅₀) was equivalent to 10μM non-degraded β-carotene, whereas the IC₅₀ for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 μM. Carotenoid oxidation products are more potent inhibitors of Na⁺-K⁺-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or β-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.     

The role of calcium channels in substance P-induced contractile response in the rat iris

This study was undertaken to assess the role of calcium channels in the contractile response induced by substance P in the isolated rat iris. Substance P produced graded and sustained contraction in the rat iris. Pre-incubation of preparations with thapsigargin (1 μM), verapamil (1 μM), isradipine (1 μM) or with ω-conotoxin MCIIA (0.1 μM) did not significantly inhibit substance P-mediated contraction in the isolated rat iris. However, pre-incubation of the preparations with nicardipine (1 μM) or ruthenium red (1 mM) caused parallel displacement to the right of the substance P concentration–response curve without affecting its maximal response. In contrast, amiloride (1 μM), markedly inhibited substance P-mediated contraction (73±5%), while econazole (1 mM) also significantly inhibited (44±11%) substance P-mediated contraction in the isolated rat iris. Collectively, these results suggest that substance P-mediated contractile response in the isolated rat iris depends largely on the influx of external Ca²⁺, by a mechanism which might involve the T-type calcium channels.     

Anti-allergic substances from the rhizomes of Dioscorea membranacea

Extracts of five species of Thai medicinal plants, locally known as Hua-Khao-Yen, were screened for anti-allergic activities using RBL-2H3 cells. Of the five species studied, the ethanolic extract of Dioscorea membranacea exhibited potent inhibitory activity against β-hexosaminidase release as a marker of degranulation in RBL-2H3 cells, with an IC₅₀ value of 37.5 μg/mL. Eight compounds were isolated from this crude ethanolic extract, [two naphthofuranoxepins (1, 2), one phenanthraquinone (3), three steroids (4–6), and two steroidal saponins (7, 8)], and tested for their anti-allergic activities. The results showed that dioscorealide B (2) possessed the highest activity with an IC₅₀ value of 5.7 μM, followed by dioscoreanone (3, IC₅₀ = 7.7 μM), dioscorealide A (1, IC₅₀ = 27.9 μM), and diosgenin (9, IC₅₀ = 29.9 μM). Structure–activity relationship studies of naphthofuranoxepins on anti-allergic activity revealed that the hydroxylation at position 8 conferred higher activity than methoxylation. For diosgenin derivatives, the aglycone was found to possess higher activity than the diglucosylated molecule; whereas substitution with rhamnoglucosides apparently results in loss of activity. Furthermore, effects of dioscorealide A, dioscorealide B, and dioscoreanone on antigen-induced release of TNF- and IL-4 in the late phase reaction were also examined.     

Dithioerythritol (DTE) prevents inhibitory effects of triphenyltin (TPT) on the key enzymes of the human sex steroid hormone metabolism

Organotins are known to induce imposex (pseudohermaphroditism) in marine neogastropods and are suggested to act as specific endocrine disruptors, inhibiting the enzyme-mediated conversion of steroid hormones. Therefore, we investigated the in vitro effects of triphenyltin (TPT) on human 5-reductase type 2 (5-Re 2), cytochrome P450 aromatase (P450arom), 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD 3), 3β-HSD type 2 and 17β-HSD type 1 activity. First, the present study demonstrates that significant amounts of TPT occurred in the blood of eight human volunteers (0.17–0.67 μg organotin cation/l, i.e. 0.49–1.92 nmol cation/l). Second, TPT showed variable inhibitory effects on all the enzymes investigated. The mean IC₅₀ values were 0.95 μM for 5-Re 2 (mean of n=4 experiments), 1.5 μM for P450arom (n=5), 4.0 μM for 3β-HSD 2 (n=1), 4.2 μM for 17β-HSD 3 (n=3) and 10.5 μM for 17β-HSD 1 (n=3). To exclude the possibility that the impacts of TPT are mediated by oxidizing essential thiol residues of the enzymes, the putative compensatory effects of the reducing agent dithioerythritol (DTE) were investigated. Co-incubation with DTE (n=3) resulted in dose-response prevention of the inhibitory effects of 100 μM deleterious TPT concentrations on 17β-HSD 3 (EC₅₀ value of 12.9 mM; mean of n=3 experiments), 3β-HSD 2 (0.90 mM; n=3), P450arom (0.91 mM; n=3) and 17β-HSD 1 (0.21 mM; n=3) activity. With these enzymes, the use of 10 mM DTE resulted in an at least 80% antagonistic effect, whereas, the effect of TPT on 5-Re 2 was not compensated. In conclusion, the present study shows that TPT acts as an unspecific, but significant inhibitor of human sex steroid hormone metabolism and suggests that the inhibitory effects are mediated by the interaction of TPT with critical cysteine residues of the enzymes.     

