microM CuSO4 Research Articles

Implication of lipoprotein associated phospholipase A2 activity in oxLDL uptake by macrophages

Recognition and uptake of oxidized LDL (oxLDL) by scavenger receptors of macrophages and foam cell formation are mediated by the oxidatively modified apolipoprotein B (ApoB) and lipid moiety of oxLDL. A great amount of oxidized phosphatidylcholine (oxPC) of oxLDL is hydrolyzed at the sn-2 position by lipoprotein associated phospholipase A(2) (Lp-PLA(2)) to lysophosphatidylcholine and small oxidation products. This study examines the involvement of Lp-PLA(2) in the uptake of oxLDL by mouse peritoneal macrophages. LDL with intact Lp-PLA(2) activity [LDL(+)] and LDL with completely inhibited Lp-PLA(2) activity [LDL(-)] were subjected to oxidation with 5 microM CuSO(4) for 6 h [moderately oxLDL (MoxLDL)], or 24 h [heavily oxLDL (HoxLDL)] and peritoneal macrophages were incubated with these preparations. The uptake of MoxLDL(-) was about 30% increased compared with that of MoxLDL(+), and HoxLDL(-) uptake was about 20% increased compared with that of HoxLDL(+). Inhibition of Lp-PLA(2) activity had no effect on the uptake of ApoB-liposomes conjugates with ApoB isolated from MoxLDL(-), MoxLDL(+), HoxLDL(-), and HoxLDL(+). Liposomes prepared from the lipid extract of MoxLDL(-), MoxLDL(+), HoxLDL(-), and HoxLDL(+) exhibited a similar pattern to that observed in the uptake of the corresponding intact lipoproteins. This study suggests that the progressive inactivation of Lp-PLA(2) during LDL oxidation leads to an increased uptake of oxLDL by macrophages, which could be primarily attributed to the increased uptake of the oxidized phospholipids enriched lipid moiety of oxLDL.

Cupric stress induces oxidative damage marked by accumulation of H2O2and changes to chloroplast ultrastructure in primary leaves of beans (Phaseolus vulgarisL.)

The effect of copper excess (CuSO 4 ) on lipid peroxidation, H2O2 content, and antioxidative enzyme activities was studied in primary leaves of bean seedlings. Fourteen-day-old bean seedlings were cultured in a nutrient solution containing Cu 2+ at various concentrations (50 and 75 microM) for 3 days. Excess of copper significantly increased malondialdehyde content and endogenous H2O2 . This radical accumulated in the intercellular spaces of palisade mesophyll cells. In addition, cupric stress induced changes in antioxidant enzyme activities. GPX (guaiacol peroxidase, EC 1.11.1.7) activity was decreased in 50 microM Cu-stressed leaves whereas 75 microM of CuSOP 4 resulted in an increase of enzyme activity. On the contrary, CAT (catalase, EC 1.11.1.6) activity was stimulated at 50 microM CuSO 4 but unaltered at 75 microM CuSO 4 . Transmission electron microscopy revealed that excess copper induced changes in the ultrastructure of chloroplasts visible in form of a deterioration in the grana structure and the accumulation and swelling of starch grains in the stroma.

Structural Changes of Cell Wall and Lignifying Enzymes Modulations in Bean Roots in Response to Copper Stress

Fourteen-day-old bean seedlings were cultured in nutrient solution containing Cu(2+) ions at various concentrations (50 and 75 microM of CuSO(4)) for 3 days. This excess of copper induced a reduction in the water volume absorbed by the plants. Moreover, this reduction was accompanied by an increase of the amount of copper taken up by the roots. Analysis by native gel electrophoresis of cell wall peroxidase activities in the roots revealed a stimulation of two anionic isoforms (A(2) and A(3)) under cupric stress conditions. Moreover, the activity of phenylalanine ammonia lyase (EC. 4.3.1.5), which plays an important role in plant defense, was enhanced. Copper-treated bean roots showed modifications in the cell walls of various tissues. Label for lignin, callose, and suberin with berberine-aniline blue revealed abnormal cell wall thickenings in the endodermis, the phloem, and the xylem, especially in plants treated with 75 microM CuSO(4).

