Protective Effect of <Emphasis Type="Italic">Nigella sativa</Emphasis> Extract and Thymoquinone on Serum/Glucose Deprivation-Induced PC12 Cells Death

The serum/glucose deprivation (SGD)-induced cell death in cultured PC12 cells represents a useful in vitro model for the study of brain ischemia and neurodegenerative disorders. Nigella sativa L. (family Ranunculaceae) and its active component thymoquinone (TQ) has been known as a source of antioxidants. In the present study, the protective effects of N. sativa and TQ on cell viability and reactive oxygen species (ROS) production in cultured PC12 cells were investigated under SGD conditions. PC12 cells were cultured in DMEM medium containing 10% (v/v) fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cells were seeded overnight and then deprived of serum/glucose for 6 and 18 h. Cells were pretreated with different concentrations of N. sativa extract (15.62–250 μg/ml) and TQ (1.17–150 μM) for 2 h. Cell viability was quantitated by MTT assay. Intracellular ROS production was measured by flow cytometry using 2′,7′-dichlorofluorescin diacetate (DCF-DA) as a probe. SGD induced significant cells toxicity after 6, 18, or 24 h (P < 0.001). Pretreatment with N. sativa (15.62–250 μg/ml) and TQ (1.17–37.5 μM) reduced SGD-induced cytotoxicity in PC12 cells after 6 and 18 h. A significant increase in intracellular ROS production was seen following SGD (P < 0.001). N. sativa (250 μg/ml, P < 0.01) and TQ (2.34, 4.68, 9.37 μM, P < 0.01) pretreatment reversed the increased ROS production following ischemic insult. The experimental results suggest that N. sativa extract and TQ protects the PC12 cells against SGD-induced cytotoxicity via antioxidant mechanisms. Our findings might raise the possibility of potential therapeutic application of N. sativa extract and TQ for managing cerebral ischemic and neurodegenerative disorders.     

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.     

Effect of bacterial growth in the complexing capacity of a culture medium supplemented with cadmium(II)

The aim of this study was to evaluate complexing capacity (CC) accompanying microbial growth in a metal supplemented culture medium. A combined strategy of square wave anodic stripping voltammetry (SWASV) monitored titration and ion-exchange resins treatment (Chelex 100) has been applied. Culture medium, supplemented with Cd(II) in excess to ligands, was inoculated with an indigenous bacterial culture; total ligand concentration and stability constants were determined at different bacterial growth stages. As far as known, determination of CC in such conditions has not been reported (usually ligands in natural or wastewaters exceed metal concentration). HIDA, (N-(2-hydroxyethyl)iminodiacetic acid), was used as a model ligand to mimic soluble products derived from the resin treatment and bacterial metabolism. Ligand concentration, Lt (1.3 ± 0.1 μM), and the conditional stability constant, K′, (log K′ = 5.7 ± 0.2) were in good agreement with expected values (1.0 ± 0.1 μM and log K′ = 6.1). In the supplemented culture medium, total ligand concentration in the micromolar range (60–80 μM) and conditional stability constants (5.5 < log K′ < 6.5) were determined. Cd(II) complexes detected in the different stages of microbial growth are labile from an electrochemical point of view. Results were compared to the case of Cd(II) non-supplemented broth.     

Effects of Chromium(III) Picolinate on Cortisol and DHEAs Secretion in H295R Human Adrenocortical Cells

Dietary chromium(III) picolinate (CrPic) effects on circulating steroid hormones have been reported in various experimental animals. However, direct effects of CrPic on adrenocortical steroidogenesis are uncertain. Therefore, the objective was to determine the effects of CrPic on cortisol and dehydroepiandrosterone sulfate (DHEAs) secretion from H295R cells. In experiment 1, a 24-h exposure to CrPic (0 to 200 μM) had both linear (p < 0.001) and quadratic (p < 0.001) effects on cortisol secretion from forskolin-stimulated cells with the highest cortisol secretion at 0.1 μM of CrPic and the lowest at 200 μM of CrPic. In experiment 2, a 48-h exposure to CrPic (200 μM) decreased cortisol (p < 0.07) release from forskolin-stimulated cells during a 24-h collection period. In experiment 3, a 48-h exposure to CrPic (100 μM) decreased cortisol (p < 0.05) and DHEAs (p < 0.01) from forskolin-stimulated cells during a 24-h sampling period. In experiment 4, a 24-h exposure to forskolin followed by a 24-h exposure to both forskolin and CrPic (100 and 200 μM) decreased both cortisol and DHEAs secretion (p < 0.01). This study suggests that at high concentrations, CrPic inhibits aspects of steroidogenesis in agonist-stimulated adrenocortical cells.     

