Samples were extracted in Erlenmeyer flasks with hexane-ethanolacetone (50:25:25). After extraction, 15 mL of water were added to separate the solution into polar and nonpolar layers. Lycopene was concentrated in the upper nonpolar phase while other components and polar pigments were contained in the lower polar phase. The lycopene phase was analyzed using isocratic HPLC with a C-18 column and methanol-THF-water (67:27:6) mobile phase. Lycopene and l3-carotene eluted as chromatographically pure peaks.

Early in mitochondria-mediated apoptosis, the mitochondrial outer membrane becomes permeable to proteins that, when released into the cytosol, initiate the execution phase of apoptosis. Proteins in the Bcl-2 family regulate this permeabilization, but the molecular composition of the mitochondrial outer membrane pore is under debate. We reported previously that at physiologically relevant levels, ceramides form stable channels in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis (Siskind, L. J., Kolesnick, R. N., and Colombini, M. (2006) Mitochondrion 6, 118 -125). Here we show that Bcl-2 proteins are not required for ceramide to form protein-permeable channels in mitochondrial outer membranes. However, both recombinant human Bcl-x L and CED-9, the Caenorhabditis elegans Bcl-2 homologue, disassemble ceramide channels in the mitochondrial outer membranes of isolated mitochondria from rat liver and yeast. Importantly, Bcl-x L and CED-9 disassemble ceramide channels in the defined system of solvent-free planar phospholipid membranes. Thus, ceramide channel disassembly likely results from direct interaction with these anti-apoptotic proteins. Mutants of Bcl-x L act on ceramide channels as expected from their ability to be anti-apoptotic. Thus, ceramide channels may be one mechanism for releasing pro-apoptotic proteins from mitochondria during the induction phase of apoptosis.Apoptosis is required for normal development and tissue homeostasis in multicellular organisms. Deregulation of apoptosis is fundamental to many diseases, such as cancer, stroke, heart disease, neurodegenerative disorders, autoimmune disorders, and viral diseases. During apoptosis, DNA fragments and other contents of the cell are packaged into apoptotic bodies that are consumed by phagocytosis. There are two main pathways for apoptosis, namely the extrinsic receptor-mediated pathway and an intrinsic mitochondria-mediated one. There is also cross-talk between these two pathways. The intrinsic pathway is initiated when one or more of a multitude of signals converge on mitochondria that ultimately result in an increase in the permeability of the mitochondrial outer membrane (MOM).2 This permeabilization leads to the release of intermembrane space proteins, including cytochrome c, procaspases, apoptosis-inducing factor, heat shock proteins, Smac/ Diablo, and endonuclease G (1). In the cytosol, these proteins activate caspases and DNases that carry out the execution phase of apoptosis.The mechanism for the increased permeability of the MOM during the induction phase of apoptosis is currently highly debated. Several mechanisms have been proposed to explain how mitochondrial intermembrane space proteins are released into the cytosol to facilitate apoptotic cell death. Some involve direct pore formation in the MOM. Several candidate pores exist, but most involve activated multidomain pro-apoptotic Bcl-2 family proteins, Bax and Bak (for example see Refs. 2-5). Another model for MOM permeabil...

Two strains of Dunaliella salina (Dunal) Teod., UTEX 1644 and UTEX 200, were cultured under different growth regimes, including 10 mM NO3− or NH4+, 1.5 or 3.0 M NaCl, and low (0.035%) or high (5%) CO2 in air. The release of 14C‐labeled dissolved organic carbon (DOC), expressed as a rate and as a percentage of photosynthetic 14CO2 assimilation, was subsequently determined. The percentage of DOC released was inversely related to cell density in the assay medium, but photosynthesis on a per‐cell basis was not. Release of DOC was low, in the range of 1–5% of photosynthesis, but during acclimation to growth on NH4+, it rose to 11%. The presence of NH4+ rather than NO3− in the growth medium increased the rate of release by both strains, but the percentage release was stimulated only in UTEX 200 cells, because their photosynthetic rate was depressed by NH4+. For UTEX 1644, high, as compared to low, CO2‐grown cells, had somewhat higher rates and percentages of DOC release, but release from UTEX 200 cells was unaffected by the growth‐CO2. The rate of DOC release by high CO2‐grown cells was not enhanced at a low concentration of dissolved inorganic carbon, indicating that the released material did not originate from the photorespiratory pathway. The effects of NaCl on DOC release varied with strain and growth conditions. For UTEX 200, the cells in NO3−, but not NH4+, exhibited a doubling or more in percentage of release with a doubling in NaCl concentration, irrespective of growth‐CO2. With UTEX 1644 the low CO2‐grown cells showed the greatest enhancement in 3.0 M NaCl. Organic matter accumulated on the external surface of the cell membrane and constituted a well‐defined cell‐coat, which was more dense in NH4+ than in NO3−‐grown cells. Microtubules, which may play a role in maintaining cell shape, were observed just below the plasma membrane. From a practical viewpoint, the presence of organic material in the hypersaline ponds of salt‐works is detrimental to salt production. When D. salina cells become abundant in such ponds, the attendant, continuous release of DOC may make a significant contribution to the problem.

No abstract