Interactions of free copper (II) ions alone or in complex with iron (III) ions with erythrocytes of marine fish Dicentrarchus labrax

As a consequence of human activity, various toxicants - especially metal ions - enter aquatic ecosystems and many fish are exposed to considerable levels. As the free ion and in some complexes, there is no doubt that copper promotes damage to cellular molecules and structures through radical formation. Therefore, we have investigated the influence of copper uptake by the red blood of the sea bass (Dicentrarchus labrax), and its oxidative action and effects on cells in the presence of complexed and uncomplexed Fe3+ ions. Erythrocytes were exposed to various concentrations of CuSO4, Fe(NO3)3, and K3Fe(CN)6 for up to 5h, and the effects of copper ions alone and in the combination with iron determined. The results show that inside the cells cupric ion interacts with hemoglobin, causing methemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+. Potassium ferricyanide as a source of complexed iron decreases Met-Hb formation induced by copper ions unlike Fe(NO3)3. We also found that incubation of fish erythrocytes with copper increased hemolysis of cells. But complexed and uncomplexed iron protected the effect of copper. CuSO4 increased the level of lipid peroxidation and a protective effect on complexed iron was observed. Incubation of erythrocytes with copper ions resulted in the loss of a considerable part of thiol content at 10 and 20 microM. This effect was decreased by potassium ferricyanide and Fe(NO3)3 only after 1 and 3h of incubation. The level of nuclear DNA damage assayed by comet assay showed that 20 microM CuSO4 as well as 20 microM Fe(NO3)3 and 10 mM K3Fe(CN)6 induce single- and double-strand breaks. The lower changes were observed after the exposure of cells to K3Fe(CN)6. The data suggest that complexed iron can act protectively against copper ions in contrast to Fe(NO3)3.

Expression of merA, amoA and hao in continuously cultured Nitrosomonas europaea cells exposed to zinc chloride additions

The effects of ZnCl2 additions on a mercuric reductase, merA, ammonia monooxygenase, amoA, and hydroxylamine (NH2OH) oxidoreductase, hao, gene expression were examined in continuously cultured Nitrosomonas europaea cells. The reactor was operated for 85 days with a 6.9 d hydraulic retention time and with four successive additions of ZnCl2 achieving maximum concentrations from 3 to 90 microM Zn2+. Continuously cultured N. europaea cells were more resistant to Zn2+ inhibition than previously examined batch cultured cells due to the presence of Mg2+ in the growth media, suggesting that Zn2+ enters the cell through Mg2+ import channels. The maximum merA up-regulation was 45-fold and expression increased with increases in Zn2+ concentration and decreased as Zn2+ concentrations decreased. Although Zn2+ irreversibly inactivated ammonia oxidation in N. europaea, the addition of either 600 microM CuSO4 or 2250 microM MgSO4 protected N. europaea from ZnCl2 inhibition, indicating a competition between Zn2+ and Cu2+/Mg2+ for uptake and/or AMO active sites. Since ZnCl2 inhibition is irreversible and amoA was up-regulated at 30 and 90 microM additions, it is hypothesized that de novo synthesis of the AMO enzyme is needed to overcome inhibition. The up-regulation of merA during exposure to non-inhibitory Zn2+ levels indicates that merA is an excellent early warning signal for Zn2+ inhibition.

High copper levels and increased elastolysis in a patient with cutis marmorata teleangiectasia congenita

Cutis marmorata telangiectatica congenita (CMTC) is a rare cutaneous vascular disease presenting at birth with levido reticularis, phlebectasia, and telangiectasia, often accompanied by skin ulcerations. Extra-dermal vascular anomalies can be also detected in 30-70% of described cases. The pathomechanism responsible for development of these phenotypic changes is not well understood. Here, we report on a 16-month-old boy with CMTC, generalized vascular abnormalities and severe, nitric oxide sensitive, pulmonary hypertension associating with markedly elevated level of blood copper. Results of laboratory investigations indicated that primary cultures (passage one) of dermal fibroblasts derived from this patient were capable of normal synthesis of tropoelastin, but their net deposition of mature elastic fibers was significantly diminished as compared with cultures of normal fibroblasts. Because the low net deposition of elastin was reversed when 1 mg/ml of alpha1-antitrypsin was added to the media, we conclude that heightened elastolysis by endogenous serine proteinase's is responsible for the low net elastogenesis by CMTC fibroblasts. Since simultaneous addition of 30 microM CuSO(4) and 1 mg/ml alpha1-antitrypsin abolished the beneficial effect of this serine proteinase's inhibitor, we concluded that this may be due to copper-dependent inactivation of alpha1-antitrypsin. Our data suggest that a high level of free copper may constitute a major triggering factor contributing to the development of the CMTC phenotype.

Effects of apoptosis and lipid peroxidation on T-lymphoblastoid phospholipid-dependent procoagulant activity.

Coagulation has an absolute requirement for macromolecular complexes to be assembled on a negatively charged phospholipid (PL) surface. Previously, we reported that malignant T-lymphoblastoid cells have the ability to support procoagulant activity (PCA) independently of tissue factor by providing such a surface. To explore the effect of two pathophysiologic processes, apoptosis and lipid peroxidation (LP), on this PL-dependent PCA. Three different assays for PL-dependent PCA (factor IXa-initiated FXa and thrombin generation and prothrombinase activity) were used to investigate this PCA after exposing three T-lymphoblastoid cell lines to either apoptotic stimuli (1 microM staurosporine) or oxidative stress (4 mm H(2)O(2) and 40 microM CuSO(4)). Surface exposure of anionic PL was measured by flow cytometry using annexin A5(FITC) and an antibody (3G4) specific for native, but not oxidized, phosphatidylserine (PS). Both apoptosis and LP significantly enhanced the PCA of cells, to a level that was greater than that observed following calcium ionophore treatment, suggesting that the increased activity was not solely due to anionic PL exposure. Whereas cells undergoing apoptosis bound both annexin A5(FITC) and 3G4, only annexin A5(FITC) bound to cells undergoing LP. This implies that apoptosis increases PCA by causing the translocation of oxidized/native PS to the outer membrane, whereas LP appears to increase the PCA, possibly due to malondialdehyde adducts altering the net charge on the cell surface, which allows PLs other than PS to participate in thrombin generation.

Declination of Copper Toxicity in Pigeon Pea and Soil System by Growth-Promoting Proteus vulgaris KNP3 Strain

The copper-resistant (1318 microM CuSO(4).5H(2)O) strain KNP3 of Proteus vulgaris was isolated from soil near the Panki power plant, Kanpur, India, and was used to inoculate pigeon pea (Cajanus cajan var. UPS-120) seeds grown in soil for 60 days in the presence of 600 microM CuSO(4).5H(2)O. A study of siderophore production (126.34 +/- 0.52 microg ml(-1)) and its subsequent effects on plant growth promotion under in situ conditions was conducted. The parameters that were monitored included the plants' wet weight, dry weight, shoot length, chlorophyll content, and concentration of copper in plant roots and shoots. The results showed that the strain caused a significant (p < 0.05) increase in wet weight, dry weight, root length, shoot growth, and chlorophyll content (57.8%, 60%, 19.7%, 47.8%, and 36.3%, respectively) in the presence of copper. Furthermore, the strain reduced accumulation of Cu in the roots and shoots to 36.8% and 60.5%, respectively. Apart from this, copper concentration in the soil was measured on 0, 7, 15, 30, and 45 days consecutively and the results indicated that the bioinoculant KNP3 causes a significant decrease in Cu concentration in soil (55.6%), which was unlikely in the control (10.5%) treatment. The data suggested that the bacterial strain has the ability to protect plants against the inhibitory effects of copper besides reducing the copper load of the soil.

