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A one-step analysis method was developed for four types of amino acids using a microfluidic paper-based analytical device fabricated from chromatography filtration paper and laminate films. Aminoacyl-tRNA synthetase was used to detect each amino acid. The obtained laminated paper-based analytical device (LPAD) contained four enzymatic reaction areas. Colorimetric detection was performed based on the molybdenum blue reaction. A model method for the simple, easy, and simultaneous detection of several amino acid concentrations was suggested, in contrast to the conventional methods such as HPLC or LC–MS. The method provided a selective quantification at the ranges of 3.6–100 μM for tryptophan, 10.1–100 μM for glycine, 5.9–100 μM for histidine and 5.6–100 μM for lysine with a detection limit of 1.1 μM, 3.3 μM, 1.9 μM and 1.8 μM, respectively. LPAD fabrication was considerably simple, and the subsequent detection process was easy and required a short period of time (within 15 min). [ABSTRACT FROM AUTHOR]

Abstract: An amino acid-sensing system with absorption spectrophotometric detection was developed. To ensure specific recognition of each amino acid, aminoacyl-tRNA synthetases were employed and the concentration of NADH produced by way of several enzymatic reactions was measured. Using this detection system, from 1.5 to 55μM of histidine and from 15 to 95μM of lysine could be measured selectively in HEPES-KOH buffer (pH8.0). [Copyright &y& Elsevier]

Abstract: The analysis of free amino acids in urine and plasma is useful for estimating disease status in clinical diagnoses. Changes in the concentration of free amino acids in foods are also useful markers of freshness, nutrition, and taste. In this study, the specific interaction between aminoacyl–tRNA synthetase (aaRS) and its corresponding amino acid was used to measure amino acid concentrations. Pyrophosphate released by the amino acid–aaRS binding reaction was detected by luminol chemiluminescence; the method provided selective quantitation of 1.0–30μM histidine and 1.0–60μM lysine. [Copyright &y& Elsevier]

A phosphate-selective molecularly imprinted polymer was prepared using 1-allyl-2-thiourea as a functional monomer, and a method of sensing for phosphate was suggested using an ion-sensitive field-effect transistor (ISFET) or pH meter as a transducer. The phosphate-selective polymer bound to phosphate selectively, and the ion concentration change or the pH shift in the analyte solutions after incubation with the imprinted polymer were measured in these systems. In using ISFET electrode, concentration of phosphate from 10 to 40 μM could be measured selectively. [ABSTRACT FROM AUTHOR]

Abstract: A biomimetic sensor for cAMP was fabricated in combination with an ion-sensitive field-effect transistor (ISFET) as a transducer and a cAMP-imprinted polymer as a molecular recognition material. The cAMP-imprinted polymer was prepared using 1-allyl-2-thiourea as a functional monomer, and the binding ability and the selectivity of the polymer were evaluated. In addition, the imprinted polymer membrane was coated on the ISFET electrode, and the response of the sensor was evaluated. The cAMP-imprinted polymer showed high binding ability to and selectivity for cAMP in aqueous media. The linear relationship was obtained from 0.1 to 1.0 mM cAMP from the calibration curve in the cAMP-sensor. [Copyright &y& Elsevier]

Phosphate-selective molecularly imprinted polymers were prepared using N-allyl-N'-methyl-thiourea or 1-allyl-2-thiourea as functional monomers, and the binding ability and selectivity of the polymers were evaluated. River water collected from agricultural use was the test sample. Though the water included some impurities, 65% of the phosphate was recovered from the imprinted polymer prepared using N-allyl-N'-methyl-thiourea as the functional monomer, while on the other hand, the binding of other anions was scarcely low. The imprinted polymer showed selective binding to phosphate and indicated the phosphate recovery material from river water. [ABSTRACT FROM AUTHOR]

Abstract: A phosphate-selective molecularly imprinted polymer was prepared using 1-allyl-2-thiourea as a functional monomer, and the binding ability and selectivity of the polymer were evaluated. The imprinted polymer showed high binding ability to and selectivity for phosphate in aqueous media. The recoverability of phosphate from the imprinted polymer was also examined, and nearly 70% of highly concentrated phosphate could be recovered. [Copyright &y& Elsevier]

