Polysaccharide of Alocasia cucullata Exerts Antitumor Effect by Regulating Bcl-2, Caspase-3 and ERK1/2 Expressions during Long-Time Administration.

OBJECTIVE: To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism. METHODS: B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR. RESULTS: In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells. CONCLUSIONS: Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.

Photosystem I in low light-grown leaves of Alocasia odora, a shade-tolerant plant, is resistant to fluctuating light-induced photoinhibition.

When intact green leaves are exposed to the fluctuating light, in which high light (HL) and low light (LL) alternate, photosystem I (PSI) is readily damaged. This PSI inhibition is mostly alleviated by the addition of far-red (FR) light. Here, we grew Alocasia odora, a shade-tolerant species, at several light levels and examined their photosynthetic traits in relation to the fluctuating light-induced PSI inhibition. We found that, even in the absence of FR, PSI in LL-grown leaves was resistant to the fluctuating light. LL leaves showed higher chlorophyll (Chl) contents on leaf area basis, lower Chl a/b ratios, lower cytochrome f/P700 ratios, and lower PSII/PSI excitation ratios assessed by the 77 K fluorescence. Also, P700 in the HL phase of the fluctuating light was more oxidized. The results of the regression analyses of the PSI photoinhibition to these traits indicate that the lower electron flow rate to P700 and more excitation energy transfer to PSI protect PSI in LL-grown leaves. Both of these contribute oxidization of P700 to the efficient quencher form P700+. These features may be common in LL-grown shade-tolerant species, which are often exposed to strong sunflecks in their natural habitats.

Using a high biomass plant Pennisetum hydridum to phyto-treat fresh municipal sewage sludge.

The study was carried out to investigate the use of a high biomass plant, Pennisetum hydridum, to treat municipal sewage sludge (MSS). An experiment composed of plots with four treatments, soil, fresh sludge, soil-sludge mixture and phyto-treated sludge, was conducted. It showed that the plant could not survive directly in fresh MSS when cultivated from stem cuttings. The experiment transplanting the incubated cutting with nurse medium of P. hydridum in soil and fresh MSS, showed that the plants grew normally in fresh MSS. The pilot experiment of P. hydridum and Alocasia macrorrhiza showed that the total yield and nutrient amount of P. hydridum were 9.2 times and 3.6 times more than that of A. macrorrhiza. After plant treatment, MSS was dried, stabilized and suitable to be landfilled or incinerated, with a calorific value of about 5.6MJ/kg (compared to the initial value of 1.9MJ/kg fresh sludge).

Phytotreatment of sewage sludge contaminated by heavy metals and PAHs by co-planting Sedum alfredii and Alocasia marorrhiza.

High concentrations of heavy metals and organic pollutants in municipal sewage sludge are key factors limiting its use in agriculture. The objectives of this study were to decrease the heavy metal and polycyclic aromatic hydrocarbon concentrations in sewage sludge by phytotreatment and to determine, in a field experiment, whether co-planting is more effective than using a mono-crop of Sedum alfredii. Four treatments were used in the plot experiment: no sludge, no plants, S. alfredii and co-planting S. alfredii and Alocasia marorrhiza. The results showed that co-planting produced tubers and shoots of A. marorrhiza that were suitable as a safe animal feed and good organic K fertilizer, respectively. Co-planting was more effective than mono-planting at reducing concentrations of total Zn and diethylenetriaminepentaacetic acid (DTPA)-extractable Zn, Cd, and Cu in the sludge. Co-planting decreased the concentrations of DTPA-extractable heavy metals and benzo[a]pyrene (B[a]P) in the sludge significantly compared with the unplanted sludge. Decreases of 87, 75, 85, 31, and 64% were obtained for B[a]P and DTPA-extractable Zn, Cd, Cu, and Pb, respectively, compared with the fresh sludge. These results indicate that co-planting can reduce significantly the environmental risks associated with heavy metals and B[a]P in sewage sludge for further disposal.

Fate of heavy metals and major nutrients in a sludge-soil-plant-leachate system during the sludge phyto-treatment process.

