De-intercalation of ethidium bromide and acridine orange by xanthine derivatives and their modulatory effect on anticancer agents: a study of DNA-directed toxicity enlightened by time correlated single photon counting

Time Correlated Single Photon Counting (TCSPC) was used for the first time to analyze the effect/changes in the mode of intercalation of ethidium bromide (EtBr) and acridine orange (AO) to calf thymus DNA brought about due to interaction of naturally occurring methylxanthines such as theophylline (X1), theobromine (X2) and caffeine (X3). UV absorption and fluorescence studies were also carried to observe the behaviour of these xanthines on the modulation of the binding mode of anticancer agents (cisplatin, novantrone, and actinomycin D) and certain intercalating dyes (EtBr and AO) to DNA. In TCSPC analysis we found that when the concentration of the drugs (X1, X2 and X3) increased from 0.025 mM to 2 mM i.e. P/D 2.4 to P/D 0.03 reduction in intercalation of EtBr and AO was observed, suggesting that xanthine derivatives could play very important role in reducing the DNA-directed toxicity in a dose dependent manner. In TCSPC, the amplitude of smaller lifetime component A(1) and higher lifetime component A(2) are attributed to free and intercalated dye concentration and their variation could indicate the process of intercalation or reduced intercalation of EtBr and AO by xanthine derivatives. We found that at the maximum drug concentration the smaller lifetime component A(1) was increased by 7-8% and 17-37% in EtBr and AO intercalated complex respectively. Also the changes in lifetime and fluorescence decay profile were observed for the DNA-intercalated dyes before and after treatment with xanthines. Especially, at maximum P/D 0.03 the lifetime of DNA-intercalated EtBr and AO reduced by 1-2 ns. The present analysis reveals that xanthines are able to interact with free dyes and also with intercalated dyes, suggesting that when they interact with free dyes they might inhibit the further intercalation of dye molecules to DNA and the interaction with intercalated dyes might lead to displacement of the dyes resulting in de-intercalation. The results obtained from UV and fluorescence spectroscopy also support the present investigation of probable interaction of xanthines with the DNA damaging agents in modulating/reducing the DNA-directed toxicity.     

Highly Selective Acridine and Ethidium Staining of Bacterial DNA and RNA

The acridine dyes acridine orange (AO) and coriphosphine O (CPO) and ethidium bromide (EtBr) were used to stain bacterial digests after electrophoresis in native and denaturing (SDS) polyacrylamide gels and were shown to stain DNA and RNA preferentially over other subcellular components in the gels. Vegetative cell digests of Bacillus subtilis, Escherichia coli, Micrococcus luteus, and Staphylococcus aureus showed intense staining of DNA with AO and CPO near the top of the gel, but little or no staining of other cellular constituents. EtBr stained both DNA and RNA in the gels. Protein standards and non-nucleic acid cellular constituents stained faintly with high concentrations (> 100 μM) of AO, lower concentrations (13.9 μM) of CPO, and did not stain with 0.5 μg/ml EtBr in denaturing gels. The complete set of cellular biochemicals was visualized by silver staining, while the protein subset was detected by Coomassie blue staining. The highest concentrations of AO (120 μM) and CPO (13.9 μM) were shown to detect purified DNA in gels with a sensitivity in the range of 25–50 ng per band. This work demonstrates the specificity of acridine and ethidium dyes for nucleic acids, while illustrating the level of non-nucleic acid-specific interactions with other cellular components by staining of electrophoretically separated cellular components in a gel matrix.     

