Comparative genomic analysis reveals novel facts about Leptospirillum spp. cytochromes

Chemolithoautotrophic acidophilic bacteria, which belong to the genus Leptospirillum, can only grow with Fe(II) as electron donor and oxygen as an electron acceptor. Members of this genus play an important role in bioleaching sulfide ores. We used nearly complete genome sequences of Leptospirillum ferrooxidans (group I), Leptospirillum rubarum, Leptospirillum '5-way CG' (group II) and Leptospirillum ferrodiazotrophum (group III) to identify cytochromes that are likely involved in electron transfer chain(s). The results show the presence of genes encoding a number of c-type cytochromes (18-20 genes were identified in each species), as well as bd and cbb? oxidases. Genes encoding cbb? oxidase are clustered, with predicted genes involved in cbb? maturation proteins. Duplication of cbb? encoding genes (ccoNO) was detected in all four genomes. Interestingly, these micro-organisms also contain genes that potentially encode bc? and b?f-like complexes organized into two putative operon structures. To date, the Leptospirillum genus includes the only organisms reported to have genes coding for two different bc complexes. This study provides detailed insights into the components of electron transfer chains of Leptospirillum spp., revealing their conservation among leptospirilla groups and suggesting that there may be a single common pathway for electron transport between Fe(II) and oxygen.     

Cryopreservation of umbilical cord mesenchymal cells in xenofree conditions

Background aimsMesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues.MethodsMSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive^® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays.ResultsPost-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure.ConclusionsA method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.     

Analysis of karyotypic stability of homoeologous-pairing (ph) mutants in allopolyploid wheats

Karyotypic analysis of wheat lines with different genotypes for the homoeologous-pairing loci Ph1 and Ph2 was carried out by means of a genomic in situ hybridization method that allowed unequivocal identification of the A, B and D genomes. Chromosomal rearrangements mainly affecting the A and D genomes were found in all plants of allohexaploid wheat (AABBDD) lacking Ph1 activity. The frequency of intergenomic exchanges per plant in ph1b mutant and nulli-5B lines was 4.31 and 3.40, respectively. In addition, an unbalanced genomic constitution was found in a few plants, some even showing a euploid chromosomal number. By contrast, rearranged karyotypes were detected neither in the ph1 mutant line (ph1c) of allotetraploid wheat (AABB) nor in the allohexaploid wheat lines lacking Ph2 activity, namely ph2b mutant and nulli-3D lines. These results were compared with the chromosomal pairing behaviour displayed by mutant lines ph1c, ph1b and ph2b at first meiotic metaphase. Despite the finding of standard, nonrearranged karyotypes in the phlc tetraploid mutant, the frequency of A-B homoeologous metaphase I association was similar to that observed in the ph1b hexaploid mutant. The results presented clearly demonstrate that inactivity of the Ph1 locus induces karyotypic instability in wheat. Intergenomic exchanges have probably been accumulating since the original ph1 mutant and aneuploid lines were obtained, which should be taken into account when it is planned to use these lines for basic research on Ph1 function or in applied wheat breeding programmes.     

Spliceosome-targeted therapies trigger an antiviral immune response in triple-negative breast cancer

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In vitro rescue of isolated embryos of Bactris major jacq. and Desmoncus orthacanthos mart., potentially useful native palms from the Yucatan Peninsula (Mexico)

Summary Bactris major and Desmoncus orthacanthos are native palms from the Yucatan Peninsula which could be used as substitutes for rattan. When their seeds were germinated in vivo and in vitro they proved to be highly recalcitrant. Therefore, the culture of isolated embryos was studied as an alternative means of producing planting material for nurseries. It was found that the in vitro germination of the isolated embryos was gradually reduced by storage, falling to zero by 5 wk. However, isolated embryos from freshly collected seeds germinated at ∼100% frequency. The presence of the endosperm, whether still attached to the embryos or separated from them but in direct contact with the nutrient medium, greatly reduced germination in both species. High concentrations of abscisic acid (ABA, 100 μM) only slightly diminished it, suggesting a different cause for the observed endosperm-induced inhibition. This embryo rescue method permits the production of sufficient plants for in vitro micropropagation and the establishment of experimental plots to evaluate the full potential of these materials.     

Cloning and functional expression of a rodent brain cDNA encoding a novel protein with agmatinase activity, but not belonging to the arginase family

A rat brain cDNA encoding for a novel protein with agmatinase activity was cloned and functionally expressed. The protein was expressed as a histidine-tagged fusion product with a molecular weight of about 63 kDa. Agmatine hydrolysis was strictly dependent on Mn(2+); K(m) and k(cat) values were 2.5+/-0.2 mM and 0.8+/-0.2 s(-1), respectively. The product putrescine was a linear competitive inhibitor (K(i)=5+/-0.5 mM). The substrate specificity, metal ion requirement and pH optimum (9.5) coincide with those reported for Escherichia coli agmatinase, the best characterized of the agmatinases. However, as indicated by the k(cat)/K(m) (320 M(-1)s(-1)), the recombinant protein was about 290-fold less efficient than the bacterial enzyme. The deduced amino sequence revealed great differences with all known agmatinases, thus excluding the protein from the arginase family. It was, however, highly identical (>85%) to the predicted sequences for fragments of hypothetical or unnamed LIM domain-containing proteins. As a suggestion, the agmatinase activity is adscribed to a protein with an active site that promiscuously catalyze a reaction other than the one it evolved to catalyze.     

Mimicking rubella virus particles by using recombinant envelope glycoproteins and liposomes.

The envelope glycoproteins E1 and E2 of rubella virus (RV) were engineered to display the FLAG epitope tag and a polyhistidine tag, at their amino and carboxy termini, respectively. These modified envelope proteins were produced in Sf9 insect cells utilizing baculovirus expression vectors, the E1 and E2 vectors giving rise to protein products of about 58 and 42 kDa, respectively. The recombinant proteins were purified by immobilized metal-ion affinity chromatography and reconstituted into liposomes via their hydrophobic transmembrane anchors. The liposomes were prepared by detergent dialysis in the presence of europium-DTPA chelate, enabling the subsequent measurement of the binding of the resultant proteoliposomes to the antibodies by time resolved fluorescence. RV mimicking proteoliposomes were recognized by antibodies specific for the E1 and E2 proteins, as well as the FLAG epitope tag. This type of virosome may prove useful for studies on the basic biological events of an RV infection or as diagnostic reagents.