ω‐Aminotransferase (ωAT) is an interesting biocatalyst for the preparation of chiral amines, which are widely used as building blocks for pharmaceuticals, agrochemicals and fine chemicals. With the ...assumption that substrate and sequence spaces are in the process of co‐evolution, we explored sequences space to screen the enzymes showing activity to new target compounds. Bacterial genome sequences (n=527) were analyzed by the profiles of subgroups in aminotransferase group II including ornithine aminotransferase (orAT), acetylornithine aminotransferase (aoAT), ωAT, γ‐aminobutyrate aminotransferase (GABAAT) and 7,8‐diaminopelargonate aminotransferase (DAPAAT). We selected the sequences having a Z score of 0‐1.8 to guarantee the ωAT reaction and to avoid the typical ωAT sequences. Among the selected sequences, we filtered out the sequences with very low Z scores for the rest of four subgroups in aminotransferase group II to consider the diversity. For the selected sequences, we performed protein‐ligand docking simulations to predict the docking pose of amino acceptor. Throughout all the analysis procedures, several candidate aminotransferase sequences for the asymmetric synthesis of chiral amines were obtained. An efficient procedure for virtual screening of novel enzymes was demonstrated.
L-Theanine, found in green tea leaves has been shown to positively affect immunity and relaxation in humans. There have been many attempts to produce L-theanine through enzymatic synthesis to ...overcome the limitations of traditional methods. Among the many genes coding for enzymes in the L-theanine biosynthesis, glutamylmethylamide synthetase (GMAS) exhibits the greatest possibility of producing large amounts of production. Thus, GMAS from
No. 9 was overexpressed in several strains including vectors with different copy numbers. BW25113(DE3) cells containing the pET24ma::
was selected for strains. The optimal temperature, pH, and metal ion concentration were 50°C, 7, and 5 mM MnCl
, respectively. Additionally, ATP was found to be an important factor for producing high concentration of L-theanine so several strains were tested during the reaction for ATP regeneration. Bakers yeast was found to decrease the demand for ATP most effectively. Addition of potassium phosphate source was demonstrated by producing 4-fold higher L-theanine. To enhance the conversion yield, GMAS was additionally overexpressed in the system. A maximum of 198 mM L-theanine was produced with 16.5 mmol/l/h productivity. The whole-cell reaction involving GMAS has greatest potential for scale-up production of L-theanine.
Naturally degradable bioplastic polyhydroxyalkanoate (PHA) is a promising biopolymer and its physical properties could be changed by introducing of different monomers such as 3-hydroxybutyrate (3HB), ...3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx). To produce poly(3-hydroxybutyrate-
co
-3-hydroxyvalerate) P(3HB-
co
-3HV)) including a high fraction of hydroxyvalerate, we introduced
ctfAB
into engineered
Escherichia coli
YJ200 possessing a pLW487 vector containing
bktB
,
phaB
, and
phaC
under control of the
trc
promoter. To enhance the HV fraction of P(3HB-
co
-3HV), the optimal concentrations of propionate, which acts as a precursor of 3HV and isopropyl β-D-1-thiogalactopyranoside, were determined and found to be 0.1 mM and 0.3%, respectively. Under the optimized conditions,
E. coli
, YJ201 produced P(3HB-
co
-3HV) containing a large amount of 3HV. Comparison with other CoA transferases showed that CtfAB produced relatively lower molecular weight copolymers. This demonstrates the necessity of identifying additional different CoA transferases, because CoA transferase can affect both the monomer fraction and molecular weight of polymers.
Retrospective study MR images for spinal cord tumors.
To analyze the characteristics of MR images for spinal cord tumors, which were then verified at surgery or biopsy.
MR images are often used as ...the primary diagnostic imaging tool and the preoperative study of choice. The need for biopsy may be obviated because of increasingly accurate preoperative histologic diagnosis by MR images.
The study group consisted of 39 patients who had undergone MR imaging for preoperative evaluation of spinal cord tumors between September 1989 and February 2008. All patients had operations for spinal cord tumors, which were confirmed at biopsy. Of the 39 patients, 18 were men, and 21 were women. The average follow-up period was 23.8 months. The mean patient age was 46.6 years.
Diagnoses included neurilemmoma (19 cases), neurofibroma (4 cases), meningioma (5 cases), hemangioma (3 cases), giant cell tumor (1 case), ganglioneuroma (1 case), lymphoma (1 case), neuroblastoma (1 case), and metastatic tumor from the prostate (1 case). The remaining 3 cases were composed of arachnoid cysts (2 cases) and a vascular malformation (arteriovenous malformation, 1 case).
MR images are the preoperative modality of choice in the evaluation of spinal cord tumors. MR images can narrow the differential diagnosis and guide surgical resection.
AIM: To evaluate the inflammasome activation and the effect of peroxisome proliferator-activated receptors(PPAR)-δ agonist treatment in nonalcoholic fatty liver disease(NAFLD) models.METHODS: Male ...C57BL/6J mice were classified according to control or high fat diet(HFD) with or without PPAR-δ agonist(GW) over period of 12 wk control, HFD, HFD + lipopolysaccharide(LPS), HFD + LPS + GW group. Hep G2 cells were exposed to palmitic acid(PA) and/or LPS in the absence or presence of GW.RESULTS: HFD caused glucose intolerance and hepatic steatosis. In mice fed an HFD with LPS, caspase-1 and interleukin(IL)-1β in the liver were significantly increased. Treatment with GW ameliorated the steatosis and inhibited overexpression of pro-inflammatory cytokines. In Hep G2 cells, PA and LPS treatment markedly increased m RNA of several nucleotide-binding andoligomerization domain-like receptor family members(NLRP3, NLRP6, and NLRP10), caspase-1 and IL-1β. PA and LPS also exaggerated reactive oxygen species production. All of the above effects of PA and LPS were reduced by GW. GW also enhanced the phosphorylation of AMPK-α.CONCLUSION: PPAR-δ agonist reduces fatty acidinduced inflammation and steatosis by suppressing inflammasome activation. Targeting the inflammasome by the PPAR-δ agonist may have therapeutic implication for NAFLD.
