Protective Effect of Saffron Extract and Crocin on Reactive Oxygen Species-Mediated High Glucose-Induced Toxicity in PC12 Cells

Diabetic neuropathy is one of the most frequent complications of diabetes. Despite some studies, the exact mechanism of glucose neurotoxicity has not been fully elucidated. Increased reactive oxygen species (ROS) has proposed as a possible mechanism. Crocus sativus L. (saffron) has been known as a source of antioxidants. Therefore, neuroprotective effect of saffron extract, its active component crocin and γ-glutamylcysteinylglycine (GSH) was studied in glucose-induced neurotoxicity, using PC12 cells as a suitable in vitro model of diabetic neuropathy. Cell viability was quantitated by MTT assay. ROS was measured using DCF-DA by flow cytometry analysis. The result showed that glucose (13.5 and 27 mg/ml) reduced the cell viability of PC12 cells after 4 days. Saffron extract (5 and 25 mg/ml), crocin (10 and 50 μM) and GSH (10 μM) could decrease this toxicity. Glucose toxicity was consistent with increased ROS production which reduced by saffron, crocin and GSH pretreatment. These results suggest saffron and its carotenoid crocin could be potentially useful in diabetic neuropathy treatment.     

Impact of protein binding on the analytical detectability and anticancer activity of thymoquinone

Thymoquinone (TQ), an active component of Nigella sativa L., is known to have anti-cancer and anti-inflammatory effects; however, no studies on its analytical detection in serum and its protein binding have been published. Using high performance liquid chromatography analysis, we show that the average recovery of TQ from serum is 2.5% at 10 μg/ml of TQ and 72% at 100 μg/ml. The low recovery of TQ from serum is due to its extensive binding to plasma proteins, as more than 99% of TQ was bound within 30 min of incubation. The binding of TQ to the major plasma proteins, bovine serum albumin (BSA) and alpha −1 acid glycoprotein (AGP), was studied and found to be 94.5 ± 1.7% for BSA and 99.1 ± 0.1% for AGP. Mass spectrometric analysis revealed that TQ was bound covalently to BSA, specifically on Cyst-34. Using WST-1 proliferation assay, we showed that BSA plays a protective role against TQ-induced cell death; pre-incubation with BSA prevented TQ from exerting its anti-proliferative effects against DLD-1 and HCT-116 human colon cancer cells. On the other hand, binding of TQ to AGP did not alter its anti-proliferative activity against both cell lines. When TQ was pre-incubated with AGP prior to the addition of BSA, the activity of TQ against DLD-1 was maintained, suggesting that AGP prevented the binding of TQ to BSA. This is the first time the covalent binding and inhibitory effect of BSA on TQ is documented. These data offer new grounds for TQ future pharmacokinetic analysis in vivo.     

Neuroprotective Effect of Crocin on Acrylamide-induced Cytotoxicity in PC12 cells

Acrylamide (ACR) is a potent neurotoxic in human and animal models. In this study, the effect of crocin, main constituent of Crocus sativus L. (Saffron) on ACR-induced cytotoxicity was evaluated using PC12 cells as a suitable in vitro model. The exposure of PC12 cells to ACR reduced cell viability, increased DNA fragmented cells and phosphatidylserine exposure, and elevated Bax/Bcl-2 ratio. Results showed that ACR increased intracellular reactive oxygen species (ROS) in cells and ROS played an important role in ACR cytotoxicity. The pretreatment of cells with 10–50 μM crocin before ACR treatment significantly attenuated ACR cytotoxicity in a dose-dependent manner. Crocin inhibited the downregulation of Bcl-2 and the upregulation of Bax and decreased apoptosis in treated cells. Also, crocin inhibited ROS generation in cells exposed to ACR. In conclusion, our results indicated that pretreatment with crocin protected cells from ACR-induced apoptosis partly by inhibition of intracellular ROS production.     