Shoot organogenesis and plant regeneration in <Emphasis Type="Italic">Pityopsis ruthii</Emphasis>

Pityopsis ruthii is an endangered herbaceous perennial species from the United States. In vitro multiplication of this species can be valuable for germplasm conservation. Flower receptacles of P. ruthii were cultured on Murashige and Skoog medium (MS) supplemented with 11.4 μM indole-3-acetic acid (IAA) in combination with 2.2, 4.4 or 8.8 μM 6-benzyladenine (BA). Shoots were visible within 14–28 days and three plants were successfully rooted on MS medium supplemented with 5.7 μM IAA. A two tailed t-test for paired-variates revealed that shoot regeneration on MS medium amended with 11.4 μM IAA and 2.2 μM BA was significantly higher (P < 0.05) than on other treatments. Leaf explants were also cultured on MS not supplemented with growth regulators or supplemented with 11.4 μM IAA in combination with 0, 2.2, 4.4 or 8.8 μM BA. Shoots were visible within 21–35 days and one plant was successfully rooted on MS medium supplemented with 5.4 μM NAA. Shoot regeneration on MS medium augmented with 11.4 μM IAA and 2.2 μM BA was significantly higher (P < 0.05) than the other treatments according to analysis of variance (ANOVA) with a rank transformation. Hyperhydricity and rooting of shoots was problematic for explants derived from flower receptacles and leaf tissue, but viable plants were regenerated using both explants sources indicating the potential role for micropropagation in the ex situ conservation of the species.     

Docosahexaenoic acid and eicosapentaenoic acid-enriched phosphatidylcholine liposomes enhance the permeability, transportation and uptake of phospholipids in Caco-2 cells

The influence of docosahexaenoic acid (DHA)- and eicosapentaenoic acid (EPA)-enriched phosphatidylcholine (PC) on the permeability, transport and uptake of phospholipids was evaluated in Caco-2 cells. The cells were grown on permeable polycarbonate transwell filters, thus allowing separate access to the apical and basolateral chambers. The monolayers of the cells were used to measure lucifer yellow permeability and transepithelial electrical resistance (TEER). Transcellular transportation of diphenylhexatriene (DPH) labeled-PC small unilamellar vesicles (SUV) from the apical to basolateral chamber, and uptake of the same SUV was monitored in the cell monolayers. Cell-membrane perturbation was evaluated to measure the release of lactate dehydrogenase and to determine the cell viability with sodium 2-(4-iodophenyl)-3-(4-nitrophenyl) -5-(2, 4-disulfophenyl)-2H-tetrazolium dye reduction assay. The lucifer yellow flux was 1.0 and 1.5 nmol/h/cm² with 50 μM PC, and 17.0 and 23.0 nmol/h/cm² with 100 μM PC when monolayers of Caco-2 cells were treated with DHA- and EPA-enriched PC, respectively. TEER decreased to 24 and 27% with 50 and 100 μM DHA-enriched PC, and to 25 and 30% with 50 and 100 μM EPA-enriched PC, respectively. Our results show that DHA- and EPA-enriched PC increases tight junction permeability across the Caco-2 cell monolayer whereas soy PC has no effect on tight junction permeability. Transportation and uptake of DHA- and EPA-enriched PC SUV differed significantly (P < 0.01) from those of soy PC SUV at all doses. We found that PC SUV transported across Caco-2 monolayer and was taken up by Caco-2 cells with very slight injury of the cell membrane up to 100 μM PC. Lactate dehydrogenase release and cell viability did not differ significantly between the treatment and control, emphasizing that injury was minimal. Our results suggest that DHA- and EPA-enriched PC enhance the permeability, transport and uptake of PC SUV across monolayers of Caco-2 cells. (Mol Cell Biochem xxx: 1–9, 2005)     

