A bipartite sequence motif induces translation reinitiation in feline calicivirus RNA

The mechanism leading to reinitiation of translation after termination of protein synthesis in eukaryotes has not yet been resolved in detail. One open question concerns the way the post-termination ribosome is tethered to the mRNA to allow binding of the necessary initiation factors. In caliciviruses, a family of positive strand RNA viruses, the capsid protein VP2 is translated via a termination/reinitiation process. VP2 of the feline calicivirus is encoded in the 3'-terminal open reading frame 3 (ORF3) that overlaps with the preceding ORF2 by four nucleotides. In transient expression studies, the efficiency of VP2 expression was 20 times lower than that of the ORF2 proteins. The close vicinity of the ORF2 termination signal and the ORF3 AUG codon was crucial, whereas the AUG could be replaced by alternative codons. Deletion mapping revealed that the 3'-terminal 69 nucleotides of ORF2 are crucial for VP2 expression. This sequence contains two essential sequence motifs. The first motif is conserved among caliciviruses and complementary to part of the 18 S rRNA. In conclusion, VP2 is expressed in a translation termination/reinitiation process that is special because it requires a sequence element that could prevent dissociation of post-termination ribosomes via hybridization with 18 S rRNA.     

Thrombin inhibitors with novel P1 binding pocket functionality: free energy of binding analysis

The high incidence of thrombembolic diseases justifies the development of new antithrombotics. The search for a direct inhibitor has resulted in the synthesis of a considerable number of low molecular weight molecules that inhibit human α-thrombin potently. However, efforts to develop an orally active drug remain in progress as the most active inhibitors with a highly basic P1 moiety exhibit an unsatisfactory bioavailability profile. In our previous work we solved several X-ray structures of human α-thrombin in complexes with (1) novel bicyclic arginine mimetics attached to the glycylproline amide and pyridinone acetamide scaffold and (2) inhibitors with a novel aza scaffold and with charged or neutral P1 moieties. In the present contribution, we correlate the structures of the complex between these inhibitors and the protein with the calculated free energy of binding. The energy of solvation was calculated using the Poisson–Boltzmann approach. In particular, the requirements for successful recognition of an inhibitor at the protein’s active site pocket S1 are discussed. Figure We report here on free energy of binding analysis of thrombin inhibitors with novel aza scaffold and novel bicyclic arginine mimetics in S1 pocket of thrombin     

Sequence analysis of the conserved protamine gene cluster shows that it contains a fourth expressed gene

Structural data are presented on the protamine gene cluster (PGC) of human, mouse, rat, and bull. By restriction mapping we demonstrate that the organization of the protamine cluster is conserved throughout all four species, i.e., the genes are situated in a head to tail arrangement in the order: protamine l-protamine 2-transition protein 2. Further, we established the nucleotide sequence of the entire human PGC (25 kb in total) and the 3′ portion of the rat protamine cluster (PRM2 and TNP2 genes and intergenic region). In addition, a 1 kb fragment of the bovine and murine protamine cluster, situated between PRM2 and TNP2, was sequenced. This fragment is conserved regarding sequence, position, and orientation in all species examined, and was classified as likely coding region by gene recognition program GRAIL. Using the rat fragment as a probe in RNA blots, we detected a testis-specific signal of about 0.5 kb. Finally, we demonstrate a high density of Alu elements, both full and fragmented copies, in the human PGC and discuss their localization with respect to evolutionary and functional aspects. © 1996 Wiley-Liss, Inc.     

Investigation of sugar binding kinetics of the E. coli sugar/H+ symporter XylE using solid-supported membrane-based electrophysiology

Bacterial transporters are difficult to study using conventional electrophysiology because of their low transport rates and the small size of bacterial cells. Here, we applied solid-supported membrane–based electrophysiology to derive kinetic parameters of sugar translocation by the Escherichia coli xylose permease (XylE), including functionally relevant mutants. Many aspects of the fucose permease (FucP) and lactose permease (LacY) have also been investigated, which allow for more comprehensive conclusions regarding the mechanism of sugar translocation by transporters of the major facilitator superfamily. In all three of these symporters, we observed sugar binding and transport in real time to determine K_M, V_max, K_D, and k_obs values for different sugar substrates. K_D and k_obs values were attainable because of a conserved sugar-induced electrogenic conformational transition within these transporters. We also analyzed interactions between the residues in the available X-ray sugar/H⁺ symporter structures obtained with different bound sugars. We found that different sugars induce different conformational states, possibly correlating with different charge displacements in the electrophysiological assay upon sugar binding. Finally, we found that mutations in XylE altered the kinetics of glucose binding and transport, as Q175 and L297 are necessary for uncoupling H⁺ and d-glucose translocation. Based on the rates for the electrogenic conformational transition upon sugar binding (>300 s⁻¹) and for sugar translocation (2 s⁻¹ − 30 s⁻¹ for different substrates), we propose a multiple-step mechanism and postulate an energy profile for sugar translocation. We also suggest a mechanism by which d-glucose can act as an inhibitor for XylE.     