Evidence for involvement of copper ions and redox state in regulation of butane monooxygenase in Pseudomonas butanovora.

Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C(2)-C(9) alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of beta-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O(2) when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH(4)Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O(2) (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O(2), CuSO(4) (0.5 microM) repressed 1-butanol-dependent induction of beta-galactosidase activity. Under oxic conditions (20% O(2) [vol/vol]), significantly higher concentrations of CuSO(4) (2 microM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu(2+) reducing agent Na ascorbate (100 microM) and CuSO(4) (0.5 microM) repressed the induction of beta-galactosidase activity under oxic conditions to the same extent that 0.5 microM CuSO(4) alone repressed it under anoxic conditions. Under oxic conditions, 2 microM CuSO(4) repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO(4) repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains.

Cloning and characterization ofTsMT3, a type 3 metallothionein gene from salt cress (Thellungiella salsuginea)

A full-length type 3 plant metallothionein cDNA was isolated from 200 mM NaCl stressed shoots of the salt cress (Thellungiella salsuginea). The 447 bp TsMT3 cDNA sequence has a 207 bp open reading frame (ORF) and encodes a deduced 69 residue peptide of molecular weight 7.52 kDa. Southern blot analysis indicates that, there is only one copy of TsMT3 in the T. salsuginea genome. The accumulation of TsMT3 mRNA is enhanced by the stress imposed by PEG6000, 200 mM NaCl, 50 microM ABA, 4 degrees C, 40 microM CuSO(4) or 25 microM CdCl2. The expression vector pET28-TsMT3 was heterologously expressed in Escherichia coli to define the contribution of TsMT3 to heavy metal tolerance. In the presence of 2 mM CuSO4, 0.3 mM Pb(NO3)2 or 0.4 mM CdCl2, TsMT3 expressing cells exhibited enhanced metal tolerance and accumulated more metal than the controls. We believe that TsMT3 is probably involved in the processes of metal homeostasis, tolerance, and reactive oxygen species (ROS) scavenging.

Regulation of Copper‐Dependent Endocytosis and Vacuolar Degradation of the Yeast Copper Transporter, Ctr1p, by the Rsp5 Ubiquitin Ligase

The Saccharomyces cerevisiae high-affinity copper transporter, Ctr1p, mediates cellular uptake of Cu(I). We report that when copper (50 microm CuSO(4)) is added to the growth medium of copper-starved cells, Ctr1p is rapidly internalized by endocytosis, delivered to the lumen of the lysosome-like vacuole and slowly degraded by vacuolar proteases. Through analysis of the trafficking and degradation of Ctr1p mutants, two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-green fluorescent protein were substantially diminished. Surprisingly, a Ctr1p mutant that lacks Lys340 and Lys345 was still ubiquitylated in a copper-dependent manner, indicating that ubiquitylation of Ctr1p on other sites is insufficient to drive copper-dependent endocytosis and degradation. This study demonstrates that copper regulates turnover of Ctr1p by stimulating Rsp5p-dependent endocytosis and degradation of Ctr1p in the vacuole.

Over-expression of the apple spermidine synthase gene in pear confers multiple abiotic stress tolerance by altering polyamine titers

