An α-Tubulin Mutant Destabilizes the Heterodimer: Phenotypic Consequences and Interactions with Tubulin-binding Proteins

Many effectors of microtubule assembly in vitro enhance the polymerization of subunits. However, several Saccharomyces cerevisiae genes that affect cellular microtubule-dependent processes appear to act at other steps in assembly and to affect polymerization only indirectly. Here we use a mutant α-tubulin to probe cellular regulation of microtubule assembly. tub1-724 mutant cells arrest at low temperature with no assembled microtubules. The results of several assays reported here demonstrate that the heterodimer formed between Tub1-724p and β-tubulin is less stable than wild-type heterodimer. The unstable heterodimer explains several conditional phenotypes conferred by the mutation. These include the lethality of tub1-724 haploid cells when the β-tubulin–binding protein Rbl2p is either overexpressed or absent. It also explains why the TUB1/tub1-724 heterozygotes are cold sensitive for growth and why overexpression of Rbl2p rescues that conditional lethality. Both haploid and heterozygous tub1-724 cells are inviable when another microtubule effector, PAC2, is overexpressed. These effects are explained by the ability of Pac2p to bind α-tubulin, a complex we demonstrate directly. The results suggest that tubulin-binding proteins can participate in equilibria between the heterodimer and its components.     

Formation and Function of the Rbl2p–β-Tubulin Complex

The yeast protein Rbl2p suppresses the deleterious effects of excess β-tubulin as efficiently as does α-tubulin. Both in vivo and in vitro, Rbl2p forms a complex with β-tubulin that does not contain α-tubulin, thus defining a second pool of β-tubulin in the cell. Formation of the complex depends upon the conformation of β-tubulin. Newly synthesized β-tubulin can bind to Rbl2p before it binds to α-tubulin. Rbl2p can also bind β-tubulin from the α/β-tubulin heterodimer, apparently by competing with α-tubulin. The Rbl2p–β-tubulin complex has a half-life of ~2.5 h and is less stable than the α/β-tubulin heterodimer. The results of our experiments explain both how excess Rbl2p can rescue cells overexpressing β-tubulin and how it can be deleterious in a wild-type background. They also suggest that the Rbl2p–β-tubulin complex is part of a cellular mechanism for regulating the levels and dimerization of tubulin chains.     

Design,synthesis, and docking of highly hypolipidemic agents: Schizosaccharomyces pombe as a new model for evaluating α-asarone-based HMG-CoA reductase inhibitors

A series of α-asarone-based analogues was designed by conducting docking experiments with published crystal structures of human HMG-CoA reductase. Indeed, synthesis and evaluation of this series showed a highly hypocholesterolemic in vivo activity in a murine model, as predicted by previous docking studies. In agreement with this model, the polar groups attached to the benzene ring could play a key role in the enzyme binding and probably also in its biological activity, mimicking the HMG-moiety of the natural substrate. The hypolipidemic action mechanism of these compounds was investigated by developing a simple, efficient, and novel model for determining HMG-CoA reductase inhibition. The partial purification of the enzyme from Schizosaccharomyces pombe allowed for testing of α-asarone- and fibrate-based analogues, resulting in positive and significant inhibitory activity.     

Synergy of Omeprazole and Praziquantel In Vitro Treatment against Schistosoma mansoni Adult Worms

BackgroundTreatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis.MethodologyWe used a custom-designed Schistosoma mansoni expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against S. mansoni adult worms with a survival in vitro assay.ConclusionsFunctional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.     