Land application of sewage sludge usually leads to increased levels of heavy metals in soil, plants and groundwater. Pre-treatment using plants has been proposed to reduce the contents of heavy metals and water in sludge prior to land application. This study quantified the transfer of Zn, Cd, Pb and major nutrients in a sludge-soil-plant-leachate system during the treatment of sewage sludge. To accomplish this, a two year pot experiment was carried out to collect leachate, mono- and co-cropping of Sedum alfredii and feed crops was conducted in sludge with an under-layer soil support. Sludge phyto-treatment increased Zn and Cd concentrations in the under-layer soil, but not Pb. Specifically, 70%, 70% and 80% of the original Zn, Cd and Pb, respectively, remained in the sludge, while about 40%, 70% and 60% of the original N, P and K remained. Only 3% to 5% of Cd and Zn and < 1% of Pb were transferred into the under-layer soils or leachates, while more than 12% of the N and P were transferred. Co-planting S. alfredii and feed crops led to a significant reduction of heavy metals in leachates when compared with sludge without planting. Overall, sludge leachate is more appropriate than whole sludge for recycling in agriculture since it reduces the chance of heavy metal contamination in the agro-ecosystem; therefore, co-cropping phytotreatment of sludge can be coupled with sludge leachate recycling for crop production and re-collection of the sludge residue for landfilling.

The heterogeneity and spatial patterning of structure and physiology across the leaf surface in giant leaves of Alocasia macrorrhiza.

Leaf physiology determines the carbon acquisition of the whole plant, but there can be considerable variation in physiology and carbon acquisition within individual leaves. Alocasia macrorrhiza (L.) Schott is an herbaceous species that can develop very large leaves of up to 1 m in length. However, little is known about the hydraulic and photosynthetic design of such giant leaves. Based on previous studies of smaller leaves, and on the greater surface area for trait variation in large leaves, we hypothesized that A. macrorrhiza leaves would exhibit significant heterogeneity in structure and function. We found evidence of reduced hydraulic supply and demand in the outer leaf regions; leaf mass per area, chlorophyll concentration, and guard cell length decreased, as did stomatal conductance, net photosynthetic rate and quantum efficiency of photosystem II. This heterogeneity in physiology was opposite to that expected from a thinner boundary layer at the leaf edge, which would have led to greater rates of gas exchange. Leaf temperature was 8.8°C higher in the outer than in the central region in the afternoon, consistent with reduced stomatal conductance and transpiration caused by a hydraulic limitation to the outer lamina. The reduced stomatal conductance in the outer regions would explain the observed homogeneous distribution of leaf water potential across the leaf surface. These findings indicate substantial heterogeneity in gas exchange across the leaf surface in large leaves, greater than that reported for smaller-leafed species, though the observed structural differences across the lamina were within the range reported for smaller-leafed species. Future work will determine whether the challenge of transporting water to the outer regions can limit leaf size for plants experiencing drought, and whether the heterogeneity of function across the leaf surface represents a particular disadvantage for large simple leaves that might explain their global rarity, even in resource-rich environments.

Acclimation of leaves to low light produces large grana: the origin of the predominant attractive force at work.

Photosynthetic membrane sacs (thylakoids) of plants form granal stacks interconnected by non-stacked thylakoids, thereby being able to fine-tune (i) photosynthesis, (ii) photoprotection and (iii) acclimation to the environment. Growth in low light leads to the formation of large grana, which sometimes contain as many as 160 thylakoids. The net surface charge of thylakoid membranes is negative, even in low-light-grown plants; so an attractive force is required to overcome the electrostatic repulsion. The theoretical van der Waals attraction is, however, at least 20-fold too small to play the role. We determined the enthalpy change, in the spontaneous stacking of previously unstacked thylakoids in the dark on addition of Mg(2+), to be zero or marginally positive (endothermic). The Gibbs free-energy change for the spontaneous process is necessarily negative, a requirement that can be met only by an increase in entropy for an endothermic process. We conclude that the dominant attractive force in thylakoid stacking is entropy-driven. Several mechanisms for increasing entropy upon stacking of thylakoid membranes in the dark, particularly in low-light plants, are discussed. In the light, which drives the chloroplast far away from equilibrium, granal stacking accelerates non-cyclic photophosphorylation, possibly enhancing the rate at which entropy is produced.

Lead and cadmium induced alterations of cellular functions in leaves of Alocasia macrorrhiza L. Schott.