SCO0948 was found to be the single open reading frame annotated to encode an alpha-mannosidase (AM1) in Streptomyces coelicolor M145. To characterize the protein, we overexpressed SCO0948 in ...Escherichia coli BL21(DE3). Recombinant AM1, with a molecular weight of 110 kDa, exhibited alpha-mannosidase activity toward 4-nitrophenyl-alpha-d-mannopyranoside with a K ^sub m^ of 4.61 mM, a V ^sub max^ of 101.6 mM/min, and a specific activity of 47.96 U/mg. Treatment of ovalbumin, a glycoprotein, with AM1 resulted in partial deglycosylation, as assessed by glycostaining and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The S. coelicolor deletion mutant for SCO0948 failed to produce alpha-mannosidase activity, confirming AM1 as the only alpha-mannosidase in S. coelicolor M145. Interestingly, the deletion mutant and a complementation strain produced lower levels of the antibiotics actinorhodin and undecylprodigiosin in glucose minimal media. The results indicate that AM1 as an alpha-mannosidase influences deglycosylation and antibiotic production in S. coelicolor M145.PUBLICATION ABSTRACT
Substrate specificity of the ...-aminotransferase obtained from Vibrio fluvialis (...-ATvf) was rationally redesigned for the kinetic resolution of aliphatic chiral amines. ...-ATVf showed unique ...substrate specificity toward aromatic amines with a high enantioselectivity (E > 100) for (S)-enantiomers. However, the substrate specificity of this enzyme was much narrower toward aliphatic amines. To overcome the narrow substrate specificity toward aliphatic amines, we redesigned the substrate specificity of ...-ATVf using homology modeling and the substrate structure-activity relationship. The homology model and the substrate structure-activity relationship showed that the active site of ...-ATVf consists of one large substrate-binding site and another small substrate-binding site. The key determinant in the small substrate-binding site was D25, whose role was expected to mask R415 and to generate the electrostatic repulsion with the substrate's α-carboxylate group. In the large substrate-binding site, R256 was predicted to recognize the α-carboxylate group of substrate thus obeying the dual substrate recognition mechanism of aminotransferase subgroup II enzymes. Among the several amino acid residues in the large substrate-binding site, W57 and W147, with their bulky side chains, were expected to restrict the recognition of aliphatic amines. Two mutant enzymes, W57G and W147G, showed significant changes in their substrate specificity such that they catalyzed transamination of a broad range of aliphatic amines without losing the original activities toward aromatic amines and enantioselectivity. (ProQuest: ... denotes formulae/symbols omitted.)
An enzymatic resolution was carried out for the preparation of enriched bgr-heterocyclic D-alanine derivatives using Escherichia coli aromatic L- amino acid transaminase. The excess of pyrazole, ...imidazole, or 1,2,4-triazole reacted with methyl-2-acetamidoacrylate in acetonitrile in the presence of potassium carbonate at 60 degree C, directly leading to make the potassium salt of the corresponding N-acetyl-bgr-heterocyclic alanine derivatives. After the acidic deprotection of the N-acetyl group, 10 mM of racemic pyrazolylalanine, triazolylalanine, and imidazolylalanine were resolved to D- pyrazolylalanine, D-triazolylalanine, and D-imidazolylalanine with 46% (85% ee), 42% (72% ee), and 48% (95% ee) conversion yield in 18 h, respectively, using E. coli aromatic L-amino acid transaminase (EC 2.6.1.5). Although the three bgr-heterocyclic L-alanine derivatives have similar molecular structures, they showed different reaction rates and enantioselectivities. The relative reactivities of the transaminase toward the bgr-heterocyclic L-alanine derivatives could be explained by the relationship between the substrate binding energy (E, kcal/mol) to the enzyme active site and the distance (dgr, Aa) from the nitrogen of agr-amino group of the substrates to the C4prime carbon of PLP-Lys258 Schiff base. As the ratio of the substrate binding energy (E) to the distance (dgr) becomes indicative value of k sub(cat)/K sub(M) of the enzyme to the substrate, the relative reactivities of the bgr-heterocyclic L-alanine derivatives were successfully correlated with E/dgr, and the relationship was confirmed by our experiments.
AIM： To evaluate the treatment options for nephrotoxicity due to cisplatin combination chemotherapy. METHODS： We retrospectively reviewed patients who had received cisplatin combination chemotherapy ...for gastric cancer between January 2002 and December 2008. We investigated patients who had shown acute renal failure （ARF）, and examined their clinical characteristics, laboratory data, use of preventive measures, treatment cycles, the amount of cisplatin administered, recovery period, subsequent treatments, and renal status between the recovered and unrecovered groups. RESULTS： Forty-one of the 552 patients had serum creatinine （SCR） levels greater than 1.5 mg/dL. We found that pre-ARF SCR, ARF SCR, and ARF glomerular filtration rates were significantly associated with renal status post- ARF between the two groups （P = 0.008, 0.026, 0.026, respectively）. On the receiver operating characteristic curve of these values, a 1.75 mg/dL ARF SCR value had 87.5% sensitivity and 84.8% specificity （P = 0.011）.CONCLUSION： Cessation or reduction of chemotherapy should be considered for patients who have an elevation of SCR levels during cisplatin combination chemotherapy.
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