Iron ions (Fe 2+ and Fe 3+ ) are essential trace elements for the human body, and are often added to various foods, but their effects on protein glycation remain unclear. This study evaluated the differential influences of Fe 2+ and Fe 3+ on the glycation reaction of β -lactoglobulin ( β -Lg)-D-ribose system in terms of glycation degree, protein conformation and the distribution of modification sites. Free amino group contents and HPLC HCD MS/MS analyses indicated that both Fe 3+ and Fe 2+ could catalyze the glycation process and increase the glycated sites. The system contain Fe 2+ exhibited higher glycation degree and more glycation sites (8), and lesser glycation sites were identified in system contain Fe 3+ (5) and system without ferric ions (2). Additional sites (L1, K14, K135) were facilitated glycation by Fe 2+ , and most glycation sites showed higher degree of substitution per peptide (DSP) values when with Fe 2+ . In comparison with Fe 2+ , Fe 3+ caused more pronounced alterations on both secondary and tertiary protein structure, promoted the β -Lg unfolding, and changed the protein structure to a more unordered form. In conclusion, Fe 2+ at a specified concentration was a better choice to promote glycation reaction while maintain the protein structure. This study provide a theoretical basis for protein glycation modification with iron ions at different valence states participated.

CCAAT/enhancer binding protein alpha (C/EBP alpha) is the key transcription factor involved in lipid metabolism, however, the role of C/EBP alpha in milk fat synthesis of dairy goats remains unknown. The objective of the present research was to clarify the function of C/EBP alpha in goat mammary epithelial cells (GMECs) and its impact on peroxisome proliferator-activated receptor gamma (PPARG) promoter activity. In this study, C/EBP alpha overexpression increased its mRNA and protein levels by 42-fold and 6-fold, respectively. In contrast, transfecting siRNA targeting C/EBP alpha decreased its mRNA level to 20% and protein abundance to 80% of the basal level. The contents of lipid droplets, triacylglycerol (TAG), and cholesterol were increased (P < 0.05) in C/EBP alpha-overexpressing GMECs, and knockdown of C/EBP alpha led to the opposite results. Overexpression of C/EBP alpha significantly increased the expression levels of genes involved in TAG synthesis (AGPAT6, DGAT2, P < 0.01), lipid droplet formation (PLIN2, P < 0.01), and fatty acid synthesis (FADS2, P < 0.05; ELOVL6, P < 0.01). Knockdown of C/EBP alpha decreased (P < 0.05) the expression levels of AGPAT6, DGAT1, DGAT2, PLIN2, FADS2, and ELOVL6. C/EBP alpha upregulated the expression level of PPARG (P < 0.05), and four C/EBP alpha binding regions were identified in the PPARG promoter at -1,112 to -1,102 bp, -734 to -724 bp, -248 to -238 bp, and -119 to -109 bp. Knockdown of C/EBP alpha reduced (P < 0.05) the PPARG promoter activity when the C/EBP alpha binding regions were mutated at -1,112 to -1,102 bp, -734 to -724 bp, and -248 to -238 bp locations of the promoter. However, the promoter activity did not change when the mutation was located at -119 bp. In conclusion, our results suggest that C/EBP alpha can promote TAG synthesis in GMECs through its effects on mRNA abundance of genes related to lipid metabolism and regulation of the PPARG promoter activity via C/EBP alpha binding regions.

C(CO)-alkyl bonds are ubiquitous in a variety of organic molecules, and their selective activation and functionalization are important for the reconstruction of simple ketones into valuable building blocks. However, due to the thermodynamic and kinetic stability, the cleavage and transformation of the unstrained C(CO)-alkyl bonds remain a significant challenge. Herein, we report a novel silver-catalyzed scission of the unstrained C(CO)-alkyl bond of ketones by reacting with N-isocyanoiminotriphenylphosphorane (NIITP) under mild conditions. This method could transform a variety of unstrained ketones into iminophosphoranes and nitriles in high yields. Experimental and computational studies disclosed the reaction proceeded through an unprecedented [3+2]/retro-[3+2] cycloaddition mechanism.

