The use of microelectrodes to investigate compartmentation and the transport of metabolized inorganic ions in plants

Microelectrode measurements can be used to investigate both the intracellular pools of ions and membrane transport processes of single living cells. Microelectrodes can report these processes in the surface layers of root and leaf cells of intact plants. By careful manipulation of the plant, a minimum of disruption is produced and therefore the information obtained from these measurements most probably represents the 'in vivo' situation. Microelectrodes can be used to assay for the activity of particular transport systems in the plasma membrane of cells. Compartmental concentrations of inorganic metabolite ions have been measured by several different methods and the results obtained for the cytosol are compared. Ion-selective microelectrodes have been used to measure the activities of ions in the apoplast, cytosol and vacuole of single cells. New sensors for these microelectrodes are being produced which offer lower detection limits and the opportunity to measure other previously unmeasured ions. Measurements can be used to determine the intracellular steady-state activities or report the response of cells to environmental changes.     

A novel GFP nude rat model to investigate tumor-stroma interactions

The anticancer effects of ceramide have been reported in many types of cancers but less in lung cancer. In this study, we used C₂-ceramide to further investigate its possible anticancer effects and mechanisms on non-small cell lung cancer (NSCLC) H1299 cells. The result of cell proliferation in terms of trypan blue assay showed high dose of C₂-ceramide inhibited cell survival after 24 h treatment. The flow cytometry-based assays indicated the effect of apoptosis, chromatin condensation, and G₁ arrest in terms of Annexin V/propidium iodide (PI), DAPI, and PI stainings, respectively. Moreover, the decreased protein level of p-Akt, p-NFκB, survivin and cyclin A2 were detected by Western blot assay. Taken together, these results indicated the antiproliferative effect of C₂-ceramide is majorly responsible for cell apoptosis in lung cancer H1299 cells.     

Ultrasound imaging of the lactating breast: methodology and application

Ultrasound imaging has been used extensively to detect abnormalities of the non-lactating breast. In contrast, the use of ultrasound for the investigation of pathology of the lactating breast is limited. Recent studies have re-examined the anatomy of the lactating breast highlighting features unique to this phase of breast development. These features should be taken into consideration along with knowledge of common lactation pathologies in order to make an accurate diagnosis when examining the lactating breast. Scanning techniques and ultrasound appearances of the normal lactating breast will be contrasted to those of the non-lactating breast. In addition ultrasound characteristics of common pathologies encountered during lactation will be described.     

Specialized cellular arrangements in legume leaves in relation to assimilate transport and compartmentation: comparison of the paraveinal mesophyll

Leaves of eight species of Leguminosae-Papilionoideae were examined for the presence of a highly specialized cell layer called the paraveinal mesophyli (PVM). Three species, Glycine max (L.) Merr, Psophocarpus tetragonolobus D.C. and Vigna radiata L., contained PVM; five (Medicago sativa L., Phaseolus vulgaris L., Pisum sativum L., Vicia faba L., Vigna unguiculata L.) did not. The PVM of G. max and P. tetragonolobus was anatomically identical and consisted of large, interconnected, multiarmed cells forming a network, one cell thick, spanning the region between vascular bundles and abutting the bundle sheath at the level of the phloem. The PVM of V. radiata differed in that elaborate extensions of individual bundle-sheath cells comprised the entire intervascular network. The PVM cells of all three species were large, contained a dense, thin peripheral layer of cytoplasm, and a large central vacuole. The cytoplasm contained few small chloroplasts and few microbodies, but was enriched in rough endoplasmic reticulum. Plasmodesmata were common in crosswalls between adjacent PVM cells and between PVM cells and other cell types abutting them. Vacuolar material was present in all three species, but was variable in appearance. That of G. max was present in large amounts, semifibrillar and finely dispersed. That of P. tetragonolobus was also present in large amounts but primarily as large aggregates, although some fibrillar material was also present. Vigna radiata had small amounts of vacuolar material evenly distributed between small aggregates and dispersed fibrils. Removal of flowers or young pods resulted in further increase of the vacuolar material in G. max PVM and increase of the fibrillar material in P. tetragonolobus, but had no appreciable affect on the vacuolar material in V. radiata. Histochemical staining indicated the vacuolar material in G. max and P. tetragonolobus was proteinaceous.Abbreviations PTA phosphotungstic acid - PVM paraveinal mesophyll Contribution No. 3215 from the Central Research and Development Department, E.I. du Pont de Nemours & Co     

