Antioxidant properties of <Emphasis Type="Italic">Galium verum</Emphasis> L. (Rubiaceae) extracts

The antioxidant properties of methanol extracts of Lady’s Bedstraw (Galium verum L., Rubiaceae) herb from two different localities in Serbia were evaluated. Antioxidant activity was assessed in four different model systems. Free radical scavenging capacity (RSC) was examined by measuring the scavenging activity of extracts on 2,2-diphenyl-1-pycrylhydrazil (DPPH) and hydroxyl radical (OH), as well as on hydrogen peroxide. In addition, the protective effects of lipid peroxidation (LP) in corn oil were evaluated by the TBA-assay using the Fe²⁺/ascorbate system of induction. The amount of dried extract, the content of total phenolics, flavonoids and chlorophylls was also determined. Extracts from both locations expressed very strong scavenger activity, reducing the DPPH^⊙ (IC₅₀=3.10 μg/mland 8.04 μg/ml) and OH radical formation (IC₅₀=0.05 μg/ml and 0.54 μg/ml) and neutralising H₂O₂ (IC₅₀=4.98 μg/ml and 3.80 μg/ml), in a dose dependant manner. Also, examined extracts showed notable inhibition of LP (IC₅₀=11.69 μg/ml and 19.47 μg/ml). The observed differences in antioxidant activity could be partially explained by the levels of phenolics (2.44–4.65 mg and 4.57–5.16 mg gallic acid equivalents/g dry extract), flavonoids (6.38–10.70 μg and 15.56–17.96 μg quercetin equivalents/g dry extract) and chlorophylls in the investigated Lady’s Bedstraw extracts.     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of Alcaligenes xylosoxydans EMS33 in a Bench-scale Fermenter

Summary Optimization of medium composition and pH for chitinase production by the Alcaligenes xylosoxydans mutant EMS33 was carried out in the present study and the optimized medium composition and conditions were evaluated in a fermenter. The medium components screened initially using Plackett–Burman design were (NH₄)₂SO₄, MgSO₄ 7H₂O, KH₂PO₄, yeast extract, Tween 20 and chitin in shake flask experiments. The significant medium components identified by the Plackett–Burman method were MgSO₄ 7H₂O, Tween 20 and chitin. Central composite response surface methodology was applied to further optimize chitinase production. The optimized values of MgSO₄ 7H₂O, Tween 20, chitin and pH were found to be 0.6 g/l, 0.05 g/l, 11.5 g/l and 8.0, respectively. Chitinase and biomass production of Alcaligenes xylosoxydans EMS33, was studied in a 2-l fermenter containing (g/l): chitin, 11.5; yeast extract, 0.5; (NH₄)₂SO₄, 1; MgSO₄ 7H₂O, 0.6; KH₂PO₄, 1.36 and Tween 20, 0.05. The highest chitinase production was 54 units/ml at 60 h and pH 8.0 when the dissolved O₂ concentration was 60%, whereas the highest biomass production was achieved at 36 h and pH 7.5 without any dissolved O₂ control.     

Development of enzyme-linked immunosorbent assay for determination of polybrominated diphenyl ether BDE-121

Our interests are in the development of immunoassay-based fast scanning methods for persistent organic pollutants. To develop the immunoassay method of polybrominated diphenyl ether (PBDE), a model compound of PBDE, 2,3′,4,5′,6-pentabromodiphenylether (BDE-121), has been chosen to develop its antibody and the competitive indirect enzyme-linked immunosorbent assay (ELISA) is developed. The hapten of BDE-121 containing reactive carboxylic acid was synthesized and conjugated to carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]). Anti-BDE-121 polyclonal antibody was then developed in rabbits as a result of immunization with the BDE-121–BSA conjugate. The optimal amount of coating antigen BDE-121–OVA conjugate and the dilution of antiserum needed in the ELISA were determined with the checkerboard method, and the effects of the properties of PBST (phosphate-buffered saline and Tween 20) buffer (pH and salt concentration) and chemical solvent (types and concentrations) on the ELISA were investigated to achieve a rapid robust assay with high sensitivity. Under the optimized conditions, the developed indirect ELISA shows a linear detection range from 1.74 to 84.1 ng/ml, with an IC₅₀ value of 8.07 ng/ml and a detection limit of 0.644 ng/ml. In total, 11 kinds of compounds were tested for calculating the cross-reactivity, which was less than 8% for nearly all of them. Real samples were analyzed by the proposed immunoassay and gas chromatography/mass spectrometry (GC/MS).     