<Emphasis Type="Italic">Vibrio cholerae</Emphasis> in waters of the Sunderban mangrove: relationship with biogeochemical parameters and chitin in seston size fractions

Wetland dynamics are probably linked to cholera endemicity in South Asia. We focus on links between Vibrio cholerae abundance, chitin content and suspended particle load in size fractions of suspended particulate matter (SPM) along the salinity gradient of Sunderban mangrove waters. SPM decreased downstream, while salinity increased from 0.2 to 4. Particulate organic carbon (90 ± 25 μM) and nitrogen (9.1 ± 3.3 μM) highly correlated with SPM and turbidity, suggesting a significant contribution of fine particles to organic matter. Total chitin ranged 1–2 mg/l and decreased downstream. The distribution among size fractions of SPM, chitin and V. cholerae O1 (the bacterial serogroup mainly associated with cholera epidemics) was similar, with ~98% of the total in the fraction <20 μm. In comparison, the number of V. cholerae O1 attached to zooplankton and microplankton size classes >20 μm was almost negligible, in contrast to usual assumptions. Thus, microdetritus, nanoplankton and fungal cells in size classes <20 μm represent a chitinaceous substrate on which V. cholerae can grow and survive. Total bacteria, cultivable vibrios and V. cholera O1 increased 5–10 times downstream, together with salinity and nitrite concentration. Overall, nitrate and silicate concentrations were relatively constant (>22 μM N and 100 μM Si). However, nitrite increased ~9 times in the outer sector, reaching ~1.2 μM N, probably as a result of increased abundance of nitrate-reducing vibrios. A characterization of Vibrio habitats that takes account of the presence of nitrate-reducing bacteria could improve the understanding of both mangrove nitrogen cycling and cholera seasonality.     

Study of the Action of Se and Cu on the Growth Metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> by Microcalorimetry

The biological effect of Se and Cu²⁺ on Escherichia coli (E. coli) growth was studied by using a 3114/3236 TAM Air Isothermal Calorimeter, ampoule method, at 37°C. From the thermogenesis curves, the thermokinetic equations were established under different conditions. The kinetics showed that a low concentration of Se (1–10 μg/mL) promoted the growth of E. coli, and a high concentration of Se (>10 μg/mL) inhibited the growth, but the Cu²⁺ was always inhibiting the growth of E. coli. Moreover, there was an antagonistic or positive synergistic effect of Se and Cu²⁺ on E. coli in the different culture medium when Se was 1–10 μg/ml and Cu²⁺ was 1–20 μg/ml. There was a negative synergistic effect of Se and Cu²⁺ on E. coli when Se was higher than 10 μg/ml and Cu²⁺ was higher than 20 μg/ml. The antagonistic or synergistic effect between Se and Cu²⁺ on E. coli was related to the formation of Cu–Se complexes under the different experimental conditions chosen.     

The Effect of Gentian Violet on Virulent Properties of <Emphasis Type="Italic">Candida albicans</Emphasis>

The aim of this study was to evaluate the effect of gentian violet (GV) on phospholipase activity, proteinase activity and germ tube formation rate of Candida albicans. Both 12 phospholipase-positive and 12 proteinase-positive C. albicans isolates with Pz values ≤0.89 were obtained. A yeast suspension (1–3 × 10⁷ cfu/ml) of each isolate was prepared. After a brief exposure (60 min) to sub-therapeutic concentrations (0.5 or 2 μg/ml) of GV, Pz value of phospholipase, Pz value of proteinase and germ tube formation rate were determined. Phospholipase activity, proteinase activity and germ tube formation rate in two groups exposed to GV were significantly lower than those in the group unexposed (P < 0.05). The results of this study indicated that sub-therapeutic concentrations of GV may lead to reduction in phospholipase activity, proteinase activity and germ tube formation, and then may suppress virulence and pathogenicity of C. albicans.     

Neuroprotective Effects of Chalcones from <Emphasis Type="Italic">Myracrodruon urundeuva</Emphasis> on 6-Hydroxydopamine-Induced Cytotoxicity in Rat Mesencephalic Cells

In the present work, we showed that a chalcone-enriched fraction (CEF) isolated from the stem bark of a Brazilian medicinal plant, Myracrodruon urundeuva, presents neuroprotective actions on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death, in rat mesencephalic cells. In the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] assay, which is an index of cell viability, CEF (1–100 μg/ml) reversed in a concentration-dependent manner the 6-OHDA-induced cell death. While cells exposed to 6-OHDA (40 μM) showed an increased concentration of thiobarbituric acid reactive substances (TBARS), the pretreatment with CEF (10–100 μg/ml) significantly decreased the 6-OHDA-induced TBARS formation, indicative of a neuroprotection against lipoperoxidation. Furthermore, the drastic increase of nitrite levels induced by 6-OHDA, indicative of nitric oxide formation and free radicals production, was prevented by CEF. Double staining with acridine orange/ethidium bromide showed that cultures exposed to 6-OHDA (40 and 200 μM) presented an increase of apoptotic and necrotic cell numbers in a concentration-dependent manner. CEF (100 μg/ml) protected cells from apoptosis and necrosis and increased number of cells presenting a normal morphology. The immunohistochemical analysis for tyrosine hydroxylase (TH) positive neurons indicated that 6-OHDA (40 and 200 μM) caused a concentration-dependent loss of TH+ and TH− neurons. CEF protected both cells types from 6-OHDA-induced cell death. All together, our results demonstrated neuroprotective effects of chalcones, which are able to reduce oxidative stress and apoptotic injury caused by 6-OHDA. Our findings suggest that chalcones could provide benefits, along with other therapies, in neurodegenerative injuries, such as Parkinson’s disease.     

