The influence of pH on the inhibition of DNA cleavages induced by pyrogallol

Induction of DNA damage by pyrogallol has been shown at physiological pH, but mutagenesis data also suggest there is inhibition in acidic media. In the present work, the plasmid pBSK was incubated with pyrogallol, under aerobic conditions at 37 degrees C, at pH 7.4, 4.5 or 3.5, for 1, 3 or 5 h, in the absence or presence of Cu(2+). Cleavage of the supercoiled DNA form was analyzed through topology modifications by agarose gel electrophoresis and quantified by densitometry. Independently of the presence of Cu(2+), DNA cleavage at pH 7.4 was significantly (P < 0.001) induced and occurred extensively after 1-h incubation. At pH 4.5, the cleavage was significantly (P < 0.05) induced only after 5 h incubation in the absence of Cu(2+), but was extensive (P < 0.001) after 1-h incubation when the metal ion was present. At pH 3.5, DNA cleavage was inhibited (P > 0.05), after 5-h incubation, even in the presence of Cu(2+). Our results provide evidence that DNA cleavage by pyrogallol is pH-dependent, catalyzed by Cu(2+) , and extensively decreased in acidic pH. Due to the abundant presence of the pyrogallate ion in physiological media, we suggest that this conjugate base form is responsible for DNA cleavage.     

Effects of Sanionia uncinata extracts in protecting against and inducing DNA cleavage by reactive oxygen species

Abstract

When mosses are exposed to increased quantities of ultraviolet (UV) radiation, they produce more secondary metabolites. Antarctica moss Sanionia uncinata (Hedw.) Loeske has presented high carotenoid contents in response to an increase in UVB radiation. This moss has been recommended as a potential source of antioxidants. In the present work, the protective and enhancing effects of aqueous (AE) and hydroalcoholic (HE) extracts of S. uncinata on the cleavage of supercoiled DNA were evaluated through topological modifications, quantified by densitometry after agarose gel electrophoresis. Total phenolic contents reached 5.89 mg/g. Our data demonstrated that the extract does not induce DNA cleavage. Furthermore, both extracts showed antioxidant activity that protected the DNA against cleavage induced by (i) O₂^??, 89% (AE) and 94% (HE) (P < 0.05), and (ii) .OH, 17% (AE) and 18% (HE). However, the extracts intensified cleavage induced by Fenton-like reactions: (i) Cu²⁺/H₂O₂, 94% (AE) and 100% (HE) (P < 0.05), and (ii) SnCl₂, 62% (AE) and 56% (HE). DNA damages seem to follow different ways: (i) in the presence of Fenton-like reactions could be via reactive oxygen species generation and (ii) with HE/Cu²⁺ could have also been triggered by other mechanisms.     

Adsorption and degradation of zearalenone by bacillus strains

Two Bacillus strains; Bacillus subtilis 168 and Bacillus natto CICC 24640 separately adsorbed and degraded zearalenone in liquid media, in vitro. Viable, autoclaved (121°C, 20 min) and acid-treated cells of both strains separately bound more than 55% of zearalenone (ZEN, 20 μg/L) after 30 min and 1-h incubation at 37°C under aerobic conditions, and the amount of ZEN adsorbed was dependent on initial cell volume. In addition, ZEN was degraded by the culture extract of both strains. Degradation by B. subtilis 168 and B. natto CICC 24640 culture extract after 24-h aerobic incubation at 30°C was 81% and 100%, respectively. B. natto CICC 24640 culture extract comprehensively degraded ZEN and, for both strains, no oestrogenic ZEN analogues were present. ZEN degradation was accompanied by carbondioxide emission indicating a decarboxylation reaction. ZEN degradation by the salient B. natto CICC 24640 culture extract varied with initial ZEN concentration, incubation time, temperature and pH. Degradation was enhanced by Mn²⁺, Zn²⁺, Ca²⁺ and Mg²⁺ but impeded by Hg²⁺, Cu²⁺, Pb²⁺, ethylenediaminetetraacetic acid and 1,10-phenanthroline. The degradation reaction is associated with a metalloproteinase of molar mass in the range 31–43 kDa. Overall, the two generally recognised as safe Bacillus strains can, potentially, be utilised for detoxification of zearalenone in food.     

