Effects of Salt Stimulation on Lunasin Accumulation and Activity during Soybean Germination

Lunasin, a bioactive peptide, was originally found in soybeans, and it has exhibited multiple biological functions. On the basis of previous studies, salt stress was found able to induce changes in many polypeptides and translatable mRNA levels in plants. Salt stress was applied to soybean germination, with water treatment as a control group, to evaluate the effects of salt stimulation on lunasin accumulation and activity during soybean germination. Lunasin content gradually increased in the control group during germination, reached the highest level after six hours of imbibition, and then slowly decreased. Under salt exposure, lunasin content showed a similar trend to that of the control group. The lunasin content in salt-treated soybean was significantly higher than that in the control group. Lunasin peptide was purified from soybean after six hours of imbibition and it was then used for function evaluation. Purified lunasin from salt-stress-germinated soybean (6 h-LSGS) exhibited stronger antioxidant activity than lunasin from water-treatment-germinated soybean (6 h-LWGS) and soybean seed without imbibition (DRY). The 6 h-LSGS presented anti-inflammatory activity on LPS-induced macrophage cells (p < 0.05) by suppressing the release of nitric oxide (NO) and proinflammatory cytokines, including IL-1 and IL-6. The gene expression of NOS, IL-1, IL-6, and TNF-α was significantly inhibited by 6 h-LSGS. Further, 6 h-LSGS exhibited superior antiproliferation activity on human breast-cancer cells MDA-MB-231 when compared to 6 h-LWGS and DRY. Overall, this study offers a feasible elicitation strategy for enhancing lunasin accumulation and its properties in soybean for possible use in functional food.

Separating the Wheat from the Chaff: RNA Editing and Selection of Translatable mRNA in Trypanosome Mitochondria

In the mitochondria of trypanosomes and related kinetoplastid protists, most mRNAs undergo a long and sophisticated maturation pathway before they can be productively translated by mitochondrial ribosomes. Some of the aspects of this pathway (identity of the promotors, transcription initiation, and termination signals) remain obscure, and some (post-transcriptional modification by U-insertion/deletion, RNA editing, 3′-end maturation) have been illuminated by research during the last decades. The RNA editing creates an open reading frame for a productive translation, but the fully edited mRNA often represents a minor fraction in the pool of pre-edited and partially edited precursors. Therefore, it has been expected that the final stages of the mRNA processing generate molecular hallmarks, which allow for the efficient and selective recognition of translation-competent templates. The general contours and several important details of this process have become known only recently and represent the subject of this review.

A Concept for Selection of Codon-Suppressor tRNAs Based on Read-Through Ribosome Display in anIn VitroCompartmentalized Cell-Free Translation System

Here is presented a concept forin vitroselection of suppressor tRNAs. It uses a pool of dsDNA templates in compartmentalized water-in-oil micelles. The template contains a transcription/translation trigger, an amber stop codon, and another transcription trigger for the anticodon- or anticodon loop-randomized gene for tRNASer. Upon transcription are generated two types of RNAs, a tRNA and a translatable mRNA (mRNA-tRNA). When the tRNA suppresses the stop codon (UAG) of the mRNA, the full-length protein obtained upon translation remains attached to the mRNA (read-through ribosome display) that contains the sequence of the tRNA. In this way, the active suppressor tRNAs can be selected (amplified) and their sequences read out. The enriched anticodon (CUA) was complementary to the UAG stop codon and the enriched anticodon-loop was the same as that in the natural tRNASer.

Probing the circadian control of phosphoenolpyruvate carboxylase kinase expression in Kalanchoë fedtschenkoi

This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. In crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) kinase is expressed at night under the control of a circadian oscillator. We have proposed that this is an indirect effect, secondary to circadian fluctuations in the level of a metabolite, possibly cytosolic malate, resulting from a primary effect on the permeability of the tonoplast (Nimmo 2000, Trends in Plant Science 5, 75-80). Here we show that the nocturnal accumulation of PEPC kinase translatable mRNA and phosphorylation of PEPC in Kalanchoë fedtschenkoi is blocked by the protein phosphatase inhibitor cantharidin. This implicates protein dephosphorylation in the circadian pathway that regulates expression of PEPC kinase. We also show that the effect of reducing the temperature from 30 to 15 °C on CO2 fixation by detached leaves held in constant darkness and normal air is 'gated' by the circadian clock. This strongly supports the view that the effect of the clock on the expression of PEPC kinase is secondary rather than direct. We have developed a non-aqueous fractionation protocol that separates the cytosolic material in mature leaves from vacuolar material. The cytosolic malate in mature leaves represents a very small part of the total malate, and its concentration cannot be measured precisely by this method.

Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis

SUMMARY Formation of mRNA 3′ ends in eukaryotes requires the interaction of transacting factors with cis-acting signal elements on the RNA precursor by two distinct mechanisms, one for the cleavage of most replication-dependent histone transcripts and the other for cleavage and polyadenylation of the majority of eukaryotic mRNAs. Most of the basic factors have now been identified, as well as some of the key protein-protein and RNA-protein interactions. This processing can be regulated by changing the levels or activity of basic factors or by using activators and repressors, many of which are components of the splicing machinery. These regulatory mechanisms act during differentiation, progression through the cell cycle, or viral infections. Recent findings suggest that the association of cleavage/polyadenylation factors with the transcriptional complex via the carboxyl-terminal domain of the RNA polymerase II (Pol II) large subunit is the means by which the cell restricts polyadenylation to Pol II transcripts. The processing of 3′ ends is also important for transcription termination downstream of cleavage sites and for assembly of an export-competent mRNA. The progress of the last few years points to a remarkable coordination and cooperativity in the steps leading to the appearance of translatable mRNA in the cytoplasm.