<Emphasis Type="Italic">N</Emphasis><Superscript>4</Superscript>,<Emphasis Type="Italic">N</Emphasis><Superscript>9</Superscript>-Dioleoyl Spermine Is a Novel Nonviral Lipopolyamine Vector for Plasmid DNA Formulation

Purpose To study the effect of synthesized N⁴,N⁹-dioleoyl spermine on DNA condensation and then measure its transfection efficiency in cell culture.Methods The lipopolyamine was synthesized from the naturally occurring polyamine spermine. The ability of this novel compound to condense DNA was studied using ethidium bromide fluorescence quenching and light scattering assays. Transfection efficiency was studied in primary skin cells (FEK4) and in an immortalized cancer cell line (HtTA), and compared with the commercially available transfection formulations Lipofectin and Lipofectamine.Results The synthesized N⁴,N⁹-dioleoyl spermine formula is efficient at condensing calf thymus and circular plasmid DNA and effectively transfects both primary skin cells and cancer cell lines at low charge ratios of (+/– ammonium/phosphate) 2.5.Conclusions N⁴,N⁹-Dioleoyl spermine condenses DNA and achieves high transfection levels in cultured cells.     

<Emphasis Type="Italic">In Vitro</Emphasis> antioxidant and anti-inflammatory activities of Jaceosidin from <Emphasis Type="Italic">Artemisia princeps</Emphasis> Pampanini <Emphasis Type="Italic">cv.</Emphasis> Sajabal

Oxidized low-density lipoprotein (oxLDL) plays a key role in the inflammatory processes of atherosclerosis. Jaceosidin isolated from the methanolic extracts of the aerial parts of Artemisia princeps Pampanini cv. Sajabal was tested for antioxidant and anti-inflammatory activities. Jaceosidin inhibited the Cu²⁺-mediated LDL oxidation with IC₅₀ values of 10.2 μM in the thiobarbituric acid-reactive substances (TBARS) assay as well as the macrophage-mediated LDL oxidation. The antioxidant activities of jaceosidin were exhibited in the conjugated diene production, relative electrophoretic mobility, and apoB-100 fragmentation on copper-mediated LDL oxidation. Jaceosidin also inhibited the generation of reactive oxygen species (ROS) concerning in regulation of NF-κB signaling. And jaceosidin inhibited nuclear factor-kappa B (NF-κB) activity, nitric oxide (NO) production, and suppressed expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages.     

Two new antibacterial norabietane diterpenoids from cyanobacteria, <Emphasis Type="Italic">Microcoleous </Emphasis><Emphasis Type="Italic">lacustris</Emphasis>

Two abietane diterpenes were isolated from cyanobacteria Microcoleous lacustris, 20-nor-3α-acetoxyabieta-5,7,9,11,13-pentaene and 20-nor-3α-acetoxy-12-hydroxy-abieta-5,7,9,11,13-pentaene. These compounds were assayed against Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhi, Vibrio cholerae, Bacillus subtilis, Bacillus cereus, Escherichia coli, and Klebsiella pneumoniae. Both compounds showed activity against S. aureus, S. epidermidis, S. typhi, and V. cholerae, but not against the other bacteria.     

Cadmium tolerance and accumulation by two species of <Emphasis Type="Italic">Iris</Emphasis>

