Importance of Ribosomal Frameshifting for Human Immunodeficiency Virus Type 1 Particle Assembly and Replication

The recent development and use of protease inhibitors have demonstrated the essential role that combination therapy will play in the treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1). Past clinical experience suggests that due to the appearance of resistant HIV-1 variants, additional therapeutics will be required in the future. To identify new options for combination therapy, it is of paramount importance to pursue novel targets for drug development. Ribosomal frameshifting is one potential target that has not been fully explored. Data presented here demonstrate that small molecules can stimulate frameshifting, leading to an imbalance in the ratio of Gag to Gag-Pol and inhibiting HIV-1 replication at what appears to be the point of viral particle assembly. Thus, we propose that frameshifting represents a new target for the identification of novel anti-HIV-1 therapeutics.     

Mammalian expression of transmembrane receptors for pharmaceutical applications

Three mammalian expression systems suitable for expressing recombinant receptors have been described. Each is suited to a different aspect of the study of receptors and their behaviour. IRES-based vectors are ideal for creating stable mammalian cell lines suitable for screening receptors using a signalling readout. Unlike traditional vectors they result in almost 100% of cell lines generated expressing a particular receptor, thus increasing the efficiency of cell line generation and increasing the chance of higher expression-level cell lines being generated. They may also be utilized to express more than one protein of interest, for example it is possible to co-express a particular receptor with a particular signalling protein or trafficking protein from a single RNA, thus ensuring that both are expressed simultaneously in the same cell. The ecdysone-inducible expression system is ideal for studying receptor signalling and behaviour. It is possible to alter receptor expression levels in an identical cellular background thus making it possible to study phenomena such as constitutive receptor activity in the absence of agonist. The SFV expression system is ideal for expressing receptors at high levels of a mammalian cell. It is thus a good system for purifying receptors for structural analysis and for providing material for binding assays. All of the expression systems described above have been demonstrated to express seven-transmembrane receptors with the expected pharmacological and functional profile.     

Incorporation of terminal phosphorothioates into oligonucleotides.

Considerable effort has been directed towards studying the structure and function of oligonucleotides and several approaches rely on the attachment of reporter groups to oligonucleotides. We report here the introduction of 3'- and 5'-terminal phosphorothioates into heptameric oligonucleotides and their post-synthetic modification with several reporter groups. The synthesis of terminal phosphorothioates is based on the coupling of a ribonucleoside phosphoramidite at the first or last nucleotide, respectively, which, after sulphurization, is removed by sequential oxidation of the vicinal hydroxyl groups and then beta-elimination. Product formation is of the order of 95%. The ratio of phosphorothioate- versus phosphate-terminated oligodeoxynucleotides as analysed by electrophoresis on a Hg2+gel is in general 85/15. Examples for the reactivity of the terminal phosphorothioates for conjugation with cholesterol, bimane and for sulphydryl exchange are described.     

Polyamine deficiency alters EGF receptor distribution and signaling effectiveness in IEC-6 cells

Cell growth andmigration are essential processes for the differentiation, maintenance,and repair of the intestinal epithelium. Epidermal growth factor (EGF)is an important factor in the reorganization of the cytoskeletonrequired for both processes. Because we had previously foundsignificant changes in the cytoskeleton during polyamine deficiency, itwas of interest to know whether those changes could prevent EGF fromstimulating growth and migration. Polyamine biosynthesis in IEC-6 cellswas interrupted by treatment with -difluoromethylornithine (DFMO), aspecific inhibitor of ornithine decarboxylase, the primaryrate-limiting enzyme of polyamine biosynthesis. DFMO halted cellproliferation and inhibited cell migration, and neither function couldbe normally stimulated by EGF. Immunocytochemistry of the transferrinreceptor (used as a marker for the endocytic pathway) revealed anabnormal distribution of the EGF receptor (EGFR) 10 min after bindingEGF. Polyamine deficiency depleted the cells of interiormicrofilaments, thickened the actin cortex, and prevented the promptassociation of EGF-bound EGFR with actin. EGF-stimulated 170-kDaprotein tyrosine phosphorylation and the kinase activity of purifiedmembrane EGFR were reduced by 50%. Immunoprecipatated EGFR proteinconcentration, however, was not reduced by polyamine deficiency. All ofthese changes could be prevented by supplementation with putrescine.Cytoskeletal disruption, reduced EGFR phosphorylation and kinaseactivity, aberrant intracellular EGFR distribution, and delayedassociation with actin filaments suggest a partial explanation for thedependence of epithelial cell growth and migration on polyamines.

     

THE SHELL ZONE: ITS DIFFERENTIATION AND PROBABLE FUNCTION IN SOME DICOTYLEDONS

Thirty-five species belonging to various dicotyledonous families were investigated to study the origin, development, and probable function of the shell zone, which is defined as an arcuate zone of cambiform cells delimiting the early axillary bud meristem. It is present in the majority of the investigated plants and five intergrading patterns of origin are described: (i) from the parenchymatized derivatives of the cells of the peripheral meristem of the shoot apex, adaxial to the bud meristem, (ii) from the peripheral meristem of the shoot apex along with the initiation of the early bud meristem, (iii) from the adaxial cells of the bud meristem, (iv) from the derivatives of the cells of the bud meristem at its base, and (v) partly from the parenchymatized cells of the peripheral meristem adaxial to the bud and partly from the adaxial derivatives of the bud meristem. The shell zone loses its identity at different stages of bud development in various species. Its cells ultimately contribute to the ground meristem, procambium, and pith cells of the axis. In Cuminum cyminum and lpomoea cairica the shell zone contributes in bringing about the axillary position of the bud from its early lateral position. In Solarium melongena, derivatives of the shell zone initiate the internodal elongation between the flower or inflorescence and the shoot apex, ultimately shifting the bud to an extra-axillary position on the internode.