Exonuclease V from Saccharomyces cerevisiae. A 5'----3'-deoxyribonuclease that produces dinucleotides in a sequential fashion

A novel deoxyribonuclease, exonuclease V, has been purified approximately 30,000-fold from Saccharomyces cerevisiae. Exonuclease V is localized in the nucleus. The nuclease degrades single-stranded, but not double-stranded, DNA from the 5'-end. The products of exonuclease action are dinucleotides, except the 3'-terminal tri- and tetranucleotides which are not degraded. Studies with synthetic oligo- and polynucleotides with specified sequence elements showed that exonuclease V cleaves off dinucleotides as primary digestion products. Thus, the polymers (pT)9pA(pT)n and (pT)10pA(pT)n yielded pTpA and pApT as digestion products, respectively. Removal of the 5'-terminal phosphate from the DNA substrate results in reduced binding of the enzyme to the substrate. In addition, the initial hydrolytic cut by exonuclease V on the dephosphorylated substrate produces a mixture of dinucleoside monophosphates and trinucleoside diphosphates. The enzyme is processive in action.     

A Bacterial Ras-Like Small GTP-Binding Protein and Its Cognate GAP Establish a Dynamic Spatial Polarity Axis to Control Directed Motility

Regulated cell polarity is central to many cellular processes. We investigated the mechanisms that govern the rapid switching of cell polarity (reversals) during motility of the bacterium Myxococcus xanthus. Cellular reversals are mediated by pole-to-pole oscillations of motility proteins and the frequency of the oscillations is under the control of the Frz chemosensory system. However, the molecular mechanism that creates dynamic polarity remained to be characterized. In this work, we establish that polarization is regulated by the GTP cycle of a Ras-like GTPase, MglA. We initially sought an MglA regulator and purified a protein, MglB, which was found to activate GTP hydrolysis by MglA. Using live fluorescence microscopy, we show that MglA and MglB localize at opposite poles and oscillate oppositely when cells reverse. In absence of MglB, MglA-YFP accumulates at the lagging cell end, leading to a strikingly aberrant reversal cycle. Spatial control of MglA is achieved through the GAP activity of MglB because an MglA mutant that cannot hydrolyze GTP accumulates at the lagging cell end, despite the presence of MglB. Genetic and cell biological studies show that the MglA-GTP cycle controls dynamic polarity and the reversal switch. The study supports a model wherein a chemosensory signal transduction system (Frz) activates reversals by relieving a spatial inhibition at the back pole of the cells: reversals are allowed by Frz-activated switching of MglB to the opposite pole, allowing MglA-GTP to accumulate at the back of the cells and create the polarity switch. In summary, our results provide insight into how bacteria regulate their polarity dynamically, revealing unsuspected conserved regulations with eukaryots.     

Selenium addition alters mercury uptake,bioavailability in the rhizosphere and root anatomy of rice (Oryza sativa)

Background and Aims

Mercury (Hg) is an extremely toxic pollutant, especially in the form of methylmercury (MeHg), whereas selenium (Se) is an essential trace element in the human diet. This study aimed to ascertain whether addition of Se can produce rice with enriched Se and lowered Hg content when growing in Hg-contaminated paddy fields and, if so, to determine the possible mechanisms behind these effects.

Methods

Two cultivars of rice (Oryza sativa, japonica and indica) were grown in either hydroponic solutions or soil rhizobags with different Se and Hg treatments. Concentrations of total Hg, MeHg and Se were determined in the roots, shoots and brown rice, together with Hg uptake kinetics and Hg bioavailability in the soil. Root anatonmy was also studied.

Key Results

The high Se treatment (5 μg g^–1) significantly increased brown rice yield by 48 % and total Se content by 2·8-fold, and decreased total Hg and MeHg by 47 and 55 %, respectively, compared with the control treatments. The high Se treatment also markedly reduced ‘water-soluble’ Hg and MeHg concentrations in the rhizosphere soil, decreased the uptake capacity of Hg by roots and enhanced the development of apoplastic barriers in the root endodermis.

Conclusions

Addition of Se to Hg-contaminated soil can help produce brown rice that is simultaneously enriched in Se and contains less total Hg and MeHg. The lowered accumulation of total Hg and MeHg appears to be the result of reduced bioavailability of Hg and production of MeHg in the rhizosphere, suppression of uptake of Hg into the root cells and an enhancement of the development of apoplastic barriers in the endodermis of the roots.     

Evolutionary Divergence in Brain Size between Migratory and Resident Birds

Despite important recent progress in our understanding of brain evolution, controversy remains regarding the evolutionary forces that have driven its enormous diversification in size. Here, we report that in passerine birds, migratory species tend to have brains that are substantially smaller (relative to body size) than those of resident species, confirming and generalizing previous studies. Phylogenetic reconstructions based on Bayesian Markov chain methods suggest an evolutionary scenario in which some large brained tropical passerines that invaded more seasonal regions evolved migratory behavior and migration itself selected for smaller brain size. Selection for smaller brains in migratory birds may arise from the energetic and developmental costs associated with a highly mobile life cycle, a possibility that is supported by a path analysis. Nevertheless, an important fraction (over 68%) of the correlation between brain mass and migratory distance comes from a direct effect of migration on brain size, perhaps reflecting costs associated with cognitive functions that have become less necessary in migratory species. Overall, our results highlight the importance of retrospective analyses in identifying selective pressures that have shaped brain evolution, and indicate that when it comes to the brain, larger is not always better.     

Quantitative spin trapping and triplet quenching by radicals for in vivo studies. I. The chemical model

In order to determine quantitatively the free radical content and its changes affected by additives using spin trapping under in vivo conditions, an approach is suggested carrying out experiments in a completely mixed open system (CMOS). Measurements have been carried out for a chemical oxidation process as a model system, and analysis of products and of the spin trap was extended by kinetic ESR spectrometry of the spin adducts. Since in a CMOS differential equations of accumulation of all species can be transformed into algebraic expressions using available rate constants for the formation of the spin adducts, corresponding concentrations of free radicals have been calculated. In addition, it has been established that triplet excited photosensitizers have a double effect: increasing the rate of initiation by decomposing hydroperoxide-type compounds and inhibiting the overall process by interactions with free radicals. Results indicate that by changing the "reaction vessel" the method can be applied for ex vivo and in vivo systems.     

Discovery of novel aspartyl ketone dipeptides as potent and selective caspase-3 inhibitors

The discovery of a series of potent, selective and reversible dipeptidyl caspase-3 inhibitors are reported. The iterative discovery process of using combinatorial chemistry, parallel synthesis, moleculare modelling and structural biology will be discussed.     

25-Hydroxylation of vitamin D3 in the human hepatoma cell lines Hep G2 and Hep 3B

Two human hepatoma cell lines, Hep G2 and Hep 3B, were screened for vitamin D3-25-hydroxylase enzyme activity by incubation with radioactive vitamin D3. A compound co-chromatographing with 25-OH-D3 was synthesized in both cell lines but its rate of synthesis was tenfold greater in Hep 3B than in Hep G2 cells. The identity of the compound was confirmed by comparing its chromatographic properties with authentic 25-OH-D3 on three different high pressure liquid chromatography systems. Its production was suppressed by adding fetal calf serum (10%), lipoprotein-deficient fetal calf serum, or pure vitamin D-binding globulin to the medium. The mechanism of action of these plasma proteins appears to involve retardation of uptake of the substrate. These two cell lines offer considerable potential as defined in vitro models for studying the effects of physiological factors on the 25-hydroxylation of vitamin D3.