Alocasia macrorrhiza is a fast growing and propagating herbaceous species commonly found in South China. To determine its physiological responses to Pb and Cd stresses, the biochemical, histochemical and cytochemical changes under PbAC2 and CdCl2 phytotoxicity were detected using leaf discs as an experimental model. After leaf discs were infiltrated in different concentrations of PbAC2 and CdCl2 solutions (0, 50, 100, 150, 200 microM) for 72 h, the formation of reactive oxygen species (H2O2 and O2-) in plant tissue were found to be exaggerated together with elevated OH concentration and cell death. Changes in chlorophyll fluorescence (Fv/Fm, PhiPSII, qP and NPQ) imaging colours/areas of leaf discs indicated decreased photosystem II functions by both heavy metal treatments and positive reactions of antioxidants under Pb2+ stress. Results showed that fluorescent detection of hydroxylated terephthlate using terephthalic acid as OH trap is a simple, yet valuable and specific method for monitoring OH generation in plant tissue under heavy metal stresses. As compared with Cd2+, Pb2+ was found to be less toxic, indicating that A. macrorrhiza tissue might have a potential tolerance to Pb.

Recovery of photoinactivated photosystem II in leaves: retardation due to restricted mobility of photosystem II in the thylakoid membrane.

The functionality of photosystem II (PS II) following high-light pre-treatment of leaf segments at a chilling temperature was monitored as F(v)/F(m), the ratio of variable to maximum chlorophyll fluorescence in the dark-adapted state and a measure of the optimal photochemical efficiency in PS II. Recovery of PS II functionality in low light (LL) and at a favourable temperature was retarded by (1) water stress and (2) growth in LL, in both spinach and Alocasia macrorrhiza L. In spinach leaf segments, water stress per se affected neither F(v)/F(m) nor the ability of the adenosine triphosphate (ATP) synthase to be activated by far-red light for ATP synthesis, but it induced chloroplast shrinkage as observed in frozen and fractured samples by scanning electron microscopy. A common feature of water stress and growth of plants in LL is the enhanced anchoring of PS II complexes, either across the shrunken lumen in water-stress conditions or across the partition gap in larger grana due to growth in LL. We suggest that such enhanced anchoring restricts the mobility of PS II complexes in the thylakoid membrane system, and hence hinders the lateral migration of photoinactivated PS II reaction centres to the stroma-located ribosomes for repair.

Co-cropping for phyto-separation of zinc and potassium from sewage sludge.

The use of sewage sludge as a fertilizer and soil amendment has resulted in high concentrations of heavy metals in the soil limiting its use. The present study was carried out to find the possibility of phyto-separating toxic and beneficial elements from the sludge using suitable plants. Of the five plants tested the hyperaccumulator Sedum alfredii H achieved the greatest removal of Zn, while shoots of Alocasia marorrhiza accumulated high content of K. Co-cropping these two plants on the sludge verified the previous observations on A. marorrhiza and the shoots of this plant could accumulate more than 120 g K kg(-1) dry matter in the median growth stage. Zn hyperaccumulated in Sedum's shoots to an extent more than 10 g kg(-1) dry matter; K concentrated five to ten times in the Alocasia's shoots which could be used as a good organic-K-fertilizer. Hence, the two elements were simultaneously phytoseparated and could be recycled. Furthermore, cultivation of plants in the sludge resulted in significant decreases in total Zn but kept the favorable agronomic characteristics of the sludge material, such as pH, organic matter content, and NPK concentrations and ameliorated its biological stability. These results suggest that simultaneous phyto-separation of toxic and beneficial elements from sewage sludge are possible by co-cropping using specific plants without the input of any chemicals.

Isolation of a novel N-acetyl-D-lactosamine specific lectin from Alocasia cucullata (Schott.).

An N-acetyl-D: -lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on asialofetuin-linked amino activated silica. The pure lectin showed a single band in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa. It was heat stable up to 55 degrees C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected by denaturing agents such as urea (2 M: ), thiourea (2 M: ) and guanidine-HCl (0.5 M: ) and did not require Ca2+ and Mn2+ for its activity. It was a potent mitogen at 10 microg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory potential towards SiHa (human cervix ) cancer cell line at 100 microg/ml.

Granal stacking of thylakoid membranes in higher plant chloroplasts: the physicochemical forces at work and the functional consequences that ensue.

