Transfection-independent production of alphavirus replicon particles based on poxvirus expression vectors

This report describes a transfection-independent system for packaging alphavirus replicon vectors using modified vaccinia virus Ankara (MVA) vectors to express all of the RNA components necessary for the production of Venezuelan equine encephalitis (VEE) virus replicon particles (VRP). Infection of mammalian cells with these recombinant MVA vectors resulted in robust expression of VEE structural genes, replication of the alphavirus vector and high titers of VRP. In addition, VRP packaging was achieved in a cell type (fetal rhesus lung) that has been approved for the manufacturing of vaccines destined for human use.     

Influence of the length of the phosphate chain in mRNA 5' cap analogues on their interaction with eukaryotic initiation factor 4E

The recognition of the 5'mRNA cap structure m7G(5')ppp(5')N by one of the components of the initiation translation machinery, the eIF4E factor, plays a pivotal role in regulation of the protein synthesis. In the present study we have shown two opposing roles of the cap phosphate chain in the specific eIF4E-cap interaction. The extension of the phosphate chain enhances the binding of the cap to the unphosphorylated eIF4E but destabilises the eIF4E-cap complex in case of the phosphorylated protein.     

Binding studies of eukaryotic initiation factor eIF4E with novel mRNA dinucleotide cap analogues

Studies on the interaction of the murine translation initiation factor 4E with two new-synthesized cap-analogues, modified at C2' of 7-methylguanosine, have been performed by means of the fluorescence titration method. No difference in the binding affinity for eIF4E was observed compared with the "anti reversed" cap analogues, possessing the analogous modifications at C3'. Potential significance of the novel caps as research tools for examination of the nuclear cap binding complex CBC80/20 has been discussed.     

Interaction between yeast eukaryotic initiation factor eIF4E and mRNA 5' cap analogues differs from that for murine eIF4E

Measurements of interaction of 7-methyl-GTP eIF4E from S. cerevisiae were performed by means of two methods: Isothermal Titration Calorimetry (ITC) and fluorescence titration. The equilibrium association constants (Kas) derived from the two methods show significantly different affinity of yeast eIF4E for the mRNA 5' cap than those of the murine and human proteins. The observed differences in the Kas values and the enthalpy changes of the association (deltaH(o)) suggest some dissimilarity in the mode of binding and stabilization of cap in the complexes with eIF4E from various sources.     

Structure-activity relationships of novel anti-malarial agents: part 5. N-(4-acylamino-3-benzoylphenyl)-[5-(4-nitrophenyl)-2-furyl]acrylic acid amides

We have developed the [5-(4-nitrophenyl)-2-furyl]acrylic acid substituted benzophenone 4g as a novel lead for anti-malarial agents. Here, we demonstrated that the acyl residue at the 2-amino group of the benzophenone core structure has to be a phenylacetic acid substructure substituted in its para-position with methyl or other substituents of similar size. The trifluoromethyl substituted derivative displayed an IC(50) of 47 nM against the multi-drug resistant Plasmodium falciparum strain Dd2.     

Comparison of life-history traits of the two male morphs of the bulb mite, Rhizoglyphus robini

Two basic male morphs occur in several species of the family Acaridae: heteromorphic fighters, possessing a thickened and sharply terminated third pair of legs, and homeomorphic males with unmodified legs. We compared major life-history traits of the two morphs in the bulb mite, Rhizoglyphus robini. We found no significant differences in development time or virility, but homeomorphic males lived 23% longer than heteromorphs. We discuss the possibility that the trade-off between longevity and adaptation for fighting maintains genetic variation for the male morph in the studied species.     

The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer’s Disease

In Alzheimer’s disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80–90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau–microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer’s disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.