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Surface based on polyelectrolytes functionalized with amino acids onto amino-terminated solid surfaces of silicon wafers was prepared, with the purpose of evaluate the chemical functionality of the polyelectrolyte films in adsorption and catalytic activity of an enzyme. In this work, the adsorption of the enzyme glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides (LmG6PD) was studied as model. The polyelectrolytes were obtained from poly (maleic anhydride-alt-vinylpyrrolidone) [poly(MA-alt-VP)] and functionalized with amino acids of different hydropathy index: glutamine (Gln), tyrosine (Tyr) and methionine (Met). The polyelectrolytes were adsorbed onto the amino-terminated silicon wafer at pH 3.5 and 4.5 and at low and high ionic strength. At low ionic strength and pH 3.5, the largest quantity of adsorbed polyelectrolyte was on the films containing glutamine moiety as the most hydrophilic amino acid in the side chain of polymer chain (5.88 mg/m 2 ), whereas at high ionic strength and pH 4.5, the lowest quantity was in films containing tyrosine moiety in the side chain (1.88 mg/m 2 ). The films were characterized by ellipsometry, contact angle measurements and atomic force microscopy (AFM). The polyelectrolyte films showed a moderate degree of hydrophobicity, the methionine derivative being the most hydrophobic film. With the aim of evaluate the effect of the amino acid moieties on the ability of the surface to adsorb enzymes, we study the activity of the enzyme on these surfaces. We observed that the polarity of the side chain of the amino acid in the polyelectrolyte affected the quantity of LmG6PD adsorbed, as well as its specific activity, showing that films prepared from poly(MA-alt-VP) functionalized with Met provide the best enzymatic performance. The results obtained demonstrated that the surfaces prepared from polyelectrolytes functionalized with amino acids could be an attractive and simple platform for the immobilization of enzymes, which could be of interest for biocatalysis applications., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Polyphosphate (polyP) is a linear biopolymer found in all living cells. In bacteria, mutants lacking polyphosphate kinase 1 (PPK1), the enzyme responsible for synthesis of most polyP, have many structural and functional defects. However, little is known about the causes of these pleiotropic alterations. The link between ppk1 deletion and those numerous phenotypes observed can be the result of complex molecular interactions that can be elucidated via a systems biology approach., Methods: By integrating different omics levels (transcriptome, proteome and phenome), we described the functioning of various metabolic pathways among Escherichia coli polyphosphate mutant strains (Δppk1, Δppx, and ΔpolyP). Bioinformatic analyses reveal the complex metabolic and regulatory bases of the phenotypes unique to polyP mutants., Results: Our results suggest that during polyP deficiency (Δppk1 mutant), metabolic pathways needed for energy supply are up-regulated, including fermentation, aerobic and anaerobic respiration. Transcriptomic and q-proteomic contrasting changes between Δppk1 and Δppx mutant strains were observed in those central metabolic pathways and confirmed by using Phenotypic microarrays. In addition, our results suggest a regulatory connection between polyP, second messenger metabolism, alternative Sigma/Anti-Sigma factors and type-II toxin-antitoxin (TA) systems., Conclusions: We suggest a broader role for polyP via regulation of ATP-dependent proteolysis of type II toxin-antitoxin system and alternative Sigma/Anti-Sigma factors, that could explain the multiple structural and functional deficiencies described due to alteration of polyP metabolism., General Significance: Understanding the interplay of polyP in bacterial metabolism using a systems biology approach can help to improve design of novel antimicrobials toward pathogens., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hemiparkinsonism induced by 6-hydroxydopamine (6-OHDA) injected in left corpus striatum is a recognized model of motor deficits in rats. Some reports concerning motor deficits indicate a favorable response to steroid administration in hemiparkinsonian animals. However, there is no much information regarding progesterone administration in relation to cognitive and affective dysfunctions. Here we could confirm earlier reports regarding a mild deficit of memory and a noticeable depressive-like behavior 4 weeks after injecting 6-OHDA. We also present some evidence that progesterone could be - when administered 7 days after the injection of 6-OHDA - a possible neuroprotector concerning both motor deficits as well as cognitive - memory- and depression-like behaviors. The affective deficit was reverted by administering the tricyclic antidepressant imipramine. Since Parkinson's disease is a conspicuous cause of psycho-organic decline in human beings, it would be important to be able of dealing early with non-motor indicators in order to use prospective neuroprotectors to prevent the progression of the disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Phosphofructokinase-2 (Pfk-2) belongs to the ribokinase family and catalyzes the ATP-dependent phosphorylation of fructose-6-phosphate, showing allosteric inhibition by a second ATP molecule. Several structures have been deposited on the PDB for this family of enzymes. A structure-based multiple sequence alignment of a non-redundant set of these proteins was used to infer phylogenetic relationships between family members with different specificities and to dissect between globally conserved positions and those common to phosphosugar kinases. We propose that phosphosugar kinases appeared early in the evolution of the ribokinase family. Also, we identified two conserved sequence motifs: the TR motif, not described previously, present in phosphosugar kinases but not in other members of the ribokinase family, and the globally conserved GXGD motif. Site-directed mutagenesis of R90 and D256 present in these motifs, indicate that R90 participates in the binding of the phosphorylated substrate and that D256 is involved in the phosphoryl transfer mechanism., (Copyright 2010 Elsevier Inc. All rights reserved.)

