Production of Higher-quality Plastein from a Crude Single-cell Protein

From defatted n-paraffin-assimilating yeast cells, a crude protein was obtained by alkaliextraction followed by acid-precipitation. Then the protein was treated with ether until extractable substances were removed exhaustively at this stage. However, at the next stage where the ether-treated protein had been partially hydrolyzed with pepsin, when the hydrolysate was retreated with ether, it was found that ether-extractable substances totalling 270 mg/100 g were obtainable additionally. Chromatographic investigations demonstrated that the substances included significant amounts of aliphatic and aromatic hydrocarbons, some indoles, and a ubiquinone (n = 8).

From the protein hydrolysate (substrate) after the above ether-treatment, a plastein was synthesized with Bioprase under the specific conditions. The plastein was obtained as a precipitate when the whole reaction mixture was treated with aqueous ethanol or acetone. The quantity and quality (nitrogen content) of the plastein depended on the ethanol or acetone concentration. Roughly speaking, the higher the concentration, the more the plastein quantity. The converse relation held for the quality; a plastein precipitated by treatment solely with water showed a higher quality than any other case.     

His164 regulates accessibility to the active site in fungal 17beta-hydroxysteroid dehydrogenase

17beta-Hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl) is an NADPH-dependent member of the short-chain dehydrogenase/ reductase superfamily. To study the catalytic properties of this enzyme, we prepared several specific mutations of 17beta-HSDcl (Tyr167Phe, His164Trp/Gly, Tyr212Ala). Wild-type 17beta-HSDcl and the 17beta-HSDcl mutants were evaluated by chromatographic, kinetic and thermodynamic means. The Tyr167Phe mutation resulted in a complete loss of enzyme activity, while substitution of His164 with Trp and Gly both resulted in higher specificity number (V/K) for the steroid substrates, which are mainly a consequence of easier accessibility of steroid substrates to the active-site hollow under optimized conditions. The Tyr212Ala mutant showed increased activity in the oxidative direction, which appears to be a consequence of increased NADPH dissociation. The kinetic characterizations and thermodynamic analyses also suggest that His164 and Tyr212 in 17beta-HSDcl have a role in the opening and closing of the active site of this enzyme and in the discrimination between oxidized and reduced coenzyme.     

The SGLT2 inhibitor dapagliflozin promotes systemic FFA mobilization,enhances hepatic β-oxidation,and induces ketosis

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to increase ketone bodies in patients with type 2 diabetes; however, the underlying mechanisms have not been fully elucidated. Here we examined the effect of the SGLT2 inhibitor dapagliflozin (1 mg/kg/day, formulated in a water, PEG400, ethanol, propylene glycol solution, 4 weeks) on lipid metabolism in obese Zucker rats. Fasting FFA metabolism was assessed in the anesthetized state using a [9,10-³H(N)]-palmitic acid tracer by estimating rates of plasma FFA appearance (R_a), whole-body FFA oxidation (R_ox), and nonoxidative disposal (R_st). In the liver, clearance (K_β-ox) and flux (R_β-ox) of FFA into β-oxidation were estimated using [9,10-³H]-(R)-bromopalmitate/[U-¹⁴C]palmitate tracers. As expected, dapagliflozin induced glycosuria and a robust antidiabetic effect; treatment reduced fasting plasma glucose and insulin, lowered glycated hemoglobin, and increased pancreatic insulin content compared with vehicle controls. Dapagliflozin also increased plasma FFA, R_a, R_ox, and R_st with enhanced channeling toward oxidation versus storage. In the liver, there was also enhanced channeling of FFA to β-oxidation, with increased K_β-ox, R_β-ox and tissue acetyl-CoA, compared with controls. Finally, dapagliflozin increased hepatic HMG-CoA and plasma β-hydroxybutyrate, consistent with a specific enhancement of ketogenesis. Since ketogenesis has not been directly measured, we cannot exclude an additional contribution of impaired ketone body clearance to the ketosis. In conclusion, this study provides evidence that the dapagliflozin-induced increase in plasma ketone bodies is driven by the combined action of FFA mobilization from adipose tissue and diversion of hepatic FFA toward β-oxidation.     