Analysis of cadmium and lead in mice organs

Analysis and distribution of Pb and Cd in different mice organs, including the liver, kidney, spleen, heart, and blood, were evaluated before and after treatment with different aqueous concentrations of Nigella sativa (1.25–10.0 mg/L). Atomic absorption spectrometry was used for analysis of Pb and Cd in these organs. Results indicated that the Pb in the unexposed group of mice without treatment with N. sativa (black cumin) was in the following order: liver>heart>spleen>kidney, and the distribution of Pb in various organs of the unexposed group was not affected significantly by N. sativa. Moreover, results of mice exposed for Pb show that the Pb concentrations in different organs were reduced significantly (p<0.05) by 72.9%, 63.4%, 72.3%, 66.7%, and 39.5% at a dose of 10 mg/L of N. sativa for the liver, kidney, heart, spleen, and blood, respectively. Furthermore, the distribution of Cd in the unexposed Cd group of mice without treatment with N. sativa was in the following order: kidney>heart>spleen>liver. Nigella sativa at 10 mg/L reduced Cd levels in mice exposed to Cd by 75.5%, 83.3%, 47.0%, 95.3%, and 100% in the liver, kidney, heart, spleen, and blood, respectively, whereas blood Cd concentrations were lowered to below the detection limit of 0.05 μg/L. A 28-d exposure of mice to a Cd−Pb mixture at a concentration of 1 ppm in drinking water induced a highly significant inhibition (p<0.0001) of antibody response to human serum (80.5%). The suppressed immune responses in mice pretreated with the Cd−Pb mixture were reversed by 43.1% and 38.9% in the presence of 1.25 and 2.5 mg/mL of N. sativa, respectively, whereas higher concentrations (5–10 mg/mL) of N. sativa increased the immunosuppression significantly. Nigella sativa at 1.25–10 mg/mL did not induce any significant modulation of the antibody response in unexposed mice.     

Regulation of Root Growth in Lactuca sativa L. Seedlings by the Ent-Kaurane Diterpenoid Epinodosin

Epinodosin, an ent-kaurane diterpenoid isolated from Isodon japonica var. galaucocalyx, had a biphasic, dose-dependent effect on root growth and a strong inhibitory effect on root hair development in Lactuca sativa L. seedlings. Lower levels of epinodosin (25–100 μM) significantly promoted root growth, but higher concentrations (150–200 μM), by contrast, had inhibitory effects. In addition, all of the tested concentrations (20–80 μM) inhibited root hair development of lettuce seedlings in a dose-dependent manner. Further investigations on the underlying mechanism revealed that the promotion effect of epinodosin (25–100 μM) resulted from increasing the cell length in the mature region and enhancing the mitotic activity of meristematic cells in lettuce seedling root tips. On the other hand, epinodosin at higher concentrations inhibited root growth by strongly affecting both the cell length in the mature region and the division of meristematic cells. Comet assay analysis demonstrated that the decrease of mitotic activity of root meristematic cells was due to DNA damage induced by higher levels of epinodosin.     

Effects of light and temperature on the attachment and development of <Emphasis Type="BoldItalic">Gloiopeltis tenax</Emphasis> and <Emphasis Type="BoldItalic">Gloiopeltis furcata</Emphasis> tetraspores