Increased production of laccase by <Emphasis Type="Italic">Cerrena unicolor</Emphasis> in submerged liquid cultures

The wood-degrading basidiomycete Cerrena unicolor C-139 has been suggested as a potential producer of the industrially important enzyme laccase. Basic culture parameters influencing the enzyme synthesis in shaken-flask and aerated bioreactor cultures were evaluated to improve the yields of the process. Production of extracellular laccase was considerably enhanced by the addition of Cu²⁺ in the micromolar range to a carbon-sufficient and nitrogen-sufficient culture medium (C/N = 16.69). When an optimised medium containing glucose (10 g/L) and l-asparagine (1.5 g/L) was used, and enzyme synthesis was stimulated by addition of 10 μM Cu²⁺ to the culture medium on days 3, 6 and 9, maximal laccase productivity obtained after 17 days’ cultivation in shaken flask cultures was above 100,000 nkat/L. In fermenter fungal cultures, the influence of stabilisation of medium pH on laccase activity was additionally studied. The use of a bioreactor with an automatic pH control set at pH 6.5 after 48-h incubation resulted in the enzyme activity of 65,000 nkat/L after 8 days’ cultivation.     

Purification and Some Properties of a Metalloprotease from Sorghum Malt Variety KSV8-1

Summary A metalloprotease from sorghum malt variety KSV8-I was purified by a combination of 4-M sucrose fractionation, ion-exchange chromatography on Q-Sepharose (Fast flow), gel-filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. The enzyme was purified 7.9-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2128.7 U mg⁻¹ protein. SDS-PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 35 kDa. The purified enzyme had optimal activity at 60 °C and maximal temperature stability between 40 and 60 °C but retained over 77% of its initial activity after incubation at 70 °C for 30 min. Both pH optimum and maximal stability were at 7.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. Using 0.2 ml of 5 mM solution of each metal ion, the purified protease was slightly (P<0.05) inhibited by Zn²⁺, appreciably (P<0.01) inhibited by Ca²⁺ and Co²⁺ and highly significantly (P<0.001) inhibited by Ag⁺, Ba²⁺, Hg²⁺, Mn²⁺ and Pb²⁺. The enzyme was equally highly significantly (P<0.001) inhibited by EDTA and hydrolysed casein to give the following kinetic constants: K_m = 21.0 mg ml⁻¹; V_max = 8.2 μmol ml¹ min⁻¹ and K_i = 0.390 mM.     

Mechanisms involved in the in vitro modification of low density lipoprotein by human umbilical vein endothelial cells and copper ions

In this study we evaluated the time course and mechanism of low density lipoprotein (LDL) oxidation induced by human umbilical vein endothelial cells (HUVECs), cell-free medium (CFM) and Cu²⁺. After incubating LDL (200 μg/ml) with HUVECs, CFM and Cu²⁺ (concentration adjusted to obtain the same degree of LDL modification as with HUVECs), the extent of LDL lipid peroxidation and apoprotein B modification was monitored at different times from 0 to 24 h. This involved evaluating the time course of LDL conjugated diene, peroxide, malonyldialdehyde (MDA), fluorescence, relative electrophoretic mobility (REM), vitamin E and monounsaturated and polyunsaturated fatty acids. After incubation with HUVECs, the LDL REM was significantly higher than that obtained in CFM (p < 0.01). When balanced for the same degree of LDL modification as obtained with HUVECs, Cu²⁺ gave a REM similar to that obtained with HUVECs. At the different times of incubation there was no statistical difference between conjugated diene and peroxide values after incubation with HUVECs and with CFM. The values obtained with Cu²⁺ were significantly higher than those obtained with HUVECs and CFM (p < 0.01). MDA and LDL fluorescence were significantly higher after exposure to HUVECs than to CFM (p<0.01), values being similar to those obtained with Cu²⁺. There was no statistical difference between the values of LDL oleic, linoleic, arachidonic and eicosapentaenoic acids after incubation with HUVECs and CFM. Eicosatetraynoic acid (ETYA), a lipoxygenase inhibitor, determined dose-dependent reduction of MDA formation induced by the incubation of LDL with HUVECs; it did not affect LDL conjugated diene. ETYA did not have any effect on the MDA derived from LDL after incubation with Cu²⁺ or CFM. The results of this study demonstrate that, unlike Cu²⁺, the contribution of HUVECs to LDL modification does not involve only lipid peroxidation of the lipoprotein; it also includes intracellular radical and non-radical processes.     