In Vitro evaluation of the cytotoxic and anti-proliferative properties of resveratrol and several of its analogs

Resveratrol (RES), a component of red wine, possesses anti-inflammatory properties. The studies described in the present work were aimed at evaluating the potential for RES and related stilbene analogs (piceatannol, PIC; pterostilbene, TPS; trans-stilbene, TS; and trans-stilbene oxide, TSO) to exhibit toxicity towards RAW 264.7 mouse macrophages. The effect of TS, TSO, RES and TPS on RAW 264.7 macrophage viability was determined by two standard methods: (a) the MTT assay and (b) the trypan blue dye exclusion test. Whereas macrophages were more sensitive to PIC (LC_{50 trypan} ∼ 1.3 μM) and to TPS (LC_{50 trypan} ∼ 4.0 μM and LC_{50 MTT} ∼ 8.3 μM) than to RES (LC_{50 trypan} ∼ 8.9 μM and LC_{50 MTT} ∼ 29.0 μM), they were relatively resistant to TSO (LC_{50 trypan} ∼ 61.0 μM and LC_{50 MTT} > 100 μM) and to TS (LC_{50 trypan} ≥ 5.0 μM and LC_{50 MTT} ≥ 5.0 μM). The ability of selected stilbenes (RES, TPS and PIC) to exhibit growth inhibitory effects was also examined. Although RES and TPS were observed to inhibit cell proliferation in macrophages (IC₅₀ ≤ 25 μM), these cells were resistant to growth inhibition by PIC (IC₅₀ ≥ 50 μM). The data obtained in the present analysis demonstrate that substituted stilbene compounds such as RES have the capacity to exhibit cytotoxic and anti-proliferative activities in macrophages.     

Characteristics and purification of an oxygen insensitive azoreductase from Caulobacter subvibrioides strain C7-D

An azo dye-degrading bacterium, Caulobacter subvibrioides strain C7-D, semi-constitutively produces an azoreductase that reduced the azo bond of the dyes Acid Orange (AO) 6, AO7, AO8, AO12, Acid Red (AR) 88, AR151, and Methyl Red (MR). This activity was oxygen insensitive. Of the dyes tested, AO7 was the best inducer and the most rapidly reduced substrate suggesting that dye AO7 most closely mimics the natural physiological substrate for this enzyme. The K _m for AO7 was 1 μM. Purification of the azoreductase from C. subvibrioides strain C7-D was achieved through dye-ligand affinity chromatography using the dye Orange-A covalently coupled to an agarose support. The azoreductase is approximately 30 kDa and enzyme studies indicate a single azoreductase. The optimal activity, pH, cofactor usage, substrate specificity, molecular weight and K _m characteristics of the enzyme set it apart from other known oxygen-insensitive azoreductases. Received 18 May 1999/ Accepted in revised form 13 July 1999     

Ethidium bromide binding to native and denatured poly(dA)poly(dT)

Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·10⁶ M⁻¹≤K≤1.4·10⁶ M⁻¹, free energy ΔG ^o=−8.7±0.3 kcal/mol, enthalpy ΔH ^o≅0, and entropy ΔS ^o=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (T _m) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10⁻⁵ M EtBr) increased by 17°C as compared with the ΔT _m of free homopolymer, whereas the half-width of the transition (T _m) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K ₁=1.7·10⁵ M⁻¹; ΔG ^o=−8.10±0.03 kcal/mol) and weak (K ₂=2.9·10³ M⁻¹; ΔG ^o=−6.0±0.3 kcal/mol).The ΔG ^o of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.     

Inhibition of cation-induced DNA condensation by intercalating dyes

Several intercalating dyes are shown to inhibit the cation-induced condensation of λ-DNA when Co³⁺(NH₃)₆ is the condensing agent. The dyes that have been studied are ethidium, propidium, proflavin, quinacrine, and actinomycin D. Earlier work has shown that intercalating dyes inhibit ψ-DNA condensation. [Lerman, L. S. (1971) Prog. Mol. Subcell. Biol. 2 , 382–391; Cheng, S. & Mohr, S. C. (1975) Biopolymers 14 , 663–674.] Dye-induced decondensation of intramolecularly condensed DNA has been studied by making use of conditions in which Co³⁺(NH₃)₆ produces intramolecular condensation without significant aggregation. Some aggregation is caused, however, during dye-induced decondensation. Dye titration curves of DNA decondensation have been measured by excess light scattering to monitor decondensation and by fluorescence to monitor intercalation. All of the dyes studied act as competing cations in displacing the condensing cation Co³⁺(NH₃)₆ from the DNA. Competition occurs both in and below the transition zone for condensation. The effectiveness of a dye as a competing cation increases with its net positive charge. Before decondensation begins, no intercalated dye can be detected, suggesting that intercalation might be incompatible with the proper helix packing needed for cation-induced DNA condensation. To test this last point, methidium–spermine was synthesized: it contains an intercalating methidium head group combined with a polyamine tail. Methidium–spermine is found to cause λ-DNA condensation, but aggregation accompanies condensation, as has been found earlier for spermine and spermidine. Fluorescence and absorption spectra indicate that the methidium group is intercalated when the DNA is condensed, indicating that intercalation need not be incompatible with DNA condensation. The presence of aggregates among the condensed DNA molecules makes this last conclusion tentative.     