Opium poppy and Madagascar periwinkle: model non-model systems to investigate alkaloid biosynthesis in plants

Alkaloids represent a large and diverse group of compounds that are related by the occurrence of a nitrogen atom within a heterocyclic backbone. Unlike other types of secondary metabolites, the various structural categories of alkaloids are unrelated in terms of biosynthesis and evolution. Although the biology of each group is unique, common patterns have become apparent. Opium poppy ( Papaver somniferum ), which produces several benzylisoquinoline alkaloids, and Madagascar periwinkle ( Catharanthus roseus ), which accumulates an array of monoterpenoid indole alkaloids, have emerged as the premier organisms used to study plant alkaloid metabolism. The status of these species as model systems results from decades of research on the chemistry, enzymology and molecular biology responsible for the biosynthesis of valuable pharmaceutical alkaloids. Opium poppy remains the only commercial source for morphine, codeine and semi-synthetic analgesics, such as oxycodone, derived from thebaine. Catharanthus roseus is the only source for the anti-cancer drugs vinblastine and vincristine. Impressive collections of cDNAs encoding biosynthetic enzymes and regulatory proteins involved in the formation of benzylisoquinoline and monoterpenoid indole alkaloids are now available, and the rate of gene discovery has accelerated with the application of genomics. Such tools have allowed the establishment of models that describe the complex cell biology of alkaloid metabolism in these important medicinal plants. A suite of biotechnological resources, including genetic transformation protocols, has allowed the application of metabolic engineering to modify the alkaloid content of these and related species. An overview of recent progress on benzylisoquinoline and monoterpenoid indole alkaloid biosynthesis in opium poppy and C. roseus is presented.     

Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs

The General Fluorescence Plant Meter (GFP-Meter) is a portable spectrofluorometer that utilizes a fiber-optic cable and a leaf clip to gather spectrofluorescence data. In contrast to traditional analytical systems, this instrument allows for the rapid detection and fluorescence measurement of proteins under field conditions with no damage to plant tissue. Here we discuss the methodology of gathering and standardizing spectrofluorescence data from tobacco and canola plants expressing GFP. Furthermore, we demonstrate the accuracy and effectiveness of the GFP-Meter. We first compared GFP fluorescence measurements taken by the GFP-Meter to those taken by a standard laboratory-based spectrofluorometer, the FluoroMax-2. Spectrofluorescence measurements were taken from the same location on intact leaves. When these measurements were tested by simple linear regression analysis, we found that there was a positive functional relationship between instruments. Finally, to exhibit that the GFP-Meter recorded accurate measurements over a span of time, we completed a time-course analysis of GFP fluorescence measurements. We found that only initial measurements were accurate; however, subsequent measurements could be used for qualitative purposes.     

Using metalloproteomics to investigate the cellular physiology of copper in hepatocytes

Proteomics is a systems biology approach for examining proteins and their function in a given specified system. Metalloproteomics narrows the focus of proteomics to those proteins which bind a metal or are metalloproteins. An important system where metalloproteomics can be applied is the hepatocyte, the liver's parenchymal cell engaged in protein synthesis, nutrient deployment, and drug biotransformation. Hepatocellular metalloproteomics is an exciting new scientific discipline which has already advanced our understanding of certain genetic and neoplastic liver disorders. It has the potential to elucidate the action of numerous metals in hepatocytes and generate new diagnostic parameters, namely, novel biomarkers.     