Fresh green peas require blanching to terminate enzymatic reaction induced quality deterioration before frozen storage. Radio frequency (RF) heating is a novel way of dry blanching for fruits and vegetables with high processing efficiency. In this study, blanching effects of RF heating on relative activities of lipoxygenase (LOX) and peroxidase (POD), physiochemical properties as well as cellular morphology changes of green peas were investigated. Results showed relative activities of pea LOX and POD reduced to 0.90 ± 0.78% and 1.10 ± 0.71%, respectively at 85 °C by RF heating with an electrode gap 105 mm. Weight loss, color, texture and electrolyte leakage of peas changed significantly with increasing temperature (60-85 °C). Ascorbic acid, chlorophyll and mineral contents had different loss after RF processing and long term heating at 115 mm exacerbated the loss of nutrients. Microstructure features showed the deconstruction of pea cell well and starch granule gelatinization.

We report here a novel reductive radical-polar crossover reaction that is a reductive radical-initiated 1,2-C migration of 2-azido allyl alcohols enabled by an azidyl group. The reaction tolerates diverse migrating groups, such as alkyl, alkenyl, and aryl groups, allowing access ton+1 ring expansion of small to large rings. The possibility of directly using propargyl alcohols in one-pot is also described. Mechanistic studies indicated that an azidyl group is a good leaving group and provides a driving force for the 1,2-C migration.

Compared with traditional hot water (HW) blanching, radio frequency (RF) has the advantages of faster heating rate and higher penetration depth. The aim of this study was to explore the optimum conditions of RF blanching of apple slice and compare the quality of apple subjected to RF and HW. Results showed that the optimum heating rate and uniformity can be obtained when electrode gap was of 120 mm, sample geometry of 50 × 8 × 50 mm, and not superimposed of apple slices, which were used for further RF blanching. It required 111 s and 270 s to reach the target temperature (85 °C) in RF and HW blanching respectively. After treated by RF and HW for 120 s, the residual enzyme activities of PPO of apples were 0% and 61.42 ± 1.48% (P < 0.05), the hardness were 402.83 ± 11.93 g and 469.39 ± 10.24 g (P < 0.05), the weight loss were 10.23 ± 1.01% and 14.39 ± 2.58% (P > 0.05), respectively. There was no significant difference (P > 0.05) in color change.

研究了不同热处理方式(水浴、微波、射频)和处理终点温度(70、80、90℃)对玉米醇溶蛋白功能性质与结构的影响.结果表明:多种热处理均会提高玉米醇溶蛋白溶解度、乳化能力和巯基含量,且这3项指标随着处理终点温度的升高而升高,射频处理至90℃使玉米醇溶蛋白溶解度、乳化能力和巯基含量分别提高了74.65％、171.7％和53.94％.应用差示扫描量热法测定玉米醇溶蛋白变性温度,发现微波处理后蛋白质变性温度略有升高.此外,紫外光谱分析提示,玉米醇溶蛋白经微波和射频加热处理后,蛋白质分子结构舒展、基团暴露,这使得蛋白质的溶解性和乳化能力有所改善.傅里叶变换红外光谱分析发现热处理改变了玉米醇溶蛋白的二级结构,其中微波和射频处理效果更明显.

过氧化物酶增殖物激活受体γ(peroxisome proliferator-activated receptor γ,PPARγ)是核受体家族成员,作为配体激活型转录因子,在调控脂肪酸代谢和脂滴形成等方面发挥重要作用.为分析PPARγ基因启动子结构及功能,完善奶山羊(Capra hircus)乳脂代谢调控网络,本研究以西农萨能奶山羊全血DNA为模板,通过PCR技术扩增PPARγ基因5'侧翼序列,并克隆得到7个长度不同的缺失片段,分别连接pGL3-basic载体构建重组体,利用双荧光素酶报告系统检测各重组质粒活性,结合生物信息学分析确定其转录核心区域;并通过启动子定点突变和过表达CCAAT增强子结合蛋白α(CCAAT enhancer binding protein alpha,C/EBPα)研究转录因子C/EBPα对PPARγ基因的转录调控.结果表明,克隆得到的PPARγ基因启动子序列全长为2 328 bp(包含转录起始位点上游2 181 bp,GenBank登录号:MG770492.1),启动子上无典型的真核生物元件TATA框,但存在C/EBPα、肝X受体(liver X receptor,LXR)、胆固醇调节元件结合蛋白(sterol-regulatory element binding protein,SREBP)及特异性蛋白1(specificity protein 1,SP1)等多个转录因子结合位点;缺失片段分析结果表明,PPARγ基因启动子核心区域位于转录起始位点上游194～108 bp,且启动子上存在负调控元件;转录因子C/EBPα通过结合位于启动子上游119 bp处的C/EBPα结合元件而调控PPARγ基因的转录.本研究为后续开展PPARγ调控羊奶脂肪酸代谢的机理研究提供了资料.