Purification and characterization of extracellular tannin acyl hydrolase from <Emphasis Type="Italic">Aspergillus heteromorphus</Emphasis> MTCC 8818

A tannase (E.C. 3.1.1.20) producing fungal strain was isolated from soil and identified as Aspergillus heteromorphus MTCC 8818. Maximum tannase production was achieved on Czapek Dox minimal medium containing 1% tannic acid at a pH of 4.5 and 30°C after 48 h incubation. The crude enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography. Diethylaminoethyl-cellulose column chromatography led to an overall purification of 39.74-fold with a yield of 19.29%. Optimum temperature and pH for tannase activity were 50°C and 5.5 respectively. Metal ions such as Ca²⁺, Fe²⁺, Cu¹⁺, and Cu²⁺ increased tannase activity, whereas Hg²⁺, Na¹⁺, K¹⁺, Zn²⁺, Ag¹⁺, Mg²⁺, and Cd²⁺ acted as enzyme inhibitors. Various organic solvents such as isopropanol, isoamyl alcohol, benzene, methanol, ethanol, toluene, and glycerol also inhibited enzyme activity. Among the surfactants and chelators studied, Tween 20, Tween 80, Triton X-100, EDTA, and 1, 10-o-phenanthrolein inhibited tannase activity, whereas sodium lauryl sulfate enhanced tannase activity at 1% (w/v).     

Purification,characterization, and gene cloning of two laccase isoenzymes (Lac1 and Lac2) from Trametes hirsuta MX2 and their potential in dye decolorization

In this study, two laccase isoenzymes (Lac1 and Lac2) from the culture supernatant of Trametes hirsuta MX2 were purified, and the genes (Lac1 and Lac2) coding the isoenzymes were cloned. Both Lac1 and Lac2 contained an open reading frame of 1563 bp with an identity of 79%. The two isoenzymes showed significant biochemical differences. The maximal activities of Lac1 and Lac2 were at pH 2.5 with 2-2′-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS), and the optimal temperatures for the activities of Lac1 and Lac2 were 60 and 50 °C, respectively. Lac1 exhibited excellent resistance to acidic conditions and retained 62.17% of its initial activity at pH 2.5 after a 72-h incubation. Lac2 was more thermostable than Lac1 with half-lives (t_1/2) of 9.58 and 3.12 h at 50 and 60 °C, respectively; the t_1/2 of Lac1 were only 4.19 and 0.88 h, respectively. Both Lac1 and Lac2 isoenzymes have a strong tolerance to Mg²⁺, Mn²⁺, Cu²⁺, and EDTA (50 mM). At a low concentration of 0.05 U mL^?1, the enzymes could decolorize towards Remazol Brilliant Blue R, Acid Red 1, Crystal Violet, and Neutral Red in the presence of ABTS. These unusual properties demonstrated that the two laccases have strong potential for specific industrial applications.

