Structure of the L1 protuberance in the ribosome

The L1 protuberance of the 50S ribosomal subunit is implicated in the release/disposal of deacylated tRNA from the E site. The apparent mobility of this ribosomal region has thus far prevented an accurate determination of its three-dimensional structure within either the 50S subunit or the 70S ribosome. Here we report the crystal structure at 2.65 A resolution of ribosomal protein L1 from Sulfolobus acidocaldarius in complex with a specific 55-nucleotide fragment of 23S rRNA from Thermus thermophilus. This structure fills a major gap in current models of the 50S ribosomal subunit. The conformations of L1 and of the rRNA fragment differ dramatically from those within the crystallographic model of the T. thermophilus 70S ribosome. Incorporation of the L1-rRNA complex into the structural models of the T. thermophilus 70S ribosome and the Deinococcus radiodurans 50S subunit gives a reliable representation of most of the L1 protuberance within the ribosome.     

Structure of 2',5'-linked tetraribonucleotide loops: a novel RNA motif

We report on the three dimensional structure of an RNA hairpin containing a 2',5'-linked tetraribonucleotide loop, namely, 5'-rGGAC(UUCG)GUCC-3' (where UUCG = U(2'p5')U(2'p5')C(2'p5')G(2'p5')). We show that the 2',5'-linked RNA loop adopts a conformation that is quite different from that previously observed for the native 3',5'-linked RNA loop. The 2',5'-RNA loop is stabilized by (a) U:G wobble base pairing, with both bases in the anti conformation, (b) extensive base stacking, and (c) sugar-base contacts, all of which contribute to the extra stability of this hairpin structure.     

Enforced unbinding of biomembranes whose mutual adhesion is mediated by a specific interaction

The enforced unbinding of a biomembrane from a rigid substrate whose adhesion is mediated by a specific interaction has been studied theoretically. We argue that the unbinding takes place via motion of the adhesion rim. We account for the release of the elastic energy (stored in the membrane curvature) in the binding-unbinding process and obtain the dissociation constant at the rim. We further deduce an equation of motion for the rim. The solution exhibits an initial phase describing a slow motion followed by a regime of rapid motion and finally breaking of the adhesion. We show that the unbinding force depends on the rate of force application as F(*) approximately equal (F')(beta). When small forces are applied, beta=1/2, while in the case of large forces, beta=1/3.     

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     

Synthesis of the 15N-Gln11 labeled peptaibol antibiotic zervamicin-IIB

Summary Synthesis of zervamicin IIB, specifically labeled at the α-position of glutamine-11 with¹⁵N, was achieved by the Fmoc/tert.-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered α-aminoisobutyric acid residues, BOP/DMAP activation was applied. Peptide fragments were coupled by means of the coupling reagent, CF₃-PyBOP. Using the strategy developed, zervamicin IIB specifically¹⁵N labeled has been synthesized in 30% overall yield based on the isotopically labeled amino acid. From 600 MHz NMR spectroscopy the position of the¹⁵N-label was clearly detected. The isotope enrichment (98 ±2%) was determined by FAB-mass spectrometry.     

The Laptev Sea walrus Odobenus rosmarus laptevi: an enigma revisited

The walrus ( Odobenus rosmarus ) is in some current systematic schemes divided into three subspecies: O. r. rosmarus in the North Atlantic, O.   r. divergens in the North Pacific and O.   r. laptevi in the Laptev Sea. These three subspecies have been described as differing in body size, but the taxonomic status of O.   r. laptevi is disputed. The current study applies molecular and morphometric methods to assess the taxonomic status of O.   r. laptevi and to analyse the systematic and phylogeographic relationships between the three purported walrus subspecies. Tusk length and tusk circumference were measured from the few skulls available of O.   r. laptevi , and the obtained values were within the ranges reported for Pacific walruses. Thus, morphologically, subspecies status for O.   r. laptevi is not supported according to the Amadon–Mayr '75% rule'. Phylogenetic analyses and haplotype networks based on mitochondrial nucleotide sequence data of NADH dehydrogenase 1, 16S rRNA, cytochrome oxidase I and the d -loop of the control region of the historic O.   r. laptevi bone material and contemporary O.   r. rosmarus and O.   r. divergens showed that the Laptev Sea walrus groups with individuals from the North Pacific. Thus, the mitochondrial sequence data do not support the recognition of three walrus subspecies as reciprocally monophyletic evolutionary units with independent evolutionary histories. Only O.   r. rosmarus and O.   r. divergens meet this criterion with the present sampling. Accordingly, we recommend that Odobenus r. laptevi be abandoned and the Laptev walrus instead be recognized as the westernmost population of the Pacific walrus, Odobenus r. divergens. However, further research is recommended to assess whether the Laptev walrus could be considered as a significant unit in terms of conservation and management, since it is unique in several ecological parameters.     

A Humanized Leucine Zipper-TRAIL Hybrid Induces Apoptosis of Tumors both In Vitro and In Vivo

Evidence suggests that stimulating apoptosis in malignant cells without inflicting collateral damage to the host''s normal tissues is a promising cancer therapy. Chemo- and radiation therapies that, especially if combined, induce apoptosis in tumor cells have been used for treating cancer patients for decades. These treatments, however, are limited in their ability to discriminate between malignant and non-malignant cells and, therefore, produce substantial healthy tissue damage and subsequent toxic side-effects. In addition, as a result of these therapies, many tumor types acquire an apoptosis-resistant phenotype and become more aggressive and metastatic. Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been considered a promising and reliable selective inducer of apoptosis in cancerous cells. TRAIL, however, is not uniformly effective in cancer and multiple cancer cell types are considered resistant to natural TRAIL. To overcome this deficiency of TRAIL, we have earlier constructed a yeast-human hybrid leucine zipper-TRAIL in which the yeast GCN4-pII leucine zipper was fused to human TRAIL (GCN4-TRAIL). This construct exhibited a significantly improved anti-tumor apoptotic activity and safety, but is potentially immunogenic in humans. Here, we report a novel, potent, and fully human ATF7 leucine zipper-TRAIL (ATF7-TRAIL) fusion construct that is expected to have substantially lower immunogenicity. In solution, ATF7-TRAIL exists solely as a trimer with a Tm of 80°C and is active against cancer cells both in vitro and in vivo, in a mouse tumor xenograft model. Our data suggest that our re-engineered TRAIL is a promising candidate for further evaluation as an antitumor agent.     

Thiol Peroxidase Deficiency Leads to Increased Mutational Load and Decreased Fitness in Saccharomyces cerevisiae

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness.