Poly(A) polymerase from Escherichia coli adenylylates the 3'-hydroxyl residue of nucleosides, nucleoside 5'-phosphates and nucleoside(5')oligophospho(5')nucleosides (NpnN).

The capacity of Escherichia coli poly(A) polymerase to adenylylate the 3'-OH residue of a variety of nucleosides, nucleoside 5'-phosphates and dinucleotides of the type nucleoside(5')oligophospho(5')nucleoside is described here for the first time. Using micromolar concentrations of [alpha-32P]ATP, the following nucleosides/nucleotides were found to be substrates of the reaction: guanosine, AMP, CMP, GMP, IMP, GDP, CTP, dGTP, GTP, XTP, adenosine(5')diphospho(5')adenosine (Ap2A), adenosine (5')triphospho(5')adenosine (Ap3A), adenosine(5')tetraphospho(5')adenosine (Ap4A), adenosine(5')pentaphospho(5')adenosine (Ap5A), guanosine(5')diphospho(5') guanosine (Gp2G), guanosine(5')triphospho(5')guanosine (Gp3G), guanosine(5')tetraphospho(5')guanosine (Gp4G), and guanosine(5')pentaphospho(5')guanosine (Gp5G). The synthesized products were analysed by TLC or HPLC and characterized by their UV spectra, and by treatment with alkaline phosphatase and snake venom phosphodiesterase. The presence of 1 mM GMP inhibited competitively the polyadenylylation of tRNA. We hypothesize that the type of methods used to measure polyadenylation of RNA is the reason why this novel property of E. coli poly(A) polymerase has not been observed previously.     

Sph3 Is a Glycoside Hydrolase Required for the Biosynthesis of Galactosaminogalactan in Aspergillus fumigatus

Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (β/α)₈ fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.     

Progress in the prediction of pKa values in proteins

The pK(a) -cooperative aims to provide a forum for experimental and theoretical researchers interested in protein pK(a) values and protein electrostatics in general. The first round of the pK(a) -cooperative, which challenged computational labs to carry out blind predictions against pK(a) s experimentally determined in the laboratory of Bertrand Garcia-Moreno, was completed and results discussed at the Telluride meeting (July 6-10, 2009). This article serves as an introduction to the reports submitted by the blind prediction participants that will be published in a special issue of PROTEINS: Structure, Function and Bioinformatics. Here, we briefly outline existing approaches for pK(a) calculations, emphasizing methods that were used by the participants in calculating the blind pK(a) values in the first round of the cooperative. We then point out some of the difficulties encountered by the participating groups in making their blind predictions, and finally try to provide some insights for future developments aimed at improving the accuracy of pK(a) calculations.     

Involvement of eukaryotic translation initiation factor 5A (eIF5A) in skeletal muscle stem cell differentiation

The eukaryotic translation initiation factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post‐translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non‐differentiated satellite cells and skeletal muscle. The treatment with N1‐guanyl‐1,7‐diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L ‐Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration. J. Cell. Physiol. 218: 480–489, 2009. © 2008 Wiley‐Liss, Inc.     

Recombinant disintegrin domain of human ADAM9 inhibits migration and invasion of DU145 prostate tumor cells

One of the most important features of malignant cells is their capacity to invade adjacent tissues and metastasize to distant organs. This process involves the creation, by tumor and stroma cells, of a specific microenvironment, suitable for proliferation, migration and invasion of tumor cells. The ADAM family of proteins has been involved in these processes. This work aimed to investigate the role of the recombinant disintegrin domain of the human ADAM9 (rADAM9D) on the adhesive and mobility properties of DU145 prostate tumor cells. rADAM9D was able to support DU145 cell adhesion, inhibit the migration of DU145 cells, as well as the invasion of this cell line through matrigel in vitro. Overall this work demonstrates that rADAM9D induces specific cellular migratory properties when compared with different constructs having additional domains, specially those of metalloproteinase and cysteine-rich domains. Furthermore, we showed that rADAM9D was able to inhibit cell adhesion, migration and invasion mainly through interacting with α6β1 in DU145 tumor cell line. These results may contribute to the development of new therapeutic strategies for prostate cancer.     

Towards a ‘Sea‐Level Sensitive’ dynamic model: impact of island ontogeny and glacio‐eustasy on global patterns of marine island biogeography

A synthetic model is presented to enlarge the evolutionary framework of the General Dynamic Model (GDM) and the Glacial Sensitive Model (GSM) of oceanic island biogeography from the terrestrial to the marine realm. The proposed ‘Sea‐Level Sensitive’ dynamic model (SLS) of marine island biogeography integrates historical and ecological biogeography with patterns of glacio‐eustasy, merging concepts from areas as diverse as taxonomy, biogeography, marine biology, volcanology, sedimentology, stratigraphy, palaeontology, geochronology and geomorphology. Fundamental to the SLS model is the dynamic variation of the littoral area of volcanic oceanic islands (defined as the area between the intertidal and the 50‐m isobath) in response to sea‐level oscillations driven by glacial–interglacial cycles. The following questions are considered by means of this revision: (i) what was the impact of (global) glacio‐eustatic sea‐level oscillations, particularly those of the Pleistocene glacial–interglacial episodes, on the littoral marine fauna and flora of volcanic oceanic islands? (ii) What are the main factors that explain the present littoral marine biodiversity on volcanic oceanic islands? (iii) How can differences in historical and ecological biogeography be reconciled, from a marine point of view? These questions are addressed by compiling the bathymetry of 11 Atlantic archipelagos/islands to obtain quantitative data regarding changes in the littoral area based on Pleistocene sea‐level oscillations, from 150 thousand years ago (ka) to the present. Within the framework of a model sensitive to changing sea levels, we discuss the principal factors affecting the geographical range of marine species; the relationships between modes of larval development, dispersal strategies and geographical range; the relationships between times of speciation, modes of larval development, ecological zonation and geographical range; the influence of sea‐surface temperatures and latitude on littoral marine species diversity; the effect of eustatic sea‐level changes and their impact on the littoral marine biota; island marine species–area relationships; and finally, the physical effects of island ontogeny and its associated submarine topography and marine substrate on littoral biota. Based on the SLS dynamic model, we offer a number of predictions for tropical, subtropical and temperate volcanic oceanic islands on how rates of immigration, colonization, in‐situ speciation, local disappearance, and extinction interact and affect the marine biodiversity around islands during glacials and interglacials, thus allowing future testing of the theory.     

Proton pathways in a [NiFe]-hydrogenase: A theoretical study

We present here a theoretical study to investigate possible proton pathways in the [NiFe]-hydrogenase from Desulfovibrio gigas. The approach used in this study consists of a combination of Poisson-Boltzmann and Monte Carlo simulations together with a distance-based network analysis to find possible groups involved in the proton transfer. Results obtained at different pH values show a reasonable number of proton active residues distributed by the protein interior and surface, with a concentration around the metal centres. The electrostatic interactions in this protein are strong, as shown by the unusual shape of the titration curves of several sites. Some residue pairs show strongly correlated protonations, indicating the sharing and probably exchange of a proton between them. The conjugation of the PB and MC simulations with the distance-based analysis allows a detailed characterization of the possible proton pathways. We discuss previous suggestions and propose a new complete pathway for the proton transfer between the active site and the surface. This pathway is mainly composed of histidines and glutamic acid residues.     

Stability and performance of Xanthobacter autotrophicus GJ10 during 1,2-dichloroethane biodegradation

