Crystallization and Properties of N-Benzoylglycine Amidohydrolase from Pseudomonas putida

N-Benzoylgiycine amidohydrolase (hippurate hydrolase EC 3.5.1.32), which catalyzes the hydrolysis of hippuric acid to benzoic acid and glycine, was found in a cell-free extract of Pseudomonas putida C692-3 grown on a medium containing hippuric acid. The enzyme was purified from the extract by ammonium sulfate fractionation and column chromatographies on DEAE-cellulose, DEAE-Sephadex A-50, hydroxyapatite, and Sepharose CL-6B. The enzyme was finally crystallized. The crystalline enzyme was almost homogeneous on electrophoresis. The enzyme had a molecular weight of about 170,000 and consisted of four subunits identical in molecular weight (approximately 42,000). The enzyme hydrolyzed N-benzoylglycine most rapidly, and N-benzoyl-l-alanine and N-benzoyl-l-aminobutyric acid. The Km value for these substrates were 0.72 mm, 0.87 mm, and 0.87mm, respectively. The optimum pH of the enzyme reaction was 7.0 to 8.0 and the enzyme was stable from pH 6.0 to 8.0.     

Proteins Released from Myofibrils by CASF (Ca2+-activated sarcoplasmic factor) and Trypsin

The nature of the soluble proteins and peptides released from myofibrils by treatment with CASF (Ca²⁺-activated sarcoplasmic factor) was investigated by using Polyacrylamide gel electrophoresis in both a nondenaturing and a denaturing (sodium dodecyl sulfate=SDS) solvent and by using gel permeation chromatography on Sepharose 6B. Both CASF and trypsin treatment cause removal of Z-disks before causing other ultrastructurally detectable degradation of myofibrils. CASF treatment of myofibrils releases a protein that is identical to α-actinin, one of the known components of the Z-disk, on the basis of mobility in Polyacrylamide gel electrophoresis in a nondenaturing solvent and in SDS and on the basis of elution from gel permeation columns. Trypsin treatment of myofibrils releases a number of smaller molecular weight products that cannot be identified with any of the known myofibrillar proteins. Hence, CASF seems to remove Z-disks from myofibrils by means of a very specific proteolytic activity that releases α-actinin without extensively degrading it. Trypsin, on the other hand, probably seems to remove Z-disks by degrading α-actinin to peptides.     

Effect of Materials Released from Myofibrils by CAF (Ca2+-activated Factor) on Z-Disk Reconstitution

To investigate the constituents of Z-disk, reconstitution of Z-disk by incubating some proteins released from myofibrils by CAF(Ca²⁺-activated factor) with Z-disk-extracted fiber bundles was carried out and examined with electron microscope. The materials released from myofibrils by CAF have been bound in Z-disk region, and Z-disk extracted from myofibrils with a low ionic strength solution has been reconstituted. On the other hand, Z-disk removed from myofibrils by CAF has not been reconstituted by the same way.     

Synthesis of 3Z-Nonenal and 3Z,6Z-Nonadienal

3Z-Nonenal and 3Z, 6Z-nonadienal, potential biosynthetic precursors of 2E-nonenal and 2E, 6Z-nonadienal, were for the first time synthesized stereoseleclively.     

Relationship between the Circadian Variation of Uric Acid Level and Dietary Conditions

We studied the inhibitory effects of isorhamnetin on mushroom tyrosinase by inhibition kinetics and computational simulation. Isorhamnetin reversibly inhibited tyrosinase in a mixed-type manner at K _i=0.235 ± 0.013 mM. Measurements of intrinsic and 1-anilinonaphthalene-8-sulfonate(ANS)-binding fluorescence showed that isorhamnetin did not induce significant changes in the tertiary structure of tyrosinase. To gain insight into the inactivation process, the kinetics were computed via time-interval measurements and continuous substrate reactions. The results indicated that inactivation induced by isorhamnetin was a first-order reaction with biphasic processes. To gain further insight, we simulated docking between tyrosinase and isorhamnetin. Simulation was successful (binding energies for Dock6.3: ?32.58 kcal/mol, for AutoDock4.2: ?5.66 kcal/mol, and for Fred2.2: ?48.86 kcal/mol), suggesting that isorhamnetin interacts with several residues, such as HIS244 and MET280. This strategy of predicting tyrosinase interaction in combination with kinetics based on a flavanone compound might prove useful in screening for potential natural tyrosinase inhibitors.     