Akt serine/threonine kinase acts as a central mediator in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, regulating a series of biological processes. In lipid metabolism, Akt activation regulates a series of gene expressions, including genes related to intracellular fatty acid synthesis. However, the regulatory mechanisms of Akt in dairy goat mammary lipid metabolism have not been elaborated. In this study, the coding sequences of goat Akt1 gene were cloned and analyzed. Gene expression of Akt1 in different lactation stages was also investigated. For in vitro studies, a eukaryotic expression vector of Akt1 was constructed and transfected to goat mammary epithelial cells (GMECs), and specific inhibitors of Akt/mammalian target of rapamycin (mTOR) signaling were applied to GMECs. Results showed that Akt1 protein was highly conserved, and its mRNA was highly expressed in midlactation. In vitro studies indicated that Akt1 phosphorylation activated mTOR and subsequently enhanced sterol regulatory element binding protein 1 (SREBP1), thus increasing intracellular triacylglycerol content. Inhibition of Akt/mTOR signaling down-regulated the gene expression of lipogenic genes. Overall, Akt1 plays an important role in regulating de novo fatty acid synthesis in goat mammary epithelial cells, and this process probably is through the mTOR/SREBP1 axis.

In mammals, sterol regulatory element binding protein-1 (SREBP-1) is the master regulator of fatty acid and triacylglycerol synthesis. Recent gene silencing studies in mammary cells indicate that SREBP-1 has a central role in milk fat synthesis. However, SREBP-1 knockdown studies in goat mammary cells have not been performed; hence, its direct role in controlling mRNA expression of lipid metabolism genes and triacylglycerol synthesis remains unknown. Inhibition of SREBP-1 in goat mammary epithelial cells (GMEC) by small interference RNA (siRNA) markedly reduced the content of cellular triacylglycerol (~50% decrease, P < 0.05) and was partly related to downregulation of AGPAT6, LPIN1, and DGAT2 (-23%, -28% and -19%, respectively. P < 0.05), which are key enzymes involved in triacylglycerol synthesis, cellular triacylglycerol content and lipid droplet accumulation all decreased by SREBP-1 inhibition. The expression of lipid droplet formation and secretion genes was not altered in response to treatment. Although the lack of effect on expression of ACACA and FASN (rate-limiting enzymes for de novo fatty acid synthesis) with SREBP-1 knockdown was unexpected (P > 0.05), the downregulation of genes related to synthesis of acetyl-CoA and acetate activation (ACLY, ACSS2, and IDH1, P < 0.05) suggests that lipogenesis was inhibited. SREBP-1 knockdown also resulted in decreased expression of genes associated with fatty acid desaturation and elongation (SCD1 and ELOVL6, P < 0.05), long-chain fatty acid (LCFA) activation and transport (ACSL1, FABP3, and SLC27A6, P < 0.05). The results underscored the essential role of SREBP-1 not only in fatty acid synthesis but also in desaturation, elongation, and esterification in GMEC. Clearly, the lack of effect on ACACA and FASN, both of which are considered the key lipogenic enzymes, implies that there may be different regulatory mechanisms in goat compared with bovine mammary cells.