Seedlings of Iris lactea var. chinensis (Fisch.) Koidz. and I. tectorum Maxim. were subjected to 0–160 mg l⁻¹ Cd in hydroponic system and harvested after 42 days to determine effects on root and shoot dry mass. A subset of 16-day-old seedlings was exposed to 1000 mg l⁻¹ Cd to characterize sub-cellular localization of Cd in root cells. The Cd contents in the shoots of I. lactea var. chinensis reached 529 μg g⁻¹ dry weight (dw) at 80 mg l⁻¹ Cd treatment and in the shoots of I. tectorum reached 232 μg g⁻¹ dw at 40 mg l⁻¹ Cd treatment, without showing signs of visible toxicity. The Cd contents in the shoots of both two test species exceeded 100 μg g⁻¹, the critical value of Cd hyperaccumulator. The indices of tolerance (ITs) of I. lactea var. chinensis were higher than those of I. tectorum under 10–160 mg l⁻¹Cd stress. Sub-cellular localization of Cd in root cells was evaluated using transmission electron microscopy (TEM) and Cd deposits were found in the cell walls, in the cytoplasm and on the inner surface of xylem vessels in the root tip of I. lactea var. chinensis and I. tectorum. A few cells in the root tip of I. tectorum were necrotic. The results showed that the tolerance and accumulation of Cd by I. lactea var. chinensis were higher than those of I. tectorum, suggesting that I. lactea var. chinensis has potential application in phytoremediation.     

Cocaine inhibits cromakalim-activated K<Superscript>+</Superscript> currents in follicle-enclosed<Emphasis Type="Italic"> Xenopus</Emphasis> oocytes

The effect of cocaine on K⁺ currents activated by the K_ATP channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3–500 M reversibly inhibits cromakalim-induced K⁺ currents. The IC₅₀ value for cocaine was 96 M. Inhibition of the cromakalim-activated K⁺ current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca²⁺ chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K⁺ currents, suggesting that Ca²⁺-activated second messenger pathways are not involved in the actions of cocaine. Outward K⁺ currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC₅₀ and slope values for the activation of K⁺ currents by cromakalim were 184±19 M and 1.14 in the absence of cocaine as compared to 191±23 M and 1.03 in the presence of cocaine (300 M). Cocaine also blocked K⁺ currents mediated through C-terminally deleted form of Kir6.2 (KirC26) in the absence of sulfonylurea receptor with an IC₅₀ value of 87 M, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 M) did not affect the binding characteristics of the K_ATP ligand, [³H]glibenclamide. These results demonstrate that cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes are modulated by cocaine.     

<Emphasis Type="Italic">Perilla frutescens</Emphasis> var. <Emphasis Type="Italic">frutescens</Emphasis> in northern Laos

Twenty-eight samples of mericarps of Perilla frutescens var. frutescens were collected through fieldwork performed in Phongsali and Xieng Khouang provinces in northern Laos. No perilla samples were collected from Savannakhet province in the south although more than 20 sites were investigated. Perilla plants are mostly grown mixed with dry-paddy rice by slash-and-burn cultivation in Laos. The most popular local name for perilla mericarps in the area was “Ma Nga Chan”. Weight of 1,000 grains and hardness of the mericarps were measured, and all mericarps were found to be large (weight of 1,000 grains around 2 g) and soft (limit load weight under 300 g), which were preferred for culinary use in Laos. The composition of the essential oils obtained from the herbaceous plants raised from the mericarps was divided into five types, perillaketone, elemicine plus myristicine, shisofuran, piperitenon, and myristicine, and GC–MS analysis of these Laotian perilla samples showed that they were similar to those of corresponding types of known Japanese perilla strains. One of the shisofuran-type perilla contained large amounts of putative α-naginatene, which is likely to be an intermediate of the biosynthesis of naginataketone. The farmers' indifference to the oil type of the leaf seems to leave Laotian perilla as a good genetic resource for studies of the biosynthesis of oil compounds.     

Identification of <Emphasis Type="Italic">Curcuma</Emphasis> plants and curcumin content level by DNA polymorphisms in the <Emphasis Type="Italic">trn</Emphasis>S-<Emphasis Type="Italic">trn</Emphasis>fM intergenic spacer in chloroplast DNA

With the goal of developing an accurate plant identification method, molecular analysis based on polymorphisms of the nucleotide sequence of chloroplast DNA (cpDNA) was performed in order to distinguish four Curcuma species: C. longa, C. aromatica, C. zedoaria, and C. xanthorrhiza. Nineteen regions of cpDNA were amplified successfully via polymerase chain reaction (PCR) using total DNA of all Curcuma plants. Using the intergenic spacer between trnS and trnfM (trnSfM), all four Curcuma plant species were correctly identified. In addition, the number of AT repeats in the trnSfM region was predictive of the curcumin content in the rhizome of C. longa.     