The Effect of Food-Grade Low-Molecular-Weight Surfactants and Sodium Caseinate on Spray Drying of Sugar-Rich Foods

The effect of low-molecular-weight surfactants (LMS) and sodium caseinate (NaCas) on spray drying of sugar-rich foods has been studied. Sucrose and NaCas were selected as a model sugar-rich food and protein, respectively. Sodium stearoyl lactylate (SSL) and Polysorbate 80 (Tween 80) were chosen as model ionic and nonionic LMS. Sucrose–NaCas solutions with the solids ratio of 99.5:0.5 in the absence and presence (0.01% and 0.05%) of SSL and Tween 80 were prepared. The feed solutions had 25% solid concentration in all cases. The dynamic surface tension (DST) values of the solutions were measured for 100 s and the solutions were subsequently spray dried at inlet and outlet temperatures of 165 and 65 °C, respectively. The glass–rubber temperature (T _g-r), the surface elemental composition and amorphous–crystalline nature of the powders were also determined. At these concentrations and experimental time frame, it was found that the proteins preferentially migrated to the air–water interface reasonably swiftly. The addition of LMS resulted in partial or complete displacement of the proteins from the air–water interface. For spray-drying trials with the yield of 82.0%, it was found that 52.0% of the powder particle surface was covered with proteins. The powder recovery was greatly reduced by the LMS concentration and type. At 0.05% on dry solid basis, in the case of nonionic surfactant (Tween 80), the displacement of protein from the surface was such that no powder was recovered. The ionic surfactant (SSL) displaced 2.0% and 29.3% proteins from the droplet surface at concentrations of 0.01% and 0.05%, respectively, resulting in 75.5 ± 1.8% and 30.1 ± 1.4% powder yield. The T _g-r results revealed that the amount of protein required for successful spray drying of the sucrose–protein solution depends on the amount of proteins present in the droplet surface but not in the bulk. X-ray diffraction and scanning electron microscopy results confirmed that the powders of both sucrose–NaCas and sucrose–NaCas with 0.01% SSL were mostly amorphous, while those with sucrose–NaCas–Tween 80 (0.01%) and sucrose–NaCas–SSL (0.05%) were crystalline.     

Antioxidant activities of cultured <Emphasis Type="Italic">Armillariella mellea</Emphasis>

This study aimed to evaluate the antioxidant activities of a cultured medicinal fungus—Armillariella mellea (Vahl. ex Fr.) Karst. (AM). Three antioxidant assay systems, namely cytochrome c, xanthine oxidase inhibition, and FeCl₂-ascorbic acid stimulated lipid peroxidation in rat tissue homogenate tests, were used. Total flavonoid and phenol contents of AM extracts were also analyzed. Results showed that both aqueous (AM-H₂O) and ethanolic (AM-EtOH) extracts of solid state cultured AM showed antioxidant activities in a concentration-dependent manner. At concentrations 1–100 μg/ml, the free radical scavenging activity was 73.7–92.1% for AM-H₂O, and 60.0–90.8% for AM-EtOH. These extracts also showed an inhibitory effect on xanthine oxidase activity, but with a lesser potency (IC₅₀ is 9.17 μg/ml for AM-H₂O and 7.48 μg/ml for AM-EtOH). In general, AM-H₂O showed a stronger antilipid peroxidation activity on different rat’s tissues than AM-EtOH. However, both AM extracts displayed a weak inhibitory effect on lipid peroxidation in plasma. Interestingly, the antilipid peroxidation activity of AM-H₂O (IC₅₀–6.66 μg/ml) in brain homogenate was as good as IC₅₀–5.42 μg/ml. AM-H₂O (80.0 mg/g) possessed a significantly higher concentration of total flavonoids than AM-EtOH (30.0 mg/g), whereas no difference was noted in the total phenol content between these two extracts. These results conclude that AM extracts possess potent free radical scavenging and antilipid peroxidation activities, especially the AM-H₂O in the brain homogenate. Published in Russian in Prikladnaya Biokhimiya i Mikrobiologiya, 2007, Vol. 43, No. 4, pp. 495–500. The text was submitted by the authors in English.     