Novel mutations of CDKN1C in Japanese patients with Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is an imprinting-related human disease that is characterized by macrosomia, macroglossia, abdominal wall defects, and variable minor features. BWS is caused by several genetic/epigenetic alterations, such as loss of methylation at KvDMR1, gain of methylation at H19-DMR, paternal uniparental disomy of chromosome 11, CDKN1C mutations, and structural abnormalities of chromosome 11. CDKN1C is an imprinted gene with maternal preferential expression, encoding for a cyclin-dependent kinase (CDK) inhibitor. Mutations in CDKN1C are found in 40 % of familial BWS cases with dominant maternal transmission and in ~5 % of sporadic cases. In this study, we searched for CDKN1C mutations in 37 BWS cases that had no evidence for other alterations. We found five mutations—four novel and one known—from a total of six patients. Four were maternally inherited and one was a de novo mutation. Two frame-shift mutations and one nonsense mutation abolished the QT domain, containing a PCNA-binding domain and a nuclear localization signal. Two missense mutations occurred in the CDK inhibitory domain, diminishing its inhibitory function. The above-mentioned mutations were predicted by in silico analysis to lead to loss of function; therefore, we strongly suspect that such anomalies are causative in the etiology of BWS.     

Ipragliflozin Improves Hepatic Steatosis in Obese Mice and Liver Dysfunction in Type 2 Diabetic Patients Irrespective of Body Weight Reduction

Type 2 diabetes mellitus (T2DM) is associated with a high incidence of non-alcoholic fatty liver disease (NAFLD) related to obesity and insulin resistance. Currently, medical interventions for NAFLD have focused on diet control and exercise to reduce body weight, and there is a requirement for effective pharmacological therapies. Sodium-glucose cotransporter 2 (SGLT2) inhibitors are oral antidiabetic drugs that promote the urinary excretion of glucose by blocking its reabsorption in renal proximal tubules. SGLT2 inhibitors lower blood glucose independent of insulin action and are expected to reduce body weight because of urinary calorie loss. Here we show that an SGLT2 inhibitor ipragliflozin improves hepatic steatosis in high-fat diet-induced and leptin-deficient (ob/ob) obese mice irrespective of body weight reduction. In the obese mice, ipragliflozin-induced hyperphagia occurred to increase energy intake, attenuating body weight reduction with increased epididymal fat mass. There is an inverse correlation between weights of liver and epididymal fat in ipragliflozin-treated obese mice, suggesting that ipragliflozin treatment promotes normotopic fat accumulation in the epididymal fat and prevents ectopic fat accumulation in the liver. Despite increased adiposity, ipragliflozin ameliorates obesity-associated inflammation and insulin resistance in epididymal fat. Clinically, ipragliflozin improves liver dysfunction in patients with T2DM irrespective of body weight reduction. These findings provide new insight into the effects of SGLT2 inhibitors on energy homeostasis and fat accumulation and indicate their potential therapeutic efficacy in T2DM-associated hepatic steatosis.     

Identification of lysophosphatidylthreonine with an aromatic fatty acid surrogate as a potent inducer of mast cell degranulation

Upon various stimulations, mast cells (MCs) release a wide variety of chemical mediators stored in their cytoplasmic granules, which then initiates subsequent allergic reactions. Lysophosphatidylserine (LysoPS), a kind of lysophospholipid, potentiates the histamine release from MCs triggered by antigen stimulation. We previously showed through structure-activity studies of LysoPS analogs that LysoPS with a methyl group at the carbon of the serine residue, i.e., lysophosphatidylthreonine (LysoPT), is extremely potent in stimulating the MC degranulation. In this study, as our continuing study to identify more potent LysoPS analogs, we developed LysoPS analogs with fatty acid surrogates. We found that the substitution of oleic acid to an aromatic fatty acid surrogate (C3-pH-p-O-C11) in 2-deoxy-1-LysoPS resulted in significant increase in the ability to induce MCs degranulation compared with 2-deoxy-1-LysoPS with oleic acid. Conversion of the serine residue into the threonine residue further increased the activity of MC degranulation both in vitro and in vivo. The resulting super agonist, 2-deoxy-LysoPT with C3-pH-p-O-C11, will be a useful tool to elucidate the mechanisms of stimulatory effect of LysoPS on MC degranulation.