A new <Emphasis Type="Italic">Erythrina</Emphasis> alkaloid from <Emphasis Type="Italic">Erythrina herbacea</Emphasis>

A new Erythrina alkaloid, 10-hydroxy-11-oxoerysotrine (1), has been isolated from the flowers of Erythrina herbacea together with five known compounds: erytharbine (2), 10,11-dioxoerysotrine (3), erythrartine (4), erysotramidine (5) and erysotrine-N-oxide (6). The structure of the new compound was elucidated on the basis of its spectral data, including 2-D NMR and mass (MS) spectra. The new compound is a rare C-10 oxygenated Erythrina alkaloid. The antioxidant activities of the isolated compounds 1–6 were evaluated by scavenging with peroxynitrite.     

Characterisation of (<Emphasis Type="Italic">R</Emphasis>/<Emphasis Type="Italic">S</Emphasis>)-propafenone and its metabolites as substrates and inhibitors of P-glycoprotein

Digoxin is a drug with a narrow therapeutic index, which is a substrate of the ATP-dependent efflux pump P-glycoprotein. Increased or decreased digoxin plasma concentrations occur in humans due to the inhibition or induction of this drug transporter in organs with excretory function such as small intestine, liver and kidney. It is well known that serum concentrations of digoxin increase considerably in humans if propafenone is given simultaneously. However, it has not been investigated in detail whether propafenone and its metabolites are substrates and/or inhibitors of human P-glycoprotein. The aim of this study, therefore, was to investigate the P-glycoprotein-mediated transport and inhibition properties of propafenone and its major metabolites 5-hydroxypropafenone and N-desalkylpropafenone in Caco-2 cell monolayers. Inhibition of P-glycoprotein-mediated transport by propafenone and its metabolites was determined using digoxin as a P-glycoprotein substrate. No polarised transport was observed for propafenone and N-desalkylpropafenone in Caco-2 cell monolayers. However, 5-hydroxypropafenone translocation was significantly greater from basal-to-apical compared with apical-to-basal (P_app basal–apical vs. P_app apical–basal, 10.21±2.63×10^–6 vs. 4.34±1.84×10^–6 cm/s; P<0.01). Moreover, propafenone, 5-hydroxypropafenone and N-desalkylpropafenone inhibited P-glycoprotein-mediated digoxin transport with IC₅₀ values of 6.8, 19.9, and 21.3 M, respectively. In summary, whereas propafenone and N-desalkylpropafenone are not substrates of P-glycoprotein, 5-hydroxypropafenone is translocated by human P-glycoprotein across cell monolayers. In addition, propafenone and its two major metabolites 5-hydroxypropafenone and N-desalkylpropafenone are inhibitors of human P-glycoprotein and therefore contribute to the digoxin–propafenone interaction observed in humans.     

Seasonal changes of metal accumulation and distribution in common club rush (<Emphasis Type="Italic">Schoenoplectus lacustris</Emphasis>) and common reed (<Emphasis Type="Italic">Phragmites australis</Emphasis>)

In this study, two aquatic macrophytes Phragmites australis and Schoenoplectus lacustris and corresponding sediment samples were collected every three months from Lake Sapanca (Turkey) and analysed for their heavy-metal contents (Pb, Cr, Cu, Mn, Ni, Zn and Cd). Accumulation factor ratios of plant parts were calculated for all metals, and the two species were compared in terms of accumulation properties. The highest concentrations were measured in the root systems while relatively low concentrations were found in the rhizome and above-ground parts of the plants. The accumulation ratios of root for P. australis were usually higher than the ratios for S. lacustris. While the accumulation ratios of root were higher in winter than in the other seasons for P. australis, for S. lacustris the highest accumulation ratios were found in the autumn. Both plant species were found to be root accumulators of Pb, Cu, Mn, Ni, Zn and Cd.     