Intranasal Microemulsion of Sildenafil Citrate: In Vitro Evaluation and In Vivo Pharmacokinetic Study in Rabbits

The purpose of the present study was to prepare intranasal delivery system of sildenafil citrate and estimate its relative bioavailability after nasal administration in rabbits to attain rapid onset of action with good efficacy at lower doses. Sildenafil citrate saturated solubility was determined in different solvents, cosolvents, and microemulsion systems. For nasal application, sildenafil citrate was formulated in two different systems: the first was a cosolvent system (S3) of benzyl alcohol/ethanol/water/Transcutol/taurodeoxy cholate/Tween 20 (0.5:16.8:47.7:15.9:1:18.1% w/w). The second was a microemulsion system (ME6) containing Oleic acid: Labrasol/Transcutol/water (8.33:33.3:16.66:41.66% w/w). The prepared systems were characterized in relation to their clarity, particle size, viscosity, pH, and nasal ciliotoxicity. In vivo pharmacokinetic performance of the selected system ME6 (with no nasal ciliotoxicity) was evaluated in a group of six rabbits in a randomized crossover study and compared to the marketed oral tablets. The targeted solubility (>20 mg/ml) of sildenafil citrate was achieved with cosolvent systems S1, S3, and S5 and with microemulsion systems ME3–ME6. The saturated solubility of sildenafil citrate in cosolvent system S3 and microemulsion system ME6 were 22.98 ± 1.26 and 23.79 ± 1.16 mg/ml, respectively. Microemulsion formulation ME6 showed shorter t _max (0.75 h) and higher AUC_(0-∞) (1,412.42 ng h/ml) compared to the oral tablets which showed t _max equals 1.25 h and AUC_(0-∞) of 1,251.14 ng h/ml after administration to rabbits at dose level of 5 mg/kg. The relative bioavailability was 112.89%. In conclusion, the nasal absorption of sildenafil citrate microemulsion was found to be fast, indicating the potential of nasal delivery instead of the conventional oral administration of such drug.     

Fumonisins in Maize in Relation to Climate,Planting Time and Hybrids in Two Agroecological Zones in Zambia

Field experiments in the high rainfall zone (HRZ) and the medium rainfall zone (MRZ) in Zambia were designed to determine the natural occurrence of fumonisins (FB_1–2) in Zambian maize hybrids, accumulation of FB_1–2 resulting from artificial inoculation with Fusarium verticillioides and effects of climate and planting time on FB_1–2 in maize. Combined FB_1–2 concentrations varied from 0 to 13,050 ng/g, with an overall mean of 666 ng/g. Maize from the HRZ had low incidences of FB_1–2-positive samples (mean 41%) which contained FB_1–2 below 500 ng/g. In the MRZ, higher incidences (mean 97%) and concentrations (40% of samples >1,000 ng/g) were recorded in two out of three years. There was no correlation between mean location FB_1–2 concentrations in individual years and precipitation, number of rain days or monthly precipitation. Postponing the planting time with 10 or 20 days did not significantly affect FB_1–2 concentration, but it reduced the yields in some years.     

Sensitivity of human melanoma cells to adherent leukocytes depends on the ratio between them, the activation status of adherent leukocytes and the metastatic potential of tumor cells

 This study examined the interaction of the poorly metastatic human melanoma cell line M4Be and the highly metastatic clone 4 derived from M4Be, with respect to fresh adherent leukocytes (AL) isolated from 17 different healthy blood donors. These AL contained 80% (73%–93%) monocytes, 15% (6%–20%) B lymphocytes and 5% (1%–8%) T lymphocytes. The survival of these tumor cells against the stress exerted by these AL was estimated with a clonogenic assay where isolated tumor cells were co-cultured for 14 days in contact with AL and lipopolysaccharide (LPS). For a given blood donor, AL either stimulates or inhibits the colony formation of the tumor cells (T) depending on the AL/T ratio, the AL activation status and the metastatic potential of tumor cells. At low AL/T ratios (<10/1) in the presence of low (8 ng/ml) and trace (8 pg/ml) levels of LPS, hydrogen peroxide (H₂O₂) release is significantly reduced, and tumor cells significantly increase their colony formation; the feeder effect of AL is suggested to be due to low concentrations of soluble tumor necrosis factor-alpha (TNF-α). At high AL/T ratios (>10/1), whatever the characteristics of the blood donor, clone 4 is significantly more sensitive than M4Be to AL activated with medium containing low (8 ng/ml) or high (1,000 ng/ml) levels of LPS; this killing effect is suggested to be due to TNF-α, both soluble and membrane-bound, but not to be due to release of H₂O₂. These data suggest that the regulatory role of AL, which remove the majority of human melanoma cells and stimulate the colony formation of a small fraction of them, is partly due to TNF-α. Received: 2 November 2000 / Accepted: 15 February 2001     