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL, LT) is the most aggressive type of primary cutaneous B-cell lymphoma. In a recent study on 12 patients it was found that inactivation of CDKN2A by either deletion of 9p21.3 or promoter hypermethylation is correlated with a worse prognosis. In the present EORTC multicenter study, skin biopsies of 64 PCLBCL, LT patients were analyzed by multiplex ligation-dependent probe amplification to validate these previous results and to fine-map the losses in this region. Although no minimal common region of loss could be identified, most homozygous loss was observed in the CDKN2A gene (43 of 64; 67%) encoding p16 and p14ARF. Promoter hypermethylation of p16 and p14ARF was found in six and zero cases, respectively. Survival was markedly different between patients with versus without aberrations in the CDKN2A gene (5-year disease-specific survival 43 versus 70%; P=0.06). In conclusion, our results confirm that deletion of chromosome 9p21.3 is found in a considerable proportion of PCLBCL, LT patients and that inactivation of the CDKN2A gene is associated with an unfavorable prognosis. In most patients the deletion involves a large area of at least several kilobase pairs instead of a small minimal common region.

Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATP-dependent 6-phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual non-Watson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the substrate and allosteric ATPs are also participating in the dimer-dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous phosphofructokinases from E. coli.

We have developed a system that allows focal drug application for cell culture microscopy. Single-cell drug delivery is achieved through the insertion of a patch-clamp-like micropipette in a microenvironment-controlled chamber mounted on a standard 35-mm Petri dish. The system has precise control of temperature, CO(2) concentration, and humidity, while preventing contamination during experiments. The use of standard Petri dishes allows long-term experiments by alternating in situ microscopy with incubator growth. Modern biological long-term experiments such as the characterization of drug effects on cell movement, axonal guidance, mitosis, apoptosis, differentiation, or volume regulation can be performed. The chamber is compatible with any inverted microscope without significant modifications.

Binding of MgATP to the allosteric site of phosphofructokinase-2 promotes a dimer to tetramer conversion. In the presence of Fru-6-P the enzyme remains as a dimer. Limited proteolysis in the presence of MgATP completely protects the enzyme against inactivation and cleavage, while Fru-6-P provides a partial protection. A 28-kDa proteolytic fragment containing the N-terminus of the protein is inactive, but retains the ability to bind Fru-6-P and the allosteric effector MgATP. The fragment remains as a dimer but does not form a tetramer in the presence of MgATP. The results suggest major conformational changes of the enzyme upon ligand binding that confer a higher degree of compactness to the monomers in the dimer and in the tetramer, demonstrate the presence of the active and allosteric sites in this N-terminus fragment, and stress the importance of the C-terminus region of the protein for catalytic activity and ligand-induced oligomerization.