Mathematical modeling of myoglobin facilitated transport of oxygen in devices containing myoglobin-expressing cells

Low pO(2) is perhaps the most significant factor in artificial pancreas failure. In these environments, not only is the beta cell production of insulin reduced, but the cell death rate is also significantly higher. Mathematical models are developed to test the feasibility of facilitated oxygen transport in enhancing O(2) flux to genetically engineered cells in a bioartificial device such as a pancreas. For this device, it is proposed that beta cells be genetically engineered to express myoglobin throughout the cell. In addition, the significance of including myoglobin throughout the alginate matrix present to provide immuno-protection for the transplanted cells is considered. The mathematical analysis predicts that myoglobin facilitated oxygen transport has the potential of increasing the oxygen concentration at the centre of a cluster of cells (islet) with an effective radius of 100 microm by 50%. These theoretical models for myoglobin facilitated oxygen transport with homogeneous Michaelis-Menten consumption also indicate that including myoglobin in the alginate gel would beneficially improve the flux of oxygen to the transplanted cells.     

Effects of elevated ultraviolet-B radiation on native and cultivated plants of southern Africa

Seventeen herb, shrub and tree species of commercial and ecological importance in southern Africa were exposed at one location to ultraviolet-B (UV-B, 280-315 nm) radiation approx. 35 % above clear-sky background (control). The aims were to assess how UV-B affects canopy area, dry mass, and some biochemical and morphological properties of leaves, and to investigate whether differences between species are related to growth form of the plants. There was no pattern of response to UV-B related to growth form. Leaves of trees had altered chlorophyll a and b, carotenoid and flavonoid concentrations, but those of shrubs or herbs did not. Non-structural carbohydrates were unaffected. Smaller canopy areas and dry masses were observed under enhanced UV-B, but these were not statistically different among growth forms. There was a general insensitivity of species to elevated UV-B. Only five species had significantly altered leaf biochemical and morphological properties, canopy area and dry mass, the changes differing in magnitude. There was no consistent pattern of change in leaf thickness or biochemical composition with increased UV-B. Correlation analyses did not support the view that growth is less negatively affected in species with thick leaves or in those where leaf thickness increases, or in species with naturally high leaf flavonoid contents or that are able to synthesize additional flavonoids in response to UV-B enhancement. The analyses did not support the hypothesis that growth was inhibited by starch accumulation in leaves under elevated UV-B. However, changes in leaf shape did correlate with canopy area and dry mass, showing the importance of photomorphogenetic changes caused by UV-B which affect species' performance. We conclude that generalizations on plant sensitivity to UV-B based on growth form and functional type could be misleading, and that the great majority of economically important species of the region are likely to be insensitive to future UV-B increases. Notable exceptions include the Colophospermum mopane tree ecotypes chota and leslie and the arable annual Vigna unguiculata, both of which are traditional sources of livelihood to rural African populations and of importance to African industry and agriculture.     

Relationship between type and age of the inoculum cultures and betalains biosynthesis by Beta vulgaris hairy root culture

From a study of the relationship between the type and age of the inocula, and the growth and biosynthesis of betalains in a Beta vulgaris hairy root culture, the best results were achieved with a 14 d inoculum grown in submerged culture giving 42 mg betalains (16 mg betacyanins and 26 betaxanthins) and 1.5 g dry biomass in 40 ml medium.     

Identification of the hydroxyl radical and other reactive oxygen species in human neutrophil granulocytes exposed to a fragment of the amyloid beta peptide

A fragment of the amyloid beta protein, βA(25-35), was investigated for its effect on production of reactive oxygen species (ROS) in human neutrophil granulocytes. The formation and identification of ROS were examined by using a 2',7'-dichlorofluorescin (DCF) fluorescence assay, a luminol chemiluminescence assay, electron paramagnetic resonance (EPR) spectroscopy with DEPMPO as a spin trap, and hydroxylation of 4-hydroxybenzoate (4-HBA). The DCF assay showed that βA(25-35) stimulated formation of ROS in a concentration and time dependent manner. The inverted peptide, βA(35-25), gave no response. Also, luminol-amplified chemiluminescence was stimulated by βA(25-35). Incubation with diethyldithiocarbamate (a superoxide dimustase inhibitor) and salicylhydroxamate (SHA; a myeloperoxidase inhibitor) reduced the chemiluminescence. This indicates that hypochlorous acid (HOCl) is formed after exposure to βA(25-35). The EPR spectra indicated a concentration dependent formation of superoxide ( O 2 • - ) - and hydroxyl ( •OH)- radicals. Hydroxylation of 4-HBA to 3,4,-dihydroxybenzoate confirmed production of •OH. This response was attenuated by SHA, indicating involvement of HOCl in formation of •OH. The DCF fluorescence was inhibited with U0126 (an extracellular signal regulated protein kinase (ERK) inhibitor). Further analysis with western blot confirmed phosphorylation of ERK1/2 after exposure to βA(25-35). The phospholipase A 2 (PLA 2 ) inhibitor 7,7-dimethyl-(5Z,8Z)-eicosadienoic acid, and diphenyleneiodonium, which inhibits the NADPH oxidase, also led to a reduction of the DCF fluorescence. The present findings indicate that βA(25-35) stimulates the NADPH oxidase by activating the ERK pathway and PLA 2 . Production of O 2 • - can lead to HOCl and further formation of •OH, which both have a cytotoxic potential.     

Plant genome archaeology: evidence for conserved ancestral chromosome segments in dicotyledonous plant species

We have developed genetic maps, based on expressed sequence tags (ESTs) that are homologous to Arabidopsis genes, in four dicotyledonous crop plant species from different families. A comparison of these maps with the physical map of Arabidopsis reveals common genome segments that appear to have been conserved throughout the evolution of the dicots. In the four crop species analysed these segments comprise between 16 and 33% of the Arabidopsis genome. Our findings extend the synteny patterns previously observed only within plant families, and indicate that structural and functional information from the model species will be, at least in part, applicable in crop plants with large genomes.     

