Physicochemical studies of amylose and its derivatives in aqueous solutions: Thermodynamics of the iodine–triiodide complex

Thermodynamic properties of the amylose–iodine–triiodide complex have been studied by spectrophotometry and by calorimetry using previously studied samples of amylose ionic derivatives, carboxymethylamylose and diethylaminoethylamylose. The ratio of triiodide to total molecular iodine ([I₃]_b/[I]_b + [I₂]_b) in the complex is ca. 0.3 over a range of iodide concentration from 10^?5 to 10^?4M, and there is no evidence for an increasing charge at slightly higher iodide concentration. Direct calorimetric experiments have been carried out in different conditions of polymer, iodine, and iodide concentration in order to study the dependence of the heat of the complexation as a function of the above parameters. It is shown that the dependence of the measured ΔH on the iodide concentration simply derives from the rearrangement of the triiodide equilibrium because of the uptake of a fixed ratio of iodine and triiodide molecules in the complex.     

From the cradle of grapevine domestication: molecular overview and description of Georgian grapevine (Vitis vinifera L.) germplasm

Historical information and archaeological and palaeobotanical findings point Georgia, in the South Caucasus, as a cradle for grapevine (Vitis vinifera L.) domestication from its wild form (V. vinifera silvestris Beck.) and subsequent selection and development of varieties with characters suitable for human consumption. The hypothesis of Georgia being a center of domestication, combined with its distance from western countries and the importance of its viticulture and wine production, make Georgian grape germplasm particularly interesting to be investigated under the genetic point of view. Twenty nuclear microsatellite loci were used to genotype 112 Georgian grapevine accessions (V. vinifera sativa Beck.) from germplasm collections and 18 from spontaneous growing plants (V. vinifera silvestris Beck.) found in wild conditions and to compare them to a large international cultivar collection in France. Data analysis shows that Georgian grapevine germplasm has maintained distinctive traits despite arrival of international, foreign varieties and still conserve characteristics of local breeding linked to traditional wine production regions of the country. Results have identified alleles, overall loci, well represented in the Georgian germplasm (cultivated and wild) and absent or poorly represented in other countries, highlighting uniqueness and originality of traits of this viticulture. Moreover, the search for relationships between Georgian and foreign viticulture has evidenced few interesting cases linking the Georgian varieties with Western European ones and with neighboring Caucasian countries, helping to identify the real place of origin in some doubtful cases. In addition, populations or sparse individuals of wild grapevine still preserved in the Georgian natural environments present smaller genetic distances with local cultivars than in other European regions. Principal component analysis (PCA) has also identified special overlapping of the wild compartment with some cultivated varieties. This work provides a highly significant new contribution to applied aspects of Georgian grapevine genetic resources management and use. Uniqueness of the Georgian cultivated grapevine gene pool together with its close relatedness with the wild compartment makes this country a good candidate to address questions regarding domestication and grapevine genetic resource conservation.     

Lemon peel and Limoncello liqueur: A proteomic duet

Combinatorial peptide ligand libraries (CPLLs) have been adopted for investigating the proteomes of lemon peels and pulp, of a home-made alcoholic infusion of peels and of a very popular Italian liqueur called “Limoncello”, stated to be an infusion of the flavedo (the outer, yellow skin of lemons). The aim of this study was not only to perform the deepest investigation so far of the lemon peel proteome but also to assess the genuineness of the commercial liqueur via a three-pronged attack. First, different extraction techniques have been used for the characterization of the peel (and additionally of the pulp) proteome, secondly a home-made infusion has been analysed and finally the proteome of the commercial drink was checked. The peel (the flavedo, not the underlying layer called albedo) proteome has been evaluated via prior capture with CPLLs at different pH values (2.2 and 7.2). Via mass spectrometry analysis of the recovered fractions, after elution of the captured populations in 4% boiling SDS, we could identify a total of 1011 unique gene products in the peel extracts and 674 in the pulp, 264 proteins in the home-made infusion and just 8 proteins (and protein fragments), together with 12 peptides, in one Italian Limoncello produced in the Sorrento Region, thus proving the genuineness of this product. On the contrary, cheaper Limoncellos were devoid of any protein/peptide, casting doubts on their production from vegetable extracts. This could be the starting point for investigating the genuineness and natural origin of commercial drinks in order to protect consumers from adulterated products.     

