Defining a novel cis element in the 3'-untranslated region of mammalian ribonucleotide reductase component R2 mRNA: role in transforming growth factor-beta 1 induced mRNA stabilization.

Ribonucleotide reductase R2 gene expression is elevated in BALB/c 3T3 fibroblasts treated with transforming growth factor beta 1. We investigated the possibility that the 3'-UTR of ribonucleotide reductase R2 mRNA contains regulatory information for TGF-beta 1 induced message stability. Using end-labeled RNA fragments in gel shift assays and UV cross-linking analyses, we detected in the 3'-UTR a novel 9 nucleotide (nt) cis element, 5'-GAGUUUGAG-3' site, which interacted specifically with a cytosolic protease sensitive factor to form a 75 kDa complex. The cis element protein binding activity was inducible and markedly up-regulated cross-link 4 h after TGF-beta 1 treatment of mouse BALB/c 3T3 cells. Other 3'-UTRs [IRE, GM-CSF, c-myc and homopolymer (U)] were poor competitors to the cis element with regard to forming the TGF-beta 1 dependent RNA-protein complex. However, the cis element effectively competed out the formation of the R2 3'-UTR protein complex. Cytosolic extracts from a variety of mammalian cell lines (monkey Cos7, several mouse fibrosarcomas and human HeLa S3) demonstrated similar TGF-beta 1 dependent RNA-protein band shifts as cell extract from BALB/c 3T3 mouse fibroblasts. Binding was completely prevented by several different mutations within the cis element, and by substitution mutagenesis, we were able to predict the consensus sequences, 5'-GAGUUUNNN-3' and 5'-NNNUUUGAG-3' for optimal protein binding. These results support a model in which the 9 nt region functions in cis to destabilize R2 mRNA in cells; and upon activation, a TGF-beta 1 responsive protein is induced and interacts with the 9 nt cis element in a mechanism that leads to stabilization of the mRNA. This appears to be the first example of a mRNA binding site that is involved in TGF-beta 1-mediated effects.     

Tolerance ofRhizobium phaseoli to acidity,aluminium and manganese

Summary Strains ofRhizobium phaseoli were evaluated with respect to their ability to grow or survis when subjected to various stresses common to soils of the tropics. These stresses included low pH, high Al and Mn. In liquid culture 10 strains ofR. phaseoli all grew well at pH 5.0, a few strains grew at pH 4.5 and one at pH 4.0. Some strains grew at pH 4.0 after repeated transfer to medium at this pH, but this characteristic remained stable for only one strain, S-442, which combined a tolerance to low pH with an improved tolerance to Al and Mn compared to the parent strain P-442. Survival studies of S-442 and P-442 in three acidic Brazilian soils, at their natural pH (4.2–4.6) and when limed to near neutral pH, showed little difference in numbers after a 21–30 day period. Only a one log cycle decrease in numbers of P-442 occurred in the Erechim soil that had a 185 mol/l Al concentration in the soil saturation extract. Strains ofR. phaseoli were screened for their ability to grow in liquid culture at pH 5.0 in the presence of Al up to 100 mol/l (16 strains) and Mn up to 320 g/ml (13 strains). Strains differed in relative tolerance to both Al and Mn with some strains being capable of excellent growth at the highest concentrations of Al and Mn employed. With the exception ofR. phaseoli (C-12) the ability to tolerate high levels of Mn did not show any relationship to Al tolerance. It was concluded that soil stress factors need not have a serious impact on survival ofR. phaseoli in soils because sufficient variability in tolerance to these factors occurs naturally among strains.

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Resumen Se evaluaron cepas deRhizobium phaseoli en relación con su habilidad para crecer o sobrevivir bajo las condiciones de stress que son habituales de suelos tropicales. Estas condiciones comprenden bajos pH y altas concentraciones de Al y Mn. Diez cepas deR. phaseoli crecieron bien, en cultivo líquido, a pH: 5; tan solo algunas lo hicieron a pH: 4.5 y únicamente una cepa creció a pH: 4.0. Aunque varias cepas consiguieron crecer a pH: 4.0, tras transferencias sucesivas a un medio con dicho pH, esta característica solo logró establizarse en la cepa S-442 que combinó esta tolerancia a la acidez con un incremento en la tolerancia a Al y Mn comparada con la cepa parental P-442. Se realizaron estudios sobre la supervivencia de las cepas S-442 y P-442 en tres suelose ácidos del Brasil, en estado natural (pH: 4.2–4.6) y neutralizados con cal. Se observaron pocas diferencias cuantitativas al cabo de un periodo de 21–30 dias un solo ciclo Log de P-442 mostró una disminución numérica en un suelo denominado Erechim que contenía 185 mol/l de Al medido en extracto de pasta saturada. Se estudió la capacidad de cepas deR. phaseoli para crecer en cultivo liquido a pH: 5.0 en presencia de hasta 100 mol/l de Al (16 cepas) y 320 g/ml de Mn (13cepas), algunas de estas cepas fueron capaces de crecer a las máximas concentraciones de Al y Mn utilizadas. ExceptuandoR. phaseoli (C-12) la tolerancia a niveles altos de Mn no estaba correlacionada con la tolerancia al Al. Se concluyó que estos factores edáficos de stress no deberían de tener efecto alguno en la supervivencia deR. phaseoli en suelo, ya que existe una variabilidad natural de tolerancias a dichos factores suficiente para garantizar esta supervivencia.