Multicolour Fluorescence Imaging of Sugar Beet Leaves with Different Nitrogen Status by Flash Lamp UV-Excitation

Fluorescence images of leaves of sugar beet plants (Beta vulgaris L. cv. Patricia) grown on an experimental field with different fertilisation doses of nitrogen [0, 3, 6, 9, 12, 15 g(N) m^–2] were taken, applying a new multicolour flash-lamp fluorescence imaging system (FL-FIS). Fluorescence was excited by the UV-range (280–400 nm, _max = 340 nm) of a pulsed Xenon lamp. The images were acquired successively in the four fluorescence bands of leaves near 440, 520, 690, and 740 nm (F₄₄₀, F₅₂₀, F₆₉₀, F₇₄₀) by means of a CCD-camera. Parallel measurements were performed to characterise the physiological state of the leaves (nitrogen content, invert-sugars, chlorophylls and carotenoids as well as chlorophyll fluorescence induction kinetics and beet yield). The fluorescence images indicated a differential local patchiness across the leaf blade for the four fluorescence bands. The blue (F₄₄₀) and green fluorescence (F₅₂₀) were high in the leaf veins, whereas the red (F₆₉₀) and far-red (F₇₄₀) chlorophyll (Chl) fluorescences were more pronounced in the intercostal leaf areas. Sugar beet plants with high N supply could be distinguished from beet plants with low N supply by lower values of F₄₄₀/F₆₉₀ and F₄₄₀/F₇₄₀. Both the blue-green fluorescence and the Chl fluorescence rose at a higher N application. This increase was more pronounced for the Chl fluorescence than for the blue-green one. The results demonstrate that fluorescence ratio imaging of leaves can be applied for a non-destructive monitoring of differences in nitrogen supply. The FL-FIS is a valuable diagnostic tool for screening site-specific differences in N-availability which is required for precision farming.     

Monitoring gene flow from transgenic sugar beet using cytoplasmic male-sterile bait plants

One of the most discussed environmental effects associated with the use of transgenic plants is the flow of genes to plants in the environment. The flow of genes may occur through pollen since it is the reproductive system that is designed for gene movement. Pollen-mediated gene escape is hard to control in mating plants. Pollen from a wind pollinator can move over distances of more than 1000 m. To investigate the efficiency of transgenic pollen movement under realistic environmental conditions, the use of bait plants might be an effective tool. In this study, cytoplasmic male-sterile (CMS) sugar beets were tested with regard to their potential for monitoring transgene flow. As the pollen source, transgenic sugar beets were used that express recombinant DNA encoding viral (beet necrotic yellow vein virus) resistance, and antibiotic (kanamycin) and herbicide (glufosinate) tolerance genes. In a field trial, the effectiveness of a hemp (Cannabis sativa) stripe containment strategy was tested by measuring the frequency of pollinated CMS bait plants placed at different distances and directions from a transgenic pollen source. The results demonstrated the ineffectiveness of the containment strategy. Physiological and molecular tests confirmed the escape and production of transgenic offspring more than 200 m behind the hemp containment. Since absolute containment is unlikely to be effective, the CMS-bait plant detection system is a useful tool for other monitoring purposes.     

ATP-induced sucrose efflux from red-beet tonoplast vesicles

 Sucrose efflux from the vacuole of mobilizing red-beet (Beta vulgaris L.) hypocotyl cells was investigated using purified tonoplast vesicles. Tonoplast vesicle purity was assured by the immunoreactivity to antibodies raised against the vacuolar ATPase and by the strong inhibition exhibited by the H⁺-ATPase to bafilomycin-A and NO₃ ⁻. Inhibition of the H⁺-ATPase by vanadate and azide was negligible. Sucrose was loaded into tonoplast vesicles by using the pH-jump method of energization. Addition of ATP to sucrose-loaded vesicles in the presence of bafilomycin-A resulted in efflux of a significant amount of sucrose. During ATP-induced sucrose efflux, bafilomycin-insensitive ATPase activity increased significantly with no increase in H⁺-translocating activity. The additional bafilomycin-A insensitive ATPase activity observed in sucrose-loaded vesicles was completely inhibited by vanadate as was the efflux of sucrose. Similar to vanadate, thapsigargin was also inhibitory to sucrose efflux and to the bafilomycin-A insensitive ATPase activity. The data indicate that vacuolar sucrose can be actively mobilized by a specific ATP-dependent efflux mechanism. Received: 12 October 1999 / Accepted: 18 November 1999     

Involvement of 14-3-3 proteins in the osmotic regulation of H+-ATPase in plant plasma membranes

 Taking the binding of fusicoccin to plasma membranes as an indicator of complex formation between the 14-3-3 dimer and H⁺-ATPase, we assessed the effect of osmotic stress on the interaction of these proteins in suspension-cultured cells of sugar beet (Beta vulgaris L.). An increase in osmolarity of the cell incubation medium, accompanied by a decrease in turgor, was found to activate the H⁺ efflux 5-fold. The same increment was observed in the number of high-affinity fusicoccin-binding sites in isolated plasma membranes; the 14-3-3 content in the membranes increased 2- to 3-fold, while the H⁺-ATPase activity changed only slightly. The data obtained indicate that osmotic regulation of H⁺-ATPase in the plant plasma membrane is achieved via modulation of the coupling between H⁺ transport and ATP hydrolysis, and that such regulation involves 14-3-3 proteins. Received: 10 February 2000 / Accepted: 31 March 2000