The Beneficial Effect of Organic Solvents on the Enzymatic Synthesis of Nucleoside Analogues Using N-Deoxyribosyltransferases from Lactobacillus leichmannii (E.C. 2.4.2.6)

Addition of ethylene glycol (10% v/v) has a beneficial effect on the synthesis of 2'-deoxynucleosides catalysed by crude preparations of N-deoxyribosyltransferases from Lactobacillus leichmannii. In the absence of added organic solvent decomposition of products and starting materials by deamination or hydrolysis occurs giving rise to poor yields of products if the transfer reaction is slow. The glycosyl transfer reaction is unaffected by addition of organic solvent but decomposition of products and starting materials is largely suppressed. The organic solvent appears to inhibit selectively contaminating enzymes in the crude N-deoxyribosyltransferase preparation as the purified transferase does not possess hydrolytic or deaminating activity. Different concentrations of ethylene glycol and other organic solvents have been examined as inhibitors of the side reactions but 10% (v/v) appears to be the most effective. Using the N-deoxyribosyltransferase in the presence of ethylene glycol, a number of 2'-deoxynucleosides of 6-substituted nucleosides have been obtained in high yield on a preparative scale.     

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin ( ca . 8 × 10⁸ molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus . The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.     

The Genomic Basis of Color Pattern Polymorphism in the Harlequin Ladybird

1. Download high-res image (251KB)
2. Download full-size image

     

Histone H2A‐H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin

The eukaryotic replisome disassembles parental chromatin at DNA replication forks, but then plays a poorly understood role in the re‐deposition of the displaced histone complexes onto nascent DNA. Here, we show that yeast DNA polymerase α contains a histone‐binding motif that is conserved in human Pol α and is specific for histones H2A and H2B. Mutation of this motif in budding yeast cells does not affect DNA synthesis, but instead abrogates gene silencing at telomeres and mating‐type loci. Similar phenotypes are produced not only by mutations that displace Pol α from the replisome, but also by mutation of the previously identified histone‐binding motif in the CMG helicase subunit Mcm2, the human orthologue of which was shown to bind to histones H3 and H4. We show that chromatin‐derived histone complexes can be bound simultaneously by Mcm2, Pol α and the histone chaperone FACT that is also a replisome component. These findings indicate that replisome assembly unites multiple histone‐binding activities, which jointly process parental histones to help preserve silent chromatin during the process of chromosome duplication.     

Comparison of solutes, nutrients, and bacteria inputs from two types of groundwater to the Rhône river during an artificial drought

Solute, nutrient and bacterial inputs to the River Rhône from the interstitial habitat of a gravel bar and the floodplain aquifer were investigated during an artificial drought. Eight springs were investigated: four groundwater-fed springs in the floodplain, located at the bottom of the bank; and four interstitial-fed springs located at the downstream end of a gravel bar. During this period, the inflows of groundwater to the river represented an average input of 0.77 mg l^–1 of nitrogen (of which 93.3% were nitrates), 0.0187 mg l^–1 of total phosphorus (of which 42.2% was orthophosphate), 3.56 mg l^–1 of silica, 2.315 ± 0.703 mg l^–1 of dissolved organic carbon (DOC, of which 47% was biodegradable) and 7.3 × 10⁴ ± 3.7 × 10⁴ bacteria per ml (of which 8.8% were active). Silica, DOC, biodegradable DOC, and bacteria concentrations displayed temporal variations during the study, which seem to be linked to the biological activity of the groundwater biofilm. There was a strong heterogeneity between the two types of groundwater that flow to the river: concentrations of calcium and alkalinity were higher in bank springs than in gravel bars springs. In these latters, sulfate, sodium, nitrogen, phosphorus were significantly higher.     

Are all GMOs the same? Consumer acceptance of cisgenic rice in India

India has more than 215 million food‐insecure people, many of whom are farmers. Genetically modified (GM) crops have the potential to alleviate this problem by increasing food supplies and strengthening farmer livelihoods. For this to occur, two factors are critical: (i) a change in the regulatory status of GM crops, and (ii) consumer acceptance of GM foods. There are generally two classifications of GM crops based on how they are bred: cisgenically bred, containing only DNA sequences from sexually compatible organisms; and transgenically bred, including DNA sequences from sexually incompatible organisms. Consumers may view cisgenic foods as more natural than those produced via transgenesis, thus influencing consumer acceptance. This premise was the catalyst for our study—would Indian consumers accept cisgenically bred rice and if so, how would they value cisgenics compared to conventionally bred rice, GM‐labelled rice and ‘no fungicide’ rice? In this willingness‐to‐pay study, respondents did not view cisgenic and GM rice differently. However, participants were willing‐to‐pay a premium for any aforementioned rice with a ‘no fungicide’ attribute, which cisgenics and GM could provide. Although not significantly different (P = 0.16), 76% and 73% of respondents stated a willingness‐to‐consume GM and cisgenic foods, respectively.     