Potential anthelmintics: polyphenols from the tea plant <Emphasis Type="Italic">Camellia sinensis</Emphasis> L. are lethally toxic to <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

A novel gallate of tannin, (−)-epigallocatechin-(2β→O→7′,4β→8′)-epicatechin-3′-O-gallate (8), together with (−)-epicatechin-3-O-gallate (4), (−)-epigallocatechin (5), (−)-epigallocatechin-3-O-gallate (6), and (+)-gallocatechin-(4α→8′)-epigallocatechin (7), were isolated from the tea plant Camellia sinensis (L.) O. Kuntze var. sinensis (cv., Yabukita). The structure of 8, including stereochemistry, was elucidated by spectroscopic methods and hydrolysis. The compounds, along with commercially available pyrogallol (1), (+)-catechin (2), and (−)-epicatechin (3), were examined for toxicity towards egg-bearing adults of Caenorhabditis elegans. The anthelmintic mebendazole (9) was used as a positive control. Neither 2 nor 3 were toxic but the other compounds were toxic in the descending order 8, 7 ≈ 6, 9, 4, 5, 1. The LC₅₀ (96 h) values of 8 and 9 were evaluated as 49 and 334 μmol L⁻¹, respectively. These data show that many green tea polyphenols may be potential anthelmintics.     

Antitubercular triterpenes and phytosterols from <Emphasis Type="Italic">Pandanus tectorius </Emphasis>Soland. var. <Emphasis Type="Italic">laevis</Emphasis>

Bioassay-guided chromatographic purification of the antitubercular chloroform extract of Pandanus tectorius Soland. var. laevis leaves afforded a new tirucallane-type triterpene, 24,24-dimethyl-5β-tirucall-9(11),25-dien-3-one (1), squalene and a mixture of the phytosterols stigmasterol and β-sitosterol. Microplate Alamar Blue Assay (MABA) showed that 1 inhibited the growth of Mycobacterium tuberculosis H₃₇Rv with a MIC of 64 μg/mL, while squalene and the sterol mixture have MICs of 100 and 128 μg/mL, respectively.     

Determinants for the cAMP-binding site at the <Emphasis Type="Italic">S</Emphasis>-adenosylhomocysteine-hydrolase

S-Adenosylhomocysteine-hydrolase (AdoHcy-hydrolase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to adenosine (Ado) and homocysteine (Hcy). Since Ado competes with cAMP at the high affinity-binding site of the enzyme, we determined the effect of cAMP on enzyme activity and its binding characteristics to purified AdoHcy-hydrolase from bovine kidney in its native, in its fully oxidized (NAD⁺), and in its fully reduced (NADH) form. cAMP (10 μmol/l) enhanced the hydrolytic activity of native AdoHcy-hydrolase by 35%, whereas the activity of the enzyme in its NAD⁺ form was not stimulated by cAMP. In contrast to azido-Ado, binding of azido-cAMP did not inhibit the enzymatic activity of AdoHcy-hydrolase. Furthermore, cAMP did not prevent the Ado induced inhibition of the AdoHcy hydrolysis. Saturation binding experiments with the three different forms of AdoHcy-hydrolase, native, NAD⁺, and NADH showed only one binding site with high affinity. This binding site was identified after photoaffinity labeling of the enzyme with 8-azido-[2-³H]-cAMP. One photolabeled peptide was isolated as Trp³¹⁰-Val³²⁵ from each AdoHcy-hydrolase from native, NAD⁺, and NADH. The cAMP-labeled peptide is located in the NAD-binding domain of AdoHcy-hydrolase. In conclusion, our data show that the cAMP-binding site at the AdoHcy-hydrolase is independent of the NAD⁺/NADH ratio of the enzyme and is identical with the high affinity-binding site of Ado. Moreover, cAMP did not interact with the catalytic site of AdoHcy-hydrolase and did not act as an allosteric effector for the AdoHcy-hydrolase.     