Subcellular Localization and Clues for the Function of the HetN Factor Influencing Heterocyst Distribution in Anabaena sp. Strain PCC 7120

In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, heterocysts are formed in the absence of combined nitrogen, following a specific distribution pattern along the filament. The PatS and HetN factors contribute to the heterocyst pattern by inhibiting the formation of consecutive heterocysts. Thus, inactivation of any of these factors produces the multiple contiguous heterocyst (Mch) phenotype. Upon N stepdown, a HetN protein with its C terminus fused to a superfolder version of green fluorescent protein (sf-GFP) or to GFP-mut2 was observed, localized first throughout the whole area of differentiating cells and later specifically on the peripheries and in the polar regions of mature heterocysts, coinciding with the location of the thylakoids. Polar localization required an N-terminal stretch comprising residues 2 to 27 that may represent an unconventional signal peptide. Anabaena strains expressing a version of HetN lacking this fragment from a mutant gene placed at the native hetN locus exhibited a mild Mch phenotype. In agreement with previous results, deletion of an internal ERGSGR sequence, which is identical to the C-terminal sequence of PatS, also led to the Mch phenotype. The subcellular localization in heterocysts of fluorescence resulting from the fusion of GFP to the C terminus of HetN suggests that a full HetN protein is present in these cells. Furthermore, the full HetN protein is more conserved among cyanobacteria than the internal ERGSGR sequence. These observations suggest that HetN anchored to thylakoid membranes in heterocysts may serve a function besides that of generating a regulatory (ERGSGR) peptide.     

The progamic phase of an early-divergent angiosperm, Annona cherimola (Annonaceae)

Background and Aims

Recent studies of reproductive biology in ancient angiosperm lineages are beginning to shed light on the early evolution of flowering plants, but comparative studies are restricted by fragmented and meagre species representation in these angiosperm clades. In the present study, the progamic phase, from pollination to fertilization, is characterized in Annona cherimola, which is a member of the Annonaceae, the largest extant family among early-divergent angiosperms. Beside interest due to its phylogenetic position, this species is also an ancient crop with a clear niche for expansion in subtropical climates.

Methods

The kinetics of the reproductive process was established following controlled pollinations and sequential fixation. Gynoecium anatomy, pollen tube pathway, embryo sac and early post-fertilization events were characterized histochemically.

Key Results

A plesiomorphic gynoecium with a semi-open carpel shows a continuous secretory papillar surface along the carpel margins, which run from the stigma down to the obturator in the ovary. The pollen grains germinate in the stigma and compete in the stigma-style interface to reach the narrow secretory area that lines the margins of the semi-open stylar canal and is able to host just one to three pollen tubes. The embryo sac has eight nuclei and is well provisioned with large starch grains that are used during early cellular endosperm development.

Conclusions

A plesiomorphic simple gynoecium hosts a simple pollen–pistil interaction, based on a support–control system of pollen tube growth. Support is provided through basipetal secretory activity in the cells that line the pollen tube pathway. Spatial constraints, favouring pollen tube competition, are mediated by a dramatic reduction in the secretory surface available for pollen tube growth at the stigma–style interface. This extramural pollen tube competition contrasts with the intrastylar competition predominant in more recently derived lineages of angiosperms.Key words: Annona cherimola, Annonaceae, embryo sac, endosperm, Magnoliid, ovule, pollen–pistil interaction, pollen tube     

Synthesis and nootropic activity of some 2,3-dihydro-1H-isoindol-1-one derivatives structurally related with piracetam

Three 2,3‐dihydro‐1H‐isoindol‐1‐ones structurally related with piracetam (=2‐oxopyrrolidine‐1‐acetamide) have been synthesized and tested for their nootropic effects in the passive avoidance test in mice. Compounds (RS)‐ 2 , (R,R)‐ 3 , and (R,S)‐ 3 were obtained in good yields in only two steps starting from methyl dl ‐phthaloylalanine. Compound (RS)‐ 2 exhibited nootropic activity at lower doses than piracetam, used as reference drug, but it showed lower efficacy. Whereas diastereoisomers (R,R)‐ 3 and (R,S)‐ 3 were as potent as piracetam to revert amnesia induced by scopolamine, (R,S)‐ 3 showed lower efficacy than (R,R)‐ 3 . Only (R,R)‐ 3 showed myorelaxant effect at doses of 10 and 30 mg/kg; other compounds did not exhibit any anticonvulsant, sedative, myorelaxant, or impaired motor‐coordination effect in mice. These synthesized 2,3‐dihydro‐1H‐isoindol‐1‐one derivatives constitute a new kind of nootropic compounds.