DNA damage in mouse lymphocytes exposed to curcumin and copper

Dietary polyphenolics, such as curcumin, have shown antioxidant and anti-inflammatory effects. Some antioxidants cause DNA strand breaks in excess of transition metal ions, such as copper. The aim of this study was to evaluate thein vitro effect of curcumin in the presence of increasing concentrations of copper to induce DNA damage in murine leukocytes by the comet assay. Balb-C mouse lymphocytes were exposed to 50 μM curcumin and various concentrations of copper (10 μM, 100 μM and 200 μM). Cellular DNA damage was detected by means of the alkaline comet assay. Our results show that 50 μM curcumin in the presence of 100–200 μM copper induced DNA damage in murine lymphocytes. Curcumin did not inhibit the oxidative DNA damage caused by 50 μM H₂O₂ in mouse lymphocytes. Moreover, 50 μM curcumin alone was capable of inducing DNA strand breaks under the tested conditions. The increased DNA damage by 50 μM curcumin was observed in the presence of various concentrations of copper, as detected by the alkaline comet assay.     

Evaluation of antigenotoxicity effects of umbelliprenin on human peripheral lymphocytes exposed to oxidative stress

The protective properties of a prenylated coumarin, umbelliprenin (UMB), on the human lymphocytes DNA lesions were tested. Lymphocytes were isolated from blood samples taken from healthy volunteers. DNA breaks and resistance to H₂O₂-induced damage were measured using a single-cell microgel electrophoresis technique under alkaline conditions (comet assay). Human lymphocytes were incubated in UMB (10, 25, 50, 100, 200, and 400 μM) alone or a combination of different concentrations of UMB (10, 25, 50, 100, 200, and 400 μM) and 25 μM H₂O₂. Untreated cells, ascorbic acid (AA; 25, 50, 100, 200, and 400 μM) and H₂O₂ (25 μM) were considered as negative control, positive control, and the standard antioxidant agent for our study, respectively. Single cells were analyzed with “TriTek Cometscore version 1.5” software. The DNA damage was expressed as percent tail DNA. UMB exhibited a concentration-dependent increase in protection activity against DNA damage induced by 25 μM H₂O₂ (from 67.28% to 39.17%). The antigenotoxic activity of AA, in the range 0–50 μM, was greater than that of UMB. However, no significant difference (p > 0.05) in the protective activity was found between UMB and AA at concentrations of approximately higher than 50 μM.     

The ncgl1108 (PheP (Cg)) gene encodes a new L-Phe transporter in Corynebacterium glutamicum