Evaluation of <Emphasis Type="Italic">Sesbania grandiflora</Emphasis> for antiurolithiatic and antioxidant properties

In the indigenous system of medicine in India, the plant Sesbania grandiflora is claimed to be useful for various ailments, and one such use is for the treatment of renal calculi. The major purpose of this study is to investigate the potential of S. grandiflora in the treatment of renal calculi. The leaf juice of S. grandiflora was evaluated for median lethal dose, gross behavioral changes, antiurolithiatic and antioxidant activities. The antiurolithiatic activity was evaluated by a calculi-producing diet model, using gentamicin (subcutaneously) and 5% ammonium oxalate in rat feed to induce calcium oxalate-type stones. The parameters monitored in the present study are calcium and oxalate deposition in the kidney, kidney weights, urinary excretion of calcium and oxalate. The in vivo antioxidant parameters lipid peroxidation, glutathione reductase and catalase were monitored. The plant juice was also evaluated for scavenging of nitric oxide and 2-diphenyl-2-picryl hydrazyl free radicals. The leaf juice of S. grandiflora was safe orally and exhibited no gross behavioral changes except for an increase in urination. The leaf juice of S. grandiflora showed significant antiurolithiatic activity against calcium oxalate-type stones and also exhibited antioxidant properties. The results obtained in this study provide evidence for the efficacy of the leaf juice of S. grandiflora as antiurolithiatic agent.     

Combinatorial anti-<Emphasis Type="Italic">Trichophyton</Emphasis> effects of <Emphasis Type="Italic">Ligusticum chuanxiong</Emphasis> essential oil components with antibiotics

The antifungal activities of ligustilide and butylidene phthalide, which are major oil components comprising the essential oil of Ligusticum chuanxiong, in combination with antifungal drugs were evaluated. Checkerboard microtiter and microdilution tests were used to measure combined effects against Trichophyton species, T. erinacei, T. mentagrophytes, T. rubrum, T. schoenleinii, T. tonsurans and T. soudanense. Susceptibility of these Trichophyton species to L. chuanxiong essential oil differed distinctly. The fractional inhibitory concentrations (FICIs) against Trichophyton species ranged between 0.27 and 0.56 for ketoconazole combined with ligustilde or butylidene phthalide, indicating synergism or additive effects between the antibiotic and essential oil compounds. Testing by itraconazole instead of ketoconazole, similar combination effects were observed with FICIs from 0.31 to 0.63 against the tested strains of T. mentagrophytes, T. rubrum, T. schoenleinii, and T. soudanense. Thus, data reported here showed that Trichophyton susceptibility to ketoconazole and itraconazole could be notably improved by ligustilde and butylidene phthalide.     

Molecular analysis of constitutive mutations in <Emphasis Type="Italic">ermB</Emphasis> and <Emphasis Type="Italic">ermA</Emphasis> selected <Emphasis Type="Italic">In Vitro</Emphasis> from inducibly MLS<Subscript>B</Subscript>-resistant enterococci

Frequencies of spontaneous mutation from inducible resistance to constitutive resistance were determined for the four clinical isolates of erythromycin-resistant enterococci, including one isolate with ermB gene and three clinical isolates with ermA gene. The rate of ermB mutation was higher than that of ermA mutation by more than 10 fold. Sequence analysis of the regulatory regions of erm genes revealed that mutation type of ermB was just point mutation, by contraries the mutation type of ermA was either deletion or tandem duplication. These results showed distinct characteristics in mutation patterns of ermB and ermA.     