Ten steps to investigate a cellular system with mathematical modeling

Cellular and intracellular processes are inherently complex due to the large number of components and interactions, which are often nonlinear and occur at different spatiotemporal scales. Because of this complexity, mathematical modeling is increasingly used to simulate such systems and perform experiments in silico, many orders of magnitude faster than real experiments and often at a higher spatiotemporal resolution. In this article, we will focus on the generic modeling process and illustrate it with an example model of membrane lipid turnover.     

Sodium and potassium compartmentation and transport in the roots of intact lettuce plants

In this report, we consider the accumulation in roots, and transport to the shoot, of Na⁺ and K⁺ in intact lettuce plants (Lactuca sativa cv Black-seeded Simpson). Plants were grown in modified Hoagland medium supplemented with 10 moles NaCl per cubic meter. At this salinity, significant levels of Na⁺ were accumulated in roots and shoots, but there was no reduction in plant growth. Transport characteristics for both Na⁺ and K⁺ were qualitatively similar to those previously reported, for Spergularia marina, indicating that the results obtained with these experimental protocols are not limited to one unconventional experimental plant. The most pronounced difference in transport of the two ions was evident when transport was followed in a chase period after a 10 minute uptake pulse. For Na⁺, there was an initially rapid, but small, loss of label to the medium, and very little movement to the shoot. For K⁺, little label was lost from the plants, but translocation to the shoot proceeded for at least 60 minutes. The transport systems were further distinguished by treating the roots during labeling with 20 micrograms per milliliter cycloheximide. For K⁺, both uptake and translocation were reduced by about 50%. For Na⁺, root accumulation was stimulated more than five-fold, while transport to the shoot was reduced about 20%. Cycloheximide also modified the Na⁺ transport characteristics such that continued translocation occurred during the chase period of pulse-chase studies.     

Localization and compartmentation of Al in the leaves and roots of tea plants

Under acid soil conditions, solubility of aluminum (Al) increases leading to toxicity for plants. Al accumulator species such as tea, however, accumulate high levels of Al in tissues without toxicity symptoms. In this work, Al localization and compartmentation were studied in tea [Camellia sinensis (L.) O. Kuntze] grown hydroponically at 0 or 100 µM Al for eight weeks. Plant dry matter production was significantly higher in the presence of Al and accumulated up to 1.21 and 6.18 mg Al/g DW in the leaves and roots, respectively. About 40-50% of Al was partitioned into cell wall (CW)-bound fraction without any difference among leaves of different age and roots. A significant increase of the soluble phenolics fraction by Al was observed in both leaves and roots. Conventional and confocal laser scanning microscopy images of morin-stained roots indicated a high fluorescence signal in the caps and adjacent mersitematic cells. Towards basal parts, however, Al tended to accumulate mainly in the root hairs, rhizodermal and endodermal cell layers and slightly in the cortex while it was clearly excluded from the central cylinder. A high Al-morin signal was detected from the CW compared with other parts of the cells. Relatively high green fluorescence signal was emitted from the epidermal cell layer, trichomes, vascular bundle region and stomatal cells of particularly young leaves. Our study provides evidences for involvement of both avoidance and tolerance mechanisms for Al in tea plants.     

Proteases and cellular regulation in plants

Protein degradation is accomplished by a diverse collection of proteases. Recent studies have illustrated the importance of proteolysis in the control of many aspects of cellular regulation from photosynthesis to photomorphogenesis. In addition, new results point to a role for proteolysis in programmed cell death, circadian rhythm, and defense response in plants.     