Modification at the C9 position of the marine natural product isoaaptamine and the impact on HIV-1, mycobacterial, and tumor cell activity

As part of an investigation to generate optimized drug leads from marine natural pharmacophores for the treatment of neoplastic and infectious diseases, a series of novel isoaaptamine analogs were prepared by coupling acyl halides to the C9 position of isoaaptamine (2) isolated from the Aaptos sponge. This library of new semisynthetic products was evaluated for biological activity against HIV-1, Mtb, AIDS-OI, tropical parasitic diseases, and cancer. Compound 4 showed potent activity against HIV-1 (EC₅₀ 0.47 μg/mL), compound 19 proved to possess remarkable activity against Mycobacterium intracellulare with an IC₅₀ and MIC value of 0.15 and 0.31 μg/mL, while compounds 4 and 17 possessed anti-leishmanial activity with IC₅₀ values of 0.1 and 0.4 μg/mL, respectively. Compounds 16 and 17 showed antimalarial activity with EC₅₀ values of 230 and 240 ng/mL, respectively, and compound 14 exhibited an EC₅₀ of 0.05 μM against the Leukemia cell line K-562.     

A nitrite reductase with cytochrome oxidase activity from Micrococcus denitrificans

The formation of nitrite reductase and cytochrome c in Micrococcus denitrificans was repressed by O₂. The purified nitrite reductase utilized reduced forms of cytochrome c, phenazine methosulphate, benzyl viologen and methyl viologen, respectively, as electron donors. The enzyme was inhibited by KCN, NaN₃ and NH₂OH each at 1 mM, whereas CO and bathocuproin, diethyl dithiocarbamate, o-phenanthroline and ,'-dipyridyl at 1 mM concentrations were relatively ineffective. The purified enzyme contains cytochromes, probably of the c and a₂ types, in one complex. A K_m of 46 μM for NO₂⁻ and a pH optimum of 6.7 were recorded for the enzyme. The molecular weight of the enzyme was estimated to be around 130000, and its anodic mobility was 6.8·10⁻⁶ cm²·sec⁻¹·V⁻¹ at pH 4.55.

The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a K_m of 27 μM for O₂. This activity was also inhibited by KCN, NaN₃ and NH₂OH and by NO₂⁻.

A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO₂⁻. It was inhibited by smaller amounts of KCN, NaN₃ and NH₂OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a K_m for O₂ of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO₂⁻.

The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller K_m for O₂ and would therefore be expected to function more efficiently especially at low concentrations of O₂.     