Expansion of a direct shoot organogenesis system in peanut (Arachis hypogaea L.) to include US cultivars

Agrobacterium-mediated transformation, employing direct shoot organogenesis, allows for mature transgenic plants to be obtained quickly (3–4 mo). In this study, peanut (Arachis hypogaea L.) cultivars Florida-07, Georgia Green, Georgia Brown, New Mexico Valencia A, and VC-2 were selected to test their shoot induction response for use in future transformation experiments. Two types of cotyledon explants were examined, those that previously had an attached embryo axis upon cotyledon separation (explant A) and those that were embryo axis-free upon separation (explant B). Explants were placed onto a shoot induction medium with N ⁶-benzyladenine concentrations ranging from 10–80 μM for Florida-07, Georgia Green, and VC-2; 10–20 μM for Georgia Brown; and 10–640 μM for New Mexico Valencia A. Following a 4-wk culture period, explants were visually rated based on a scale of 1–4, where 1 indicated slight greening, but no growth, and 4 indicated greening, adventitious bud formation, as well as small leaf expansion. A difference in shoot induction was observed for the cotyledon explants examined (P > t = <0.0001). Explant A had greater shoot induction with a visual rating of 1.8 ± 0.1; explant B had a rating of 1.6 ± 0.1 (P > t = <0.0001). Additionally, cultivars responded to the culture conditions differently (cultivar × N ⁶-benzyladenine interaction). Georgia Green on 10 μM N ⁶-benzyladenine produced the most shoot buds (24.6%) and the highest visual rating (2.1), followed by VC-2 on 10 μM N ⁶-benzyladenine (22.1%, 1.8), New Mexico Valencia A on 640 μM N ⁶-benzyladenine (21.4%, 1.8), Georgia Brown on 80 μM N ⁶-benzyladenine (9.0%, 1.7), and Florida-07 on 40 μM N ⁶-benzyladenine (7.1%, 1.8). Of the tested varieties, Georgia Green, New Mexico Valencia A, and VC-2 were best suited for future transformation experiments based on their shoot bud production.     

Changes in the Growth, Chemical Composition, and Antioxidant Activity in the Aquatic Plant Wolffia arrhiza (L.) Wimm. (Lemnaceae) Exposed to Jasmonic Acid

The present study was undertaken to test the influence of exogenously applied jasmonic acid (JA) at concentrations of 0.01–100 μM upon the growth and metabolism of the aquatic plant Wolffia arrhiza (Lemnaceae). JA acted in a concentration-dependent manner. JA at 0.1 μM stimulated plant growth and accumulation of cellular components (proteins, monosaccharides, chlorophylls, phaeophytins, and carotenoids). Treatment with JA at 0.1 μM enhanced W. arrhiza viability by the induction of biomass production and increased the level of photosynthetic pigments, monosaccharides, and soluble proteins. Moreover, JA at 0.1 μM activated the enzymatic (catalase, ascorbate peroxidase, NADH peroxidase) and nonenzymatic antioxidant (ascorbate, glutathione) system in W. arrhiza and, therefore, suppressed lipid peroxidation. In contrast, decreases in fresh weight, major photosynthetic pigments, monosaccharides, and soluble protein content were observed in W. arrhiza exposed to 100 μM JA. JA applied at 100 μM also stimulated the formation of lipid peroxides which are responsible for membrane damage. In the presence of 100 μM JA, antioxidant enzyme (catalase, ascorbate peroxidase, NADH peroxidase) activity and ascorbate as well as glutathione content were inhibited. The data support the hypothesis that JA plays an important role in W. arrhiza growth and metabolism, regulating oxidative status by direct influence on the enzymatic as well as nonenzymatic antioxidant machinery.     