Tolerance and accumulation of lead by species of <Emphasis Type="Italic">Iris</Emphasis> L.

Seedling development, accumulation and distribution of lead (Pb) in Iris lactea var. chinensis (Fisch.) Koidz. and I. tectorum Maxim. were studied using plants grown in sand culture and exposed to 0–10 mmol l⁻¹ concentrations of Pb supplied as Pb(NO₃)₂ for 28 days. A significant reduction in dry weight (dw) of shoots and roots of I. lactea var. chinensis was observed at 6 and 10 mmol l⁻¹, respectively, and a significant reduction in dw of shoots and roots of I. tectorum was observed at 6 mmol l⁻¹. Concentration of Pb in the shoots and roots of I. lacteal var. chinensis exposed to 4 mmol l⁻¹ Pb reached 1,109 μg g⁻¹ and 2,408 μg g⁻¹ dw, respectively. The index of tolerance (IT) of I. lactea var. chinensis among 0–8 mmol l⁻¹ Pb treatments were not significantly different, while those of I. tectorum at 6 mmol l⁻¹ Pb were significantly decreased. The results indicated that I. lactea var. chinensis was more tolerant to Pb than I. tectorum. Sub-cellular localization of Pb in root cells was evaluated using transmission electron microscopy (TEM) and Pb deposits were found along the plasma membrane of some root tip cells of I. lactea var. chinensis treated at 10 mmol l⁻¹ Pb. Deposits of Pd were also observed along the surface, in the root tip cell wall and in the cytoplasm of a few malformed cells of I. tectorum exposed at 10 mmol l⁻¹ Pb treatment. One possible mechanism to explain these observations may be that most cells can maintain normal activities in the plant by sacrificing a small number of cells that accumulate a large amount Pb and show toxicity. Future studies should be designed to test this hypothesis.     

Sub-lethal effects of acetone on <Emphasis Type="Italic">Daphnia magna</Emphasis>

There is increasing concern about the sub-lethal effect of hydrophobic chemicals in the water medium. Even though acetone is a commonly used solvent in toxicity testing, few studies have focussed on its chronic toxicity to Daphnia magna and the available results are often contradictory. In this study, acetone was tested on D. magna in a 21-day exposure experiment and the effects on mortality, fertility and morphology of exposed organisms (F₀) and offspring (F₁–F₂, reared without acetone) were evaluated. No significant reduction of survival was observed with increasing concentrations, and no significant reduction in fecundity in any treatment group in terms of average number of daphnids per mother was observed. Abnormal development of second antennae was observed on F₁ from F₀ exposed to 79 mg l⁻¹ solvent. The ET50 of acetone on the number of mothers that produced deformed offspring over time was 12.5 days. Our results suggest that the acetone concentration should not exceed 7.9 mg l⁻¹, which is 10 times less than the allowed concentration as determined by OECD chronic assays on D. magna. More attention should be paid to small, water-soluble molecules usually considered of low concern for chronic toxicity because they might affect other metabolic pathways.     

Chemical composition and antibacterial activity of essential oils of <Emphasis Type="Italic">Eugenia</Emphasis> species

The essential oils of the leaves of Eugenia brasiliensis, Eugenia beaurepaireana, and Eugenia umbelliflora were analyzed by GC–MS. The major compounds found in the oil of E. brasiliensis were spathulenol (12.6%) and τ-cadinol (8.7%), of E. beaurepaireana were β-caryophyllene (8.0%) and bicyclogermacrene (7.2%), and of E. umbelliflora were viridiflorol (17.7%) and β-pinene (13.2%). These oils were assayed to determine their antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. All of the oils analyzed showed antibacterial activity, ranging from moderate to strong, which was most accentuated for the E. umbelliflora and E. brasiliensis oils, which strongly inhibited the growth of S. aureus giving values of MIC = 119.2 and 156.2 μg/mL, respectively.