Corynebacterium glutamicum played a central role in the establishment of fermentative production of amino acids, and it is a model for genetic and physiological studies. The general aromatic amino acid transporter, AroP _Cg , was the sole functionally identified aromatic amino acid transporter from C. glutamicum. In this study, the ncgl1108 (named as pheP _Cg ), which is located upstream of the genetic cluster (ncgl1110 ∼ ncgl1113) for resorcinol catabolism, was identified as a new l-Phe specific transporter from C. glutamicum RES167. The disruption of pheP _Cg resulted in RES167∆ncgl1108, and this mutant showed decreased growth on l-Phe (as nitrogen source) but not on l-Tyr or l-Trp. Uptake assays with unlabeled and ¹⁴C-labeled l-Phe and l-Tyr indicated that the mutants RES167∆ncgl1108 showed significant reduction in l-Phe uptake than RES167. Expression of pheP _Cg in RES167∆ncgl1108/pGXKZ1 or RES167∆(ncgl1108-aroP _Cg )/pGXKZ1 restored their ability to uptake for l-Phe and growth on l-Phe. The uptake of l-Phe was not inhibited by nine amino acids but by l-Tyr. The K _m and V _max values of RES167∆(ncgl1108-aroP _Cg )/pGXKZ1 for l-Phe were determined to be 10.4 ± 1.5 μM and 1.2 ± 0.1 nmol min⁻¹ (mg DW)⁻¹, respectively, which are different from K _m and V _max values of RES167∆(ncgl1108-aroP _Cg ) for l-Phe [4.0 ± 0.4 μM and 0.6 ± 0.1 nmol min⁻¹ (mg DW)⁻¹]. In conclusion, this PheP _Cg is a new l-Phe transporter in C. glutamicum.     

In vitro study of the tocolytic effect of oroxylin A from Scutellaria baicalensis root

Scutellariae Radix is one of the well-known tocolytic Chinese herbs. Oroxylin A is isolated from the root of Scutellaria baicalensis. The main syndrome of preterm birth is caused by uterus contractions from excitatory factors. Administration of tocolytic agents is a strategy to prevent the occurrence of preterm births. The aim of this study was to investigate the effects of oroxylin A on contractions of uterine strips isolated from non-pregnant female Wistar rats (250~350 g). Contractions of the uterus were induced with acetylcholine (Ach) (1 μM), PGF_2α (0.1 μM), oxytocin (10^-3 U/ml), KCl (56.3 mM), tetraethylammonium (TEA; 1 and 10 mM), 4-aminopyridine (4-AP; 5 mM), glipizide (30 μM), a nitric oxide synthase (NOS) inhibitor (LNNA; 10^-3M), a β-receptor blocker (propranolol; 10 μM), and a cyclooxygenase inhibitor (indomethacin; 60 μM). The inhibitory effects of the amplitude and frequency of spontaneous contractions by oroxylin A were antagonized with Ach (IC₅₀ 22.85 μM), PGF_2α (IC₅₀27.28 μM), oxytocin (IC₅₀ 12.34 μM), TEA; 1 and 10 mM (IC₅₀ 52.73 and 76.43 μM), 4-AP (IC₅₀ 67.16 μM), and glipizide (IC₅₀27.53 μM), but oroxylin A was not influenced by Ca²⁺-free medium, LNNA, propranolol, or indomethacin. Otherwise, oroxylin A-mediated relaxation of the rat uterus might occur through opening of uterine calcium-dependent potassium channels or adenosine triphosphate potassium channel activation. This suggests that oroxylin A is the tocolytic principle constituent of Scutellariae Radix, and oroxylin A may provide a lead compound for new tocolytic drug development in the future.     

Effects of DNA charge and length on the electrophoretic mobility of intercalated DNA

DNA molecules ranging in size from 1 to 630 kilobase pair and intercalated with either ethidium bromide (EtBr) or propidium iodide (PI) were electrophoresed in 1% agarose at four different electric field strengths. The extent of intercalation of EtBr under the conditions of our electrophoresis experiments was determined by a spectroscopic technique, whereas the extent of intercalation of PI was inferred from previous studies. The effects of the increase in DNA contour length and the concomitant decrease of linear charge density were separated based on our analysis of the mobility data. We conclude that the main factor responsible for the reduced electrophoretic mobility of intercalated DNA is the diminished linear charge density and not the increased contour length. © 1994 John Wiley & Sons, Inc.     