Two 60-day experiments were conducted to study the influence of photon flux density (PFD) and temperature on the attachment and development of Gloiopeltis tenax and Gloiopeltis furcata tetraspores. In the first experiment, tetraspores of the two Gloiopeltis species were incubated at five temperature ranges (8°C, 12°C, 16°C, 20°C, 24°C) under a constant PFD of 80 μmol photons m⁻² s⁻¹ with a photoperiod of 12:12. In a second experiment, tetraspores were incubated under five PFD gradients (30, 55, 80, 105, 130 μmol photons m⁻² s⁻¹) at a constant temperature of 16°C with a photoperiod of 12:12. Maximum density of attached tetraspores was observed at 16°C for both species. Maximum per cent of spore germinating into disc was recorded at 12–16°C for G. tenax and 8–12°C for G. furcata. Maximum per cent of discs producing erect axes for G. tenax and G. furcata were recorded at 24°C and 20°C, respectively. Light had no significant effect on tetraspore attachment and developing into disc, but it affected the growth, sprouting and survival of its discs. Under 30–55 μmol photons m⁻² s⁻¹, the discs of the two species of Gloiopeltis did not form thallus until the end of the experiment. Optimum PFD range for G. tenax discs was 80–105 μmol photons m⁻² s⁻¹, whilst it was 80–130 μmol photons m⁻² s⁻¹ for G. furcata. Results presented in this study are expected to assist the progress of artificial seeding of Gloiopeltis.     

Measurement of particle diameter of <Emphasis Type="Italic">Lactobacillus acidophilus</Emphasis> microcapsule by spray drying and analysis on its microstructure

Lactobacillus acidophilus, as a probiotic, is widely used in many functional food products. Microencapsulation not only increases the survival rate of L. acidophilus during storage and extends the shelf-life of its products, but also optimal size microcapsule makes L. acidophilus have an excellent dispersability in final products. In this paper, L. acidophilus was microencapsulated using spray drying (inlet air temperature of 170°C; outlet air temperature of 85–90°C). The wall materials used in this study were β-cyclodextrin and acacia gum in the proportion of 9:1 (w/w), and microcapsules were prepared at four levels of wall materials (15, 20, 25 and 30% [w/v]) with a core material concentration of 6% (v/v). The microcapsule diameters were measured by Malvern’s Mastersizer-2000 particle size analyzer. The results showed that the particle diameters of microcapsule were mostly within 6.607 μm and 60.256 μm and varied with 2.884–120.226 μm (the standard smaller microcapsule designated as <350 μm). Through comparison of microcapsule size and uniformity with different concentration of wall materials, we concluded that the optimal concentration of wall material was 20% (w/v), which gave microcapsule with a relatively uniform size (averaging 22.153 μm), and the number of surviving encapsulated L. acidophilus was 1.50 × 10⁹ c.f.u./ml. After 8 weeks storage at 4°C, the live bacterial number was above 10⁷ c.f.u./ml, compared with unencapsulated L. acidophilus, 10⁴–10⁵ c.f.u./ml. Through the observation of scanning electron microscopy, we found that the shapes of microcapsule were round and oval, and L. acidophilus cells located in the centre of microcapsule.     

Antibacterial effect of silver nanoparticles on <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The antibacterial activity and mechanism of silver nanoparticles (Ag-NPs) on Staphylococcus aureus ATCC 6538P were investigated in this study. The experiment results showed the minimum bactericidal concentration (MBC) of Ag-NPs to S. aureus was 20 μg/ml. Moreover, when bacteria cells were exposed to 50 μg/ml Ag-NPs for 6 h, the cell DNA was condensed to a tension state and could have lost their replicating abilities. When S. aureus cells were exposed to 50 μg/ml Ag-NPs for 12 h, the cell wall was breakdown, resulting in the release of the cellular contents into the surrounding environments, and finally became collapsed. And Ag-NPs could reduce the enzymatic activity of respiratory chain dehydrogenase. Furthermore, the proteomic analysis showed that the expression abundance of some proteins was changed in the treated bacterial cell with Ag-NPs, formate acetyltransferase increased 5.3-fold in expression abundance, aerobic glycerol-3-phosphate dehydrogenase decreased 6.5-fold, ABC transporter ATP-binding protein decreased 6.2-fold, and recombinase A protein decreased 4.9-fold.     

Effects of polysaccharides from <Emphasis Type="Italic">Morchella conica</Emphasis> on nitric oxide production in lipopolysaccharide-treated macrophages

