Low-molecular weight protein profiling of genetically modified maize using fast liquid chromatography electrospray ionization and time-of-flight mass spectrometry

In this work, the use of liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC-TOFMS) has been evaluated for the profiling of relatively low-molecular weight protein species in both genetically modified (GM) and non-GM maize. The proposed approach consisted of a straightforward sample fractionation with different water and ethanol-based buffer solutions followed by separation and detection of the protein species using liquid chromatography with a small particle size (1.8 μm) C(18) column and electrospray-time-of-flight mass spectrometry detection in the positive ionization mode. The fractionation of maize reference material containing different content of transgenic material (from 0 to 5% GM) led to five different fractions (albumins, globulins, zeins, zein-like glutelins, and glutelins), all of them containing different protein species (from 2 to 52 different species in each fraction). Some relevant differences in the quantity and types of protein species were observed in the different fractions of the reference material (with different GM contents) tested, thus revealing the potential use of the proposed approach for fast protein profiling and to detect tentative GMO markers in maize.     

Tailoring the interface using thiophene small molecules in TiO(2)/P3HT hybrid solar cells

In this paper we focus on the effect of carboxylated thiophene small molecules as interface modifiers in TiO(2)/P3HT hybrid solar cells. Our results show that small differences in the chemical structure of these molecules, for example, the presence of the -CH(2)- group in the 2-thiopheneacetic acid (TAA), can greatly increase the TiO(2) surface wettability, improving the TiO(2)/polymer contact. This effect is important to enhance exciton splitting and charge separation.     

The manganese ion of the heterodinuclear Mn/Fe cofactor in Chlamydia trachomatis ribonucleotide reductase R2c is located at metal position 1

The essential catalytic radical of Class-I ribonucleotide reductase is generated and delivered by protein R2, carrying a dinuclear metal cofactor. A new R2 subclass, R2c, prototyped by the Chlamydia trachomatis protein was recently discovered. This protein carries an oxygen-activating heterodinuclear Mn(II)/Fe(II) metal cofactor and generates a radical-equivalent Mn(IV)/Fe(III) oxidation state of the metal site, as opposed to the tyrosyl radical generated by other R2 subclasses. The metal arrangement of the heterodinuclear cofactor remains unknown. Is the metal positioning specific, and if so, where is which ion located? Here we use X-ray crystallography with anomalous scattering to show that the metal arrangement of this cofactor is specific with the manganese ion occupying metal position 1. This is the position proximal to the tyrosyl radical site in other R2 proteins and consistent with the assumption that the high-valent Mn(IV) species functions as a direct substitute for the tyrosyl radical.     

Rationale for the opposite stereochemistry of the major monoadducts and interstrand crosslinks formed by mitomycin C and its decarbamoylated analogue at CpG steps in DNA and the effect of cytosine modification on reactivity

Mitomycin C (MMC) is a potent antitumour agent that forms a covalent bond with the 2-amino group of selected guanines in the minor groove of double-stranded DNA following intracellular reduction of its quinone ring and opening of its aziridine moiety. At some 5'-CG-3' (CpG) steps the resulting monofunctional adduct can evolve towards a more deleterious bifunctional lesion, which is known as an interstrand crosslink (ICL). MMC reactivity is enhanced when the cytosine bases are methylated (5 MC) and decreased when they are replaced with 5-F-cytosine (5FC) whereas the stereochemical preference of alkylation changes upon decarbamoylation. We have studied three duplex oligonucleotides of general formula d(CGATAAXGCTAACG) in which X stands for C, 5MC or 5FC. Using a combination of molecular dynamics simulations in aqueous solution, quantum mechanics and continuum electrostatics, we have been able to (i) obtain a large series of snapshots that facilitate an understanding in atomic detail of the distinct stereochemistry of monoadduct and ICL formation by MMC and its decarbamoylated analogue, (ii) provide an explanation for the altered reactivity of MMC towards DNA molecules containing 5MC or 5FC, and (iii) show the distinct accommodation in the DNA minor groove of the different covalent modifications, particularly the most cytotoxic C1α and C1β ICLs.     

Biosynthesis of mycobacterial methylglucose lipopolysaccharides

Mycobacterial pathogenesis is closely associated with a unique cell envelope rich in complex carbohydrates and unique lipids, among which are the mycolic acids. Mycobacteria also synthesize unique intracellular polymethylated polysaccharides (PMPSs), namely methylglucose lipopolysaccharides (MGLPs), which are acylated with short-chain fatty acids, and methylmannose polysaccharides (MMPs). Since PMPSs modulate the synthesis of long-chain fatty acids in vitro, the possibility of a similar role in vivo and the regulation of mycolic acids assembly have been anticipated. Unlike MGLPs, MMPs have been identified in M. smegmatis and other fast-growing mycobacteria but not in M. tuberculosis, implying an essential role for MGLPs in this pathogen and turning the biosynthetic enzymes into attractive drug targets. The genome of M. tuberculosis was decoded 14 years ago but only recently has the identity of the genes involved in MGLPs biosynthesis been investigated. Two gene clusters (Rv1208-Rv1213 and Rv3030-Rv3037c) containing a few genes considered to be essential for M. tuberculosis growth, have initially been proposed to coordinate MGLPs biosynthesis. Among these genes, only the product of Rv1208 for the first step in the MGLPs pathway has, so far, been crystallized and its three-dimensional structure been determined. However, recent results indicate that at least three additional clusters may be involved in this pathway. The functional assignment of authentic roles to some of these M. tuberculosis H37Rv genes sheds new light on the intricacy of MGLPs biogenesis and renewed interest on their biological role.     

Coenzyme Regeneration in Hexanol Oxidation Catalyzed by Alcohol Dehydrogenase

The enzymatic ways of coenzyme regeneration include the addition of a second enzyme to the system or the addition of the co-substrate. In the present study, both methods of enzymatic coenzyme (NAD⁺) regeneration were studied and compared in the reaction of hexanol oxidation catalyzed by alcohol dehydrogenase (ADH). As a source of ADH, commercial isolated enzyme and the whole baker??s yeast cells were used. First, coenzyme regeneration was employed in the reaction of acetaldehyde reduction catalyzed by the same enzyme that catalyzed the main reaction, and then NAD⁺ regeneration was applied in the reaction of pyruvate reduction catalyzed by l-lactate dehydrogenase (l-LDH). Hexanal was obtained as the product of hexanol oxidation catalyzed by isolated ADH while hexaonic acid was detected as a product of the same reaction catalyzed by baker??s yeast cells. All of the used biocatalysts were kinetically characterized. The mass reactions were described by the mathematical models. All models were validated in the batch reactor. One hundred percent hexanol conversion was obtained using permeabilized yeast cells using both methods of cofactor regeneration. By using isolated enzyme ADH, the higher conversion was achieved in a system with cofactor regeneration catalyzed by l-LDH.