A novel lectin from the wild mushroom Polyporus adusta

A lectin with antiproliferative activity toward tumor cell lines and mitogenic activity toward splenocytes was isolated from the mushroom Polyporus adusta. The lectin was composed of two identical subunits each with a molecular weight of 12 kDa. It was adsorbed on both DEAE-cellulose and Q-Sepharose and unadsorbed on CM-Sepharose. The hemagglutinating activity of the lectin was inhibited by turanose and by a large variety of other carbohydrates. It was adversely affected in the presence of NaOH or HCl at a concentration of 7.5mM and above, and when the ambient temperature was raised above 70 degrees C. All divalent and trivalent metallic chlorides tested at 1.25-10mM including CaCl(2), MgCl(2), ZnCl(2), MnCl(2), and AlCl(3), did not alter the hemagglutinating activity of the lectin. FeCl(3) at 10mM caused the hemagglutinating activity to increase by 100%, but it did not change the lectin activity when tested at lower concentrations up to 5mM.     

Anti-proliferative lichen compounds with inhibitory activity on 12(S)-HETE production in human platelets

Several lichen compounds, i.e. lobaric acid (1), a β-orcinol depsidone from Stereocaulon alpinum L., (+)-protolichesterinic acid (2), an aliphatic -methylene-γ-lactone from Cetraria islandica Laur. (Parmeliaceae), (+)-usnic acid (3), a dibenzofuran from Cladonia arbuscula (Wallr.) Rabenh. (Cladoniaceae), parietin (4), an anthraquinone from Xanthoria elegans (Link) Th. Fr. (Calaplacaceae) and baeomycesic acid (5), a β-orcinol depside isolated from Thamnolia vermicularis (Sw.) Schaer. var. subuliformis (Ehrh.) Schaer. were tested for inhibitory activity on platelet-type 12(S)-lipoxygenase using a cell-based in vitro system in human platelets. Lobaric acid (1) and (+)-protolichesterinic acid (2) proved to be pronounced inhibitors of platelet-type 12(S)-lipoxygenase, whereas baeomycesic acid (5) showed only weak activity (inhibitory activity at a concentration of 100 μg/ml: 1 93.4±6.62%, 2 98,5±1.19%, 5 14.7±2.76%). Usnic acid (3) and parietin (4) were not active at this concentration. 1 and 2 showed a clear dose–response relationship in the range of 3.33–100 μg/ml. According to the calculated IC₅₀ values the highest inhibitory activity was observed for the depsidone 1 (IC₅₀=28.5 μM) followed by 2 (IC₅₀=77.0 μM). The activity of 1 was comparable to that of the flavone baicalein, which is known as a selective 12(S)-lipoxygenase inhibitor (IC₅₀=24.6 μM).     

Effect of clomiphene on Ca movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca²⁺ indicator. Clomiphene at concentrations between 10-50 μM increased [Ca²⁺]_i in a concentration-dependent manner. The [Ca²⁺]_i signal was biphasic with an initial rise and a slow decay. Ca²⁺ removal inhibited the Ca²⁺ signal by 41%. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with clomiphene in Ca²⁺-free medium, confirming that clomiphene induced Ca²⁺ entry. In Ca²⁺-free medium, pretreatment with 50 μM brefeldin A (to permeabilize the Golgi complex), 1 μM thapsigargin (to inhibit the endoplasmic reticulum Ca²⁺ pump), and 2 μM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 μM clomiphene-induced store Ca²⁺ release. Conversely, pretreatment with 50 μM clomiphene in Ca²⁺-free medium abolished the [Ca²⁺]_i increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 μM clomiphene-induced Ca²⁺release was unaltered by inhibiting phospholipase C with 2 μM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 μM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca²⁺]_i increases in PC3 cells by releasing store Ca²⁺ from multiple stores in an phospholipase C-independent manner, and by activating Ca²⁺ influx; and clomiphene was of mild cytotoxicity.     