     

Effects of metal ions on activity of plasmin

The effects of some metal ions on amidolytic and fibrinogenolytic activities of highly purified human plasmin were investigated in vitro. In the presence of Zn²⁺, Cu²⁺, Cd²⁺, and Au⁺ in the incubation mixture at the concentrations of 1×10⁻⁵−1×10⁻³ M, the anidolytic plasmin activity was strongly inhibited, whereas Ca²⁺ and Mg²⁺ at the same concentrations were not effective. The analysis of the kinetic study has shown that Zn²⁺ or Cu²⁺ acts as mixed-type inhibitors of plasmin activity. The inhibition of amidolytic plasmin activity by Zn²⁺ and Cu²⁺ was reduced in the presence of EDTA, histidine, or albumin. Incubation of plasmin with Zn²⁺ or Cu²⁺ (at the concentration of 5×10⁻⁴ M) resulted in complete loss of its proteolytic action on fibrinogen, whereas Cd²⁺ and Au⁺ under the same conditions only partially inhibited this process.     

Effect of Cd and Hg on the Activity of Na/K-ATPase and Mg-ATPase Adsorbed on Polystyrene Microtiter Plates

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4°C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na⁺/K⁺-ATPase and Mg²⁺-ATPase, retained 70–80% activity after the adsorption. In addition, adsorption stabilizes Na⁺/K⁺-ATPase and Mg²⁺-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na⁺/K⁺-ATPase and Mg²⁺-ATPase and their sensitivity to and mechanism of Cd²⁺- or Hg²⁺-induced inhibition. The only exception is the “high affinity” Mg²⁺-ATPase moiety, whose affinity for ATP and sensitivity toward Cd²⁺ were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

Fermentative production of extracellular amylase from novel amylase producer,Tuber maculatum mycelium,and its characterization

Abstract

Truffles are symbiotic hypogeous edible fungi (form of mushroom) that form filamentous mycelia in their initial phase of the growth cycle as well as a symbiotic association with host plant roots. In the present study, Tuber maculatum mycelia were isolated and tested for extracellular amylase production at different pH on solid agar medium. Furthermore, the mycelium was subjected to submerged fermentation for amylase production under different culture conditions such as variable carbon sources and their concentrations, initial medium pH, and incubation time. The optimized conditions after the experiments included soluble starch (0.5% w/v), initial medium pH of 7.0, and incubation time of 7 days, at room temperature (22?±?2?°C) under static conditions which resulted in 1.41?U/mL of amylase. The amylase thus obtained was further characterized for its biocatalytic properties and found to have an optimum activity at pH 5.0 and a temperature of 50?°C. The enzyme showed good thermostability at 50?°C by retaining 98% of the maximal activity after 100?min of incubation. The amylase activity was marginally enhanced in presence of Cu²⁺ and Na⁺ and slightly reduced by K⁺, Ca²⁺, Fe²⁺, Mg²⁺, Co²⁺, Zn²⁺, and Mn²⁺ ions at 1?mM concentration.     

Bacterial Degradation of EDTA

Degradation of EDTA (ethylenediaminetetraacetic acid) or metal–EDTA complexes by cell suspensions of the bacterial strain DSM 9103 was studied. The activity of EDTA degradation was the highest in the phase of active cell growth and decreased considerably in the stationary phase, after substrate depletion in the medium. Exponential-phase cells were incubated in HEPES buffer (pH 7.0) with 1 mM of uncomplexed EDTA or EDTA complexes with Mg²⁺, Ca²⁺, Mn²⁺, Pb²⁺, Co²⁺, Cd²⁺, Zn²⁺, Cu²⁺, or Fe³⁺. The metal–EDTA complexes (Me–EDTA) studied could be divided into three groups according to their degradability. EDTA complexes with stability constants K below 10¹⁶ (log K < 16), such as Mg–EDTA, Ca–EDTA, and Mn–EDTA, as well as uncomplexed EDTA, were degraded by the cell suspensions at a constant rate to completion within 5–10 h of incubation. Me–EDTA complexes with log K above 16 (Zn–EDTA, Co–EDTA, Pb–EDTA, and Cu–EDTA) were not completely degraded during a 24-h incubation, which was possibly due to the toxic effect of the metal ions released. No degradation of Cd–EDTA or Fe(III)–EDTA by cell suspensions of strain DSM 9103 was observed under the conditions studied.     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Effects of Anions on the Capacity and Affinity of Copper Adsorption in Two Variable Charge Soils

