Spectroscopic properties of two 5′-(4-dimethylamino)azobenzene conjugated G-quadruplex forming oligonucleotides

The synthesis of two 5'-end (4-dimethylamino)azobenzene conjugated G-quadruplex forming aptamers, the thrombin binding aptamer (TBA) and the HIV-1 integrase aptamer (T30695), was performed. Their structural behavior was investigated by means of UV, CD, fluorescence spectroscopy, and gel electrophoresis techniques in K+-containing buffers and water-ethanol blends. Particularly, we observed that the presence of the 5'-(4-dimethylamino)azobenzene moiety leads TBA to form multimers instead of the typical monomolecular chair-like G-quadruplex and almost hampers T30695 G-quadruplex monomers to dimerize. Fluorescence studies evidenced that both the conjugated G-quadruplexes possess unique fluorescence features when excited at wavelengths corresponding to the UV absorption of the conjugated moiety. Furthermore, a preliminary investigation of the trans-cis conversion of the dye incorporated at the 5'-end of TBA and T30695 showed that, unlike the free dye, in K+-containing water-ethanol-triethylamine blend the trans-to-cis conversion was almost undetectable by means of a standard UV spectrophotometer

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

New Dehydrogenase/Reductase as Biocatalysts for Stereoselective Reduction of Prochiral Ketones

Abstract Biocatalysis is well-matched to chemical synthesis in the pharmaceutical as well as the agrochemical and flavours industry. A significant number of products are chiral in at least one center, and many of them have multiple chiral centers. These compounds are usually manufactured in single isomer form since the current Food and Drug Administration regulations demand proof that the non-therapeutic isomer is non-toxic. The synthesis of a chiral center requires enantio and regioselective catalysts, and enzymes are consistently the most selective catalysts available being able to perform reactions under mild conditions of pH value, temperature, and pressure in aqueous solutions, a greener approach to chemistry, with remarkable chemo-, regio-, and stereoselectivity. Chiral alcohols are very important precursors for a large number of drugs, agrochemical and flavours. Their production by asymmetric bioreduction of a prochiral carbonyl precursor is becoming well-established in the field of biocatalysis. Enzymes that catalyze ketone reductions (known as ketoreductases) are a reliable source of high enantiomeric excess chiral alcohols. The aim of the present PhD research is to characterize new alcohol dehydrogenases (ADHs) for the synthesis of chiral secondary alcohols and the structural study of the isolated enzymes for the understanding of the structure-function relationships. The basic approach was to identify oxidoreductases with these distinctive features, operational stability and NAD dependency, by looking for in the genome of thermophilic microorganisms for genes coding putative short-chain dehydrogenases/reductases (SDR) with an acidic residue which determines NADH specificity in a particular position of the sequence. In the present work an applicative development of Thermus termophilus ADH in the asymmetric synthesis of two valuable chiral building blocks methyl (R)-mandelato and methyl (R)-o-chloromandelate at lab scale was carried out, and adaption of two different enzyme-coupled systems for the cofactor regeneration system was studied. Two novel NADH dependent-ADHs identified in the crenarchaeon Sulfolobus acidocaldarius SaADH and SaADH2 were biochemically characterized, a suitable cofactor regeneration system for the analytical study of the stereoselectivity both enzymes was utilized, moreover, the solved SaADH2 3D structure was discussed. Finally, an applicative utilization of Bacillus stearothermophilus ADH both as an isolated enzyme, and in recombinant E. coli whole cells in the synthesis of cinnamyl alcohol, a versatile fine chemical, by chemoselective reduction of cinnamaldehyde at gram scale was described

Alteration of ecdysone metabolism in Heliothis Virescens (F.) (Lepidoptera, Noctuidae) larvae induced by Cardiochiles Nigriceps Viereck (Hymenoptera, Braconidae) teratocytes

The haemolymph titres of ecdysteroids in last instar Heliothis virescens (F.) larvae parasitized by Cardiochiles nigriceps Viereck, or in larvae injected with teratocytes obtained from this parasitoid, were determined and compared to those of nonparasitized larvae. Ecdysteroids were extracted, purified by high performance liquid chromatography (HPLC), and the titres assessed by radioimmunoassay. The total ecdysteroid title of parasitized day 5 of 5th instar larvae was consistently lower than in nonparasitized larvae of the same age. The main ecdysteroid inactivation products were both polar and C-26 hydroxylated compounds, which were detected as soon as the 20-hydroxyecdysone appeared to be produced. The subsequent increase of the total ecdysteroid titre registered on day 7 of 5th instar parasitized larvae was almost completely due to polar compounds, whereas 20-hydroxyecdysone remained at low levels. In contrast, the total ecdysteroid titre of nonparasitized H. virescens larvae was higher and inactive metabolites increased in concentration only the day before pupation, after the typical 20-hydroxyecdysone burst. When C. nigriceps teratocytes, obtained from embryos hatched in vitro on a semi-defined medium, were injected into day 1 of nonparasitized host 5th instar larvae (new-slender stage), the total ecdysteroid titres on day 5 and 6 of these larvae were much higher than in nonparasitized controls. However, this high ecdysteroid titre was associated with low levels of 20-hydroxyecdysone and with a high titre of ecdysone and other unidentified ecdysteroids. The 20-hydroxyecdysone was the most abundant ecdysteroid released after enzymatic digestion of the polar ecdysteroids isolated from the haemolymph of parasitized day 7 of 5th instar larvae. In vitro incubation of teratocytes with radiolabelled 20-hydroxyecdysone resulted in the recovery of 34% of the total radioactivity in the HPLC fractions corresponding to polar metabolites. In contrast, only 4.5% of the total radioactivity was recovered in the same polar fractions when ecdysone was co-incubated with teratocytes. These results, along with those presented above, suggest that the inhibition of host pupation observed in parasitized H. virescens larvae is due in part to 20-hydroxyecdysone inactivation through the formation of polar metabolites and C. nigriceps teratocytes seem to play an important role in this biochemical transformation