Multiple shoot induction and callus regeneration in Sarcostemma brevistigma Wight & Arnott, a rare medicinal plant

An efficient micropropagation protocol based on multiple shoot induction and callus regeneration has been standardized in Sarcostemma brevistigma, a rare medicinal plant. The nodal cuttings were cultured on MS medium supplemented with BA (0.5–8 μM) or Kn (0.5–8 μM) alone or in combination with NAA (0.5–1.5 μM). Maximum multiple shoot induction was observed on MS medium supplemented with 4 μM BA. On this medium, 100% cultures responded with an average number of 11.3 shoots per explant. However, the average shoot length was limited to only 0.9 cm on this medium. The addition of 1 μM NAA along with 4 μM BA gave rise to an average number of 10.9 shoots with an average shoot length of 1.8 cm. Luxuriantly growing callus was obtained on MS medium supplemented with BA (5 μM) and 2,4-D (2 μM). The callus was subcultured on MS medium supplemented with BA (2–15 μM) or Kn (2–15 μM) alone or in combination with NAA (0.5–2 μM) for shoot organogenesis. Optimum callus regeneration was obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. On this medium, 100% cultures responded with an average number of 13.4 shoots per culture. The shoots obtained via multiple shoot induction and organogenesis were rooted on half-strength MS medium supplemented with NAA (1–7 μM) or IBA (1–7 μM). IBA was better than NAA in terms of both the percentage of cultures that responded and the average number of roots per explant. The rooted shoots were successfully transplanted to soil with 86% success. This standardized protocol will help to conserve this rare medicinal plant.     

Dietary zinc mediates inflammation and protects against wasting and metabolic derangement caused by sustained cigarette smoke exposure in mice

In mouse asthma models, inflammation can be modulated by zinc (Zn). Given that appetite loss, muscle wasting and poor nutrition are features of chronic obstructive pulmonary disease (COPD) and that poor dietary Zn intake is in itself accompanied by growth retardation and appetite loss, we hypothesised that dietary Zn limitation would not only worsen airway inflammation but also exaggerate metabolic effects of cigarette smoke (CS) exposure in mice. Conversely, Zn supplementation would lessen inflammation. Mice were exposed to CS [2× 2RF, 3×/day; 15 min/cigarette] and fed diets containing 2, 20 or 140 mg/kg Zn ad libitum. Airway cells were collected by bronchoalveolar lavage (BAL). Plasma Zn was measured by fluorometric assay. Inflammatory, metabolic and Zn transport markers were measured by real-time RT-PCR. Mice fed low Zn diets had less plasma labile zinc (0–0.18 μM) than mice fed moderate (0.61–0.98 μM) or high (0.77–1.1 μM) Zn diets (SDs 0.1–0.4, n = 8–10). Smoke exposure increased plasma and BAL labile Zn (1.5–2.5 fold, P < 0.001), bronchoalveolar macrophages (2.0 fold, P < 0.0001) and MT-1 (1.5 fold), MIP-2 (2.3 fold) and MMP-12 (3.5 fold) mRNA. Zn supplementation reduced alveolar macrophage numbers by 62 and 52% in sham and smoke-exposed mice, respectively (Zn effect: P = 0.011). Gastrocnemius, soleus and tibialis anterior muscle mass were affected by both smoke and dietary Zn in the order of 3–7%. The 50–60% reduction in alveolar macrophages in Zn-supplemented mice supports our evolving hypothesis that Zn is an important anti-inflammatory mediator of airway inflammation. Restoring airway Zn levels through dietary supplementation may lessen the severity of lung inflammation when Zn intake is low.     

Selenium concentration in liver and kidney of free living animals (roe and red deer) from West Pomerania (Poland)

The aim of this study was to determine concentrations of selenium in the liver and kidneys of roe deer and red deer from West Pomerania, depending on the season. Altogether, samples from 169 animals were collected (96 from roe deer and 73 from red deer) in 2003–2007. The mean concentration of selenium in the liver of red deer and roe deer was 0.37 μg/g and 0.62 μg/g dry weight, respectively. In kidneys, Se concentration was 2.72 μg/g d.w. in red deer and 2.99 μg/g d.w. in roe deer. In roe deer, liver selenium concentration in autumn was significantly higher than in winter (P < 0.05) and spring (P < 0.01) and significantly lower in spring than in summer (P < 0.05); likewise, kidney selenium concentration was higher in autumn than in summer. In deer, no statistically significant season-related differences were observed for liver selenium concentrations. In red deer kidneys, selenium concentration was the lowest in summer, significantly lower than in autumn and winter. Low selenium concentrations in the analyzed tissues show that the animals live in areas deficient in this element.     

