A Molecular Probe with Both Chromogenic and Fluorescent Units for Detecting Serine Proteases.

A molecular probe with l-phenylalanine p-nitroanilide and l-lysin 4-methylcoumaryl-7-amide, in which these amino acid derivatives are connected through a succinic-acid spacer, was prepared. Trypsin and papain were detected by blue-fluorescence emission of generated 7-amino-4-methylcoumarin (AMC). α-Chymotrypsin and nattokinase were detected from both the blue-fluorescence emission of AMC and the UV absorbance of p-nitroaniline. In addition, different time courses of p-nitroaniline and AMC were observed between the reaction of P1 with α-chymotrypsin and that with nattokinase. In the case of nattokinase, both the fluorescence emission and UV absorbance slowly increased. In contrast, the increasing UV absorbance was saturated at the early stage of the reaction of the present probe with chymotrypsin, whereas the fluorescence emission continuously increased in the following stages.

Dynamic Nuclear Polarization using Photoexcited Triplet Electron Spins in Eutectic Mixtures.

Dynamic nuclear polarization using photoexcited triplet electrons (Triplet-DNP) is a method to significantly enhance nuclear spin polarization even in a low magnetic field and at room temperature. Pentacene has been practically used as an efficient polarizing agent for Triplet-DNP. In this study, we demonstrate room temperature 1H and 13C hyperpolarization of eutectic mixtures of deuterated benzoic acid doped with pentacene and a target molecule such as salicylic acid, nicotinic acid, or 2-naphthoic acid. These molecules are otherwise difficult to hyperpolarize by Triplet-DNP due to the low pentacene dopabilities of these molecules. The highest 1H polarization of 1.2% has been obtained for the eutectic mixture of salicylic acid in 0.39 T. The present sample preparation is a crucial method to widen the range of applications of Triplet-DNP to chemical and biomedical analyses.

High-field NMR with dissolution triplet-DNP.

Dissolution dynamic nuclear polarization (DNP) has wide variety of important applications such as real-time monitoring of chemical reactions and metabolic imaging. We construct DNP using photoexcited triplet electron spins (Triplet-DNP) apparatus combined with dissolution apparatus for solution NMR in a high magnetic field. Triplet-DNP enables us to obtain high nuclear polarization at room temperature. Solid-state samples polarized by Triplet-DNP are transferred to a superconducting magnet and dissolved by injecting aqueous solvents. The 13C polarization of 0.22% has been obtained for [caryboxy-13C]benzoic acid-d in the liquid state. Our results show that Triplet-DNP can be applied to real-time monitoring with solution NMR.

A formal anti-Markovnikov hydroamination of allylic alcohols via tandem oxidation/1,4-conjugate addition/1,2-reduction using a Ru catalyst.

A formal anti-Markovnikov hydroamination of allylic alcohols using a Ru catalyst via tandem oxidation/1,4-conjugate addition/1,2-reduction was developed. Thus, the reaction of allylic alcohols with amines was performed in the presence of the catalyst generated from RuClH(CO)(PPh3)3 and 2,6-bis(n-butyliminomethyl)pyridine in situ to afford the corresponding γ-amino alcohols efficiently.

Rapid sensitive molecular diagnosis of pyogenic spinal infections using methicillin-resistant Staphylococcus-specific polymerase chain reaction and 16S ribosomal RNA gene-based universal polymerase chain reaction.

BACKGROUND CONTEXT: Rapid diagnosis and accurate detection of etiological agents in pyogenic spinal infection (PSI) patients are important. PURPOSE: The purpose of this study was to evaluate the clinical usefulness of methicillin-resistant Staphylococcus-specific polymerase chain reaction (MRS-PCR) and broad-range universal PCR (U-PCR) for diagnosing PSI. STUDY DESIGN: A prospective diagnostic study. PATIENTS: Thirty-two clinically suspect PSI patients and six control patients who underwent computerized tomography-guided biopsy and/or surgical treatment were enrolled. METHODS: Tissue samples were examined by microbiological culture, histopathology, and real-time PCR (MRS-PCR and U-PCR). The diagnostic accuracy of real-time PCR was analyzed based on the definitive diagnosis of infection, defined as a positive result from microbiological culture or histopathology. RESULTS: All six control subjects were negative for PSI for all analyses. Twelve clinically suspect PSI subjects received definitive diagnoses (PSI group). The non-PSI group consisted of six control subjects plus the remaining 20 patients from the PSI clinically suspect group. MRS-PCR results were positive for all MRS-cultured PSI subjects. U-PCR was positive for all subjects in the PSI group with one discrepancy between real-time PCR and microbiological culture results in differentiation between gram-positive and gram-negative bacteria. In the non-PSI group, MRS-PCR and U-PCR were positive in three and seven cases, respectively. The sensitivity, specificity, positive predictive value, and negative predictive value of MRS-PCR for diagnosing MRS infection were 1.00, 0.91, 0.57, and 1.00, respectively; those for the diagnosis of bacterial infection with U-PCR were 1.00, 0.73, 0.63, and 1.00, respectively. CONCLUSION: Identification of MRS infection and ability to differentiate between gram-positive and gram-negative bacteria is rapidly achieved using MRS-PCR and U-PCR. Real-time PCR provides a sensitive molecular diagnosis of PSI and may contribute to antibiotic selection.