Effects of nitrate,(NO₃⁻) chloride (Cl⁻), sulfate (SO₄^2-, and acetate (Ac⁻) on Cu²⁺ adsorption and affinity of the adsorbed Cu²⁺ were evaluated in two Fe and Al enriched variable charge soils from Southern China. The maximum adsorption of Cu²⁺ (M, a parameter from the Langmuir isotherm model) in the presence of different anions decreased in the order Cl⁻ > Ac⁻ > NO₃⁻ > SO₄^2- for both soils. The clayey loamy soil (mixed siliceous thermic Typic Dystrochrept, TTD), developed on the Arenaceous rock, adsorbed less Cu²⁺ than the clayey soil (kaolinitic thermic Plinthudults, KTP), derived from the Quaternary red earths, regardless of anion type present in the medium. The affinity of adsorbed Cu²⁺ to both soils could be characterized by the Kd (distribution coefficient) values and successive extraction of the adsorbed Cu²⁺ with 1-mol NH₄Ac L⁻¹. The log₁₀Kd value was smaller for the TTD soil than for the KTP soil and decreased in the order of Cl⁻ > NO₃⁻ > SO₄^2- > Ac⁻ at low initial Cu²⁺ concentrations (≤40 mg Cu²⁺L⁻¹), whereas at 80 mg Cu²⁺L⁻¹, the log₁₀Kd value was similar for NO₃⁻, SO₄^2-, and Ac⁻, but was slightly higher for Cl⁻. Complete extraction of Cu²⁺ adsorbed in the presence of Ac⁻ was achieved. Influence of NO₃⁻ and SO₄^2- on the affinity of adsorbed Cu²⁺ was similar, but the effects of Cl⁻ depended on the initial Cu²⁺ concentrations. The extracted percentage of the adsorbed Cu²⁺ in the presence of NO₃⁻ or SO₄^2- increased with increasing Cu²⁺ adsorption saturation. The presence of Cl⁻, NO₃⁻, or SO₄^2- markedly decreased the equilibrium solution pH for both soils with increasing initial Cu²⁺ concentrations, and the delta pH values at the highest Cu²⁺ level were 0.5, 0.63, and 0.55 U for the TTD soil and 0.79, 0.84, and 0.93 U for the KTP soil, respectively for the three anions. The presence of Ac⁻ had a minimal influence on the equilibrium solution pH because of the buffering nature of the NaAc/HAc medium which buffered the released protons. The effects of anions on Cu²⁺ adsorption and affinity of the adsorbed Cu²⁺ were dependent on anion types and were apparently related to the altered surface properties caused by anion adsorption and/or the formation of anion– Cu²⁺ complexes.     

Association of aureolic acid antibiotic, chromomycin A3 with Cu2+ and its negative effect upon DNA binding property of the antibiotic

Here we have examined the association of an aureolic acid antibiotic, chromomycin A3 (CHR), with Cu²⁺. CHR forms a high affinity 2:1 (CHR:Cu²⁺) complex with dissociation constant of 0.08 × 10⁻¹⁰ M² at 25°C, pH 8.0. The affinity of CHR for Cu²⁺ is higher than those for Mg²⁺ and Zn²⁺ reported earlier from our laboratory. CHR binds preferentially to Cu²⁺ in presence of equimolar amount of Zn²⁺. Complex formation between CHR and Cu²⁺ is an entropy driven endothermic process. Difference between calorimetric and van’t Hoff enthalpies indicate the presence of multiple equilibria, supported from biphasic nature of the kinetics of association. Circular dichroism spectroscopy show that [(CHR)₂:Cu²⁺] complex assumes a structure different from either of the Mg²⁺ and Zn²⁺ complex reported earlier. Both [(CHR)₂:Mg²⁺] and [(CHR)₂:Zn²⁺] complexes are known to bind DNA. In contrast, [(CHR)₂:Cu²⁺] complex does not interact with double helical DNA, verified by means of Isothermal Titration Calorimetry of its association with calf thymus DNA and the double stranded decamer (5′-CCGGCGCCGG-3′). In order to interact with double helical DNA, the (antibiotic)₂ : metal (Mg²⁺ and Zn²⁺) complexes require a isohelical conformation. Nuclear Magnetic Resonance spectroscopy shows that the Cu²⁺ complex adopts a distorted octahedral structure, which cannot assume the required conformation to bind to the DNA. This report demonstrates the negative effect of a bivalent metal upon the DNA binding property of CHR, which otherwise binds to DNA in presence of metals like Mg²⁺and Zn²⁺. The results also indicate that CHR has a potential for chelation therapy in Cu²⁺ accumulation diseases. However cytotoxicity of the antibiotic might restrict the use.     