A heterogeneous biotin–streptavidin-amplified enzyme-linked immunosorbent assay for detecting tris(2,3-dibromopropyl) isocyanurate in natural samples

Tris(2,3-dibromopropyl) isocyanurate (TBC) is a novel brominated flame retardant (BFR) that is widely used to substitute the prohibited BFRs throughout the world. With the development of research, the potential environmental and ecological harms of TBC have been revealed. For sensitive and selective detecting TBC, an indirect competitive biotin–streptavidin-amplified enzyme-linked immunosorbent assay (BA–ELISA) has been established in this study. The small molecular TBC–hapten was synthesized first; it mimicked the chemical structure of TBC and possessed a secondary amine group. The as-obtained hapten was then conjugated with carrier proteins to prepare artificial antigen. After immunization, the anti-TBC polyclonal antibody was obtained from separating rabbit serum. The procedures of this BA–ELISA were optimized. Under the optimal conditions, the limit of detection (IC₁₀) was 0.0067 ng/ml and the median inhibitory concentration (IC₅₀) was 0.66 ng/ml. Cross-reactivity values of the BA–ELISA with the tested TBC analogues were ?5%. This immunoassay was successfully applied to determine the TBC residue in river water samples that were collected near a BFR manufacturing plant. Satisfactory recoveries (92.1–109.2%) were obtained. The results indicated that this proposed BA–ELISA is suitable for the rapid and sensitive determining of TBC in environmental monitoring.     

Preparation of magnetic latexes and their use for the immunodetection of microbial antigens

The possibility of detecting antigens of plague, tularemia, and brucellosis microbes with magnetic latex (ML)-based test systems has been demonstrated. MLs were prepared from latexes (polyacroleine microspheres, 1.2–1.8 ± 0.1 μm) by exposing the particles to a 25–35%-solution of ferrous sulfate for 0.5 h and then to a 15–25%-aqueous solution of ammonia for 0.5 h in a 100°C water bath and dehydrating after each operation. The possibility of preparing magnetic latex immunosorbents (MLIS) by ligand immobilization on ML and using them in magnetic latex ELISA (ML-ELISA) for the detection of microbial antigens was demonstrated. The detection limit in ML-ELISA equaled 10²–10³ microbial cells in 1 ml (cells/ml). Relative experimental error was not higher than 8%.     

Inhibition of human platelet aggregation and membrane lipid peroxidation by food spice, saffron

The inhibitory activity of saffron extract was studied on human platelets. Platelet aggregation and lipid peroxidation were evaluated with platelet rich plasma (PRP) and platelet membranes respectively obtained from blood of healthy human volunteers. Human platelets were subjected to stimulation with a variety of agonists like ADP (61 μM), epinephrine (76 μM), collagen (11 μg/ml), calcium ionophore A 23187 (6 μM) and ristocetin (1.25 μg/ml) in the presence and absence of saffron extract with IC₅₀ being 0.66, 0.35, 0.86 and 0.59 mg respectively and no inhibition with ristocetin. The inhibitory effect was dose dependent with concentrations varying between 0.16 to 0.80 mg and time dependent at IC₅₀. A significant decrease was observed in malondialdehyde (MDA) formed, one of the end products of arachidonic acid metabolism and of serotonin released from dense granules of platelets at respective IC₅₀. Lipid peroxidation in platelet membranes induced by iron-ascorbic acid system was inhibited by saffron extract significantly with IC₅₀ of 0.33 mg. Hence, it may be said that aqueous extract of saffron may have component(s), which protect platelets from aggregation and lipid peroxidation. (Mol Cell Biochem 278: 59–63, 2005)     

Development of a direct competitive enzyme-linked immunosorbent assay for parathion residue in food samples