Chlorella vulgaris as a lipid source: Cultivation on air and seawater‐simulating medium in a helicoidal photobioreactor

The freshwater microalga Chlorella vulgaris was cultured batchwise on the seawater‐simulating Schlösser medium either in a 1.1‐L‐working volume helicoidal photobioreactor (HeP) or Erlenmeyer flask (EF) as control and continuously supplying air as CO₂ source. In these systems, maximum biomass concentration reached 1.65 ± 0.17 g L^?1 and 1.25 ± 0.06 g L^?1, and maximum cell productivity 197.6 ± 20.4 mg L^?1 day^?1 and 160.8 ± 12.2 mg L^?1 day^?1, respectively. Compared to the Bold's Basal medium, commonly employed to cultivate this microorganism on a bench‐scale, the Schlösser medium ensured significant increases in all the growth parameters, namely maximum cell concentration (268% in EF and 126% in HeP), maximum biomass productivity (554% in EF and 72% in HeP), average specific growth rate (67% in EF and 42% in HeP), and maximum specific growth rate (233% in EF and 22% in HeP). The lipid fraction of biomass collected at the end of runs was analyzed in terms of both lipid content and fatty acid profile. It was found that the seawater‐simulating medium, despite of a 56–63% reduction of the overall biomass lipid content compared to the Bold's Basal one, led in HeP to significant increases in both the glycerides‐to‐total lipid ratio and polyunsaturated fatty acid content compared to the other conditions taken as an average. These results as a whole suggest that the HeP configuration could be a successful alternative to the present means to cultivate C. vulgaris as a lipid source. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:279–284, 2016     

Identification and characterization of carbapenem binding sites within the RND-transporter AcrB

Understanding the molecular determinants for recognition, binding and transport of antibiotics by multidrug efflux systems is important for basic research and useful for the design of more effective antimicrobial compounds. Imipenem and meropenem are two carbapenems whose antibacterial activity is known to be poorly and strongly affected by MexAB-OprM, the major efflux pump transporter in Pseudomonas aeruginosa. However, not much is known regarding recognition and transport of these compounds by AcrAB-TolC, which is the MexAB-OprM homologue in Escherichia coli and by definition the paradigm model for structural studies on efflux pumps. Prompted by this motivation, we unveiled the molecular details of the interaction of imipenem and meropenem with the transporter AcrB by combining computer simulations with biophysical experiments. Regarding the interaction with the two main substrate binding regions of AcrB, the so-called access and deep binding pockets, molecular dynamics simulations revealed imipenem to be more mobile than meropenem in the former, while comparable mobilities were observed in the latter. This result is in line with isothermal titration calorimetry, differential scanning experiments, and binding free energy calculations, indicating a higher affinity for meropenem than imipenem at the deep binding pocket, while both sharing similar affinities at the access pocket. Our findings rationalize how different physico-chemical properties of compounds reflect on their interactions with AcrB. As such, they constitute precious information to be exploited for the rational design of antibiotics able to evade efflux pumps.     