The formation of grana in chloroplasts of higher plants is examined in terms of the subtle interplay of physicochemical forces of attraction and repulsion. The attractive forces between two adjacent membranes comprise (1) van der Waals attraction that depends on the abundance and type of atoms in each membrane, on the distance between the membranes and on the dielectric constant, (2) depletion attraction that generates local order by granal stacking at the expense of greater disorder (i.e. entropy) in the stroma, and (3) an electrostatic attraction of opposite charges located on adjacent membranes. The repulsive forces comprise (1) electrostatic repulsion due to the net negative charge on the outer surface of thylakoid membranes, (2) hydration repulsion that operates at small separations between thylakoid membranes due to layers of bound water molecules, and (3) steric hindrance due to bulky protrusions of Photosystem I (PSI) and ATP synthase into the stroma. In addition, specific interactions may occur, but they await experimental demonstration. Although grana are not essential for photosynthesis, they are ubiquitous in higher plants. Grana may have been selected during evolution for the functional advantages that they confer on higher plants. The functional consequences of grana stacking include (1) enhancement of light capture through a vastly increased area-to-volume ratio and connectivity of several PSIIs with large functional antenna size, (2) the ability to control the lateral separation of PSI from PSII and, therefore, the balanced distribution of excitation energy between two photosystems working in series, (3) the reversible fine-tuning of energy distribution between the photosystems by State 1-State 2 transitions, (4) the ability to regulate light-harvesting via controlled thermal dissipation of excess excitation energy, detected as non-photochemical quenching, (5) dynamic flexibility in the light reactions mediated by a granal structure in response to regulation by a trans-thylakoid pH gradient, (6) delaying the premature degradation of D1 and D2 reaction-centre protein(s) in PSII by harbouring photoinactived PSIIs in appressed granal domains, (7) enhancement of the rate of non-cyclic synthesis of adenosine triphosphate (ATP) as well as the regulation of non-cyclic vs. cyclic ATP synthesis, and (8) the potential increase of photosynthetic capacity for a given composition of chloroplast constituents in full sunlight, concomitantly with enhancement of photochemical efficiency in canopy shade. Hence chloroplast ultrastructure and function are intimately intertwined.

Photoacoustic analysis indicates that chloroplast movement does not alter liquid-phase CO2 diffusion in leaves of Alocasia brisbanensis.

Light-mediated chloroplast movements are common in plants. When leaves of Alocasia brisbanensis (F.M. Bailey) Domin are exposed to dim light, mesophyll chloroplasts spread along the periclinal walls normal to the light, maximizing absorbance. Under high light, the chloroplasts move to anticlinal walls. It has been proposed that movement to the high-light position shortens the diffusion path for CO(2) from the intercellular air spaces to the chloroplasts, thus reducing CO(2) limitation of photosynthesis. To test this hypothesis, we used pulsed photoacoustics to measure oxygen diffusion times as a proxy for CO(2) diffusion in leaf cells. We found no evidence that chloroplast movement to the high-light position enhanced gas diffusion. Times for oxygen diffusion were not shorter in leaves pretreated with white light, which induced chloroplast movement to the high-light position, compared with leaves pretreated with 500 to 700 nm light, which did not induce movement. From the oxygen diffusion time and the diffusion distance from chloroplasts to the intercellular gas space, we calculated an oxygen permeability of 2.25 x 10(-)(6) cm(2) s(-)(1) for leaf cells at 20 degrees C. When leaf temperature was varied from 5 degrees C to 40 degrees C, the permeability for oxygen increased between 5 degrees C and 20 degrees C but changed little between 20 degrees C and 40 degrees C, indicating changes in viscosity or other physical parameters of leaf cells above 20 degrees C. Resistance for CO(2) estimated from oxygen permeability was in good agreement with published values, validating photoacoustics as another way of assessing internal resistances to CO(2) diffusion.

Selective precipitation of haptoglobin and alpha2-macroglobulin from human serum using Alocasia macrorhiza tuber protein.

Treatment of human serum with ammonium sulfate fraction (0-50%) of Alocasia macrorhiza tuber extract resulted in precipitation at neutral pH. The precipitate was dissolved at pH 10.5 and chromatographed on Sephadex G-100 column. Two protein peaks were resolved. While the first peak represented alpha2-macroglobulin and haptoglobin, the second peak accounted for specific Alocasia protein. Incidentally the Alocasia protein was shown to be responsible for selective and specific precipitation of alpha2-macroglobulin and haptoglobin from serum. Thus the plant protein in its pure form or in crude stage could be used for the rapid isolation of two of the prominent alpha2-globulins.