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.     

Changes in the canopy structure of the Mediterranean shrub Lavandula stoechas after disturbance

Abstract. This study attempts to show the dynamics of the canopy structure of the Mediterranean pioneer shrub Lavandula stoechas after man-made perturbation (i.e. grazing). The development of the vertical structure of the shrub was studied by harvesting the canopy of plants of 2–6 yr old in horizontal layers. The supportive biomass of the canopy was concentrated near the base at all ages. Leaf biomass was evenly distributed all over the vertical profile in 2- and 3-yr old plants. In 4-yr old plants it presented a maximum near the top of the canopy. For 5-yr old plants a structural transition started with leaf profiles showing a bimodal distribution. Leaf biomass predominated near the base in 6-yr old plants, suggesting that the transition was completed. Three canopy stages in the growth processes of the plant were recognized after the first year of growth: in the first one (from 2 to 3 yr old) both leaf and supportive biomass increased; in the second one (from 3 to 4 yr) leaf biomass remained stable and there was an increase in supportive biomass until the plants reached a ‘mature stage’, in 4-yr old plants; finally, in 5- and 6-yr old plants there was a decrease both in leaf and supportive biomass and plant structure showed evidence of senescence. Early transitions from seedling to 1-yr old plant and from this to 2- to 3-yr old plants were less obvious. The leaf/supportive biomass ratio always decreased with plant age, from 1.88 in seedlings to 0.01 in 6-yr old plants. Biomass density followed the pattern of supportive biomass, with an increase from 1.7 g/dm³ (2-yr old plants) to 2.4 g/dm³ (4-yr old plants). Thereafter, biomass density decreased to 0.6 g/dm³ (6-yr old plants).     

Accumulation of Trehalose by Overexpression of tps1, Coding for Trehalose-6-Phosphate Synthase, Causes Increased Resistance to Multiple Stresses in the Fission Yeast Schizosaccharomyces pombe

Recent studies have shown that heat shock proteins and trehalose synthesis are important factors in the thermotolerance of the fission yeast Schizosaccharomyces pombe. We examined the effects of trehalose-6-phosphate (trehalose-6P) synthase overexpression on resistance to several stresses in cells of S. pombe transformed with a plasmid bearing the tps1 gene, which codes for trehalose-6P synthase, under the control of the strong thiamine-repressible promoter. Upon induction of trehalose-6P synthase, the elevated levels of intracellular trehalose correlated not only with increased tolerance to heat shock but also with resistance to freezing and thawing, dehydration, osmostress, and toxic levels of ethanol, indicating that trehalose may be the stress metabolite underlying the overlap in induced tolerance to these stresses. Among the isogenic strains transformed with this construct, one in which the gene coding for the trehalose-hydrolyzing enzyme, neutral trehalase, was disrupted accumulated trehalose to a greater extent and was more resistant to the above stresses. Increased trehalose concentration is thus a major determinant of the general stress protection response in S. pombe.     

Expansion strategies for human mesenchymal stromal cells culture under xeno‐free conditions

Choosing the culture system and culture medium used to produce cells are key steps toward a safe, scalable, and cost‐effective expansion bioprocess for cell therapy purposes. The use of AB human serum (AB HS) as an alternative xeno‐free supplement for mesenchymal stromal cells (MSC) cultivation has increasingly gained relevance due to safety and efficiency aspects. Here we have evaluated different scalable culture systems to produce a meaningful number of umbilical cord matrix‐derived MSC (UCM MSC) using AB HS for culture medium supplementation during expansion and cryopreservation to enable a xeno‐free bioprocess. UCM MSC were cultured in a scalable planar (compact 10‐layer flasks and roller bottles) and 3‐D microcarrier‐based culture systems (spinner flasks and stirred tank bioreactor). Ten layer flasks and roller bottles enabled the production of 2.6 ± 0.6 × 10⁴ and 1.4 ± 0.3 × 10⁴ cells/cm². UCM MSC‐based microcarrier expansion in the stirred conditions has enabled the production of higher cell densities (5.5–23.0 × 10⁴ cells/cm²) when compared to planar systems. Nevertheless, due to the moderate harvesting efficiency attained, (80% for spinner flasks and 46.6% for bioreactor) the total cell number recovered was lower than expected. Cells maintained the functional properties after expansion in all the culture systems evaluated. The cryopreservation of cells (using AB HS) was also successfully carried out. Establishing scalable xeno‐free expansion processes represents an important step toward a GMP compliant large‐scale production platform for MSC‐based clinical applications. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1358–1367, 2017     

Distribution and antifungal susceptibility of Candida clinical isolations coming from six health care centers in the metropolitan area of Caracas, Venezuela (years 2003-2005)

The aim of this study was to investigate the frequency and antifungal susceptibility of Candida clinical isolations coming from patients with candidiasis in six health care centers of Caracas, Venezuela metropolitan area. The laboratory reports were retrospectively revised from January 2003 through August 2005. The isolated yeasts identification was carried out by conventional methods and antifungal susceptibility was evaluated by ATB-fungus (bioMérieux, France) and Etest (AB Biodisk, Solna, Sweden). One thousand nine hundred seventy seven (1.977) yeasts were studied and their susceptibility testing were carried out only in 1,414 of them. C. albicans was the most isolated yeast (46.7%) and none-albicans Candida-species represented more than half of the isolations (53.4%). All the isolated yeasts evaluated presented CMIs<1 microg/ml to anfotericina B and showed variable susceptibility percentages to fluconazole (91.5%), itraconazole (80%) and voriconazole (98.6%).     

Genetic Diversity Within and among Disjunct Populations of the Mediterranean Island Endemic Delphinium pictum and D. requienii (Ranunculaceae)

Allozyme diversity was assessed in 13 populations of Delphinium pictum and D. requienii, two short-lived and closely related insular Mediterranean endemics. While D. pictum has scattered distribution in Corsica, Sardinia and Majorca, D. requienii is found in a few small populations restricted to the Hyères Archipelago. Eleven enzyme systems were assayed, and 15 loci were resolved. Both species harboured moderate levels of genetic diversity, comparable to the values expected for endemic plants. All genetic parameters suggested higher diversity in D. pictum (A?=?1.93, P?=?40, H _e?=?0.106) than in D. requienii (A?=?1.30, P?=?26.7, H _e?=?0.096); F _IS values revealed higher inbreeding in D. requienii. Although the two species did not harbour species-specific alelles in the surveyed loci, the UPGMA dendrogram based on Nei’s genetic similarity index supported divergence between them. These results, together with significant morphological similarity and evidence of successful hybridization between the species, support the hypothesis of a recent speciation event. The moderate levels of both genetic variability and population divergence observed for D. pictum are likely attributable to its ability to establish seed banks. Given that the allozyme variation detected for D. pictum was significantly partitioned among islands, we suggest the implementation of conservation programs throughout its distribution range, and not only in Corsica as it occurs nowadays. Management strategies are also desirable for D. requienii to maintain population size and control inbreeding.