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Résumé Des souches deRhizobium phaseoli ont été téstées pour leur aptitude à se développer et à survivre lorsqu'elles soumises à diverses contraintes habituelles dans les sols tropicaux, c'est à dire des pH acides et à de fortes teneurs en Al et Mn. En culture liquide, 10 souches deR. phaseoli ont toutes poussé à pH 5,0, un petit nombre à pH 4,5 et une seule à pH 4,0. Certaines souches parviennent, après repiquages répétés, à se developper à pH 4,0; mais ce caractère ne s'est stabilisé que dans le cas d'une seule souche (S-442), qui présente à la fois une résistance à l'acidité et une tolérance accrue à Al et Mn par rapport à la souche originelle P-442. L'étude de la survie de S-442 et P-442 dans trois sols brésiliens acides, à leur pH naturel (4,2–4,6) et après chaulage à pH neutre, n'a montré au bout de 21 à 30 jours que de faibles différences numériques. Dans le sol d'Erechim, où la concentration en Al est de 185 mol/l, on n'a constaté dans l'extrait de sol à saturation qu'une diminution de la population d'un seul facteur logarithmique. Des souches deR. phaseoli ont été téstées pour leur aptitude à pousser en milieu liquide à pH 5,0 et en présence de concentrations allant jusqu'à 100 mol/l d'Al (16 souches) et 320 g/l de Mn (13 souches). Les souches différent en ce qui concerne leur résistance à la fois à Al et à Mn, certaines étant capables de se développer très bien aux concentrations en Al et Mn les plus élevées. A l'exception deR. phaseoli C-12, la tolérance aux fortes concentrations en Mn ne présente aucune corrélation avec la résistance à Al. En conclusion, les facteurs d'agression n'ont pas d'effet important sur la survie deR. phaseoli dans le sol, étant donné que le tolérance des souches à ces facteurs peut varier de façon suffisante.

     

Environmental Control on Fish and Macrocrustacean Spring Community-Structure,on an Intertidal Sandy Beach

The inter-annual variability of the fish and macrocrustacean spring community on an intertidal sandy beach near the Canche estuary (North of France) was studied from 2000 to 2013 based on weekly spring sampling over an 11-year period. Twenty-eight species representing 21 families were collected during the course of the study. The community was dominated by a few abundant species accounting for > 99% of the total species densities. Most individuals caught were young-of-the-year indicating the importance of this ecosystem for juvenile fishes and macrocrustaceans. Although standard qualitative community ecology metrics (species composition, richness, diversity, evenness and similarity) indicated notable stability over the study period, community structure showed a clear change since 2009. Densities of P. platessa, P. microps and A. tobianus decreased significantly since 2009, whereas over the period 2010-2013, the contribution of S. sprattus to total species density increased 4-fold. Co-inertia and generalised linear model analyses identified winter NAO index, water temperature, salinity and suspended particular matter as the major environmental factors explaining these changes. Although the recurrent and dense spring blooms of the Prymnesiophyte Phaeocystis globosa is one of the main potential threats in shallow waters of the eastern English Channel, no negative impact of its temporal change was detected on the fish and macrocrustacean spring community structure.     

The structure of the periplasmic nickel-binding protein NikA provides insights for artificial metalloenzyme design

Understanding the interaction of a protein with a relevant ligand is crucial for the design of an artificial metalloenzyme. Our own interest is focused on the synthesis of artificial monooxygenases. In an initial effort, we have used the periplasmic nickel-binding protein NikA from Escherichia coli and iron complexes in which N(2)Py(2) ligands (where Py is pyridine) have been varied in terms of charge, aromaticity, and size. Six "NikA/iron complex" hybrids have been characterized by X-ray crystallography, and their interactions and solution properties have been studied. The hybrids are stable as indicated by their K (d) values, which are all in the micromolar range. The X-ray structures show that the ligands interact with NikA through salt bridges with arginine residues and π-stacking with a tryptophan residue. We have further characterized these interactions using quantum mechanical calculations and determined that weak CH/π hydrogen bonds finely modulate the stability differences between hybrids. We emphasize the important role of the tryptophan residues. Thus, our study aims at the complete characterization of the factors that condition the interaction of an artificial ligand and a protein and their implications for catalysis. Besides its potential usefulness in the synthesis of artificial monooxygenases, our approach should be generally applicable in the field of artificial metalloenzymes.     