Modulation of Glutamate and Glycine Transporters by Niflumic,Flufenamic and Mefenamic Acids

Three fenamates—niflumic, flufenamic and mefenamic acids—were tested for effects on substrate-induced currents of glutamate and glycine transporters (EAAT1, EAAT2, GLYT1b and GLYT2a) expressed in Xenopus laevis oocytes. All fenamates inhibited EAAT1 currents; 100 μM flufenamic acid produced the most inhibition, decreasing the I _max by 53 ± 4% (P < 0.001). EAAT2 currents were less sensitive, but 100 μM flufenamic acid inhibited the I _max by 34 ± 5% (P = 0.006). All fenamates inhibited GLYT1b currents; 100 μM flufenamic acid produced the most inhibition, decreasing the I _max by 61 ± 1% (P < 0.001). At 100 μM, effects on the GLYT2a I _max were mixed: 13 ± 2% inhibition by flufenamic acid (P = 0.002), 30 ± 6% enhancement by niflumic acid (P = 0.002), and no effect by mefenamic acid. Minor effects on substrate affinity suggested non-competitive mechanisms. These data could contribute to the development of selective transport modulators.     

Disturbances of energetic metabolism in rat epididymal epithelial cells as a consequence of chronic lead intoxication

Adult male Wistar rats were intoxicated with 1% lead acetate (PbAc) administered in drinking water for nine months, which amounts to a period five times longer than the duration of one spermatogenesis. There were mitochondrial ultrastructure disorders of epididymal epithelial cells observed in PbAc-treated rats; also a significant lead-induced decrease in ATP concentration in epididymal epithelial cells (by 32%, P < 0.05), Adenylate Energy Charge value (AEC) (by 8%, P < 0.05) and an increase in ADP (28.5%, P < 0.05), AMP (27%, P < 0.05) and adenosine (by 56%, P < 0.05). The results were measured using high performance liquid chromatography (HPLC) and detected even at low lead concentrations in whole blood (M:7.03 μg/dL; Q1–Q3: 2.99–7.65). The function of mitochondria in cultured epididymal epithelial cells of control and PbAc-treated animals were evaluated using fluorophores: Mitotracker Green FM and JC-1. After incubation with Mitotracker Green FM, we observed active mitochondria producing bright green fluorescence in the cytoplasm of cultured epididymal epithelial cells, both in the control group and the Pb-treated animals. Incubation of cultured epididymal epithelial cells of animals from both groups produced red-orange fluorescence with the mitochondrial JC-1 probe indicating mitochondria with high membrane potential (ΔΨm > 80–100 mV) and green fluorescence in the mitochondria with low membrane potential (ΔΨm <80 mV). The results showed that a chronic low-level exposure to lead, even without severe clinical symptoms of contamination, disrupted the ultrastructure and energy metabolism of mitochondria in epididymal epithelial cells.     

Plant regeneration from in vitro leaves of mature black cherry (Prunus serotina)

A regeneration system was developed for Prunus serotina from a juvenile (F) and two mature genotypes (#3 and #4). Adventitious shoots regenerated from leaves of in vitro cultures on woody plant medium with thidiazuron (TDZ) and naphthaleneacetic acid (NAA). The best regeneration for genotype F (91.4%) was observed on medium with 9.08 μM TDZ and 1.07 μM NAA. The highest mean number of shoots (8.2) was obtained on medium containing 9.08 μM TDZ and 0.54 μM NAA. Genotype #3 had the highest regeneration (41.7%) with a mean number of shoots (4.8) on 9.08 μM TDZ and 1.07 μM NAA, whereas genotype #4 had a 38.8% regeneration with a mean of 3.3 shoots. Genotype #4 had the highest mean number of shoots (4.8) on 4.54 μM TDZ and 1.07 μM NAA. Silver thiosulphate at 60 or 80 μM increased the percent regeneration of the mature genotypes #3 (75%) and #4 (58%). Adventious shoots were rooted (70–76%) and rooted plantlets survived after acclimatization to the greenhouse. The effect of kanamycin concentration on adventitious shoot regeneration was also evaluated.     