Morchella conica is a species of rare edible mushroom whose multiple medicinal functions have been proven. However, reports barely mention the mechanisms of these functions. In this study, the effects of two polysaccharides from M. conica (PMCs) on nitric oxide (NO) production in lipopolysaccharide (LPS)-treated macrophages were investigated. The results showed that 50–200 μg/ml of the extracellular polysaccharide (EPMC) and 25–200 μg/ml of the intracellular polysaccharide (IPMC) significantly inhibited NO production. Accordingly, the signal mechanisms were also explored. It was found that 100 μg/ml of EPMC and 25 μg/ml of IPMC could efficiently down-regulate the inducible nitric oxide synthase (iNOS) expression and nuclear factor-κB (NF-κB) DNA-binding activity and up-regulate heme oxygenase 1 (HO-1) expression. Moreover, by using a HO-1 inhibitor NaPP to treat the cells, the PMC-inhibited NO production and iNOS expression, rather than NF-κB activation, were released partially, indicating that HO-1 probably medicates the inhibition of PMCs on iNOS and NO. Besides, EPMC also significantly suppressed the phosphorylation of p38 mitogen-activated protein kinase (p38), c-jun N-terminal kinase, mitogen-activated protein kinase kinase 4, and expression of NF-κB inducing kinase, while IPMC seemed to show no regular effect on p38. In conclusion, PMCs inhibited NO production in LPS-induced macrophages through regulating a series of signal pathways, suggesting that PMCs play a potential role on immunomodulation and treating related diseases.     

Isolation,callus formation and plantlet regeneration from mesophyll protoplasts of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. f.) Merrill: an important medicinal plant

Protoplast culture and plant regeneration of an important medicinal plant Tylophora indica were achieved through callus regeneration. Protoplasts were isolated from leaf mesophyll cells and cultured at a density of 5 × 10⁵ protoplasts per gram fresh weight, which is required for the highest frequency of protoplast division (33.7%) and plating efficiency (9.3%). The first division was observed 2 d after plating and the second division after 4 d. Culture medium consists of Murashige and Skoog (MS) liquid medium with 4 μM 2,4-D, 0.4 M mannitol and 3% (w/v) sucrose with pH adjusted to 5.8. After 45 d of culture at 25°C in the dark, protoplasts formed colonies consisting of about 100 cells. The protoplast-derived microcalli were visible to the naked eye within 60 d of culture and reached a size of 0.2–0.4 mm in diameter after 90 d. Calli of 0.2–0.4-mm size were transferred to MS medium supplemented with 2,4-D (4 μM), 3% (w/v) sucrose and 0.8% (w/v) agar, formed friable organogenic calli (7-8 mm size) after 8 wk under incubation in normal light period supplemented with 200 μmol m⁻² S⁻¹ of day light fluorescent illumination. The calli were transferred to MS medium supplemented with thidiazuron (TDZ) (1–7 μM) and naphthalene acetic acid (NAA) (0.2–0.4 μM) for regeneration. The calli developed shoot buds after 3–4 wk, and the frequencies of calli-forming shoots varied from 5% to 44%. Optimum shoot regeneration occurred on MS medium supplemented with 5 μM TDZ and 0.4 μM NAA. On this medium, 44% cultures responded with an average number of 12 shoots per callus. Whole plants were recovered following rooting of shoots in 1/2 MS medium supplemented with 3 μM indole 3-butyric acid.     

Rhizosphere competent <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> enhances maize (<Emphasis Type="Italic">Zea mays</Emphasis>) and chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) growth,without altering the rhizosphere functional diversity

Plant growth promoting Pantoea agglomerans NBRISRM (NBRISRM) was able to produce 60.4 μg/ml indole acetic acid and solubilize 77.5 μg/ml tri-calcium phosphate under in vitro conditions. Addition of 2% NaCl (w/v) in the media induced the IAA production and phosphate solubilization by 11% and 7%, respectively. For evaluating the plant growth promotory effect of NBRISRM inoculation a micro plot trial was conducted using maize and chickpea as host plants. The results revealed significant increase in all growth parameters tested in NBRISRM inoculated maize and chickpea plants, which were further confirmed by higher macronutrients (N, P and K) accumulation as compared to un-inoculated controls. Throughout the growing season of maize and chickpea, rhizosphere population of NBRISRM were in the range 10⁷–10⁸ CFU/g soil and competing with 10⁷–10⁹ CFU/g soil with heterogeneous bacterial population. Functional richness, diversity, and evenness were found significantly higher in maize rhizosphere as compared to chickpea, whereas NBRISRM inoculation were not able to change it, in both crops as compared to their un-inoculated control. To the best of our knowledge this is first report where we demonstrated the effect of P. agglomerans strain for improving maize and chickpea growth without altering the functional diversity.     