Insights into Maize LEA proteins: from proteomics to functional approaches

LEA (late embryogenesis abundant) proteins participate in plant stress tolerance responses, but the mechanisms by which protection occurs are not fully understood. In the present work the unfolded proteins from maize dry embryos were analyzed by mass spectrometry. Twenty embryo proteins were identified, and among them 13 corresponded to LEA-type proteins. We selected three major LEA proteins, Emb564, Rab17 and Mlg3, belonging to groups 1, 2 and 3, respectively, and we undertook a comparative study in order to highlight differences among them. The post-translational modifications of native proteins were analyzed and the anti-aggregation properties of recombinant Emb564, Rab17 and Mgl3 proteins were evaluated in vitro. In addition, the protective effects of the LEA proteins were assessed in living cells under stress in Escherichia coli cells and in Nicotiana bentamiana leaves agroinfiltrated with fluorescent LEA-green fluorescent protein (GFP) fusions. Protein visualization by confocal microscopy indicated that cells expressing Mg3-GFP showed reduced cell shrinkage effects during dehydration and that Rab17-GFP co-localized to leaf oil bodies after heat shock. Overall, the results highlight differences and suggest functional diversity among maize LEA groups.     

Structural rearrangements at the translocation pore of the human glutamate transporter, EAAT1

Structure-function studies of mammalian and bacterial excitatory amino acid transporters (EAATs), as well as the crystal structure of a related archaeal glutamate transporter, support a model in which TM7, TM8, and the re-entrant loops HP1 and HP2 participate in forming a substrate translocation pathway within each subunit of a trimer. However, the transport mechanism, including precise binding sites for substrates and co-transported ions and changes in the tertiary structure underlying transport, is still not known. In this study, we used chemical cross-linking of introduced cysteine pairs in a cysteine-less version of EAAT1 to examine the dynamics of key domains associated with the translocation pore. Here we show that cysteine substitution at Ala-395, Ala-367, and Ala-440 results in functional single and double cysteine transporters and that in the absence of glutamate or dl-threo-beta-benzyloxyaspartate (dl-TBOA), A395C in the highly conserved TM7 can be cross-linked to A367C in HP1 and to A440C in HP2. The formation of these disulfide bonds is reversible and occurs intra-molecularly. Interestingly, cross-linking A395C to A367C appears to abolish transport, whereas cross-linking A395C to A440C lowers the affinities for glutamate and dl-TBOA but does not change the maximal transport rate. Additionally, glutamate and dl-TBOA binding prevent cross-linking in both double cysteine transporters, whereas sodium binding facilitates cross-linking in the A395C/A367C transporter. These data provide evidence that within each subunit of EAAT1, Ala-395 in TM7 resides close to a residue at the tip of each re-entrant loop (HP1 and HP2) and that these residues are repositioned relative to one another at different steps in the transport cycle. Such behavior likely reflects rearrangements in the tertiary structure of the translocation pore during transport and thus provides constraints for modeling the structural dynamics associated with transport.     

Effect of progesterone administration on the distribution of oviductal carbohydrates in <Emphasis Type="Italic">Rana tigrina</Emphasis>

Our aim has been to determine whether carbohydrate distribution in the oviducts of progesterone-treated animals is comparable with that of seasonal breeders in Rana tigrina. Like many other anurans, R. tigrina oviduct exhibits a short straight portion (pars recta, pr) at the beginning followed by a long, highly coiled portion (pars convoluta, pc). Histologically, the oviduct of this species revealed some unique features, one of which was intense toluidine blue staining, specifically in the upper mucosal glands of pc4. Based on lectin reactivities in the epithelial cells and mucosal glands, patterns of lectin staining in the seasonal breeders were classified into seven types: R1-R3 (for pr) and C1-C4 (for pc). Typically, some lectins reacted selectively either with ciliated cells (concanavalin A) or non-cialiated cells (Ricinus communis agglutinin I and wheatgerm agglutinin); however, Bandeiraea simplicifolia agglutinin I reacted with both cell types. These staining patterns were different in the progesterone-treated animals. Differences in glycan distribution in the oviductal secretions were revealed by lectin blotting. Compared with the seasonal breeders, an enhanced staining of some lectins was noted in the hormone-treated animals: either an increased staining intensity of existing protein bands or additional staining of new protein bands. Inversely, the staining of wheatgerm agglutinin was markedly diminished in the hormone-treated animals, suggesting the inhibitory effect of progesterone on oviductal glycan distribution. Whether alteration in glycan distribution upon progesterone treatment affects the physiological properties of the released jelly substances remains to be addressed. This research was supported by Thailand Research Funds (to W.W.), a Research Initiate Grant from Kasetsart University (to A.T.), and Mahidol University.