An apple spermidine synthase (SPDS) gene (MdSPDS1) was verified to encode a functional protein by the complementation of the spe3 yeast mutant, which lacks the SPDS gene. To justify our hypothesis that apple SPDS is involved in abiotic stress responses and to obtain transgenic fruit trees tolerant to abiotic stresses as well, MdSPDS1-over-expressing transgenic European pear (Pyrus communis L. 'Ballad') plants were created by Agrobacterium-mediated transformation. A total of 21 transgenic lines showing various spermidine (Spd) titers and MdSPDS1 expression levels were obtained. Selected lines were exposed to salt (150 mM NaCl), osmosis (300 mM mannitol), and heavy metal (500 microM CuSO4) stresses for evaluating their stress tolerances. Transgenic line no. 32, which was revealed to have the highest Spd accumulation and expression level of MdSPDS1, showed the strongest tolerance to these stresses. When growth increments, electrolyte leakage (EL), and values of thiobarbituric acid reactive substances (TBARS) were monitored, line no. 32 showed the lowest growth inhibition and the least increase in EL or TBARS under stress conditions. Spd titers in wild-type and transgenic lines showed diverse changes upon stresses, and these changes were not consistent with the changes in MdSPDS1 expressions. Moreover, there were no differences in the sodium concentration in the shoots between the wild type and line no. 32, whereas the copper concentration was higher in the wild type than in line no. 32. Although the mechanism(s) underlying the involvement of polyamines in stress responses is not known, these results suggest that the over-expression of the SPDS gene substantially increased the tolerance to multiple stresses by altering the polyamine titers in pear. Thus, MdSPDS1-over-expressing transgenic pear plants could be used to improve desert land and/or to repair polluted environments.

Antioxidative responses of duckweed (Lemna minor L.) to short-term copper exposure

Elevated concentrations of copper in the environment result in accumulation of the metal in plants and cause an increase in reactive oxidative species (ROS). The first response to elevated amounts of ROS is increased levels of enzymatic and non-enzymatic antioxidants that reduce oxidative stress. The aim of our study was to evaluate the early stages of antioxidative responses to the low copper concentrations usually present in moderately polluted environments. In addition, some other parameters were examined to evaluate the effect of copper on plants. Duckweed (Lemna minor L.) was exposed to different concentrations of copper sulphate for up to 24 hours. Glutathione concentration and enzymatic activities of catalase, guaiacol peroxidase and glutathione reductase were measured spectrophotometrically. Additionally, delayed and prompt chlorophyll fluorescence was measured by luminometry and fluorometry, respectively. The accumulation of copper in plants exposed for 24 hours to various concentrations of copper sulphate was measured by flame atomic absorption spectrophotometry. The treatment of plants with copper sulphate resulted in an immediate decrease of the glutathione pool, which was replenished after 24 hours at CuSO4 concentrations lower than 2 microM. Higher CuSO4 concentrations caused a decrease of reduced glutathione. The responses of the antioxidant enzymes glutathione reductase, guaiacol peroxidase and catalase to CuSO4 differed during the first six hours of exposure, but their enzyme activities all increased after 24 hours of exposure. All these enzymes displayed biphasic activity curves with maximum values between 0.5 microM and 1 microM CuSO4. The response of guaiacol peroxidase was the most pronounced and statistically significantly specific and that of catalase the least. Delayed chlorophyll fluorescence decreased after exposure to 1 microM CuSO4, but no significant effect on maximum quantum yield of photosystem II (Fv/Fm) was observed. L. minor accumulated relatively high concentrations of copper. The accumulation rate was higher at lower concentrations of copper in the test medium (up to 2 microM CuSO4) than at concentrations above 2 microM CuSO4. One of the most pronounced antioxidative responses to copper exposure was modified levels of oxidized and reduced forms of glutathione. The decrease of the glutathione pool is most probably coupled with induced production of phytochelatins. Antioxidative enzymes showed the biphasic enzyme activity characteristic of stress response. Guaiacol peroxidase exhibited the greatest significant increase of activity, even at higher CuSO4 concentrations at which the activity of catalase and glutathione reductase dropped. The intensity of delayed chlorophyll fluorescence decreased, indicating reduced photosynthesis of plants under stress. All the measured parameters showed that plants respond to even low copper concentrations very soon after exposure. The accumulation rate of copper in duckweed tissues indicates that L. minor is an accumulator species. The synchronized and prompt inducibility of antioxidants indicates their involvement in a general plant defence strategy for coping with metal-induced oxidative stress. Glutathione concentration and guaiacol peroxidase activity were found to be the most sensitive of the early indicators of exposure to copper concentrations present in polluted water bodies. The experimental design of the present study allowed us to compare the sensitivity of various methods and parameters for detecting plant responses to heavy metal-induced oxidative stress. The level of glutathione and the enzyme activities of guaiacol peroxidase and glutathione reductase could be used as a rapidly determined early warning system in toxicity studies.