Hepatic overexpression of sterol carrier protein-2 inhibits VLDL production and reciprocally enhances biliary lipid secretion

We examined in vivo a role for sterol carrier protein-2 (SCP-2) in the regulation of lipid secretion across the hepatic sinusoidal and canalicular membranes. Recombinant adenovirus Ad.rSCP2 was used to overexpress SCP-2 in livers of mice. We determined plasma, hepatic, and biliary lipid concentrations; hepatic fatty acid (FA) and cholesterol synthesis; hepatic and biliary phosphatidylcholine (PC) molecular species; and VLDL triglyceride production. In Ad.rSCP2 mice, there was marked inhibition of hepatic fatty acids and cholesterol synthesis to <62% of control mice. Hepatic triglyceride contents were decreased, while cholesterol and phospholipids concentrations were elevated in Ad.rSCP2 mice. Hepatic VLDL triglyceride production fell in Ad.rSCP2 mice to 39% of control values. As expected, biliary cholesterol, phospholipids, bile acids outputs, and biliary PC hydrophobic index were significantly increased in Ad.rSCP2 mice. These studies indicate that SCP-2 overexpression in the liver markedly inhibits lipid synthesis as well as VLDL production, and alters hepatic lipid contents. In contrast, SCP-2 increased biliary lipid secretion and the proportion of hydrophobic PC molecular species in bile. These effects suggest a key regulatory role for SCP-2 in hepatic lipid metabolism and the existence of a reciprocal relationship between the fluxes of lipids across the sinusoidal and canalicular membranes.     

The conserved glutamate residue adjacent to the Walker-B motif is the catalytic base for ATP hydrolysis in the ATP-binding cassette transporter BmrA

ATP-binding cassette (ABC) proteins constitute one of the widest families in all organisms, whose P-glycoprotein involved in resistance of cancer cells to chemotherapy is an archetype member. Although three-dimensional structures of several nucleotide-binding domains of ABC proteins are now available, the catalytic mechanism triggering the functioning of these proteins still remains elusive. In particular, it has been postulated that ATP hydrolysis proceeds via an acid-base mechanism catalyzed by the Glu residue adjacent to the Walker-B motif (Geourjon, C., Orelle, C., Steinfels, E., Blanchet, C., Deléage, G., Di Pietro, A., and Jault, J. M. (2001) Trends Biochem. Sci. 26, 539-544), but the involvement of such residue as the catalytic base in ABC transporters was recently questioned (Sauna, Z. E., Muller, M., Peng, X. H., and Ambudkar, S. V. (2002) Biochemistry, 41, 13989-14000). The equivalent glutamate residue (Glu504) of a half-ABC transporter involved in multidrug resistance in Bacillus subtilis, BmrA (formerly known as YvcC), was therefore mutated to Asp, Ala, Gln, Ser, and Cys residues. All these mutants were fully devoid of ATPase activity, yet they showed a high level of vanadate-independent trapping of 8-N3-alpha-32P-labeled nucleotide(s), following preincubation with 8-N3-[alpha-32P]ATP. However, and in contrast to the wild-type enzyme, the use of 8-N3-[gamma-32P]ATP unequivocally showed that all the mutants trapped exclusively the triphosphate form of the analogue, suggesting that they were not able to perform even a single hydrolytic turnover. These results demonstrate that Glu504 is the catalytic base for ATP hydrolysis in BmrA, and it is proposed that equivalent glutamate residues in other ABC transporters play the same role.     

Hypertrophic cardiomyopathy: two homozygous cases with "typical" hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy

About 10% of cases of hypertrophic cardiomyopathy (HCM) evolve into dilated cardiomyopathy (DCM) with unknown causes. We studied 11 unrelated patients (pts) with HCM who progressed to DCM (group A) and 11 who showed "typical" HCM (group B). Mutational analysis of the beta-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), and cardiac troponin T (TNNT2) genes demonstrated eight mutations affecting MYH7 or MYBPC3 gene, five of which were new mutations. In group A-pts, the first new mutation occurred in the myosin head-rod junction and the second occurred in the light chain-binding site. The third new mutation leads to a MYBPC3 lacking titin and myosin binding sites. In group B, two pts with severe HCM carried two homozygous MYBPC3 mutations and one with moderate hypertrophy was a compound heterozygous for MYBPC3 gene. We identified five unreported mutations, potentially "malignant" defects as for the associated phenotypes, but no specific mutations of HCM/DCM.