Medicinal plants in the fight against leishmaniasis

Despite the tremendous progress mode in the understanding o f the molecular biology of Leishmania and the clinical possibilities presented by some experimental chemotherapeutic agents, no new drugs have been developed for the treatment of leishmaniasis since the introduction of the pentovalent antimoniols more than 50 years ago. As reviewed here by Maurice M. Iwu, Joan E. Jackson and Brion G. Schuster, recognition of the current extensive use of herbal therapy in Leishmania-endemic regions has renewed interest in evaluation of plant remedies used in traditional medicine as sources of potential antileishmanials.     

Biophysical characterization of a designed TMV coat protein mutant, R46G, that elicits a moderate hypersensitivity response in Nicotiana sylvestris

The hypersensitivity resistance response directed by the N' gene in Nicotiana sylvestris is elicited by the tobacco mosaic virus (TMV) coat protein R46G, but not by the U1 wild-type TMV coat protein. In this study, the structural and hydrodynamic properties of R46G and wild-type coat proteins were compared for variations that may explain N' gene elicitation. Circular dichroism spectroscopy reveals no significant secondary or tertiary structural differences between the elicitor and nonelicitor coat proteins. Analytical ultracentrifugation studies, however, do show different concentration dependencies of the weight average sedimentation coefficients at 4 degrees C. Viral reconstitution kinetics at 20 degrees C were used to determine viral assembly rates and as an initial assay of the rate of 20S formation, the obligate species for viral reconstitution. These kinetic results reveal a decreased lag time for reconstitution performed with R46G that initially lack the 20S aggregate. However, experiments performed with 20S initially present reveal no detectable differences indicating that the mechanism of viral assembly is similar for the two coat protein species. Therefore, an increased rate of 20S formation from R46G subunits may explain the differences in the viral reconstitution lag times. The inferred increase in the rate of 20S formation is verified by direct measurement of the 20S boundary as a function of time at 20 degrees C using velocity sedimentation analysis. These results are consistent with the interpretation that there may be an altered size distribution and/or lifetime of the small coat protein aggregates in elicitors that allows N. sylvestris to recognize the invading virus.     

Regulation of p53 mediated transactivation by the beta-subunit of protein kinase CK2

The growth suppressor protein p53 plays a main part in cellular growth control. Two of its key functions are sequence specific DNA binding and transactivation. Functions of p53 in growth control are regulated at least in part by its interaction with protein kinases. p53 binds to protein kinase CK2, formerly known as casein kinase 2, and it is phosphorylated by this enzyme. CK2 is composed of two regulating beta-subunits and two catalytic alpha- or alpha'-subunits and the interaction with p53 is mediated by the regulatory beta-subunit of CK2. Recently we showed that the beta-subunit could inhibit the sequence specific DNA binding activity of p53 in vitro. Based on this finding, we asked if a coexpression of the beta-subunit of CK2 with p53 in mammalian cells could inhibit the DNA binding activity of p53 in a physiological context. We found that the coexpression of the beta-subunit showed the same inhibitory effect as in the previous assays with purified proteins. Then, we investigated the effects of the coexpression of the beta-subunit of CK2 on the transactivation and transrepression activity of p53. We found that transactivation of the mdm2, p21(WAF1/CIP1) and cyclin G promoter was inhibited in three different cell lines whereas transactivation of the bax promoter was not affected in COS1 cells but down-regulated in MCO1 and SaosS138V21 cells. p53 mediated transrepression of the fos promoter was not influenced by coexpression of the CK2 beta-subunit. Taken together we propose a cell type dependent fine regulation of the p53 transactivation function by the CK2 beta-subunit in vivo, which does not affect p53 mediated transrepression.     

Specific binding of protein kinase CK2 catalytic subunits to tubulin

Protein kinase CK2 is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits. To analyse these subunits individually we generated antibodies against unique peptides derived from the alpha-, alpha'- and beta-subunit. Immunofluorescence studies with these antibodies revealed the presence of all three CK2 subunits in the cytoplasm and weakly in the nucleus with strong signals around the nuclear membrane. Double staining experiments revealed a co-localisation of all three subunits with tubulin. A direct association between the CK2 alpha- and the alpha'-subunit and tubulin was confirmed by co-immunoprecipitation experiments as well as by Far Western analysis. There was no binding of the CK2 beta-subunit to tubulin. Thus, with tubulin we have identified a new binding partner specific for the catalytic subunits of CK2.     

Anti-plasmodial sesquiterpenoids from the African Reneilmia cincinnata

A new isodaucane sesquiterpenoid, 6,7,10-trihydoxyisodaucane, was isolated from the fruits of Reneilmia cincinnata, together with the known sesquiterpenoids oplodiol, oplopanone, 5E,10(14)-germacradien-1 beta, 4 beta-diol, 1(10)E,5E-germacradien-4 alpha-ol and eudesman-1,4,7-triol. A large amount of 5-hydroxy-3,7,4'-trimethoxyflavone was also isolated. Their structures were established by NMR techniques using 1D and 2D experiments. Three of the known sesquirernenoids exhibited noteworthy anti-plasmodial activity against Plasmodium falciparum strains.     

Altering the 3′ UTR endonucleolytic cleavage site of a Chlamydomonas chloroplast mRNA affects 3′-end maturation in vitro but not in vivo

The 3 ends of chloroplast mRNAs are produced by the processing of longer precursors. The 3 ends of most plastid mRNAs are located at, or several nucleotides downstream of, stem-loop structures, which act as 3-end-processing signals and RNA stability elements. In chloroplasts of the green alga Chlamydomonas reinhardtii, 3-end maturation of atpB mRNA involves endonucleolytic cleavage of the pre-mRNA at an AU-rich site located about 10 nucleotides downstream of the stem-loop structure. This cleavage is followed by exonucleolytic resection to generate the mature 3 end. In order to define critical nucleotides of the endonucleolytic cleavage site, we mutated its sequence. Incubation of synthetic atpB pre-RNAs containing these mutations in a chloroplast protein extract resulted in the accumulation of 3-end-processed products. However, in two cases where the AU-rich sequence of this site was replaced with a GC-rich one, the 3 end of the stable processing product differed from that of the wild-type product. To examine whether these mutations affected atpB mRNA processing or accumulation in vivo, the endogenous 3 UTR was replaced with mutated sequences by biolistic transformation of Chlamydomonas chloroplasts. Analysis of the resulting strains revealed that the accumulation of atpB mRNA was approximately equal to that of wild-type cells, and that a wild-type atpB 3 end was generated. These results imply that Chlamydomonas atpB 3 processing parallels the situation with other endonucleases such as Escherichia coli RNAse E, where specific sequences are required for correct in vitro processing, but in vivo these mutations can be overcome.     

Detection of elementary flux modes in biochemical networks: a promising tool for pathway analysis and metabolic engineering

Rational metabolic engineering requires powerful theoretical methods such as pathway analysis, in which the topology of metabolic networks is considered. All metabolic capabilities in steady states are composed of elementary flux modes, which are minimal sets of enzymes that can each generate valid steady states. The modes of the fructose-2,6-bisphosphate cycle, the combined tricarboxylic-acid-glyoxylate-shunt system and tryptophan synthesis are used here for illustration. This approach can be used for many biotechnological applications such as increasing the yield of a product, channelling a product into desired pathways and in functional reconstruction from genomic data.