A simple and sensitive high-throughput GFP screening in woody and herbaceous plants

Green fluorescent protein (GFP) has been used widely as a powerful bioluminescent reporter, but its visualization by existing methods in tissues or whole plants and its utilization for high-throughput screening remains challenging in many species. Here, we report a fluorescence image analyzer-based method for GFP detection and its utility for high-throughput screening of transformed plants. Of three detection methods tested, the Typhoon fluorescence scanner was able to detect GFP fluorescence in all Arabidopsis thaliana tissues and apple leaves, while regular fluorescence microscopy detected it only in Arabidopsis flowers and siliques but barely in the leaves of either Arabidopsis or apple. The hand-held UV illumination method failed in all tissues of both species. Additionally, the Typhoon imager was able to detect GFP fluorescence in both green and non-green tissues of Arabidopsis seedlings as well as in imbibed seeds, qualifying it as a high-throughput screening tool, which was further demonstrated by screening the seedlings of primary transformed T₀ seeds. Of the 30,000 germinating Arabidopsis seedlings screened, at least 69 GFP-positive lines were identified, accounting for an approximately 0.23% transformation efficiency. About 14,000 seedlings grown in 16 Petri plates could be screened within an hour, making the screening process significantly more efficient and robust than any other existing high-throughput screening method for transgenic plants.     

Cytokinesis in flowering plants: cellular process and developmental integration

In phragmoplast-assisted cytokinesis of somatic cells, vesicle fusion generates a cell plate that matures into a new cell wall and its flanking plasma membranes. Insight into this dynamic process has been gained in the past few years and additional molecular components of the basic machinery of cytokinesis have been identified. Specialized modes of cytokinesis occur in meiosis and gametophyte development, and recent studies indicate that they are genetically distinct from somatic cytokinesis.     

An in vitro approach to investigate medicinal chemical synthesis by three herbal plants

Ever since regulatory changes introduced herbals into mainstream supermarkets and pharmacies, there has been an explosion of demand for herbal plants and extracts which can be used to improve human health and well being. Science still lacks a basic mechanistic understanding of how environmental triggers regulate phytochemical accumulation, but this gap can be bridged using in vitro models to examine herbal species responses. For St. John's wort (Hypericum perforatum), uniform in vitro shoot cultures were set up as a parallel to a previously established sand culture system for investigation of physical and chemical environmental factors that control hypericin accumulation. Cytokinin supplementation of shoot culture medium resulted in a proliferation of abundant leaf glands with enhanced levels of hypericin, as compared to controls. Cell cultures of echinacea (Echinacea angustifolia) were established, and hydrophilic pharmacological components (caffeic acid derivatives) were detected. A protocol of rigorous explant pretreatment, and use of newly emerging vegetative shoots permitted establishment of axenic kava (Piper methysticum) callus, which was used to regenerate roots (organogenesis). Kavapyrone synthesis was achieved in both undifferentiated cell cultures and in cultured roots, although at lower levels than found in in vivo root systems. The predominance of kavain and methysticin in both forms of the in vitro cultures was parallel to the relative proportions from kava roots in vivo. The cell and organ cultures of all three herbal medicinals provide advantageous, easily-manipulated models to decipher environmental controls of phytochemical biosynthesis.     

Multi-scale imaging techniques to investigate solute transport across articular cartilage

As articular cartilage is an avascular tissue, the transport of nutrients and cytokines through the tissue is essential for the health of cells, i.e. chondrocytes. Transport of specific contrast agents through cartilage has been investigated to elucidate cartilage quality. In laboratory, pre-clinical and clinical studies, imaging techniques such as magnetic imaging resonance (MRI), computed tomography (CT) and fluorescent microscopy have been widely employed to visualize and quantify solute transport in cartilage. Many parameters related to the physico-chemical properties of the solute, such as molecular weight, net charge and chemical structure, have a profound effect on the transport characteristics. Information on the interplay of the solute parameters with the imaging-dependent parameters (e.g. resolution, scan and acquisition time) could assist in selecting the most optimal imaging systems and data analysis tools in a specific experimental set up. Here, we provide a comprehensive review of various imaging systems to investigate solute transport properties in articular cartilage, by discussing their potentials and limitations. The presented information can serve as a guideline for applications in cartilage imaging and therapeutics delivery and to improve understanding of the set-up of solute transport experiments in articular cartilage.