High-Resolution Denitrification Kinetics in Pasture Soils Link N2O Emissions to pH,and Denitrification to C Mineralization

Denitrification in pasture soils is mediated by microbial and physicochemical processes leading to nitrogen loss through the emission of N₂O and N₂. It is known that N₂O reduction to N₂ is impaired by low soil pH yet controversy remains as inconsistent use of soil pH measurement methods by researchers, and differences in analytical methods between studies, undermine direct comparison of results. In addition, the link between denitrification and N₂O emissions in response to carbon (C) mineralization and pH in different pasture soils is still not well described. We hypothesized that potential denitrification rate and aerobic respiration rate would be positively associated with soils. This relationship was predicted to be more robust when a high resolution analysis is performed as opposed to a single time point comparison. We tested this by characterizing 13 different temperate pasture soils from northern and southern hemispheres sites (Ireland and New Zealand) using a fully automated-high-resolution GC detection system that allowed us to detect a wide range of gas emissions simultaneously. We also compared the impact of using different extractants for determining pH on our conclusions. In all pH measurements, soil pH was strongly and negatively associated with both N₂O production index (IN₂O) and N₂O/(N₂O+N₂) product ratio. Furthermore, emission kinetics across all soils revealed that the denitrification rates under anoxic conditions (NO+N₂O+N₂ μmol N/h/vial) were significantly associated with C mineralization (CO₂ μmol/h/vial) measured both under oxic (r² = 0.62, p = 0.0015) and anoxic (r² = 0.89, p<0.0001) conditions.     

Executive Functions in Tobacco Dependence: Importance of Inhibitory Capacities

Background

Executive functions are linked to tobacco dependence and craving. In this cross-sectional study, we assessed the impact of three executive functions: updating, inhibition and shifting processes on tobacco craving and dependence.

Method

134 tobacco consumers were included in this study: 81 moderately (Fagerström score <7) and 53 heavily dependent (Fagerström score >7). Dependence was assessed with the Fagerström test and craving with the tobacco craving questionnaire (TCQ 12). We used the Stroop test and the Hayling test to measure inhibition, the Trail Making Test to measure shifting processes and the n-back test to measure updating processes. A multivariate logistic model was used to assess which variables explained best the level of nicotine dependence.

Results

Inhibition (p = 0.002) and updating (p = 0.014) processes, but not shifting processes, were associated with higher tobacco dependence. Inhibition capacity had a significant effect on the nicotine dependence level independently of age, education, time since last cigarette, intellectual quotient, craving, updating and shifting process.

Conclusions

Nicotine dependence level seems better explained by inhibition capacities than by craving and updating effects. The capacity to inhibit our behaviours is a good predictor of the severity of tobacco dependence. Our results suggest a prefrontal cortex dysfunction affecting the inhibitory capacities of heavy tobacco dependent smokers. Further studies are needed to investigate the application of these findings in the treatment of tobacco dependence.     

Pump-1 cDNA codes for a protein with characteristics similar to those of classical collagenase family members

Pump-1 cDNA has recently been isolated by screening a human tumor cDNA library with a transin (rat stromelysin) probe under low-stringency hybridization conditions. The cDNA codes for a potential protein with significant sequence similarity to the metalloproteinases collagenase and stromelysin, but which lacks the hemopexin-like domain characteristic of these enzymes. Expression of pump-1 cDNA in cos cells using an expression vector leads to secretion of a protein of Mr 28,000 with latent, organomercurial-activatable proteinase activity. Cos cells transfected with a partial pump-1 cDNA in the vector pPROTA secrete a fusion protein between the IgG-binding domains of staphylococcal protein A and pump-1. The fusion protein binds to IgG-Sepharose, and the bound fusion protein undergoes apparent autocleavage in the presence of 4-aminophenylmercuric acetate with elution of active pump-1 species of Mr 21,000 and 19,000. Active pump-1 degrades casein, gelatins of types I, III, IV, and V, and fibronectin and can activate collagenase. Active pump-1 is inhibited by EDTA, 1,10-phenanthroline, and the tissue inhibitor of metalloproteinases. These results show that, despite the absence of a hemopexin-like domain, pump-1 is a latent secreted metalloproteinase. Postpartum rat uteri contain elevated levels of rat pump-1 mRNA. On the basis of this observation, its size, and its substrate specificity, we suggest that pump-1 might correspond to a previously described uterine metalloproteinase, matrix metalloproteinase 7.     

The human VAV proto-oncogene maps to chromosome region 19p12→19p13.2

Summary A novel human oncogene, designated VAV, has been recently characterized. This oncogene was generated by a rearrangement within the 5 coding sequences of a normal cellular gene, the VAV proto-oncogene. The normal VAV gene is specifically expressed in hematopoietic cells regardless of their differentiation lineage. We now report that the VAV locus has been localized in the human genome at chromosome 19p12 19p13.2 by analysis of its segregation pattern in rodenthuman somatic cell hybrids and by chromosomal in situ hybridization. The VAV locus might be closely linked to the insulin receptor (INSR) locus, as suggested by comigration of INSR and VAV high-molecular-weight DNA fragments after pulsed-field gel electrophoresis. The VAV chromosomal assignment is of interest because chromosome region 19p13 is involved in different karyotypic abnormalities in a variety of malignancies including melanomas and leukemias. The identification of a novel proto-oncogene that maps to that region will enable us to define whether VAV is involved in any of the translocations observed.