Seasonal variability of inorganic and organic nitrogen in the North Sea

This study considers the cycling of nitrogen in the waters of the North Sea, particularly focussing on organic nitrogen. Dissolved inorganic nitrogen (DIN), dissolved organic nitrogen (DON) and particulate organic nitrogen (PON) were measured in the North Sea over a one-year period (autumn 2004–summer 2005). The surface water concentrations of nitrate, ammonium, DON and PON during the present study ranged from <0.1–7.2 μM, <0.1–2.0 μM, 1.9–11.2 μM and 0.3–5.6 μM, respectively, with DON the dominant fraction of total nitrogen at all times. These nutrients concentrations were significantly lower compared to previous studies in the southern North Sea. The seasonal variations showed high mean surface concentrations of nitrate (4.7 ± 0.6 μM) and DON (8.9 ± 0.9 μM), low ammonium (<0.1 μM) and PON (0.8 ± 0.1 μM) in winter, shifting to low nitrate (0.3 ± 0.3 μM) and DON (4.2 ± 1.2 μM) in summer, with high ammonium (0.8 ± 0.4 μM) in autumn and PON (2.5 ± 1.2 μM) in spring. Highest mean surface DON concentration was measured in winter and may be due to resuspension of the organic matter from the bottom sediments. For autumn and spring, phytoplankton DON release was likely to be the most significant source of DON as shown by high concentrations of low molecular weight (LMW) DON and its positive correlation to chlorophyll a. Low total and LMW DON concentrations during summer were likely to be due to the uptake of the LMW DON fraction by phytoplankton and bacteria and the stratification of the water column. DON is therefore shown to be a potentially important source of nitrogen in shelf seas especially after the spring bloom has depleted nitrate to limiting concentrations. Handling editor: L. Naselli-Flores     

Plant regeneration through direct shoot formation from leaf cultures and from protocorm-like bodies derived from callus of <Emphasis Type="Italic">Encyclia mariae</Emphasis> (Orchidaceae), a threatened Mexican orchid

Two procedures for the in vitro propagation of Encyclia mariae, a threatened Mexican orchid, were developed. In the first procedure, leaves from in vitro germinated seedlings were cultured on Murashige and Skoog medium (MS) supplemented with the range of 2.21–4.4 μM 6-benzylaminopurine (BA) in combination with 2.69–10.74 μM naphthalene acetic (NAA), 2.07–8.29 μM indole-3-butyric (IBA), or 2.85–11.42 μM indole-3-acetic acid (IAA) to determine the best medium for the induction of shooting. Maximum direct shoot formation from leaves was observed on MS containing 22.21 μM BA and 10.74 μM NAA (25 shoots/explant). The second procedure began with the culture of protocorms on media containing NAA, IBA, or IAA, which induced callus formation with high regenerative potential in the form of protocorm-like-bodies (PLBs) that eventually differentiated into shoots. The optimal response was attained when these structures were cultured on medium with 4.14 μM IBA (30 shoots/PLB). To promote the elongation of shoots derived from PLBs, the material was subcultured onto MS medium containing 22.21 μM BA and 5.37 μM NAA. Through the exploration of the effects of auxins and matrix on the rooting of shoots, it was determined that the optimal rooting occurred on media supplemented either with 5.71 μM IAA or 4.14 μM IBA either on agar-gelled medium or in liquid media with coir as the matrix. Rooting was found to be 20% higher in liquid media than in agar-gelled medium.     

In vitro propagation from axenic explants of <Emphasis Type="Italic">Lilium oxypetalum</Emphasis> (D. Don) Baker,an endemic bulbous plant of high altitude Himalaya

In vitro propagation protocol for Lilium oxypetalum, a high altitude Himalayan endemic lily, has been developed. Effect of explant types (i.e., callus and in vitro bulblet scales) and sucrose concentration [0–6.0% (w/v)] on in vitro bulblet regeneration of L. oxypetalum was tested in previously optimized Murashige and Skoog basal medium supplemented with 2.0 μM 6-benzyladenine and 0.1 μM α-naphthaleneacetic acid. Callus explants produced significantly (P < 0.01) higher number of bulblets per explant than bulblet scale explants. Of the different concentrations of sucrose tested, 4.5% (w/v) sucrose showed significantly (P < 0.01) higher percentage regeneration (i.e., 70.8 ± 4.2 and 79.2 ± 4.2% regeneration on callus and bulblet scale explants, respectively), and produced higher number of bulblets per explant (i.e., 9.0 ± 0.4 and 5.4 ± 0.5 bulblets on callus and bulblet scale explants, respectively). Regenerated bulblets developed 2–3 leaves when subcultured for 4 weeks and were subsequently transferred ex vitro with a survival rate of 66.7% after 6 weeks. Leaves of the survived plantlets became dry after growing ex vitro for 10 weeks, amongst which 86.4% re-sprouted after remaining dormant for 5–6 weeks and produced 1.5 bulblets per explant. Findings of the present study hold promise for efficiently multiplying the target species in view of its potential economic and conservation significance.     