Early production of activated oxygen species in root apoplast of wheat following copper excess

Wheat seedlings (Triticum durum Desf.) were incubated in a solution containing 100 microM CuSO(4) for increasing time ranging from 1 min to 6h. Copper rapidly accumulated into the roots, and its amount increased significantly until 360 min. During the experiment, copper did not cause any lipid peroxidation and K(+) leakage. Up to 60 min of copper treatment the superoxide (O2(*-)) production in root apoplast decreased concomitantly with increase in superoxide dismutase (SOD) activity. In contrast, after 60 min of incubation, SOD decreased and this facilitated an increase in O2(*-) production. In the presence of the SOD inhibitor diethyldithiocarbamic acid, O2(*-) production was more than two times higher and showed a biphasic increase. Very high SOD activity in the apoplast, due to the presence of three different isozymes, one Mn-SOD and two CuZn-SODs, dismutated the radical giving rise, at least in part, to an increase in hydrogen peroxide. The highest value of H(2)O(2) was detected at 15 min, when peroxidase (POD) activity reached the lowest value. Root apoplast showed the presence of at least five different isoforms of PODs, whose pattern did not change during the entire treatment.

Production, properties and application to biocatalysis of a novel extracellular alkaline phenol oxidase from the thermophilic fungus Scytalidium thermophilum

Scytalidium thermophilum produces an extracellular phenol oxidase on glucose-containing medium. Certain phenolic acids, specifically gallic acid and tannic acid, induce the expression of the enzyme. Production at 45 degrees C in batch cultures is growth-associated and is enhanced in the presence of 160 microM CuSO4 x 5 H2O and 3 mM gallic acid. The highest enzyme activity is observed at pH 7.5 and 65 degrees C, on catechol. When incubated for 1 h at pH 7 and pH 8, 95% and 86% of the activity is retained. Thermostability decreases gradually from 40 degrees C to 80 degrees C. Estimated molecular mass is c. 83 kDa, and pI is acidic at c. 5.4. Substrate specificity and inhibition analysis in culture supernatants suggest that the enzyme has unique properties showing activity towards catechol; 3,4-dihydroxy-L-phenylalanine (L-DOPA); 4-amino-N, N-diethylaniline (ADA); p-hydroquinone; gallic acid; tannic acid and caffeic acid, and no activity towards L-tyrosine, guaiacol, 2,2'-azino-bis(3-ethyl-benzthiazoline-6-sulphonic acid) (ABTS) and syringaldazine. Inhibition is observed in the presence of salicyl hydroxamic acid (SHAM) and p-coumaric acid. Enzyme activity is enhanced by cetyltrimethylammonium bromide (CTAB) and polyvinylpyrrolidone (PVP), and the organic solvents dimethyl sulfoxide (DMSO) and ethanol. No inhibition is observed in the presence of carbon monoxide. Benzoin, benzoyl benzoin and hydrobenzoin are converted into benzil, and stereoselective oxidation is observed on hydrobenzoin. The reported enzyme is novel due to its catalytic properties resembling mainly catechol oxidases, but displaying some features of laccases at the same time.

