Thai Phung

Bilayer lipid membranes supported on Teflon filters: a functional environment for ion channels.

Many ion channel proteins have binding sites for toxins and pharmaceutical drugs and therefore have much promise as the sensing entity in high throughput technologies and biosensor devices. Measurement of ionic conductance changes through ion channels requires a robust biological membrane with sufficient longevity for practical applications. The conventional planar BLM is 100-300 μm in diameter and typically contains fewer than a dozen channels whereas pharmaceutical screening methods in cells use current recordings for many ion channels. We present a new, simple method for the fabrication of a disposable porous-supported bilayer lipid membrane (BLM) ion channel biosensor using hydrated Teflon (polytetrafluoroethylene, PTFE) filter material (pore size 5 μm, filter diameter=1 mm). The lipid layer was monitored for its thickness and mechanical stability by electrical impedance spectroscopy. The results showed membrane capacitances of 1.8±0.2 nF and membrane resistances of 25.9±4.1 GΩ, indicating the formation of lipid bilayers. The current level increased upon addition of the pore-forming peptide gramicidin. Following addition of liposomes containing voltage-gated sodium channels, small macroscopic sodium currents (1-80 pA) could be recorded. By preloading the porous Teflon with sodium channel proteoliposomes, prior to BLM formation, currents of 1-10 nA could be recorded in the presence of the activator veratridine that increased with time, and were inhibited by tetrodotoxin. A lack of rectification suggests that the channels incorporated in both orientations. This work demonstrates that PTFE filters can support BLMs that provide an environment in which ion channels can maintain their functional activity relevant for applications in drug discovery, toxin detection, and odour sensing.

Transporter genes to enhance nutrient uptake: Opportunities and challenges

Recently, there has been very rapid progress in understanding the transport of mineral nutrients across plant membranes. Genes for a number of primary ion pumps, cotransporters and ion channels have been cloned and the characteristics of their function are being investigated. While these advances have yet to produce cultivars that are better able to cope with nutrient deficient soils, they provide powerful tools to address some important gaps in our knowledge, particularly the regulation of transporter genes. While the current focus is on nutrient influx into roots, other processes are also significant in determining nutrient acquisition. This is illustrated by research on the relationship between the high affinity phosphate absorption mechanism and phosphate transport to the shoot, and with data on relating to the importance of phosphate efflux relative to influx. Molecular biology and plant physiology are providing information and technology that will be valuable in improving the suitability of crops for nutrient deficient environments. However, there is an urgent need for new ways to integrate this information so that the significance of individual processes to the performance of the whole system can be understood.

Physiological responses of wheat phosphorus-efficient and -inefficient genotypes in field and effects of mixing other nutrients on mobilization of insoluble phosphates in hydroponics

Abstract To elucidate the mechanism of mobilization of insoluble phosphates, a field trial and a split root experiment in a growth chamber were conducted. The results showed that a phosphorus (P)‐efficient wheat genotype, Yanzhong 144 (YZ), transpired 50% less than a P‐inefficient genotype, 80‐55. At the grain‐filling stage, the free water content in YZs rhizosphere was 2.4 times of that of 80‐55s. When either tricalcium phosphate [Ca3(PO4)2] or ferric phosphate (FePO4) were combined with the other nutrients in the same solution, total dry‐matter production was 140% greater for Ca3(PO4)2 and 60% greater for FePO4 than when these P sources were each supplied alone to one half of the root system while the other nutrients were supplied to the other half. The excess absorption of cations over anions by the roots facilitated mobilization of insoluble phosphates. The P‐efficient genotype was also water efficient, and the insoluble phosphates TCP and FP were phyto‐available in water culture.

Porous Materials to Support Bilayer Lipid Membranes for Ion Channel Biosensors

To identify materials suitable as membrane supports for ion channel biosensors, six filter materials of varying hydrophobicity, tortuosity, and thickness were examined for their ability to support bilayer lipid membranes as determined by electrical impedance spectroscopy. Bilayers supported by hydrophobic materials (PTFE, polycarbonate, nylon, and silanised silver) had optimal resistance (14–19 GΩ) and capacitance (0.8–1.6 μF) values whereas those with low hydrophobicity did not form BLMs (PVDF) or were short-lived (unsilanised silver). The ability of ion channels to function in BLMs was assessed using a method recently reported to improve the efficiency of proteoliposome incorporation into PTFE-supported bilayers. Voltage-gated sodium channel activation by veratridine and inhibition by saxitoxin showed activity for PTFE, nylon, and silanised silver, but not polycarbonate. Bilayers on thicker, more tortuous, and hydrophobic materials produced higher current levels. Bilayers that self-assembled on PTFE filters were the longest lived and produced the most channel activity using this method.

Efflux and Influx as Factors in the Relative Abilities of Ryegrass and White Clover to Compete for Phosphate

Influx and efflux of phosphate has been measured for roots of white clover and perennial ryegrass. Efflux was consistently and substantially lower in ryegrass. For plants deprived of phosphate for 5 days, the initial rates of influx were significantly higher for ryegrass. It is concluded that higher rates of efflux may be a significant factor in the poorer ability of white clover to compete for phosphate.

hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping

The purpose of this study was to obtain functional hERG ion channel protein for use in a non-cell-based ion channel assay. hERG was expressed in Sf9 insect cells. Attempts to solubilise the hERG protein from Sf9 insect cell membranes using non-ionic detergents (NP40 and DDM) were not successful. We therefore generated liposomes from the unpurified membrane fraction and incorporated these into porous Teflon-supported bilayer lipid membranes. Macroscopic potassium currents (1 nA) were recorded that approximated those in whole-cell patch-clamping, but the channels were bidirectional in the bilayer lipid membrane (BLM). Currents were partially inhibited by the hERG blockers E4031, verapamil, and clofilium, indicating that the protein of interest is present at high levels in the BLM relative to endogenous channels. Cell liposomes produced from Sf9 insect cell membranes expressing voltage-gated sodium channels also gave current responses that were activated by veratridine and inhibited by saxitoxin. These results demonstrate that purification of the ion channel of interest is not always necessary for liposomes used in macro-current BLM systems.

Exotically long and prolific root hairs of Arabidopsis, corn and wheat grown in a phosphate nutrient buffer system

Root hairs are model cells for biological researches and very significant for efficient utilization of water and fertilizer resources for crop production. Plants can basically not develop root hairs in classical hydroponics with high P concentration but without buffer ability. Corn is completely unable to form hairs in water. Here our adjustable controlled release system of phosphate in water was able to form a P nutrient buffer solution with low available P as soil solution. Our approach made both of monocotyledon and dicotyledon grow very exotically long and prolific root hairs, called “hedgehog roots”, which were as long as 3 mm in maximum length. This system is able to present a production platform for plant model cells. The environmental condition could control or regulate the expression of the gene(s) governing hairs.

Regulation of phosphate absorption by phosphorus nutritional status in Arabidopsis thaliana seedlings

Absorption of 32P by roots of intact plants grown on different levels of PO4 was measured by depletion curves. Growth on limiting PO4 increased the rate of absorption which was reflected in the values obtained for Km. Depriving high P status plants of PO4 for 5 days increased the rate of absorption by increasing Vmax. The % of 32P translocated to the shoots decreased as the P status increased but the plants deprived of PO4 for 5 days had values similar to the high P status plants. These results suggest there is more than one factor regulating absorption by roots and translocation to the shoot.