Curcumin modulates eukaryotic initiation factors in human lung adenocarcinoma epithelial cells

Curcumin, a polyphenolic compound, is the active component of Curcuma longa and has been extensively investigated as an anticancer drug that modulates multiple pathways. Eukaryotic initiation factors (eIFs) have been known to play important roles in translation initiation, which controls cell growth and proliferation. Little is known about the effects of curcumin on eIFs in lung cancer. The objective of this study was to exam the curcumin cytotoxic effect and modulation of two major rate-limiting translation initiation factors, including eIF2α and eIF4E protein expression levels in lung adenocarcinoma epithelial cell line A549. Cytotoxicity was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and protein changes were determined by Western blot. A549 cells were treated with 0–240 μM curcumin for 4–96 h. The inhibitory effects of curcumin on cytotoxicity were dose- and time-dependent (P < 0.001). The 50% inhibitory curcumin concentrations (IC50s) at 24, 48, 72, and 96 h were 93, 65, 40, and 24 μM, respectively. Protein expressions of eIF2α, eIF4E, Phospho-4E-BP1 were down-regulated, while Phospho-eIF2α and Phospho-eIF4E were up-regulated after A549 cells were treated with 20 and 40 μM curcumin for 24 h. In addition, the effects of curcumin on these protein expression changes followed a significant dose-response (P < 0.05, trend test). These findings suggest that curcumin could reduce cell viability through prohibiting the initiation of protein synthesis by modulating eIF2α and eIF4E.     

Effect of Hydrogen Sulfide on Cyclic AMP Production in Isolated Bovine and Porcine Neural Retinae

Hydrogen sulfide (H₂S) has been reported to exert pharmacological effects on neural and non-neural tissues from several mammalian species. In the present study, we examined the role of the intracellular messenger, cyclic AMP in retinal response to H₂S donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na₂S) in cows and pigs. Isolated bovine and porcine neural retinae were incubated in oxygenated Krebs buffer solution prior to exposure to varying concentrations of NaHS, Na₂S or the diterpene activator of adenylate cyclase, forskolin. After incubation at different time intervals, tissue homogenates were prepared for cyclic AMP assay using a well established methodology. In isolated bovine and porcine retinae, the combination of both phosphodiesterase inhibitor, IBMX (2 mM) and forskolin (10 μM) produced a synergistic increase (P < 0.001) in cyclic AMP concentrations over basal levels. NaHS (10 nM–100 μM) produced a time-dependent increase in cyclic AMP concentrations over basal levels which reached a maximum at 20 min in both bovine and porcine retinae. At this time point, both NaHS and Na₂S (10 nM–100 μM) caused a significant (P < 0.05) dose-dependent increase in cyclic AMP levels in bovine and porcine retinae. For instance, NaHS (100 nM) elicited a four-fold and three-fold increase in cyclic AMP concentrations in bovine and porcine retinae respectively whilst higher concentrations of Na₂S (100 μM) produced a much lesser effect in both species. In bovine and porcine retinae, the effects caused by forskolin (10 μM) on cyclic AMP production were not potentiated by addition of low or high concentrations of both NaHS and Na₂S. We conclude that H₂S donors can increase cyclic AMP production in isolated neural retinae from cows and pigs. Bovine retina appears to be more sensitive to the stimulatory effect of H₂S donors on cyclic nucleotide production than its porcine counterpart indicating that species differences exist in the magnitude of this response. Furthermore, effects produced by forskolin on cyclic AMP formation were not additive with those elicited by H₂S donors suggesting that these agents may share a common mechanism in their action on the adenylyl cyclase pathway.     

<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.