Re-investigation of in vitro activity of acetohydroxyacid synthase I holoenzyme from Escherichia coli.

Acetohydroxyacid synthase (AHAS) is one of the key enzymes of the biosynthesis of branched-chain amino acids, it is also an effective target for the screening of herbicides and antibiotics. In this study we present a method for preparing Escherichia coli AHAS I holoenzyme (EcAHAS I) with exceptional stability, which provides a solid ground for us to re-investigate the in vitro catalytic properties of the protein. The results show EcAHAS I synthesized in this way exhibits similar function to Bacillus subtilis acetolactate synthase in its catalysis with pyruvate and 2-ketobutyrate (2-KB) as dual-substrate, producing four 2-hydroxy-3-ketoacids including (S)-2-acetolactate, (S)-2-aceto-2-hydroxybutyrate, (S)-2-propionyllactate, and (S)-2-propionyl-2-hydroxybutyrate. Quantification of the reaction indicates that the two substrates almost totally consume, and compound (S)-2-aceto-2- hydroxybutyrate forms in the highest yield among the four major products. Moreover, the protein also condenses two molecules of 2-KB to furnish (S)-2-propionyl-2-hydroxybutyrate. Further exploration manifests that EcAHAS I ligates pyruvate/2-KB and nitrosobenzene to generate two arylhydroxamic acids N-hydroxy-N-phenylacetamide and N-hydroxy-N-phenyl- propionamide. These findings enhance our comprehension of the catalytic characteristics of EcAHAS I. Furthermore, the application of this enzyme as a catalyst in construction of C-N bonds displays promising potential.

Determination of α-Dicarbonyl compounds in traditional Chinese herbal medicines.

α-DCs (α-dicarbonyls) have been proven to be closely related to aging and the onset and development of many chronic diseases. The wide presence of this kind of components in various foods and beverages has been unambiguously determined, but their occurrence in various phytomedicines remains in obscurity. In this study, we established and evaluated an HPLC-UV method and used it to measure the contents of four α-DCs including 3-deoxyglucosone (3-DG), glyoxal (GO), methylglyoxal (MGO), and diacetyl (DA) in 35 Chinese herbs after they have been derivatized with 4-nitro-1,2-phenylenediamine. The results uncover that 3-DG is the major component among the α-DCs, being detectable in all the selected herbs in concentrations ranging from 22.80 µg/g in the seeds of Alpinia katsumadai to 7032.75 µg/g in the fruit of Siraitia grosuenorii. The contents of the other three compounds are much lower than those of 3-DG, with GO being up to 22.65 µg/g, MGO being up to 55.50 µg/g, and DA to 18.75 µg/g, respectively. The data show as well the contents of the total four α-DCs in the herbs are generally in a comparable level to those in various foods, implying that herb medicines may have potential risks on human heath in view of the α-DCs.

Renal elasticity and perfusion changes on ultrasonography in an early-stage diabetic rat model.

Background: Renal hemodynamic changes in early diabetes occur before the onset of significant structural abnormalities or clinical manifestations, and timely detection of these changes has clinical significance. This study aimed to evaluate renal elasticity and perfusion changes in an early-stage diabetic rat model by shear wave elastography (SWE) and contrast-enhanced ultrasound (CEUS), and to explore the potential correlations between renal elasticity and perfusion parameters. Methods: A total of 18 male Sprague-Dawley rats were randomly divided into three groups: a control group (group 1, n=6), a diabetic group (group 2, n=6), and a diabetic group receiving drug therapy (group 3, n=6). An intraperitoneal injection of streptozotocin (STZ) for 2 days combined with a high-fat diet (HFD) was used as the early-stage diabetic rat model. The diabetic rats in group 3 were treated with canagliflozin and losartan for 6 weeks, whereas the rats in groups 1 and 2 were given equal amounts of purified water. Renal stiffness on SWE and perfusion parameters on CEUS were measured and compared among the three groups, then the rats were sacrificed, and serum, urine, and renal histopathology were evaluated to confirm the development of early diabetes. Results: The early-stage diabetic rats without significant pathological changes exhibited bigger kidneys and higher blood glucose (all P<0.05). Among the CEUS parameters, peak enhancement (PE), wash-in area under the curve (WiAUC), wash-in perfusion index (WiPI), wash-out AUC (WoAUC), wash-in and wash-out AUC (WiWoAUC), rise time (RT), and time to peak (TTP) of diabetic rats in group 2 were significantly increased (all P<0.05), and the hyperperfusion ameliorated significantly after drug treatment. The renal elasticity measured by SWE varied in accordance with certain perfusion parameters, and was strongly positively correlated with WiAUC (r=0.701, P<0.001), WoAUC (r=0.647, P<0.001), and WiWoAUC (r=0.655, P<0.001), and moderately positively correlated with PE (r=0.539, P=0.001), WiPI (r=0.555, P<0.001), RT (r=0.425, P=0.010), and TTP (r=0.439, P=0.007). Conclusions: Renal elasticity and perfusion changes in the early stage of diabetes, and renal elasticity was positively associated with delayed and increased perfusion.