Complement Protein C1q Recognizes a Conformationally Modified Form of the Prion Protein

Several studies have suggested the implication of the classical complement pathway in the early stages of prion disease pathogenesis. To explore this hypothesis, surface plasmon resonance spectroscopy was used to test the ability of human C1q to recognize mouse PrP immobilized on a sensor chip. In this configuration, C1q bound avidly to PrP, with a K(D) of 5.4 nM (k(on) = 2.4 x 10(5) M(-1) s(-1); k(off) = 1.3 x 10(-3) s(-1)). The isolated C1q globular domain also bound to immobilized PrP, although with a higher K(D) (238 nM), due to a decreased k(on) (4.2 x 10(3) M(-1) s(-1)). Interaction was strongly enhanced by Cu(2+) ions, with a 10-fold increase in overall binding in the presence of 10 microM CuSO(4), without significant modification of the kinetic parameters. In contrast, using the same technique, no interaction was detected between immobilized C1q and soluble PrP. Likewise, gel filtration and chemical cross-linking analyses yielded no evidence for an interaction between these proteins in solution. Comparative analysis of the antigenic reactivity of soluble and immobilized PrP was performed by ELISA and surface plasmon resonance spectroscopy, respectively, using anti-PrP monoclonal antibodies. This analysis provides evidence that immobilized PrP undergoes a major conformational change in the sequence stretch 141GNDWEDRYYRENMYRYPNQ159 located in its C-terminal globular domain. It is concluded that immobilized PrP undergoes structural modifications that possibly mimic the conformational changes occurring during conversion to the pathological isoform and that C1q represents a natural sensor of these changes. Pathological implications of this recognition property are discussed in light of recent reports.

Induction of laccase activity in the edible straw mushroom, Volvariella volvacea.

Volvariella volvacea, strain V14, produces multiple forms of extracellular laccase when grown in submerged culture in a defined medium with glucose as sole carbon source, and on cotton waste 'compost' representative of the conditions used for industrial-scale mushroom cultivation. In liquid culture, enzyme synthesis is associated with the onset of secondary growth, and is positively regulated by copper (up to 200 microM CuSO(4)) and by various aromatic compounds. In solid-state systems, only low levels of laccase are detectable during the vegetative growth phase but enzyme activity increases sharply at the onset of fruiting and during sporophore development.

Differential sensitivity of four Lobaria lichens to copper in vitro

Treatment of Lobaria amplissima, Lobaria pulmonaria, Lobaria scrobiculata, and Lobaria virens with 0 to 500 microM CuSO4 for 0 to 120 min at 25.0 degrees C resulted in a time- and copper-concentration-dependent decrease in the thallus total potassium content and a release of K- from the thallus, indicating that copper damaged the cytoplasmic membrane of the fungal hyphae. Lobaria pulmonaria was the most sensitive species, L virens the most resistant to copper treatment. Lobaria amplissima and L. scrobiculata displayed intermediate sensitivity. Treatment with copper also resulted in a time-dependent increase in the thallus copper content. For all four Lobaria species, the thallus potassium content decreased linearly with increasing thallus copper content, suggesting that, as cupric ions bound to the thallus, they reach target sites in the membrane. Acetone extracts contained low concentrations of chlorophyll a, amino acids, polyols, and sugars, but detectable or considerable amounts of lichen substances, i.e.. constictic, norstictic, stictic, and usnic acids and scrobiculin. Titration of the extracts with copper and assay of free Cu2+ revealed the presence of copper-binding ligands, most probably corresponding to the lichen substances detected in the extracts. Acetone extracts of L. amplissima and L. virens bound small or very small amounts of Cu2+; binding was monophasic and probably involved scrobiculin. Acetone extracts of L. pulmonaria and L. scrobiculata bound high or intermediate amounts of copper; binding was polyphasic and probably involved the lichen acids detected in the extracts. It was concluded that, in the studied Lobaria species, acetone extracts containing lichen substances could bind, in vitro, considerable amounts of Cu2+, but this binding did not prevent membrane damage in the thallus. Several hypotheses are presented to explain these results.

Chylomicron-remnant-like particles inhibit receptor-mediated endothelium-dependent vasorelaxation in pig coronary arteries.

