Relationship between molecular and enological features of Patagonian wine yeasts: relevance in selection protocols

Summary In this paper, a study of the relationship between genetic patterns, obtained by the combination of mtDNA-RFLP and PCR-amplified inter-δ sequence DNA polymorphism analysis, and relevant enological phenotypic data (fermentative power, specific productivity, volatile and total acidity) was carried out on Argentinean Saccharomyces cerevisiae isolates from north Patagonia. The use of a powerful statistical tool, Generalized Procrustes analysis, allowed us to weigh the relationship for each isolate in particular, denoting a good enough degree of agreement between molecular and physiological data for most of the population analysed. The inclusion of a physiological feature, as the killer sensitivity biotype, within identification methods resulted in a higher degree of discrimination among isolates and in better correlation between both characterizations. The combined use of methods based on molecular polymorphisms and killer biotype could be applied so as not to miss any isolate with differential enological properties in selection protocols.     

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.     

Alginate beads provide a beneficial physical barrier against native microorganisms in wastewater treated with immobilized bacteria and microalgae

When the freshwater microalga Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense were deployed as free suspensions in unsterile, municipal wastewater for tertiary wastewater treatment, their population was significantly lower compared with their populations in sterile wastewater. At the same time, the numbers of natural microfauna and wastewater bacteria increased. Immobilization of C. sorokiniana and A. brasilense in small (2–4 mm in diameter), polymer Ca-alginate beads significantly enhanced their populations when these beads were suspended in normal wastewater. All microbial populations within and on the surface of the beads were evaluated by quantitative fluorescence in situ hybridization combined with scanning electron microscopy and direct measurements. Submerging immobilizing beads in wastewater created the following sequence of events: (a) a biofilm composed of wastewater bacteria and A. brasilense was created on the surface of the beads, (b) the bead inhibited penetration of outside organisms into the beads, (c) the bead inhibited liberation of the immobilized microorganisms into the wastewater, and (d) permitted an uninterrupted reduction of ammonium and phosphorus from the wastewater. This study demonstrated that wastewater microbial populations are responsible for decreasing populations of biological agents used for wastewater treatment and immobilization in alginate beads provided a protective environment for these agents to carry out uninterrupted tertiary wastewater treatment.     

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.     

Properties of D-Xylose Isomerase from Streptomyces albus

A partially purified D-xylose isomerase has been isolated from cells of Streptomyces albus NRRL 5778 and some of its properties have been determined. D-Glucose, D-xylose, D-ribose, L-arabinose, and L-rhamnose served as substrates for the enzyme with respective Km values of 86, 93, 350, 153, and 312 mM and Vmax values measuring 1.23, 2.9, 2.63, 0.153, and 0.048 mumol min per mg of protein. The hexose D-allose was also isomerized. The enzyme was strongly activated by 1.0 mM Mg2+ but only partially activated by 1.0 mM Co2+. The respective Km values for Mg2+ and Co2+ were 0.3 and 0.003 mM. Mg2+ and Co2+ appear to have separate binding sites on the isomerase. These cations also protect the enzyme from thermal denaturation and from D-sorbitol inhibition. The optimum temperature for ketose formation was 70 to 80 C at pH values ranging from 7 to 9. D-Sorbitol acts as a competitive inhibitor with a Ki of 5.5 mM against D-glucose, D-xylose, and D-ribose. Induction experiments, Mg2+ activation, and D-sorbitol inhibition indicated that a single enzyme (D-xylose isomerase) was responsible for the isomerization of the pentoses, methyl pentose, and glucose.     

Comparative analysis reveals amino acids critical for anticancer activity of peptide CIGB‐552

Because of resistance development by cancer cells against current anticancer drugs, there is a considerable interest in developing novel antitumor agents. We have previously demonstrated that CIGB‐552, a novel cell‐penetrating synthetic peptide, was effective in reducing tumor size and increasing lifespan in tumor‐bearing mice. Studies of protein–peptide interactions have shown that COMMD1 protein is a major mediator of CIGB‐552 antitumor activity. Furthermore, a typical serine‐protease degradation pattern for CIGB‐552 in BALB/c mice serum was identified, yielding peptides which differ from CIGB‐552 in size and physical properties. In the present study, we show the results obtained from a comparative analysis between CIGB‐552 and its main metabolites regarding physicochemical properties, cellular internalization, and their capability to elicit apoptosis in MCF‐7 cells. None of the analyzed metabolites proved to be as effective as CIGB‐552 in promoting apoptosis in MCF‐7. Taking into account these results, it seemed important to examine their cell‐penetrating capacity and interaction with COMMD1. We show that internalization, a lipid binding‐dependent process, is impaired as well as metabolite–COMMD1 interaction, key component of the apoptotic mechanism. Altogether, our results suggest that features conferred by the amino acid sequence are decisive for CIGB‐552 biological activity, turning it into the minimal functional unit. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.