Identification,cloning, and functional characterization of EmrD-3, a putative multidrug efflux pump of the major facilitator superfamily from <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O395

A putative multidrug efflux pump, EmrD-3, belonging to the major facilitator superfamily (MFS) of transporters and sharing homology with the Bcr/CflA subfamily, was identified in Vibrio cholerae O395. We cloned the emrD-3 gene and evaluated its role in antimicrobial efflux in a hypersensitive Escherichia coli strain. The efflux activity of this membrane protein resulted in lowering the intracellular concentration of ethidium. The recombinant plasmid carrying emrD-3 conferred enhanced resistance to several antimicrobials. Among the antimicrobials tested, the highest relative increase in minimum inhibitory concentration (MIC) of 102-fold was observed for linezolid (MIC = 256 μg/ml), followed by an 80.1-fold increase for tetraphenylphosphonium chloride (TPCL) (156.2 μg/ml), 62.5-fold for rifampin (MIC = 50 μg/ml), >30-fold for erythromycin (MIC = 50 μg/ml) and minocycline (MIC = 2 μg/ml), 20-fold for trimethoprim (MIC = 0.12 μg/ml), and 18.7-fold for chloramphenicol (MIC = 18.7 μg/ml). Among the fluorescent DNA-binding dyes, the highest relative increase in MIC of 41.7-fold was observed for ethidium bromide (125 μg/ml) followed by a 17.2-fold increase for rhodamine 6G (100 μg/ml). Thus, we demonstrate that EmrD-3 is a multidrug efflux pump of V. cholerae, the homologues of which are present in several Vibrio spp., some members of Enterobacteriaceae family, and Gram-positive Bacillus spp.     

Antivibrio compounds produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. W3: characterisation and assessment of their safety to shrimps

In order to explore compounds naturallly inhibitory to shrimp pathogenic vibrios, a culture filtrate of Pseudomonas sp. W3 at a pH of 2 was extracted with ethyl acetate (EtOAc) to produce 82.15 mg/l of a yellow–brown extract (EtOAc-W3) that had MIC values of 225-450 μg/ml against the growth of 18 shrimp pathogenic Vibrio harveyi strains. The MIC of EtOAc-W3 against the most pathogenic strain PSU 2015 was 450 μg/ml and this strain had the lowest LD₅₀ (50% lethal dose) to pacific white shrimp (Litopenaeus vannamei, PL 21). At this MIC value, EtOAc-W3 in artificial sea water (ASW) killed strain PSU 2015; however in natural sea water, only a partial growth inhibition was observed. The toxicity to pacific white shrimp and antivibrio activity of the EtOAc-W3 were investigated by conducting an experiment with 4 sets; native control (commercial ASW), EtOAc-W3 control (MIC/10, 45 μg/ml), challenge (inoculation 6.0 × 10⁶ c.f.u./ml PSU 2015) and treatment (6.0 × 10⁶ c.f.u./ml PSU 2015 + 45 μg/ml EtOAc-W3). The same experiment was repeated by increasing the dose of EtOAc-W3 to 90 μg/ml (MIC/5). Both concentrations of EtOAc-W3 tested had no toxicity to postlarval shrimps. A significant decrease in shrimp mortality was observed over a 72 h period as approximately 80% of the shrimps died in each challenge set but only 63 and 23% died in the presence of 45 and 90 μg/ml EtOAc-W3. The major component of EtOAc-W3 was supposed to be 2-heptyl-4-quinolone (HHQ) by FAB-MS and ¹H-NMR analyses of the purified fraction.     

An efficient protocol for genetic transformation of watercress (<Emphasis Type="Italic">Nasturtium officinale</Emphasis>) using <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>

Watercress (Nasturtium officinale) is a member of the Brassicaceae family and a rich source of glucosinolate, which has been shown to possess anticancer properties. To extract these compounds from N. officinale for study, a method was developed in which Agrobacterium rhizogenes was used to transfer DNA segments into plant genomes in order to produce hairy root cultures, which are a reliable source of plant compounds. The A. rhizogenes strain R1000 had the highest infection frequency and induces the most hairy roots per explant. Polymerase chain reaction and cytohistochemical staining methods were used to validate transgenic hairy roots from N. officinale. Glucosinolate from watercress hairy roots was separated and analyzed using high-performance liquid chromatography coupled to electrospray ionization mass spectrometry. Indolic glucosinolates, including glucobrassicin (0.01–0.02 μmol/g of DW) and 4-methoxyglucobrassicin (0.06–0.18 μmol/g of DW), as well as aromatic glucosinolate (gluconasturtiin) (0.06–0.21 μmol/g of DW), were identified virtually identical or more in transformed than wild type roots of N. officinale. Hairy root culture of watercress is a valuable approach for future efforts in the metabolic engineering of glucosinolate biofortification in plants, particularly, because indolic glucosinolates are the precursors of a potent cancer chemopreventive agent (indole-3-carbinol).