The influence of native and oxidized chylomicron-remnant-like particles (CMR-LPs) on endothelium-dependent relaxation in pig coronary arteries was studied. Artificial lipid particles of a size and lipid composition resembling chylomicron remnants and containing pig apolipoprotein E were used to investigate the effects of chylomicron remnants on the relaxation of isolated segments of pig coronary arteries in response to three endothelium dilators: 5-hydroxytryptamine (5-HT), bradykinin and the calcium ionophore A23187. CMR-LPs caused significant inhibition of the maximum relaxation response of the vessels to 5-HT, but not that to bradykinin or A23187 ( P <0.05). In contrast, CMR-LPs that had been oxidized by incubation with 10 microM CuSO(4) (oxidized CMR-LPs) were found to significantly reduce maximal relaxation to bradykinin by 13% ( P <0.05) and to reduce the sensitivity of the tissue to A23187 by 1.7-fold ( P <0.05). In experiments in which either the L-arginine/nitric oxide (NO) pathway or the endothelium-derived hyperpolarizing factor (EDHF) pathway was selectively inhibited, leaving the other intact, the inhibitory effect of oxidized CMR-LPs was observed only in vessels in which the -arginine/NO-mediated pathway was operative. Furthermore, the oxidized particles had no inhibitory effect on the relaxation of the vessel segments to the non-endothelium-dependent agonists S -nitro- N -acetylpenicillamine, 5'-( N -ethylcarboxamido)adenosine or pinacidil. These results demonstrate that CMR-LPs inhibit vascular relaxation in pig coronary arteries by an endothelium-dependent mechanism involving the L-arginine/NO pathway, but not the EDHF pathway, and provide evidence in support of a role for chylomicron remnants in the endothelial dysfunction associated with hypercholesterolaemia and atherogenesis.

The protective effects of tetrahydrocurcumin on oxidative stress in cholesterol-fed rabbits.

Tetrahydrocurcumin (THC) is an antioxidative substance which is derived from curcumin by hydrogenation. Curcumin is the main component of turmeric and is responsible for the yellow color of curried foods.First, LDL derived from a normal human volunteer was incubated in the presence of an antioxidant with 10 microM CuSO(4) at 37 degrees C for 2 hours.All antioxidants tested (THC, curcumin, probucol, and alpha-tocopherol) dose-dependently (1-10 microM) inhibited the oxidative modification of LDL. Probucol was the strongest, followed by THC, alpha-tocopherol, and curcumin.Next, in order to evaluate the antioxidative activity of THC in vivo, we fed rabbits diets containing 1% cholesterol with or without 0.5% THC and examined their effects on oxidative stress and atherosclerosis. Animals were divided into two groups: the control group rabbits (n = 12) were fed a normal chow diet and the experimental group (n = 12) was fed a diet containing 0.5% THC for one week.Then, 1% cholesterol was added to the diets and the animals were allowed to feed further for either 6 (n = 4 for each group) or 12 weeks (n = 8 for each group). Although serum cholesterol levels rapidly increased after starting the high cholesterol diet, no difference was observed between the control and THC groups.TBARS formation in the absence of added copper ion was inhibited in the LDL separated from THC-treated animals compared with that from control animals.THC treatment tended to inhibit the area covered with atherosclerotic lesions compared with the control, although this was not significant (28.8 +/- 17.5% vs. 40.0 +/- 23.7%, p = 0.2). Formation of N(epsilon)-(hexanoyl) lysine, 4-hydroxynonenal and dityrosine in liver and kidney also had a tendency to be inhibited by THC treatment. Although free THC was not detected in serum and liver, THC was detected in samples treated with beta-glucuronidase and sulfatase, suggesting that THC is present as a conjugate with glucuronic acid or sulfate. In conclusion, the present results suggest that curcuminoids, particularly THC, which are contained in turmeric, may be useful as a functional food factor.