Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis.

d-Amino acids are important building blocks for various compounds, such as pharmaceuticals and agrochemicals. A more cost-effective enzymatic method for d-amino acid production is needed in the industry. We improved a one-pot enzymatic method for d-amino acid production by the dynamic kinetic resolution of N-succinyl amino acids using two enzymes: d-succinylase (DSA) from Cupriavidus sp. P4-10-C, which hydrolyzes N-succinyl-d-amino acids enantioselectively to their corresponding d-amino acid, and N-succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503. In this study, DSA and NSAR were purified and their properties were investigated. The optimum temperature of DSA was 50°C and it was stable up to 55°C. The optimum pH of DSA and NSAR was around 7.5. In d-phenylalanine production, the optical purity of product was improved to 91.6% ee from the examination about enzyme concentration. Moreover, 100 mM N-succinyl-dl-tryptophan was converted to d-tryptophan at 81.8% yield with 94.7% ee. This enzymatic method could be useful for the industrial production of various d-amino acids.

Inhibiting IκB kinase-ß downregulates inflammatory cytokines in injured discs and neuropeptides in dorsal root ganglia innervating injured discs in rats.

STUDY DESIGN: Quantitative and immunohistological analysis of the efficacy of an IκB kinase-ß (IKKß) inhibitor in an injured intervertebral disc (IVD) model. OBJECTIVE: To elucidate the efficacy of an IKKß inhibitor on inflammatory cytokine levels in injured IVDs or on neuropeptide levels in the dorsal root ganglia (DRG) neurons innervating injured IVDs in rats. SUMMARY OF BACKGROUND DATA: Multiple studies have suggested that upregulation of inflammatory cytokines in damaged IVDs causes discogenic low back pain. The efficacy of blocking individual inflammatory cytokines is limited; however, inflammatory cytokine stimuli often require IKKß to activate nuclear factor-k B. METHODS: Sprague-Dawley rats were divided into 3 groups: sham, saline (disc-injury plus saline), and IKKß (disc-injury plus anti-IKKß). To induce injury, IVDs were repeatedly punctured.Experiment 1: Four, 7, and 14 days postinjury, coccygeal (Co) 5/6, Co6/7, and Co7/8 IVDs were resected and tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 levels were quantified by enzyme-linked immunosorbent assay. Experiment 2: The neurotracer Fluoro-Gold was injected into injured L5-L6 IVDs and uninjured sham group IVDs to detect DRG neurons. One week postsurgery, L1-L6 DRGs were immunolabeled with the neuropeptide calcitonin gene-related peptide. The proportions of Fluoro-Gold-labeled calcitonin gene-related peptide-immunoreactive DRG neurons were assessed. RESULTS: Experiment 1: IVD levels of tumor necrosis factor-α (through 2 wk), IL-1ß (at 4 d), and IL-6 (at 4 d) were significantly higher in the saline group than in the sham group, and significantly lower in the IKKß group than in the saline group (P < 0.05). Experiment 2: The percentage of calcitonin gene-related peptide-immunoreactive Fluoro-Gold-labeled DRG neurons was significantly higher in the saline group than in the sham group, and significantly lower in the IKKß group than in the saline group (P < 0.05). CONCLUSION: Injury-induced upregulation of inflammatory cytokines within IVDs and increased levels of neuropeptides within DRG neurons can be suppressed by inhibiting IKKß. LEVEL OF EVIDENCE: N/A.

Tag-assisted liquid-phase peptide synthesis using hydrophobic benzyl alcohols as supports.

A soluble tag-assisted liquid-phase peptide synthesis was successfully established based on simple hydrophobic benzyl alcohols, which can be easily prepared from naturally abundant materials. Excellent precipitation yields can be obtained at each step, combining the best properties of solid-phase and liquid-phase techniques. This approach can also be applied efficiently to fragment couplings, allowing chemical synthesis of several bioactive peptides.

Biophysic evaluation of bone quality-application of Fourier transform infrared spectroscopy and phosphorus-31 solid-state nuclear magnetic resonance spectroscopy.

In this review, we focus on findings obtained with biophysic techniques, Fourier transformed infrared (FTIR) spectroscopy and phosphorus-31 solid-state nuclear magnetic resonance (31P solid-state NMR) spectroscopy, which may allow us to evaluate bone quality and to predict bone strength. FTIR measures the absorption energy that produces an increase in the vibrational or rotational energy of atoms or groups of atoms within the molecule. FTIR spectroscopy allows us to examine the relative amount of minerals and matrix content and the arrangement of apatite and organic matrix. FTIR spectroscopy should become an important tool, because the relative amount of minerals and the arrangement of apatite and organic matrix could be a measure for evaluating bone quality. 31P solid-state NMR spectroscopy is useful for evaluating the quality of bone and predicting bone strength by calculating the spine-lattice relaxation time (T1) of bone. 31P solid-state NMR imaging can be used to measure quantitatively the mass of hydroxyapatite. The T1 relaxation time of both bone and deficient hydroxyapatite was much longer than that of pure hydroxyapatite. T1 relaxation time is one of the promising indices of bone quality.