In chloroplasts, carotenoids are essential pigments involved in photosynthesis. During-photomorphogenesis, a coordinated increase in the amounts of chlorophylls and carotenoids, in conjugation with other components, leads to the formation of a functional photosynthetic apparatus. To investigate the regulation of carotenoid biosynthesis during this process at the molecular level, GGPS, PSY and PDS cDNAs have been cloned from white mustard (Sinapis alba L). GGPS encodes a key enzyme in plastid isoprenoid metabolism, while the products of PSY and PDS catalyse the subsequent steps in carotenoid biosynthesis. Due to the low mRNA levels of the genes involved, the use of a RT-PCR protocol was necessary to measure gene expression during photomorphogenesis. With light, there is an up-regulation of PSY expression, the first gene within the carotenoid biosynthetic pathway, while PDS and GGPS expression levels remain constant. Treatment with different light qualities reveals a phytochrome-mediated regulation of PSY expression in developing white mustard seedlings. To obtain more detailed information on the light-regulation, Arabidopsis thaliana wild-type and phytochrome mutants were utilized. Continuous far-red and red light both increase the expression of PSY in wild-type seedlings, demonstrating that both light-labile and light-stable phytochromes are involved in PSY regulation. The response to far-red light is completely abolished in the phyA mutant, showing that PHYA mediates the increase in PSY transcript levels under these light conditions. In the phyB mutant, the red light response is normal, indicating that PSY expression is not controlled by PHYB but by other light-stable phytochromes. Measurement of chlorophylls and carotenoids under the same light regimes shows that the up-regulation of PSY expression does not necessarily result in an increase of the carotenoid content. Only those light conditions which allow chlorophyll biosynthesis lead to a significant increase of the carotenoid content. Therefore, it is proposed that up-regulation of PSY mRNA levels leads to an increased capacity for the formation of carotenoids. However, this only takes place under light conditions leading to protochlorophyllide photoconversion.

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The precursor proteins of the carotenogenic enzymes geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase and lycopene cyclase were imported into isolated pea chloroplasts. Geranylgeranyl diphosphate synthase remained soluble in the stroma in a free form and phytoene synthase associated to thylakoid membranes upon import, both as expected. Surprisingly, phytoene desaturase and lycopene cyclase, which strongly depend on membrane association for enzymatic activity, also remained soluble in the chloroplast stroma. The soluble forms of these enzymes were, however, still competent for membrane-association, e.g. with protein-free liposomal membranes. Indeed the soluble forms of phytoene synthase, phytoene desaturase and lycopene cyclase occurred as ATP-and cold-sensitive high-molecularmass complexes. Gel-filtration experiments and blue native-PAGE plus autoradiography and western blot analysis indicated a participation of the chloroplast 60-kDa chaperonin (Cpn60) in the soluble highmolecular-mass complexes of imported carotenogenic enzymes. Finally, it was inferred that a membranebound regulatory factor plays a decisive role in membrane-binding.Keywords: carotenoid; chloroplast import; membrane binding; chaperone ; daffodil.In plants, carotenoids are synthesized in plastids by nuclearencoded enzymes. Those participating in the formation of isopentenyl diphosphate are still obscure and presumably follow an alternative reaction sequence (Liitke-Brinkhaus and Kleinig, 1987;Schwender et al., 1996), different from the so-called mevalonate pathway operating in the cytoplasm and transiently in immature chloroplasts (Heintze et a]., 1994). The five subsequent enzymes, catalyzing the reactions from isopentenyl diphosphate to p-carotene, have been molecularly identified by several groups (for review, see Bartley and Scolnik, 1995). Of these, geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase and lycopene cyclase were investigated here. Only some of the plant enzymes functioning later in the pathway forming xanthophylls have been identified, such as zeaxanthine epoxidase (Marin et al., 1996) and capsanthin capsorubin synthase (Bouvier et al., 1994).Carotenoid biosynthesis is not an independent pathway, having instead numerous off-branching prenyllipid-forming activities competing for intermediates. At the stage of geranyl diphosphate, monoterpenes are formed (Mettal et al., 1988) for the geranylgeranyl diphosphate substrate. Many of these prenyllipid products are vitally important, so the metabolite flow must be reliably regulated. For example, carotenoids and chlorophylls must be synthesized in a quantitatively and qualitatively coordinated manner to form and maintain a functional photosynthetic apparatus. Constitutive overexpression of phytoene synthase leads to withdrawal of geranylgeranyl diphosphate from gibberellin biosynthesis and thus to deleterious effects on plant growth (Fray et al., 1995), and interference with quinone biosynthesis results in bleaching effects due to inh...

Muscle atrophy as detected by the rate of change in RF-CSA moderately correlated with strength one week after sepsis admission.

Premature termination codons (PTCs) in cystic fibrosis transmembrane conductance regulator (CFTR) gene result in nonfunctional CFTR protein and are the proximate cause of ~11% of CF causing alleles. Aminoglycosides and other novel agents are known to induce translational readthrough of PTCs, a potential therapeutic approach. Among PTCs, W1282X CFTR is unique, as it is a C-terminal CFTR mutation that can exhibit partial activity, even in the truncated state. The potentiator ivacaftor (VX-770) is approved for treating CF patients with G551D and other gating mutations. Based on previous studies demonstrating beneficial effect of ivacaftor for PTC mutations following readthrough in vitro, we hypothesized that ivacaftor may enhance CFTR activity in CF patients expressing W1282X CFTR, and could be further enhanced by readthrough. Ivacaftor significantly increased CFTR activity in W1282X expressing cells compared to R1162X CFTR cells, and was further enhanced by readthrough with the aminoglycoside G418. Primary nasal epithelial cells from a W1282X homozygous patient showed improved CFTR function in the presence of ivacaftor. Upon ivacaftor administration to the same patient, there was significant improvement in pulmonary exacerbation frequency, BMI and, insulin requirement, whereas FEV1 remained stable over 3 years. These studies suggest that ivacaftor may have moderate clinical benefit in patients with preserved expression of the W1282X CFTR mutation by stimulating residual activity of the truncated protein, suggesting the need for further studies including the addition of efficacious readthrough agents.