Influence of daylight and incubation interval on water column respiration in tropical fish ponds

Water column respiration (WCR) was measured in dark BOD bottles for 2, 4 and 8 h intervals during 22 h periods in two 1000 m² ponds stocked withOreochromis niloticus at 1 m^–2, and fertilized weekly with chicken litter at 750 kg total solids ha^–1. Mean WCR ranged from a low of 0.39 mg l^–1 for 8 h nocturnal intervals to a high of 0.62 mg l^–1 for 2-h diurnal intervals. WCR was significantly influenced by daylight and time into the diurnal or nocturnal period when it was determined. Mean WCR was significantly greater during the day than during the night (P<0.01). During the day, 2 h incubation intervals resulted in significantly higher WCR than 4 h or 8 h intervals (P<0.01); length of incubation interval did not significantly influence nocturnal WCR (P>0.05). Higher WCR during the day and during short diurnal incubation intervals was attributed to greater availability of photosynthetic respiration substrate. Diurnal, diel, or nocturnal WCR could be best estimated by a single 2, 4 or 8 h incubation interval, respectively, beginning at 0800 h.     

Reduction of Cupric Ions with Elemental Sulfur by Thiobacillus ferrooxidans

In anaerobic or aerobic conditions in the presence of 5 mM sodium cyanide, an inhibitor of iron oxidase, cupric ion (Cu²⁺) was reduced enzymatically with elemental sulfur (S⁰) by washed intact cells of Thiobacillus ferrooxidans AP19-3 to give cuprous ion (Cu⁺). The rate of Cu²⁺ reduction was proportional to the concentrations of S⁰ and Cu²⁺ added to the reaction mixture. The pH optimum for the cupric ion-reducing system was 5.0, and the activity was completely destroyed by 10-min incubation of cells at 70°C. The activity of Cu²⁺ reduction with S⁰ by this strain was strongly inhibited by inhibitors of hydrogen sulfide: ferric ion oxidoreductase (SFORase), such as α,α′-dipyridyl, 4,5-dihydroxy-m-benzene disulfonic acid disodium salts, and diazine dicarboxylic acid bis-(N, N-dimethylamide). A SFORase purified from this strain, which catalyzes oxidation of both hydrogen sulfide and S⁰ with Fe³⁺ or Mo⁶⁺ as an electron acceptor in the presence of glutathione, catalyzed a reduction of Cu²⁺ by S⁰, and the Michaelis constant of SFORase for Cu²⁺ was 7.2 mM, indicating that a SFORase catalyzes the reduction of not only Fe³⁺ and Mo⁶⁺ but also Cu²⁺.     

Influence of metal addition on ethanol production with <Emphasis Type="Italic">Pichia stipitis</Emphasis> ATCC 58784

