Rational protein design of ThDP-dependent enzymes-engineering stereoselectivity

Benzoylformate decarboxylase (BFD) from Pseudomonas putida is an exceptional thiamin diphosphate-dependent enzyme, as it catalyzes the formation of (S)-2-hydroxy-1-phenylpropan-1-one from benzaldehyde and acetaldehyde. This is the only currently known S-selective reaction (92 % ee) catalyzed by this otherwise R-selective class of enzymes. Here we describe the molecular basis of the introduction of S selectivity into ThDP-dependent decarboxylases. By shaping the active site of BFD through the use of rational protein design, structural analysis, and molecular modeling, optimal steric stabilization of the acceptor aldehyde in a structural element called the S pocket was identified as the predominant interaction for adjusting stereoselectivity. Our studies revealed Leu461 as a hot spot for stereoselectivity in BFD. Exchange to alanine and glycine resulted in variants that catalyze the S-stereoselective addition of larger acceptor aldehydes, such as propanal with benzaldehyde and its derivatives-a reaction not catalyzed by the wild-type enzyme. Crystal structure analysis of the variant BFDL461A supports the modeling studies.     

Systematic investigation of optimal aptamer immobilization for protein-microarray applications

Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to a wide range of target molecules with high affinity and specificity. As nucleic acids, aptamers can undergo denaturation, but the process is reversible. As a result of this stability and the possibility of automated selection of aptamers, these oligonucleotides are highly promising capture molecules in microarray formats. In this study, his-tagged proteins and an aptamer directed against the his-tag were chosen as a model system. Different factors affect the activity of aptamers immobilized on a solid support like a microarray surface. The orientation of the immobilized aptamer plays an important role in correct aptamer folding and, thus, in effective binding of the corresponding target. Other important parameters identified in this work are the microarrays' surface charge as well as the length of the spacer between aptamer and solid support. These parameters were investigated systematically, resulting in the development of an aptamer-based microarray for detection of his-tagged proteins. The general applicability of the developed immobilization strategy was demonstrated by utilization of three different aptamers.     

Assay for glucosamine 6-phosphate using a ligand-activated ribozyme with fluorescence resonance energy transfer or CE-laser-induced fluorescence detection

A naturally occurring aptazyme, the glmS ribozyme, is adapted to an assay for glucosamine 6-phosphate, an effector molecule for the aptazyme. In the assay, binding of analyte allosterically activates aptazyme to cleave a fluorescently labeled oligonucleotide substrate. The extent of reaction, and hence analyte concentration, is detected by either fluorescence resonance energy transfer (FRET) or capillary electrophoresis with laser-induced fluorescence (CE-LIF). With FRET, assay signal is the rate of increase in FRET in presence of analyte. With CE-LIF, the assay signal is the peak height of cleavage product formed after a fixed incubation time. The assay has a linear response up to 100 (CE-LIF) or 500 microM (FRET) and detection limit of approximately 500 nM for glucosamine 6-phosphate under single-turnover conditions. When substrate is present in excess of the aptazyme, it is possible to amplify the signal by multiple turnovers to achieve a 13-fold improvement in sensitivity and detection limit of 50 nM. Successful signal amplification requires a temperature cycle to alternately dissociate cleaved substrate and allow fresh substrate to bind aptazyme. The results show that aptazymes have potential utility as analytical reagents for quantification of effector molecules.     

A pH-based method for measuring gaseous ammonia

Cumulative emission or gaseous concentrations of ammonia (NH₃) are commonly measured by trapping gaseous NH₃ in an acidic solution that is later analyzed for total ammonia content. This traditional acid trap method is inexpensive, reliable, and accurate, but it is labor-intensive and inconvenient for high-frequency sampling. This paper describes a new acid trap method in which total ammonia concentration in a citric acid solution is calculated from measured pH by applying a speciation model. With this new method, the quantity of captured ammonia in a single acid trap can be determined repeatedly over time. Testing through titrations, laboratory emission measurements, and field measurement of gaseous NH₃ showed that the method is accurate and reasonably precise. For the most sensitive case standard deviation was 8 μmol kg⁻¹ in titration results, and 10−20 μmol kg⁻¹ in the field trials. The lower quantitation limits from emission measurements and field trials were around 100 and 300 μmol kg⁻¹, respectively. Speciation modeling was used to assess the importance of interferences. Results showed that error due to minor contamination of chemicals and evaporation of water can be kept at low levels. Acidic gases may be a significant interference, but their presence is easy to recognize, and significant error can be avoided by selecting an appropriate acid concentration.     