A nucleic acid-based approach was used to investigate the dynamics of a microbial community dominated by Xanthobacter autotrophicus GJ10 in the degradation of synthetic wastewater containing 1,2-dichloroethane (DCE). This study was performed over a 140-day period in a nonsterile continuous stirred-tank bioreactor (CSTB) subjected to different operational regimens: nutrient-limiting conditions, baseline operation, and the introduction of glucose as a cosubstrate. The microbial community was analyzed by a combination of fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Under nutrient-limiting conditions, DCE degradation was restricted, but this did not affect the dominance of strain GJ10, determined by FISH to comprise 85% of the active population. During baseline operation, DCE degradation improved significantly to over 99.5% and then remained constant throughout the subsequent experimental period. DGGE profiles revealed a stable, complex community, while FISH indicated that strain GJ10 remained the dominant species. During the addition of glucose as a cosubstrate, DGGE profiles showed a proliferation of other species in the CSTB. The percentage of strain GJ10 dropped to 8% of the active population in just 5 days, although this did not affect the DCE biodegradation performance. The return to baseline conditions was accompanied by the reestablishment of strain GJ10 as the dominant species, suggesting that this system responds robustly to external perturbations, both at the functional biodegradation level and at the individual strain level.     

Is prnt a Pseudogene? Identification of Ram Prt in Testis and Ejaculated Spermatozoa

A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrP(C)), expressed by the prion gene (prnp), into an abnormally folded isoform (PrP(Sc)) with amyloid-like features that causes scrapie in sheep among other diseases. prnp together with prnd (which encodes a prion-like protein designated as Doppel), and prnt (that encodes the prion protein testis specific - Prt) with sprn (shadow of prion protein gene, that encodes Shadoo or Sho) genes, constitute the "prion gene complex". Whereas a role for prnd in the proper functioning of male reproductive system has been confirmed, the function of prnt, a recently discovered prion family gene, comprises a conundrum leading to the assumption that ruminant prnt is a pseudogene with no protein expression. The main objective of the present study was to identify Prt localization in the ram reproductive system and simultaneously to elucidate if ovine prnt gene is transcribed into protein-coding RNA. Moreover, as Prt is a prnp-related protein, the amyloid propensity was also tested for ovine and caprine Prt. Recombinant Prt was used to immunize BALB/c mice, and the anti-Prt polyclonal antibody (APPA) immune response was evaluated by ELISA and Western Blot. When tested by indirect immunofluorescence, APPA showed high avidity to the ram sperm head apical ridge subdomain, before and after induced capacitation, but did not show the same behavior against goat spermatozoa, suggesting high antibody specificity against ovine-Prt. Prt was also found in the testis when assayed by immunohistochemistry during ram spermatogenesis, where spermatogonia, spermatocytes, spermatids and spermatozoa, stained positive. These observations strongly suggest ovine prnt to be a translated protein-coding gene, pointing to a role for Prt protein in the ram reproductive physiology. Besides, caprine Prt appears to exhibit a higher amyloid propensity than ovine Prt, mostly associated with its phenylalanine residue.     

New insights into adaptation and population structure of cork oak using genotyping by sequencing

Species respond to global climatic changes in a local context. Understanding this process, including its speed and intensity, is paramount due to the pace at which such changes are currently occurring. Tree species are particularly interesting to study in this regard due to their long generation times, sedentarism, and ecological and economic importance. Quercus suber L. is an evergreen forest tree species of the Fagaceae family with an essentially Western Mediterranean distribution. Despite frequent assessments of the species’ evolutionary history, large‐scale genetic studies have mostly relied on plastidial markers, whereas nuclear markers have been used on studies with locally focused sampling strategies. In this work, “Genotyping by sequencing” is used to derive 1,996 single nucleotide polymorphism markers to assess the species’ evolutionary history from a nuclear DNA perspective, gain insights into how local adaptation is shaping the species’ genetic background, and to forecast how Q. suber may respond to global climatic changes from a genetic perspective. Results reveal (a) an essentially unstructured species, where (b) a balance between gene flow and local adaptation keeps the species’ gene pool somewhat homogeneous across its distribution, but still allowing (c) variation clines for the individuals to cope with local conditions. “Risk of Non‐Adaptedness” (RONA) analyses suggest that for the considered variables and most sampled locations, (d) the cork oak should not require large shifts in allele frequencies to survive the predicted climatic changes. Future directions include integrating these results with ecological niche modeling perspectives, improving the RONA methodology, and expanding its use to other species. With the implementation presented in this work, the RONA can now also be easily assessed for other organisms.