Reexamination of the Ergothioneine Biosynthetic Methyltransferase EgtD from Mycobacterium tuberculosis as a Protein Kinase Substrate

Ergothioneine has emerged as a crucial cytoprotectant in the pathogenic lifestyle of Mycobacterium tuberculosis. Production of this antioxidant from primary metabolites may be regulated by phosphorylation of Thr213 in the active site of the methyltransferase EgtD. The structure of mycobacterial EgtD suggests that this post-translational modification would require a large-scale change in conformation to make the active-site residue accessible to a protein kinase. In this report, we show that, under in vitro conditions, EgtD is not a substrate of protein kinase PknD.     

Characteristics and nutritional value of biscuits fortified with debittered salmon (Salmo salar) frame hydrolysate

Effect of debittered salmon frame hydrolysate (DSFH) at various levels (0, 5, 10, 15, 20 and 25 g/100 g) on physicochemical, textural, sensory and nutritional properties of biscuits was investigated. The highest thickness was obtained for the sample with 25 g/100 g DSFH (P < 0.05). There was no difference in diameter among all the biscuit samples (P > 0.05). The samples added with DSFH had lower weight, water activity and moisture content than the control (CONT, without DSFH) (P < 0.05). DSFH at 15 g/100 g showed no detrimental effect on sensory properties of resulting biscuits (DSFH-15). The DSFH-15 biscuit showed reduction in cutting force and fracturability. Scanning electron microscopic and cross-sectional images showed that DSFH-15 biscuit had more porous structure, compared to the CONT. The biscuits fortified with 15 g/100 g DSFH had higher protein but had lower energy value, fat and carbohydrate content than the CONT.     

Proton-Detected Solid-State NMR of the Cell-Free Synthesized α-Helical Transmembrane Protein NS4B from Hepatitis C Virus

Proton-detected 100 kHz magic-angle-spinning (MAS) solid-state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell-free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid-environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D ¹H-detected ¹H,¹⁵N and 3D ¹H,¹³C,¹⁵N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino-acid-selectively labeled NS4B can be readily obtained using cell-free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies.     

Nutrient digestibility and endogenous protein losses in the foregut and small intestine of weaned dairy calves fed calf starters with conventional or enzyme-treated soybean meal

The aims of this experiment were (1) to compare the effects of a soybean meal with an enzymatic treatment (ESBM) to reduce the concentration of antinutritional factors versus a standard soybean meal (SBM) on foregut and small intestine digestion in weaned dairy calves and (2) to estimate the endogenous losses of crude protein (CP) in the small intestine. Our hypothesis was that a diet containing ESBM instead of SBM would improve ruminal and small intestine digestion and absorption of nutrients. A T-cannula was placed in the duodenum, and a second T-cannula was installed in the distal ileum of 12 Holstein calves at approximately 3 wk of age. Calves were weaned on d 42, and on d 50 they were assigned randomly to a quadruplicated 3 × 3 Latin square with 10-d periods. Digesta samples were collected on d 7 and 8 from the ileum and d 9 and 10 from the duodenum. The diets were fed for ad libitum intake and consisted of a calf starter (CS) of 20% CP with SBM as the main source of protein (CTRL), and an isonitrogenous CS with an ESBM instead of SBM (ENZT). A third diet with a low content of CP (10%) and no soy protein was fed to estimate endogenous N losses and digestibilities of test ingredients. Flows and digestibilities of nutrients were compared between CTRL and ENZT and their test ingredients (SBM vs. ESBM, respectively). Duodenal net flows of CP and total AA as well as ruminal microbial protein synthesis per kilogram of digested CP were greater, and flow of nonprotein N and CP true (corrected by endogenous and microbial flows) foregut digestibility were lower with ENZT than CTRL. The apparent small intestine digestibilities of CP and total AA were greater for ESBM than SBM, but there were no differences between the CTRL and ENZT diets. We observed no differences in digestibilities at the duodenum or ileum of starch or NDF, but true small intestine digestibilities of CP and all AA were greater with ENZT than CTRL. Total endogenous protein losses in the small intestine estimated from calves fed the low-CP with no soy protein diet were 37 ± 1.5 g of CP and 29 ± 1.4 g of AA/kg of DMI. These values may be considered the basal endogenous losses as they are similar to values obtained with the regression method, which estimates N losses when dietary N is null. Our results indicated that the inclusion of an ESBM improved the efficiency of ruminal microbial protein synthesis per digested kilogram of organic matter and CP, and increased CP and AA absorption in the small intestine despite a greater proportion of undigested dietary protein entering the duodenum.     