Fruktifikationen der Gattung Hemitrapa Miki (Trapellaceae) in den Ablagerungen der Oberen Süßwasser-Molasse Bayerns (mit Bemerkungen zu den fossilen Vorkommen Eurasiens)

In contrast to the opinion of Miki (1952 b, p. 349) the genus Hemitrapa Miki (Trapellaceae) is not only found in Asia and America but also in Europe. Wrongly determined fossils of the type „Trapa silesiaca Goeppert”︁ (the original species from Schoßnitz in Poland is under research by M. Lancucka-Srodoniowa, Krakow) belong to the genus Hemitrapa Miki. Some other Bavarian fossils are newly decribed here as Hemitrapa heissigii sp. nov. Hemitrapa fossils grew not only in the Senftenberg area (Menzel 1906), in Silesia (Kräusel 1920) or the Niederlausitz area (Menzel in Gothan & Sapper 1933), at Konin (Raniecka-Bobrowska 1954), but possibly also near Cologne (Kilpper 1969 and Kramer 1974) and at Ponholz (Gregor 1980). Especially in the Middle Miocene Upper Freshwater Molasse of Bavaria the fossil species Hemitrapa heissigii is found in numerous specimens near Eberstetten, Haag a. d. Amper and Rauscheröd (all in southern Bavaria). Hemitrapa heissigii can be used as an index-fossil and signs Uppermost Miocene sediments (Badenian, Samartian). The occurence in Pliocene localities is to be prooved. The whole group around Hemitrapa is considered to be a “late element” (Upper Miocene) in Europe.     

Alkaline phosphatase and phosphoamino acid phosphatases in normal and cancerous tissues of the human larynx

The activities of alkaline phosphatase and phosphoamino acid phosphatases were measured in normal and cancerous regions of the human larynx. For each larynx, alkaline phosphatase and phosphotyrosine phosphatase activities were higher in the tumor than in the corresponding normal tissue. Phosphothreonine and phosphoserine phosphatase activities were relatively low and there were no consistent trends. The increased alkaline phosphatase activity in the tumors supports histological observations that ossification of cartilage seems to occur at the site of invasion; the phosphatase acting on phosphotyrosine could serve as a regulator of cell differentiation during tumorigenesis.     

Clinical stage drugs targeting inhibitor of apoptosis proteins purge episomal Hepatitis B viral genome in preclinical models

A major unmet clinical need is a therapeutic capable of removing hepatitis B virus (HBV) genome from the liver of infected individuals to reduce their risk of developing liver cancer. A strategy to deliver such a therapy could utilize the ability to target and promote apoptosis of infected hepatocytes. Presently there is no clinically relevant strategy that has been shown to effectively remove persistent episomal covalently closed circular HBV DNA (cccDNA) from the nucleus of hepatocytes. We used linearized single genome length HBV DNA of various genotypes to establish a cccDNA-like reservoir in immunocompetent mice and showed that clinical-stage orally administered drugs that antagonize the function of cellular inhibitor of apoptosis proteins can eliminate HBV replication and episomal HBV genome in the liver. Primary human liver organoid models were used to confirm the clinical relevance of these results. This study underscores a clinically tenable strategy for the potential elimination of chronic HBV reservoirs in patients.Subject terms: Target validation, Hepatitis B, Preclinical research, Translational research     

A fault detection service for wide area distributed computations

The potential for faults in distributed computing systems is a significant complicating factor for application developers. While a variety of techniques exist for detecting and correcting faults, the implementation of these techniques in a particular context can be difficult. Hence, we propose a fault detection service designed to be incorporated, in a modular fashion, into distributed computing systems, tools, or applications. This service uses well-known techniques based on unreliable fault detectors to detect and report component failure, while allowing the user to trade off timeliness of reporting against false positive rates. We describe the architecture of this service, report on experimental results that quantify its cost and accuracy, and describe its use in two applications, monitoring the status of system components of the GUSTO computational grid testbed and as part of the NetSolve network-enabled numerical solver. This revised version was published online in July 2006 with corrections to the Cover Date.