Dual Binding Mode of the Nascent Polypeptide-associated Complex Reveals a Novel Universal Adapter Site on the Ribosome

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of βNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.     

Regulation of the human leukocyte 5-lipoxygenase: stimulation by micromolar Ca2+ levels and phosphatidylcholine vesicles

Human leukocyte 5-lipoxygenase (EC 1.13.11.12) is unique among the human lipoxygenase not only in its requirement for free ionized calcium, but also in its regulation by a membrane-associated stimulatory factor, the 100,000 x g pellet. In the present study, phosphatidylcholine (PC) vesicles, in the absence of 100,000 x g pellet, exhibited a dose-dependent stimulatory activity on the 5-lipoxygenase, which was at least as effective as the 100,000 x g pellet. Furthermore, the enzyme was activated by isolated human neutrophil plasma membranes and to a lesser degree by endoplasmic reticulum. The chemoattractant peptide fMet-Leu-Phe (0.1 microM), GTP (10 microM), toxin from bacterium Bordetella pertussis (islet activating protein, 5 micrograms/ml) and their various combinations were unable to modulate the enzymatic activity of the 5-lipoxygenase. Stimulation of the 5-lipoxygenase by relatively low levels of free ionized calcium was observed both in the presence of the pellet and PC vesicles: maximal stimulation was seen at about 10 microM Ca2+. The human leukocyte leukotriene A4 synthase activity also exhibited a similar requirement for free calcium ions. The present study indicates that the membrane-associated stimulatory factor of the human leukocyte 5-lipoxygenase may be replaced by PC vesicles. Moreover, the 5-lipoxygenase and leukotriene A4 synthase activities require significantly lower Ca2+ levels for maximal activation than has been reported previously.     

Role of endothelial cells and their products in the modification of low-density lipoproteins

A majority of the LDL preparations from various donors could be modified by incubation with endothelial cells from human arteries, veins and microvessels. These alterations comprise changes in electrophoretic mobility, buoyant density and lipid composition of LDL, the generation of thiobarbituric acid reactive substances in the medium, and a decrease in primary amino groups of LDL. Furthermore, the association of endothelial cell proteins with LDL was demonstrated by [35S]methionine incorporation and trichloroacetic acid precipitation of reisolated endothelial cell-modified LDL. After SDS-polyacrylamide gel electrophoresis of the reisolated modified LDL particles, radioactivity was mainly found at a molecular mass of 48 kDa and at one or two bands with a molecular mass of more than 100 kDa. The 48 kDa protein was identified as a latent plasminogen activator inhibitor. Cell viability was necessary for the cell-mediated LDL modification, which indicates that endothelial cells are actively involved in this process. The Ca2+ ionophore A23187 and monensin did not influence LDL modification. LDL modification was markedly inhibited by antioxidants. It was not prevented by cyclooxygenase and lipoxygenase inhibitors, which indicates that non-enzymatic lipid peroxidation is involved. Transition metal- (copper-) induced lipid peroxidation results in similar physiochemical alterations of the LDL particle as found with endothelial cells; it is prevented by the presence of superoxide dismutase. In contrast, endothelial cell LDL modification was not influenced by superoxide dismutase. Catalase or singlet oxygen and hydroxyl radical scavengers also did not affect it. We suggest that yet unidentified radicals or lipid peroxides are generated in the cells or on the cell membrane and that these reactive molecule(s) will react with LDL after leaving the cell. HDL and lipoprotein-depleted serum prevented LDL modification markedly, and to a larger extent than that by copper ions. We speculate that LDL modification by endothelial cells will only occur under those conditions in which the balance between the generation of reactive oxygen molecules and the cellular protection against these reactive species is disturbed.     

Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments

Indole-3-methanol is a product of indole-3-acetic acid metabolism in wheat leaves ( Triticum compactum Host., cv. Little Club). It leads either to the production of the corresponding aldehyde and carboxylic acid, to the production of a polar glucoside which releases indole-3-methanol on β-glucosidase treatment, or to an unidentified apolar product on mild alkaline hydrolysis in aqueous methanol. With reference to a published pathway of indole-3-acetic acid degradation, the results provide evidence for a prominent role of indole-3-methanol and also for the occurrence of co-oxidation processes in wheat leaves involving indole-3-acetic acid and phenolic cosubstrates.     

Effect of the Specific Toxin in Helminthosporium victoriae on Host Cell Membranes

Helminthosporium victoriae toxin, which affects only hosts of the toxin-producing fungus, causes loss of electrolytes from roots, leaves, and coleoptiles of treated plants. Root hair cells lost the ability to plasmolyze after 20 minutes exposure to toxin in solution; comparable resistant cells retained plasmolytic ability during 3 hours exposure. Toxin stopped uptake of exogenous amino acids and Pi by susceptible but not by resistant tissue. Incorporation of ³²P into organic-P and ¹⁴C-amino acids into protein was blocked in susceptible but not in resistant tissue. Apparent free space increased in susceptible but not in resistant roots. The increase was evident within 30 minutes, and reached 80% free space after 2 hours exposure to toxin. When cell wall-free protoplasts were exposed to 0.16 μg toxin/ml, protoplasmic streaming stopped and all plasma membranes of susceptible protoplasts broke within 1 hour. Resistant protoplasts were not affected significantly. Data support the hypothesis of a primary lesion of toxin in the plasma membrane. Effects on synthesis could result from lack of transport of exogenous solutes to sites of synthesis. It is possible that all other observed effects of toxin are secondary to membrane damage.     

Determination of Root Exudates in a Steril Continuous Flow Culture. II. Short-Term and Long-Term Variations of Exudation Intensity

The exudate production of alfalfa under the conditions of the sterile flow culture was quantitatively measured. In the first 40 days 3.10⁻³ μmoles amino-N, 2.5 μequivalents of organic acids and approximately 10⁻⁴ μmoles of reducing sugars were liberated per plant and per day into the percolating nutrient solution. The amino acid concentration in the outflow varies according to a daily periodicity. The exudation of a colored substance also shows daily periodical variations. This pattern is different from the pattern of the amino acid exudation, however, and directly coupled to shoot illumination. Short-term 2,4-dinitrophenol additions to the nutrient lower the liberation of amino acids into the percolating solution.     

Biotransformation of monoterpenes and sesquiterpenes by cell suspension cultures of Achillea millefolium L. ssp. millefolium

The transformation capacity of Achillea millefolium L. ssp. millefolium (yarrow) cell suspension cultures was investigated using geraniol (50mg/l) and borneol, menthol, thymol and farnesols (25mg/l) as substrates. Apart from converting these substrates into several biotransformation products, the cell suspension cultures were also able to glycosylate both the substrates and the biotransformation products. aa]Key Words bb]Achillea millefolium L. ssp. millefolium bb]Yarrow bb]Compositae bb]Biotransformation bb]Glycosylation bb]Geraniol bb]Borneol bb]Menthol bb]Thymol bb]Farnesols