A heterologous direct competitive enzyme-linked immunosorbent assay (ELISA) for parathion residue determination is described based on a monoclonal antibody and a new competitor. The effects of several physicochemical factors, such as methanol concentration, ionic strength, pH value, and sample matrix, on the performance of the ELISA were optimized for the sake of obtaining a satisfactory assay sensitivity. Results showed that when the assay medium was in the optimized condition (phosphate buffer solution [PBS] containing 10% [v/v] methanol and 0.2 mol/L NaCl at a pH value of 5.0), the sensitivity (estimated as the IC₅₀ value) and the limit of detection (LOD, estimated as the IC₁₀ value) were 1.19 and 0.08 ng/ml, respectively. The precision investigation indicated that the intraassay precision values all were below 10% and that the interassay precision values ranged from 4.89 to 19.12%. In addition, the developed ELISA showed a good linear correlation (r² = 0.9962) to gas chromatography within the analyte’s concentration range of 0.1 to 16 ng/ml. When applied to the fortified samples (parathion adding level: 5-15 μg/kg), the developed ELISA presented mean recoveries of 127.46, 122.52, 91.92, 124.01, 129.72, 99.37, and 87.17% for tomato, cucumber, banana, apple, orange, pear, and sugarcane, respectively. Results indicated that the established ELISA is a potential tool for parathion residue determination.     

Antimicrobial Butyrolactone I Derivatives from the Ecuadorian Soil Fungus Aspergillus terreus Thorn. var terreus

Summary The fungus Aspergillus terreus Thorn var. terreus isolated from an Ecuador soil sample was cultured in liquid and solid media and yielded three main metabolites identified as terreic acid (1), butyrolactone I (2) and lovastatin (3). The natural products as well as three synthetic butyrolactone I derivatives were assessed for antimicrobial activity against Gram-positive and Gram-negative bacteria and fungi as well as for seed germination and seedling growth. Furthermore, the compounds were assessed as inhibitors towards the enzymes acetylcholinesterase, β-glucosidase, and β-glucuronidase. Terreic acid, butyrolactone I, butyrolactone 4′,4′′-diacetate (2.1), and 3′-(3-methylbutyl)-butyrolactone II (2.2) were active towards the phytopathogenic bacteria Erwinia carotovora with IC₅₀ of 5 and 4–18 μg/ml, respectively. Under the same experimental conditions, the IC₅₀ of streptomycin was 1.9 μg/ml. 3′-(3-Methylbutyl)-butyrolactone II was moderately active against Pseudomonas syringae and Botrytis cinerea with IC₅₀ of 21μg/ml and MIC of 15.6 μg/ml, respectively. Butyrolactone I also inhibited germination of the dicot Lactuca sativa with an IC₅₀ of 5 × 10⁻⁵ M. The IC₅₀ of reference herbicide acetochlor was 1 × 10⁻⁵ M. The effect of 2.2 and 2.3, known as butyrolactone III on Panicum millaceum germination and growth was stronger than that of 2 and 2.1. Reduction of the double bond in the isoprenyl side chain of butyrolactone I increased the antibacterial effect against E. carotovora as well as acetylation. To our best knowledge, this is the first report on the antibacterial effect of butyrolactone derivatives towards Erwinia carotovora and the phytopathogenic fungus Botrytis cinerea. The butyrolactone I derivative 2.2 presented a moderate inhibitory effect against the enzyme acetylcholinesterase with an IC₅₀ of 47 μg/ml. Under the same experimental conditions, the reference inhibitor galanthamine had an IC₅₀ of 3 μg/ml.     

Mechanism of action of aflatoxin B1

The inhibitory effects of aflatoxin B₁ were found to be related to the gram character in procaryotes, used in this study. Ethylene diamine tetra chloroacetic acid (0.05% w/v) or Tween-80 (0.05 % v/v) addition accentuated the aflatoxin B₁ growth inhibition inSalmonella typhi andEscherichia coli at different pH values. The inhibition of lipase production was only 5–20 % inPseudomonas fluorescence ca. 25–48% inStaphylococcus aureus andBacillus cereus at different aflatoxin B₁ concentrations (4–16μg/ml).However, inhibition of α-amylase induction was complete in1Bacillus megaterium whereas the inhibition was partial inPseudomonas fluorescence (27–40%) at 32μg aflatoxin B₁ concentration. An increase in leakage of cell contents and decreased inulin uptake were observed in toxin incubated sheep red blood cell suspension (1 %) with increased aflatoxin B₁ concentration     

Evaluation of the bioactivities of extracts of endophytes isolated from Taiwanese herbal plants