Trace metals always act as cofactors or coenzymes in many cellular processes. Deficiency or excess of some metals will affect the fermentation of lignocellulosic hydrolysate. In order to make sure the deficient or excessive states of metals in culture medium, metal contents analysis was conducted in Pichia stipitis ATCC 58784 cells, synthetic medium, and diluted acid hydrolysate of rice straw. The results showed that Cu, Ni, and Co were deficient, and Al was a little excessive. So the influences of Cu²⁺, Al³⁺, Ni²⁺, and Co²⁺ additions on the growth and ethanol production of ATCC 58784 were further researched. Low concentration additions of Cu²⁺ and Al³⁺ (<0.24 mM and <0.23 mM, respectively) improved biomass growth of ATCC 58784 by 34 and 13%, respectively; however, higher concentrations decreased biomass growth. On the other hand, addition of Cu²⁺ (0.39 mM) did not affect volumetric ethanol production significantly (P = 0.05) and addition of Al³⁺ (0.38 mM) showed no influence on volumetric ethanol production (P = 0.68). Addition of 0.074 mM Co²⁺ inhibited biomass growth of ATCC 58784 by 13% and volumetric ethanol production by 10%. The biomass growth and volumetric ethanol production of ATCC 58784 was arrested by the addition of 0.33 mM of Ni²⁺ by 53 and 65%, respectively.     

Mechanism of UV-Induced Apoptosis in Human Leukemia Cells: Roles of Ca/Mg-Dependent Endonuclease, Caspase-3, and Stress-Activated Protein Kinases

Ultraviolet light (UV) induced rapid apoptosis of U937 leukemia cells, concurrent with DNA fragmentation and cleavage of poly(ADP-ribose)polymerase (PARP) by activated caspase-3. Thein vitroreconstitution of intact HeLa S3 nuclei and apoptotic U937 cytosolic extract (CE) revealed that (i) Ca²⁺/Mg²⁺-dependent, Zn²⁺-sensitive endonuclease activated in the apoptotic CE induced DNA ladder in HeLa nuclei at pH 6.8–7.4, (ii) activated caspase-3 cleaved PARP in HeLa nuclei, and (iii) when the apoptotic CE was treated with the caspase-3 inhibitor (1 μM Ac-DEVD-CHO) or the caspase-1 inhibitor (10 μM Ac-YVAD-CHO), the former, but not the latter, caused a 50% inhibition of DNA fragmentation and the complete inhibition of PARP cleavage in HeLa nuclei. Similarly, Ac-DEVD-CHO (100 μM) inhibited apoptosis and DNA ladder by 50% and PARP cleavage completely in UV-irradiated U937 cells, but Ac-YVAD-CHO (100 μM) did not. Thus, UV-induced apoptosis of U937 cells involves the Ca²⁺/Mg²⁺-dependent endonuclease pathway and the caspase-3–PARP cleavage–Ca²⁺/Mg²⁺-dependent endonuclease pathway. The former pathway produced directly 50% of apoptotic DNA ladder, and the latter involved activated caspase-3 and PARP cleavage, followed by formation of the remaining 50% DNA ladder by the activated endonuclease. In UV-irradiated B-cell lines, further, p53-dependent increase of Bax resulted in a greater caspase-3 activation compared to its absence. However, UV-induced activation of JNK1 and p38 was not affected by the caspase-1 and -3 inhibitors in U937 cells, so that caspases-1 and -3 do not function upstream of JNK1 and p38.     

Purification of Oat Debranching Enzyme and Occurrence of Inactive Debranching Enzyme in Cereals

The interaction of some anthracycline antibiotics (adriamycin, daunomycin, aclacinomycin-A) with bacteriophage ?X174 was investigated. Adriamycin and daunomycin inactivated the infectivity of both free ?X174 phage and naked single-stranded ?X174 DNA without DNA strand scission, but aclacinomycin-A did not show this action. The phage inactivation reaction was reversibly inhibited by Superoxide dismutase, catalase or other oxygen radical scavengers. The inactivation of ?X174 by adriamycin and aclacinomycin-A was stimulated by the addition of Cu²⁺, while the ?X174 inactivation by daunomycin was inhibited by the addition of Cu²⁺. The ?X174 inactivation by adriamycin and aclacinomycin-A in the presence of Cu²⁺ was caused by degradation of DNA, and this inactivation reaction was inhibited irreversibly by oxygen radical scavengers. These results indicate that anthracycline antibiotics bind to ?X174 DNA in the form of free radicals and that during the auto-oxidation of these antibiotics in the presence of Cu²⁺, oxygen radicals were generated to cause the degradation of ?X174 DNA.