Characterization of Na+-Coupled Glutamate/Aspartate Transport by a Rat Brain Astrocyte Line Expressing GLAST and EAAC1

d-Aspartate (d-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. d-Asp influx is Na⁺-dependent with K _m = 5 μm and V _max= 0.7 nmoles · min⁻¹· mg protein⁻¹. Influx is sigmoidal as f[Na⁺] with _Na+ K _m ∼ 12 μm and Hill coefficient of 1.9. The cells establish steady-state d-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in V _max, with no change in K _m . At initial [d-Asp] = 10 μm, RBA take up more than 90% of total d-Asp, and extracellular levels are reduced to levels below 1 μm. Ionophores that dissipate the Δμ_Na+ inhibit gradient formation. Genistein (GEN, 100 μm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4α-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na⁺-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), l-Asp, and l-Glu, but not by d-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells. Received: 11 January 2001/Revised: 26 March 2001     

Recombinant DNA-methyltransferase M1.BspACI from <Emphasis Type="Italic">Bacillus psychrodurans</Emphasis> AC: Purification and properties

A restriction-modification system from Bacillus psychrodurans AC (recognition sequence 5′-CCGC-3′) comprises two DNA methyltransferases: M1.BspACI and M2.BspACI. The bspACIM1 gene was cloned in the pJW2 vector and expressed in Escherichia coli cells. High-purity M1.BspACI preparation has been obtained by chromatography on different carriers. M1.BspACI has a temperature optimum of 30°C and demonstrates maximum activity at pH 8.0. M1.BspACI modifies the first cytosine in the recognition sequence 5′-CCGC-3′. The kinetic parameters of M1.BspACI DNA methylation are as follows: K _m for phage λ DNA is 0.053 μM and K _m for S-adenosyl-L-methionine is 5.1 μM. The catalytic constant (k _cat) is 0.095 min⁻¹.     

Copper-induced changes in the growth, oxidative metabolism, and saponin production in suspension culture roots of Panax ginseng in bioreactors

Roots of Panax ginseng exposed to various concentrations of Cu (0.0, 5, 10.0, 25.0, and 50.0 μM) accumulated high amounts of Cu in a concentration-dependent and duration-dependent manner. Roots treated with 50 μM Cu resulted in 52% and 89% growth inhibition after 20 and 40 days, respectively. Saponin synthesis was stimulated at a Cu concentration between 5 and 25 μM but decreased at 50 μM Cu. Malondialdehyde content (MDA), lipoxygenase activity (LOX), superoxide ion (O₂ ^•−) accumulation, and H₂O₂ content at 5 and 10 μM Cu-treated roots were not increased but strongly increased at 50 μM Cu resulting in the oxidation of ascorbate (ASC) and glutathione (GSH) to dehydroascorbate (DHA) and glutathione disulfide (GSSG), respectively indicating a clear oxidative stress. Seven well-resolved bands of superoxide dismutase (SOD) were detected in the gel and an increase in SOD activity seemed to be mainly due to the induction of Fe-SOD 3. Five to 10 μM Cu slightly induced activity of ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR), guaiacol peroxidase (G-POD) but inhibited monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) enzyme activities. No changes in catalase (CAT) activity and in activity gel were found up to 25 μM Cu, but both G-POD and CAT activities were inhibited at 50 μM Cu. Glutathione metabolism enzymes such as γ-glutamylcysteine synthetase (γ-GCS), glutathione-S-transferase (GST), and glutathione peroxidase activities (GPx) were activated at 5 and 10 μM Cu but were strongly inhibited at 50 μM Cu due to the Cu accumulation in root tissues. The strong depletion of GSH at 50 μM Cu was associated to the strong induction of γ-glutamyltranspeptidase (γ-GGT) activity. These results indicate that plant could grow under Cu stress (5–25 μM) by modulating the antioxidant defense mechanism for combating Cu induced oxidative stress.     

Cloning and analysis of biochemical and catalytic properties of DNA methyltransferase M1.BspACI

DNA methyltransferases genes of the BspACI restriction-modification system from Bacillus psychrodurans AC have been cloned in E. coli cells. Analysis of amino acid sequences of the proteins showed that both of these genes belong to C5 DNA methyltransferases. Gene M1.BspACI has been subcloned in pJW2 vector. A high-purity recombinant enzyme has been obtained using chromatography on different carriers. It has been shown that M1.BspACI modifies the first cytosine residue in the sequence 5′-CCGC-3′. Kinetic parameters of DNA methylation by the enzyme have been determined. Catalytic constant appears to be 0.095 ± 0.002 min⁻¹. K _{mphage is λ DNA}—0.053 ± 0.007 μM, and K _mSAM is 5.1 ± 0.3 μM.     