Characterization of Two NMN Deamidase Mutants as Possible Probes for an NMN Biosensor

Nicotinamide mononucleotide (NMN) is a key intermediate in the nicotinamide adenine dinucleotide (NAD+) biosynthesis. Its supplementation has demonstrated beneficial effects on several diseases. The aim of this study was to characterize NMN deamidase (PncC) inactive mutants to use as possible molecular recognition elements (MREs) for an NMN-specific biosensor. Thermal stability assays and steady-state fluorescence spectroscopy measurements were used to study the binding of NMN and related metabolites (NaMN, Na, Nam, NR, NAD, NADP, and NaAD) to the PncC mutated variants. In particular, the S29A PncC and K61Q PncC variant forms were selected since they still preserve the ability to bind NMN in the micromolar range, but they are not able to catalyze the enzymatic reaction. While S29A PncC shows a similar affinity also for NaMN (the product of the PncC catalyzed reaction), K61Q PncC does not interact significantly with it. Thus, PncC K61Q mutant seems to be a promising candidate to use as specific probe for an NMN biosensor

Hooked on α-d-galactosidases: from biomedicine to enzymatic synthesis

<div><p></p><p>α-d-Galactosidases (EC 3.2.1.22) are enzymes employed in a number of useful bio-based applications. We have depicted a comprehensive general survey of α-d-galactosidases from different origin with special emphasis on marine example(s). The structures of natural α-galactosyl containing compounds are described. In addition to 3D structures and mechanisms of action of α-d-galactosidases, different sources, natural function and genetic regulation are also covered. Finally, hydrolytic and synthetic exploitations as free or immobilized biocatalysts are reviewed. Interest in the synthetic aspects during the next years is anticipated for access to important small molecules by green technology with an emphasis on alternative selectivity of this class of enzymes from different sources.</p></div

Purification and Characterization of a Novel Recombinant Highly Enantioselective Short-Chain NAD(H)-Dependent Alcohol Dehydrogenase from Thermus thermophilus▿

The gene encoding a novel alcohol dehydrogenase (ADH) that belongs to the short-chain dehydrogenase/reductase (SDR) superfamily was identified in the extremely thermophilic, halotolerant gram-negative eubacterium Thermus thermophilus HB27. The T. thermophilus ADH gene (adhTt) was heterologously overexpressed in Escherichia coli, and the protein (ADHTt) was purified to homogeneity and characterized. ADHTt is a tetrameric enzyme consisting of identical 26,961-Da subunits composed of 256 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to ∼73°C and a 30-min half-inactivation temperature of ∼90°C, as well as good tolerance to common organic solvents. ADHTt has a strict requirement for NAD(H) as the coenzyme, a preference for reduction of aromatic ketones and α-keto esters, and poor activity on aromatic alcohols and aldehydes. This thermophilic enzyme catalyzes the following reactions with Prelog specificity: the reduction of acetophenone, 2,2,2-trifluoroacetophenone, α-tetralone, and α-methyl and α-ethyl benzoylformates to (S)-(−)-1-phenylethanol (>99% enantiomeric excess [ee]), (R)-α-(trifluoromethyl)benzyl alcohol (93% ee), (S)-α-tetralol (>99% ee), methyl (R)-(−)-mandelate (92% ee), and ethyl (R)-(−)-mandelate (95% ee), respectively, by way of an efficient in situ NADH-recycling system involving 2-propanol and a second thermophilic ADH. This study further supports the critical role of the D37 residue in discriminating NAD(H) from NADP(H) in members of the SDR superfamily

A Fluorescence Polarization Assay to Detect Steroid Hormone Traces in Milk

Steroids are a class of hormones improperly used in livestock as growth-promoting agents.  Due to their high risk for human health, the European Union (EU) has strictly forbidden the administration of all natural and synthetic steroid hormones to food-producing animals, and the development of new rapid detection methods are greatly encouraged.  This work reports a novel fluorescence polarization assay, ready to use, capable of detecting 17β-estradiol directly in milk samples with a low limit of detection of <10 pmol.  It is based on the coupling of monospecific antibodies against 17β-estradiol and fluorophores, capable of modulating the fluorescence polarization emission on the basis of the specific binding of antibodies to fluorescence-labeled 17β-estradiol deriv.  The successful detection of 17β-estradiol has disclosed the development of an efficient method, easily extensible to any food matrix and having the potential to become a milestone in food quality and safety