Agronomic and chemical performance of field‐grown tobacco engineered for triterpene and methylated triterpene metabolism

Squalene is a linear intermediate to nearly all classes of triterpenes and sterols and is itself highly valued for its use in wide range of industrial applications. Another unique linear triterpene is botryococcene and its methylated derivatives generated by the alga Botryococcus braunii race B, which are progenitors to fossil fuel deposits. Production of these linear triterpenes was previously engineered into transgenic tobacco by introducing the key steps of triterpene metabolism into the particular subcellular compartments. In this study, the agronomic characteristics (height, biomass accumulation, leaf area), the photosynthetic capacity (photosynthesis rate, conductance, internal CO₂ levels) and triterpene content of select lines grown under field conditions were evaluated for three consecutive growing seasons. We observed that transgenic lines targeting enzymes to the chloroplasts accumulated 50–150 times more squalene than the lines targeting the enzymes to the cytoplasm, without compromising growth or photosynthesis. We also found that the transgenic lines directing botryococcene metabolism to the chloroplast accumulated 10‐ to 33‐fold greater levels than the lines where the same enzymes were targeted to in the cytoplasm. However, growth of these high botryococcene accumulators was highly compromised, yet their photosynthesis rates remained unaffected. In addition, in the transgenic lines targeting a triterpene methyltransferase (TMT) to the chloroplasts of high squalene accumulators, 55%–65% of total squalene was methylated, whereas in the lines expressing a TMT in the cytoplasm, only 6%–13% of squalene was methylated. The growth of these methylated triterpene‐accumulating lines was more compromised than that of nonmethylated squalene lines.     

Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells.

BACKGROUND: We report on the potential DNA binding modes and spectral characteristics of the cell-permeant far red fluorescent DNA dye, DRAQ5, in solution and bound within intact cells. Our aim was to determine the constraints for its use in flow cytometry and bioimaging. METHODS: Solution characteristics and quantum yields were determined by spectroscopy. DRAQ5 binding to nuclear DNA was analyzed using fluorescence quenching of Hoechst 33342 dye, emission profiling by flow cytometry, and spectral confocal laser scanning microscopy of the complex DRAQ5 emission spectrum. Cell cycle profiling utilized an EGFP-cyclin B1 reporter as an independent marker of cell age. Molecular modeling was used to explore the modes of DNA binding. RESULTS: DRAQ5 showed a low quantum yield in solution and a spectral shift upon DNA binding, but no significant fluorescence enhancement. DRAQ5 caused a reduction in the fluorescence intensity of Hoechst 33342 in live cells prelabeled with the UV excitable dye, consistent with molecular modeling that suggests AT preference and an engagement of the minor groove. In vivo spectral analysis of DRAQ5 demonstrated shifts to longer wavelengths upon binding with DNA. Analysis of spectral windows of the dual emission peaks at 681 and 707 nm in cells showed that cell cycle compartment recognition was independent of the far red-near IR emission wavelengths monitored. CONCLUSIONS: The study provides new clues to modes of DNA binding of the modified anthraquinone molecule in vivo, and its AT base-pair selectivity. The combination of low quantum yield but high DNA affinity explains the favorable signal-to-noise profile of DRAQ5-nuclear fluorescence. The robust nature of cell cycle reporting using DRAQ5, even when restricted spectral windows are selected, facilitates the analysis of encroaching spectral emissions from other fluorescent reporters, including GFP-tagged proteins.     

Amiloride does not alter NaCl avoidance in Fischer-344 rats

Fischer-344 (F-344) rats differ from other common rat strains in that they fail to show any preference for NaCl at any concentration in two- bottle preference tests. Because 100 microM amiloride partially blocks the NaCl-evoked chorda tympani (CT) response in electrophysiological studies, we tested NaCl preference (0.068-0.273 M) in F-344 rats with and without 100 microM amiloride solution as the solvent. A third group was tested with unadulterated NaCl solutions following CT transection. Amiloride had no significant effect on the NaCl preference-aversion function, whereas CT transection significantly reduced NaCl avoidance. These results suggest that the amiloride-sensitive component of the NaCl response is not necessary for F-344 rats to display avoidance of NaCl, but the entire CT input is.      

Neural Organization of the Median Ocellus of the Dragonfly : II. Synaptic structure

Two types of presumed synaptic contacts have been recognized by electron microscopy in the synaptic plexus of the median ocellus of the dragonfly. The first type is characterized by an electron-opaque, button-like organelle in the presynaptic cytoplasm, surrounded by a cluster of synaptic vesicles. Two postsynaptic elements are associated with these junctions, which we have termed button synapses. The second synaptic type is characterized by a dense cluster of synaptic vesicles adjacent to the presumed presynaptic membrane. One postsynaptic element is observed at these junctions. The overwhelming majority of synapses seen in the plexus are button synapses. They are found most commonly in the receptor cell axons where they synaptically contact ocellar nerve dendrites and adjacent receptor cell axons. Button synapses are also seen in the ocellar nerve dendrites where they appear to make synapses back onto receptor axon terminals as well as onto adjacent ocellar nerve dendrites. Reciprocal and serial synaptic arrangements between receptor cell axon terminals, and between receptor cell axon terminals and ocellar nerve dendrites are occasionally seen. It is suggested that the lateral and feedback synapses in the median ocellus of the dragonfly play a role in enhancing transients in the postsynaptic responses.     