Minimal levels of cold-adaptation in postsynaptic currents of a subantarctic teleost, <Emphasis Type="Italic">Notothenia rossii</Emphasis> (Perciformes: Notothenioidei: Nototheniidae)

As a part of the ICEFISH04 project on the RVIB Nathaniel B. Palmer, miniature end plate currents (MEPCs) were recorded from the extraocular muscles of Notothenia rossii captured at King Edward Point, South Georgia. A total of 1,176 MEPCs were recorded from the inferior oblique extraocular muscles of four specimens, over a temperature range of 1–12°C. The MEPCs were normal in form, with a rapid quasi-linear increase in inward current (typically <500 μs), followed by a slower exponential decay of the inward current to baseline. Exponential decay rates were calculated for individual MEPCs by linear regression of the log-transformed data, and converted to exponential time constants (τ). Only those MEPCs that fit the exponential model well, with r ² ≥ 0.95 (or in some cases r ² ≥ 0.99) were used for further calculations. At temperatures between 1 and 2°C, τ ranged from about 2,000 to 4,000 μs, similar to values extrapolated for temperate teleosts at the same temperature, but significantly longer than τ from MEPCs of high-latitude Antarctic nototheniids. Between 11 and 12°C, τ values for the N. rossii MEPCS were mainly between 1,100 and 1,700 μs, giving a Q ₁₀ of 2.05. An Arrhenius plot and linear regression were used to describe the effect of changing temperature on the decay phase of the N. rossii MEPCs: −ln τ = 27.887−6078/K, yielding an Arrhenius temperature coefficient (μ or apparent E _a) of −50.5 ± 2.9 (95% CL) kJ mol⁻¹ deg⁻¹. When compared with other nototheniids, these results showed that the neuromuscular junctions of N. rossii are compensated for low temperature, but not to the same degree as those of high Antarctic species. The ICEFISH Cruise (International Collaborative Expedition to collect and study Fish Indigenous to Sub-antarctic Habitats) was conducted on board the RVIB Nathaniel B. Palmer in May to July 2004. For further information, please visit .     

Is photocleavage of DNA by YOYO-1 using a synchrotron radiation light source sequence dependent?

The photocleavage of double-stranded and single-stranded DNA by the fluorescent dye YOYO-1 was investigated in real time by using the synchrotron radiation light source ASTRID (ISA, Denmark) both to initiate the reaction and to monitor its progress using Couette flow linear dichroism (LD) throughout the irradiation period. The dependence of LD signals on DNA sequences and on time in the intense light beam was explored and quantified for single-stranded poly(dA), poly[(dA-dT)₂], calf thymus DNA (ctDNA) and Micrococcus luteus DNA (mlDNA). The DNA and ligand regions of the spectrum showed different LD kinetic behaviors, and there was significant sequence dependence of the kinetics. However, in contrast to expectations from the literature, we found that poly(dA), mlDNA, low salt ctDNA and low salt poly[(dA-dT)₂] all had significant populations of groove-bound YOYO. It seems that this mode was predominantly responsible for the catalysis of DNA cleavage. In homopolymeric DNAs, intercalated YOYO was unable to cleave DNA. In mixed-sequence DNAs the data suggest that YOYO in some but not all intercalated binding sites can cause cleavage. It is also likely that cleavage occurs at transient single-stranded regions. The reaction rates for a 100 mA beam current of 0.5-μW power varied from 0.6 h⁻¹ for single-stranded poly(dA) to essentially zero for low salt poly[(dG-dC)₂] and high salt poly[(dA-dT)₂]. At the conclusion of the experiments with each kind of DNA, uncleaved DNA with intercalated YOYO remained.     