Metal-Sulfate Induced Generation of ROS in Human Brain Cells: Detection Using an Isomeric Mixture of 5- and 6-Carboxy-2',7'-Dichlorofluorescein Diacetate (Carboxy-DCFDA) as a Cell Permeant Tracer

Evolution of reactive oxygen species (ROS), generated during the patho-physiological stress of nervous tissue, has been implicated in the etiology of several progressive human neurological disorders including Alzheimer's disease (AD) and amylotrophic lateral sclerosis (ALS). In this brief communication we used mixed isomers of 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA; C(25)H(14)C(l2)O(9); MW 529.3), a novel fluorescent indicator, to assess ROS generation within human neuronal-glial (HNG) cells in primary co-culture. We introduced pathological stress using the sulfates of 12 environmentally-, industrially- and agriculturally-relevant divalent and trivalent metals including Al, Cd, Cu, Fe, Hg, Ga, Mg, Mn, Ni, Pb, Sn and Zn. In this experimental test system, of all the metal sulfates analyzed, aluminum sulfate showed by far the greatest ability to induce intracellular ROS. These studies indicate the utility of using isomeric mixtures of carboxy-H(2)DCFDA diacetates as novel and highly sensitive, long-lasting, cell-permeant, fluorescein-based tracers for quantifying ROS generation in intact, metabolizing human brain cells, and in analyzing the potential epigenetic contribution of different metal sulfates to ROS-generation and ROS-mediated neurological dysfunction.     

Ecological and aromatic impact of two Gram-negative bacteria (Psychrobacter celer and Hafnia alvei) inoculated as part of the whole microbial community of an experimental smear soft cheese

The impact of the growth of two Gram-negative bacteria, Psychrobacter celer and Hafnia alvei, inoculated at 10² and 10⁶ cfu/g, on the dynamics of a multispecies community as well as on volatile aroma compound production during cheese ripening was investigated. Results showed that P. celer was able to successfully implant itself in cheese, regardless of its inoculation level. However, when it was inoculated at a high level, the bacterial biodiversity was drastically lowered from day 25 to the end of ripening. Overall, the presence of P. celer led to the higher production of volatile aroma compounds such as aldehydes, ketones and sulfur compounds. Regardless of its inoculation level, H. alvei barely affected the growth of the bacterial community and was subdominant at the end of ripening. It influenced total volatile aroma compound production with volatile sulfur compounds being the most abundant. Overall, these two bacteria were able to implant themselves in a cheese community and significantly contributed to the aromatic properties of the cheese. Their role in flavoring and their interactions with the technological microorganisms must be considered during cheese ripening and should be further investigated.     

Physiologically based persistent organic pollutant accumulation in pig tissues and their edible safety differences: An in vivo study

As one of the most important animal food sources, pigs are an important model in the assessment of human exposure to persistent organic pollutants (POPs). In the present study, the distribution of the administrated polychlorodibenzo-p-dioxin/furan (PCDD/F)–polychlorinated biphenyl (PCB) mixture and polybrominated diphenyl ethers (PBDEs) in the different tissues of pigs, including liver, lung, kidney, subcutaneous fat, mesentery and muscle, for understanding the physiologically based pollutant accumulation in these tissues and their edible safety was investigated. It was found that liver had a much higher potential to accumulate PCDD/Fs and dioxin-like pollutants like PCB-126 than the other tissues, but it did not specifically concentrate PBDE congeners as compared to the other tissues. The different PCDD/F congeners and PCB-126 followed a similar distribution pattern in the different tissues; also the different PBDE congeners have the similar pattern in these tissues. The liver’s higher concentrating potency for dioxin-like pollutants may result from its detoxification function, however, it can concentrate dioxin-like pollutants but not PBDEs may suggest that it is the toxicity but not lipophilic property of these POPs dominated their accumulation in liver tissues. Also the result suggested that liver is a high-risk edible tissue for dioxin-like pollutants. In conclusion, the present study suggested that physiologically based assessments are necessary for evaluating edible tissue safety in animal source foods.     

Physico‐chemical characterization of starches extracted from potatoes of the group Phureja

Starch was extracted from twenty‐four accessions of Group Phureja cultivated diploid potatoes, and from two commercial potato (Solanum tuberosum) varieties. Extracted starch samples were characterized and compared to industrial potato starch. Starch from Phureja generally exhibited smaller granule sizes and lower phosphorus content than starch from commercial potatoes. Amylose content and thermal properties (gelatinization temperature and enthalpy) were however in the same range for both groups. Starches from Phureja displayed very distinct pasting behavior from that of commercial potato. The former exhibited lower initial pasting viscosity but higher shear resistance. This may be related to lower starch granule size, causing lower swelling power and solubility. Iodine complexation results seem to indicate that phureja potatoes have higher proportion of amylopectin long chains. Phureja thus appears to be a promising new source of starch with specific physico‐chemical and functional properties intermediate between industrial potato and cereal starches.