The endophytic extracts from 19 endophytes, isolated from 13 species of Taiwanese plants, were evaluated for biological activity, including cytotoxicity, anti-platelet aggregation, and anti-inflammatory activity. The extracts of 12 endophytes exhibited inhibitory effects on collagen-induced platelet aggregation with IC₅₀ values of 19.85–87.64 μg/ml. Four strains, Rahnella aquatilis, Pantoea agglomerans, Rhodotorula sp., and Penicillium paxilli, also showed inhibitory effects on thrombin-induced platelet aggregation with IC₅₀ values of 42.80–61.54 μg/ml. Additionally 12 extracts of endophytes exhibited cytotoxicities with IC₅₀ values of 0.12–19.83 μg/ml. However, eight extracts revealed inhibitory effects on superoxide anion generation induced by fMLP (N-formyl-l-methionyl-l-leucyl-l-phenylalanine) in human neutrophils. The extract of Rahnella aquatilis showed anti-platelet aggregation activity, and bioassay-directed fractionation led to the isolation of six compounds, including one isoalloxazine: lumichrome (1); two isoflavones: genistein (2) and daidzein (3); two cyclic peptides: cyclo-Pro-Val (4) and cyclo-Pro-Phe (5); and one benzenoid: methyl 2,4,5-trimethoxybenzoate (6). These results indicated that endophytes from Taiwanese herbal plants could be useful sources for research and development of bioactive lead compounds from nature.     

Effects of some chemical factors on flagellation and swarming ofVibrio alginolyticus

Vibrio alginolyticus strains recently isolated from Dutch coastal seawater changed flagellar organization when cultivated in the presence of certain chemical agents. On agar media with more than 4.0% (w/v) NaCl the number of lateral flagella per cell decreased with increasing salt concentration. Both on agar media and in broth cultures with 6.0–9.0% (w/v) NaCl, cells with polar tufts of 2–4 sheathed or unsheathed flagella were frequently found. Cells grown on agar media with 7.3–9.8% (w/v) Na₂SO₄ had drastically reduced numbers of lateral flagella, but lacked polar tufts. EDTA suppressed growth, but did not affect flagellar arrangement. In the presence of 0.1–0.3% boric acid or 0.05–0.1% aluminium hydroxide, cells in liquid media tended to produce lateral, in addition to the polar flagella normally observed in broth cultures. Of a number of surface-active agents tested, Tween 80 and Na-taurocholate, even in high concentrations, did not affect flagellation. Bile salts (0.1%) and Na-deoxycholate (0.05%) strongly reduced the number of both polar and lateral flagella. In agar cultures, Na-lauryl sulphate (0.01–0.1%) inhibited the formation of lateral, but increased the incidence of polar flagella. Teepol (0.05–0.2%) had a similar effect and also it had a deteriorating effect on the sheaths of the polar flagella. Concomitant with the reduction in the number of lateral flagella, induced by these agents, swarming on agar media was inhibited.     

Endophytic fungi with anti-microbial,anti-cancer and anti-malarial activities isolated from Thai medicinal plants

A total of 81 Thai medicinal plant species collected from forests in four geographical regions of Thailand were examined for the presence of endophytic fungi with biological activity. Of 582 pure isolates obtained, 360 morphologically distinct fungi were selected for cultivation on malt Czapek broth and yeast extract sucrose broth, from which extracts were tested for biological activity. Extracts of 92 isolates could inhibit Mycobacterium tuberculosis (MIC 0.0625–200 μg ml⁻¹) when tested by the microplate Alamar blue assay, while extracts of six inhibited Plasmodium falciparum (IC₅₀ of 1.2–9.1 μg ml⁻¹) as determined by the [³H]hypoxanthine incorporation method. Strong anti-viral activity against Herpes simplex virus type 1 was observed in 40 isolates (IC₅₀ of 0.28–50 μg ml⁻¹). The sulphorhodamine B assay for activity against cancer cell lines revealed that 60 were active against human oral epidermoid carcinoma cells (EC₅₀ 0.42–20 μg ml⁻¹) and 48 against breast cancer cells (EC₅₀ 0.18–20 μg ml⁻¹). Bioactivity profile was affected by the type of culture medium. Given the high incidence of bioactive extracts and the fact that most of the isolated fungi could not be identified due to lack of spore formation, the results suggested that Thai medicinal plants can provide a wide variety of endophytes that might be a potential source of novel bioactive compounds. This revised version was published online in August 2006 with corrections to the Cover Date.     

Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media

Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone. This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255.