Conditions for activity of glutaminase in kidney mitochondria

1. Rat kidney mitochondria oxidize glutamate very slowly. Addition of glutamine stimulates this respiration two- to three-fold. Addition of glutamate also stimulates respiration in the presence of glutamine. 2. By measuring mitochondrial swelling in iso-osmotic solutions of glutamine or of ammonium glutamate it was shown that glutamine penetrates the mitochondrial membrane rapidly whereas ammonium glutamate penetrates very slowly. 3. Experiments in which reduction of NAD(P)⁺ was measured in preparations of intact and broken mitochondria indicated that glutamate dehydrogenase shows the phenomenon of `latency'. On the addition of glutamine rapid reduction of nicotinamide nucleotides in intact mitochondria was obtained. 4. During the action of glutaminase there is an accumulation of glutamate inside the mitochondria. 5. When the mitochondria were suspended in a medium containing glutamine, P_i and rotenone the rate of production of ammonia was stimulated by the addition of a substrate, e.g. succinate. Addition of an uncoupler or antimycin A abolished this stimulation. 6. The effects of succinate and uncoupler were especially pronounced in the presence of glutamate, which is an inhibitor of glutaminase activity by competition with P_i. 7. Determination of the enzyme activity in media at different pH values showed that the optimum pH for glutaminase activity in the preparation of broken mitochondria was 8, whereas for intact mitochondria it was dependent on the energy state. In the presence of succinate as an energy source it was pH 8.5, but in the presence of uncoupler or antimycin A it was 9. This displacement of the pH optimum to a higher value was especially pronounced in the presence of both glutamate and uncoupler. 8. If nigericin was present in potassium chloride medium the pH optimum for enzyme activity in intact non-respiring mitochondria was nearly the same as in the preparation of broken mitochondria; however, its presence in K⁺-free medium displaced the pH optimum for glutaminase activity to a very high value. 9. It is postulated that because of low permeability of the kidney mitochondrial membrane to glutamate the latter accumulates inside the mitochondria, and that this leads to the inhibition of the enzyme by competition with P_i and also by lowering the pH of the intramitochondrial space. With succinate as substrate for respiration there is an outward translocation of H⁺ ions, which together with accumulation of P_i increases glutaminase activity. Translocation of K⁺ ions inward increases the enzyme activity, perhaps by increasing the pH of the internal spaces and causing an accumulation of P_i. 10. The importance of the location of the enzyme in the mitochondria in relation to its biological function and conditions for activity is discussed.     

PERMEABILITY OF MITOCHONDRIA FROM RAT BRAIN AND RAT LIVER TO GABA

Abstract— For the GABA shunt to operate in rivo , GABA must be able to enter brain mitochondria. GABA causes reduction of intra-mitochondrial NAD⁺; glutamate or 2-oxoglutarate stimulate this reduction at concentrations at which they do not themselves cause reduction. This stimulation is not abolished by Triton X-100. The rates of swelling of brain and liver mitochondria are similar in iso-osmotic GABA and in several analogues. The rate of swelling is proportional to the concentration of GABA in the iso-osmotic suspension medium. GABA penetrates 60% of the mitochondrial matrix volume, this value is unaffected by energizing the mitochondria. The activity of GABA-oxoglutarate aminotransferase is not latent. We conclude that GABA diffuses into both brain and liver mitochondria as a species with no net charge at rates which are able to sustain maximum activity of the GABA shunt.     

Biogenesis of mitochondria. The effects of altered membrane lipid composition on cation transport by mitochondria of Saccharomyces cerevisiae

1. The fatty acid composition of the membrane lipids of a fatty acid desaturase mutant of Saccharomyces cerevisiae was manipulated by growing the organism in a medium containing defined fatty acid supplements. 2. Mitochondria were obtained whose fatty acids contain between 20% and 80% unsaturated fatty acids. 3. Mitochondria with high proportions of unsaturated fatty acids in their lipids have coupled oxidative phosphorylation with normal P/O ratios, accumulate K(+) ions in the presence of valinomycin and an energy source, and eject protons in an energy-dependent fashion. 4. If the unsaturated fatty acid content of the mitochondrial fatty acids is lowered to 20%, the mitochondria simultaneously lose active cation transport and the ability to couple phosphorylation to respiration. 5. The loss of energy-linked reactions is accompanied by an increased passive permeability of the mitochondria to protons. 6. Free fatty acids uncouple oxidative phosphorylation in yeast mitochondria and the effect is reversed by bovine serum albumin. 7. The free fatty acid contents of yeast mitochondria are unaffected by depletion of unsaturated fatty acids, and free fatty acids are not responsible for the uncoupling of oxidative phosphorylation in organelles depleted in unsaturated fatty acids. 8. It is suggested that the loss of energy-linked reactions in yeast mitochondria that are depleted in unsaturated fatty acids is a consequence of the increased passive permeability to protons, and is caused by a change in the physical properties of the lipid phase of the inner mitochondrial membrane.     

Individual differences in children's innovative problem-solving are not predicted by divergent thinking or executive functions

Recent studies of children''s tool innovation have revealed that there is variation in children''s success in middle-childhood. In two individual differences studies, we sought to identify personal characteristics that might predict success on an innovation task. In Study 1, we found that although measures of divergent thinking were related to each other they did not predict innovation success. In Study 2, we measured executive functioning including: inhibition, working memory, attentional flexibility and ill-structured problem-solving. None of these measures predicted innovation, but, innovation was predicted by children''s performance on a receptive vocabulary scale that may function as a proxy for general intelligence. We did not find evidence that children''s innovation was predicted by specific personal characteristics.