Anti-cancer study and whey protein complexation of new lanthanum(III) complex with the aim of achieving bioactive anticancer metal-based drugs

In this study, a new lanthanum (III)-amino acid complex utilizing cysteine has been synthesized and characterized. The anticancer activities of the prepared La(III) complex against MCF-7 cell lines were studied. Results of MTT assay showed that at all three incubation times, the cytotoxic effect of prepared La(III) complex on MCF-7 breast cancer cell lines displays a time- and dose-dependent inhibitory effects. The interactions of the La(III) complex with two whey proteins (bovine serum albumin, BSA, and Bovine β-lactoglobulin, βLG) have been explored by using spectroscopic and molecular dicking methods. The obtained results indicated that La(III) complex strongly quenched the fluorescence of two carrier proteins in static quenching mode and also, BSA hah stronger binding affinity toward studied complex than βLG whit binding constant values of K_{BSA-La?Complex}?～?0.11?×?10⁴ M^?1 and K_{βLG-La?Complex}?～?0.63?×?10³ M^?1 at 300 K. The thermodynamic parameters revealed the contribution of hydrogen bond and Vander Waals interactions in both systems. The distances of the La(III) complex whit whey proteins were calculated using Förster energy transfer theory and proved existence of the energy transfer between two proteins and prepared La(III) complex with a high probability. FT-IR and UV–Vis absorption measurements indicated that the binding of the La(III) to BSA and βLG may induce conformational and micro-environmental changes of the proteins. The docking results indicate that the La(III) complex bind to residues located in the site II of BSA and second site of βLG.

Communicated by Ramaswamy H. Sarma     

Sensitive genome-wide screen for low secondary enzymatic activities: the YjbQ family shows thiamin phosphate synthase activity

Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions. If selectively advantageous, secondary reactions could be the basis for the evolution of new fully functional enzymes. Here, we investigated if Escherichia coli has cryptic enzymatic activities related to thiamin biosynthesis. We selected this pathway because this vitamin is essential, but the cell's requirements are very small. Therefore, enzymes with very low activity could complement the auxotrophy of strains deleted of some bona fide thiamin biosynthetic genes. By overexpressing the E. coli's protein repertoire, we selected yjbQ, a gene that complemented a strain deleted of the thiamin phosphate synthase (TPS)-coding gene thiE. In vitro studies confirmed TPS activity, and by directed evolution experiments, this activity was enhanced. Structurally oriented mutagenesis allowed us to identify the putative active site. Remote orthologs of YjbQ from Thermotoga, Sulfolobus, and Pyrococcus were cloned and also showed thiamin auxotrophy complementation, indicating that the cryptic TPS activity is a property of this protein family. Interestingly, the thiE- and yjbQ-coded TPSs are analog enzymes with no structural similarity, reflecting distinct evolutionary origin. These results support the hypothesis that the enzymatic repertoire of a cell such as E. coli has the potential to perform vast amounts of alternative reactions, which could be exploited to evolve novel or more efficient catalysts.     

60Coγ射线与EMS复合处理对山黧豆抗氧化酶活力及ODAP含量的影响

Lathyrus sativus seeds were treated with ⁶⁰Coγ-ray and EMS(ethyl methane sulfonate), and their emergence rate and SOD,POD and CAT activities were determined. The result indicated that the treatment decreased the emergence rate. The activities of SOD and POD were changed in accordance with the increase of irradiation dose and EMS concentration, while that of CAT had no obvious change. After treatment, the ODAP content in Lathyrus sativus decreased. Amutant was developed, with toxin content of 0.1%, compared to 0.2% in control.     

microRNA-338-3p promotes ox-LDL-induced endothelial cell injury through targeting BAMBI and activating TGF-β/Smad pathway

microRNAs (miRNAs) have been revealed to participate in the pathological process of atherosclerosis (AS). However, the exact role of miR-338-3p, a target miRNA of BMP and activin membrane-bound inhibitor (BAMBI), and its possible molecular mechanism in AS remain unidentified. In this study, we found that BAMBI was significantly decreased, whereas miR-338-3p increased in patients with AS and oxidized low-density lipoprotein (ox-LDL)-induced HUVEC cells. Furthermore, overexpression of miR-338-3p significantly decreased cell viability and elevated cell apoptosis, whereas its inhibition significantly promoted cell viability and inhibited cell apoptosis in ox-LDL-induced HUVEC cells. Moreover, miR-338-3p overexpression increased TGF-β/Smad pathway activation in ox-LDL-induced HUVEC cells. A dual-luciferase reporter assay confirmed the direct interaction between miR-338-3p and the 3′-untranslated region of BAMBI messenger RNA. Furthermore, the suppression of BAMBI ameliorated the effect of miR-338-3p inhibition against ox-LDL-induced HUVEC cell injury. In conclusion, our study thus suggests that miR-338-3p promoted ox-LDL-induced HUVEC cell injury by targeting BAMBI and activating the TGF-β/Smad pathway, which may provide a novel and promising therapeutic target for AS.