氢化物原子荧光光谱法测定酱油中微量铅的方法研究

建立了一种快速、高效的QuEChERS-高效液相色谱-质谱联用法（QuEChERS-HPLC-MS/MS）测定谷类杂粮制品中脱氧雪腐镰刀菌烯醇（DON）、3-乙酰脱氧雪腐镰刀菌烯醇（3-ADON）、15-乙酰脱氧雪腐镰刀菌烯醇（15-ADON）和玉米赤霉烯酮（ZON）共4种真菌毒素。样品前处理采用乙腈-水溶剂提取，经Florisil+C18+无水硫酸镁净化后检测。以0.10%甲酸-乙腈作为流动相，在质谱检测器的多反应监测模式下进行分析。结果表明，4种真菌毒素在各自的线性范围内线性关系良好，相关系数R2均大于0.999，回收率在85.1%～102.0%，相对标准偏差（RSD）为2.11%～6.22%。该方法具有前处理简单、净化效果好、灵敏度高和检测速度快的优点，适用于谷类杂粮制品中DON、3-ADON、15-ADON 和ZON的分析和定量检测。     

Combination of vegetable soup and glucan demonstrates synergistic effects on macrophage-mediated immune responses

Vegetable soup (VS), a plant-based functional food, has been used as a traditional folk medicine and is attracting attention for its ability to enhance the immune response. β-Glucan, a well-established and effective immunomodulator, has synergistic effects when used in combination with some bioactive compounds. In the present study, we aimed to evaluate the synergistic immunomodulatory effects of the combination of VS and β-glucan on macrophage-mediated immune responses. β-Glucan was demonstrated to synergistically enhance the VS-stimulated immune response, including the production of interleukin-6, tumor necrosis factor-α, and nitric oxide, mainly through the mitogen-activated protein kinase pathway in macrophages. In addition, this combination has the potential for further development in functional foods with immune-enhancing activity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10068-021-00888-x.     

Effect of select parameters on the properties of edible film from water-soluble fish proteins in surimi wash-water

Edible film from water-soluble fish proteins were developed by casting film solution on leveled trays and effects of pH (9.5, 10.0 and 10.5), heating temperature (60, 70 and 80 °C), and heating time (10, 20 and 30 min) of the film solution on various film properties were determined using Response Surface Methodology (RSM). The impact of pH and heating temperature of film solution was more significant, overall, on the film's properties than heating time. Contour plots of tensile strength and elongation at break was highest at pH of 10.0 at 70 °C (2.75-3.02 MPa) but low in elongation at break (6.35-9.16%), while water vapor permeability and oxygen permeability were at their lowest (58.55-65.96 g mm/m² d kPa and 351.33-624.18 cm³ μm/m² d kPa). There was a direct correlation between the films’ and proteins’ solubility on one hand, and heating temperature of film solution on the other, which reversed with change in pH of film solution. Film color was darker and more yellowish with increase in the pH of film solution.     

抗真菌蛋白Rs-AFP2基因定点突变及其对大肠杆菌中表达的影响

为了研究遗传密码子对表达调控的影响,利用PCR重叠延伸法,对萝卜抗真菌蛋白Rs-AFP2基因编码序列区的部分核苷酸进行沉默突变,构建突变体Rs-AFPm.序列分析表明,PCR产物全长240bp,有一个阅读框,编码的蛋白由29个氨基酸的信号肽和51个氨基酸的抗真菌蛋白组成.突变体与突变前的Rs-AFP2基因相比,在编码区第3号氨基酸Lys相差一个碱基(TTG→TTA),第5号氨基酸Gln相差一个碱基(CAG→CAA),第6号稀有密码子Arg相差两个碱基(CAG→CGA).重新合成引物,将切除信号肽的Rs-AFP2基因和Rs-AFPm基因与原核表达载体pET-21b(+)分别重组到大肠杆菌BL21菌株.IPTG诱导后,二者均得到了表达.软件分析显示,突变前pETAFPo表达产物占全菌蛋白的3%,突变后pETAFPm的表达产物占全菌蛋白含量的8%;表达蛋白主要以包涵体的形式存在,包涵体经超声波破碎后,蛋白质复性,抑菌结果表明,pETAFPm表达产物的抑菌半径大于pETAFP2表达产物的抑菌半径.这些都说明改造后的Rs-AFPm基因与Rs-AFP2基因相比,已有效地提高表达量.