Aviglycine and propargylglycine inhibit conidial germination and mycelial growth of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>f. sp. <Emphasis Type="Italic">luffae</Emphasis>

Two inhibitors, aviglycine and propargylglycine, were tested for their ability to suppress methionine synthesis thus inhibit conidial germination and mycelial growth of Czapek-Dox liquid medium grown Fusarium oxysporum f. sp. luffae μM. The linear inhibition range for mycelial growth was about 7.6–762.9 μM. Although aviglycine did not completely inhibit both conidial germination and mycelial growth, it showed significant inhibitory effect at 1.5 μM. The inhibition range for propargylglycine against conidial germination and mycelial growth were from 0.08 to 8841 μM and from 0.8 to 884.1 μM, respectively. Propargylglycine inhibited conidial germination and mycelial growth at a concentration of 8841 μM. The EC₅₀ values of aviglycine were 1 μM for conidial growth and 122 μM for mycelial growth, and the EC₅₀ values of propargylglycine were 47.7 μM for conidial growth and 55.6 μM for mycelial growth. Supplement of methionine released inhibition of aviglycine or propargylglycine to conidial germination. In addition, a mixture of aviglycine (1.5 μM) and propargylglycine (8841 μM) showed additive inhibitive effect than applied alone on 10 isolates. From these results, both aviglycine and propargylglycine exhibited inhibitory activity, and suggest that they can provide potential tools to design novel fungicide against fungal pathogens.     

Preparation and characterization of epoxy-functionalized magnetic chitosan beads: laccase immobilized for degradation of reactive dyes

Cross-linked magnetic chitosan beads were prepared by phase-inversion technique in the presence of epichlorohydrin under alkaline condition, and used for covalent immobilization of laccase. The activity of the immobilized laccase on the magnetic chitosan was about 260 U (g/dry beads) with an enzyme loading of about 16.33 ± 0.39 mg [(g/dry beads) mg/g]. Kinetic parameters, V _max and K _m values were determined as 21.7 U/mg protein and 9.4 μM for free enzyme, and 15.6 U/mg protein and 19.7 μM for the immobilized laccase, respectively. The operational and thermal stabilities of the immobilized laccase were improved compared to free counterpart. The immobilized laccase was operated in a batch reactor for the decolorization of reactive dyes from aqueous solution. The laccase immobilized on magnetic chitosan beads was very effective for removal of textile dyes from aqueous solution which creates an important environmental problem in the discharged textile dying solutions.     

Optimization of <Emphasis Type="Italic">Renealmia mexicana</Emphasis> (Klotzsch ex. Petersen) cultivation <Emphasis Type="Italic">in vitro</Emphasis>

Renealmia mexicana (Klotzsch ex. Petersen) is a tropical plant found in southern México with an ornamental value and a potential source of curcuminoids. Its distribution in Chiapas has decreased because of deforestation and low propagation and germination rate, so a protocol for in vitro propagation was developed. An orthogonal experimental design of L₉ (3⁴) in triplicate was used to investigate the effect of 6-benzyl adenine (BA), indole butyric acid (IBA), silver nitrate (AgNO₃), and sucrose on shoot, root, and leaf development of plantlets grown in vitro. Plantlets with well-developed shoots and roots were transferred to pots containing a mixture of peat moss and agrolite for hardening before transfer to soil. The Murashige and Skoog (Physiol. Plant. 15:473–497, 1962) mineral medium (MS) supplemented with 4.4 μM BA, 2.5 μM IBA, 11.7 μM AgNO_3y and 5.5% (w/v) sucrose gave most shoots, 8.9 μM BA, 2.5 μM IBA, 17.7 μM AgNO₃ and 5.5% (w/v) sucrose most roots, and 8.9 μM BA, 4.9 μM IBA, 11.7 μM AgNO₃ and 3.0% (w/v) sucrose most leaves, although other combinations were statistically equivalent in each case. Sucrose was the factor that most explained the variation in the promotion of shoots, roots, and leaves. The protocol developed resulted in up to 100% survival when plantlets were transferred to soil using AgNO₃, confirming that hardening of plantlets in vitro using hormonal stimulation was a suitable strategy to improve acclimatization.