Abstract: Molecularly imprinted polymers selective for phosphate were prepared with the two types of functional monomers, 1-allyl-2-thiourea and N-methyl-N′-(4-vinylphenyl)-thiourea, and the binding abilities of the polymers were evaluated. Phenylphosphonic acid or diphenyl phosphate were used as the template molecules and the imprinted polymers prepared with 1-allyl-2-thiourea as functional monomer showed high binding ability to phosphate in aqueous media and nearly 90% of phosphate could be recovered. Also, the imprinted polymer prepared with N-methyl-N′-(4-vinylphenyl)-thiourea as functional monomer had a high binding ability and specific interaction with phosphate in acetonitrile solution and over 90% of phosphate-derivatives could be recovered selectively. [Copyright &y& Elsevier]

Abstract: The detection of amino acids using aminoacyl–tRNA synthetases (ARSs) as the molecular recognition element was proposed, and the binding activity and specificity of ARSs were evaluated. Using this rapid and easy method, from 15 to 50μM tyrosine could be measured specifically. The method suggested in this article could be realized without an amino acid labeling process or a large volume of organic solvents, and the time for measurement was reasonable. [Copyright &y& Elsevier]

PG16 is a broadly neutralizing antibody to the human immunodeficiency virus (HIV). A crystal structure of PG16 revealed that the unusually long 28-residue complementarity determining region (CDR) H3 forms a unique subdomain, referred to as a "hammerhead", that directly contacts the antigen. The hammerhead apparently governs the function of PG16 while a previous experimental assay showed that the mutation of TyrH100Q to Ala, which does not directly contact the antigen, decreased the neutralization ability of PG16. However, the molecular mechanism by which a remote mutation from the hammerhead or contact paratope affects the neutralization potency has remained unclear. Here, we performed molecular dynamics simulations of the wild-type and variants (TyrH100Q to Ala, and TyrH100Q to Phe) of PG16, to clarify the effects of these mutations on the dynamics of CDR-H3. Our simulations revealed that the structural rigidity of the CDR-H3 in PG16 is attributable to the hydrogen bond interaction between TyrH100Q and ProH99, as well as the steric support by TyrH100Q. The loss of both interactions increases the intrinsic fluctuations of the CDR-H3 in PG16, leading to a conformational transition of CDR-H3 toward an inactive state. [ABSTRACT FROM AUTHOR]

To measure amino acid concentrations with high sensitivity, the pyrophosphate amplification reaction conditions of histidyl-tRNA synthetase (HisRS) and tyrosyl-tRNA synthetase (TyrRS) were examined. The amount of pyrophosphate produced by reactions involving HisRS and TyrRS was amplified compared with the amount of the initial substrate L-amino acid after the addition of excess adenosine-5′-triphosphate and magnesium ions, with incubation at 50°C in an alkaline pH. The amount of pyrophosphate produced in the HisRS and TyrRS reactions was approximately 24- and 16-fold higher than the initial amount of L-His and L-Tyr, respectively. The pyrophosphate amplification reactions involving HisRS and TyrRS showed high substrate specificity for L-His and L-Tyr, respectively. Products of pyrophosphate amplification were identified as p1, p4-di(adenosine) 5′-tetraphosphate, and adenosine-5′-monophosphate using high-performance liquid chromatography. A strong positive correlation was observed for 0 to 50 μM of L-His and L-Tyr in the pyrophosphate amplification reaction (R = 0.98 and R = 1.00, respectively). Abbreviations: L-His: L-histidine; L-Tyr: L-tyrosine; aaRSs: aminoacyl-tRNA synthetases; ATP: adenosine-5′-triphosphate; aminoacyl-AMP-aaRS: aminoacyl-adenylate intermediate; Ap4A, P1, P4-di(adenosine) 5ʹ-tetraphosphate; AMP: adenosine-5′-monophosphate; PAR: pyrophosphate amplification rate Pyrophosphate amplification reaction for measuring amino acid concentrations with high sensitivity using aminoacyl-tRNA synthetase from Escherichia coli. [ABSTRACT FROM AUTHOR]