Formaldehyde-sensitive sensor based on recombinant formaldehyde dehydrogenase using capacitance versus voltage measurements

A new formaldehyde-selective biosensor was constructed using NAD⁺- and glutathione-dependent recombinant formaldehyde dehydrogenase as a bio-recognition element immobilised on the surface of Si/SiO₂/Si₃N₄ structure. Sensor's response to formaldehyde was evaluated by capacitance measurements. The calibration curves obtained for formaldehyde concentration range from 10 μM to 20 mM showed a broad linear response with a sensitivity of 31 mV/decade and a detection limit about 10 μM. It has been shown that the output signal decreases with the increase of borate buffer concentration and the best sensitivity is observed in 2.5 mM borate buffer, pH 8.40. The response of the created formaldehyde-sensitive biosensor has also been examined in 2.5 mM Tris–HCl buffer, and the shift to the positive bias of the C(V) curves along with the potential axis has been observed, but the sensitivity of the biosensor in this buffer is decreased dramatically to the value of 2.4 mV/decade.     

Photosynthetic photon flux effects on bean response to nitrogen dioxide

Phaseolus vulgaris L. cv. Kinghorn Wax seedlings, supplied with nutrient solution containing either 0 or 5 mM nitrate as sole N source, were exposed to 0.25 μl/l NO₂ for 6 hr each day for 10 days at continuous photosynthetic photon flux (PPF) of 100, 300, 500 or 700 μmol m⁻² sec⁻¹. There was a significant interaction of PPF and nitrate. Shoot and root dry weights increased with increasing PPFs only when nitrate was supplied. The main effects of NO₂ on plant growth were significant; none of the interactions involving NO₂ were significant. Exposure to NO₂ decreased shoot and root dry weight in both the presence and absence of nutrient N and at all PPF levels. All interactions were significant for in vitro leaf nitrate reductase activity (NRA), which increased markedly at PPFs above 100 μmol m⁻² sec⁻¹ when nitrate was supplied. Treatment with NO₂ strongly inhibited enzyme activity in the presence of nitrate, particularly at the 300 μmol m⁻² sec⁻¹ PPF level. These experiments demonstrated that PPF level does not modify the effect of NO₂ on growth but does have a major effect on NRA and on NO₂ effects on NRA in the presence of nutrient nitrate.     

Effect of lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17β-estradiol, tamoxifen and clomiphene)-induced Ca²⁺ mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca²⁺–containing medium, the lignans (50–100 μM) inhibited 10 μM 17β-estradiol- and 5 μM tamoxifen-induced increases in intracellular free Ca²⁺ levels ([Ca²⁺]_i) without changing 25 μM clomiphene-induced [Ca²⁺]_i increase. 17β-estradiol and tamoxifen increased [Ca²⁺]_i by causing Ca²⁺ influx and Ca²⁺ release because their responses were partly reduced by removing extracellular Ca²⁺. In contrast, clomiphene solely induced Ca²⁺ release. The effect of the lignans on these two Ca²⁺ movement pathways underlying 17β-estradiol- and tamoxifen-induced [Ca²⁺]_i increases was explored. All the lignans (50–100 μM) inhibited 10 μM 17β-estradiol-and 5 μM tamoxifen-induced Ca²⁺ release, and 17β-estradiol-induced Ca²⁺ influx. However, only 100 μM epi-aschantin was able to reduce tamoxifen-induced Ca²⁺ influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca²⁺ signaling in human neutrophils in a multiple manner.     

Caspases are reversibly inactivated by hydrogen peroxide

Hydrogen peroxide (H₂O₂) is known to both induce and inhibit apoptosis, however the mechanisms are unclear. We found that H₂O₂ inhibited the activity of recombinant caspase-3 and caspase-8, half-inhibition occurring at about 17 μM H₂O₂. This inhibition was both prevented and reversed by dithiothreitol while glutathione had little protective effect. 100–200 μM H₂O₂ added to macrophages after induction of caspase activation by nitric oxide or serum withdrawal substantially inhibited caspase activity. Activation of H₂O₂-producing NADPH oxidase in macrophages also caused catalase-sensitive inactivation of cellular caspases. The data suggest that the activity of caspases in cells can be directly but reversibly inhibited by H₂O₂.