Yogurt is one of the most popular dairy products fermented by Lactobacillus bulgaricus and Streptococcus thermophilus. The present study introduced dielectric properties as a new technique for yogurt fermentation monitoring. Dielectric properties of cow milk and yogurt at different time of fermentation were measured from 10 to 3000 MHz at 42 °C using open-ended coaxial-line probe technology by an impedance analyzer. Meanwhile, pH and titratable acidity of yogurt at different fermentation time were measured. Results showed that the dielectric loss factor of yogurt was positively correlated with fermentation time and had an irregular change at the endpoint of fermentation (7 h, pH ≈ 4.6). The polynomial determination coefficients of dielectric loss factor with pH and titratable acidity decreased with increasing frequency and were found highest at 10 MHz (0.927 and 0.963, respectively). The linear coefficients of determination between measured values and calculated values were 0.990 for titratable acidity and 0.965 for pH at 10 MHz, respectively. Accordingly, dielectric loss factor can be a promising indicator for online monitoring of industrial yogurt fermentation process.

Lactoferrin (LF) is a prominent protein in milk, and is present in many other secretory fluids and white blood cells. The variation of LF content in milk is possibly regulated by multiple environmental factors such as milk composition and endogenous factors. In this study, we analysed the variations of LF in both milk and plasma of 20 Xinong Saanen goats and its related factors. Firstly, LF content in colostrum was 222.6 ± 41.57 μg/mL, and then throughout the lactation period the mean concentrations remained stable, ranged between 34.61 and 51.94 μg/mL. The LF concentration in plasma fluctuated between 173.56 and 246.20 μg/mL during the entire lactational period. There was no correlation between the milk and plasma LF concentrations (P > .05). However, LF in milk was closely correlated with milk lactose, protein and SNF contents (P < .01), but not with milk fat and total solids (P > .05). In addition, LF concentration in milk was correlated with the lactation stage (P < .01). Further studies are required to investigate these factors that cause LF variations in the milk of Xinong Saanen goat.

MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression at the post‐transcriptional level to cause translational repression or degradation of targets. The profiles of miRNAs across stages of lactation in small ruminant species such as dairy goats is unknown. A small RNA library was constructed using tissue samples from mammary gland of Saanen dairy goats harvested at mid‐lactation followed by sequencing via Solexa technology. A total of 796 conserved miRNAs, 263 new miRNAs, and 821 pre‐miRNAs were uncovered. After comparative analyses of our sequence data with published mammary gland transcriptome data across different stages of lactation, a total of 37 miRNAs (including miR‐145) had significant differences in expression over the lactation cycle. Further studies revealed that miR‐145 regulates metabolism of fatty acids in goat mammary gland epithelial cells (GMEC). Compared with nonlactating mammary tissue, lactating mammary gland had a marked increase in expression of miR‐145. Overexpression of miR‐145 increased transcription of genes associated with milk fat synthesis resulting in greater fat droplet formation, triacylglycerol accumulation, and proportion of unsaturated fatty acids. In contrast, silencing of miR‐145 impaired fatty acid synthesis. Inhibition of miR‐145 increased methylation levels of fatty acid synthase (FASN), stearoyl‐CoA desaturase 1 (SCD1), peroxisome proliferator‐activated receptor gamma (PPARG), and sterol regulatory element binding transcription factor 1 (SREBF1). Luciferase reporter assays confirmed that insulin induced gene 1 (INSIG1) is a direct target of miR‐145. These findings underscore the need for further studies to evaluate the potential for targeting miR‐145 for improving beneficial milk components in ruminant milk. J. Cell. Physiol. 232: 1030–1040, 2017. © 2016 Wiley Periodicals, Inc.

Sterol regulatory element binding protein 1 (SREBP‐1) is well‐known as the master regulator of lipogenesis in rodents. Acyl‐CoA synthetase short‐chain family member 2 (ACSS2) plays a key role in lipogenesis by synthesizing acetyl‐CoA from acetate for lipogenesis. ATP citrate lyase (ACLY) catalyzes the conversion of citrate and coenzyme A to acetyl‐CoA, hence, it is also important for lipogenesis. Although ACSS2 function in cancer cells has been elucidated, its essentiality in ruminant mammary lipogenesis is unknown. Furthermore, ACSS2 gene promoter and its regulatory mechanisms have not known. Expression of ACSS2 was high in lipid synthesizing tissues, and its expression increased during lactation compared with non‐lactating period. Simultaneous knockdown of both ACSS2 and ACLY by siRNA in primary goat mammary epithelial cells decreased (p < 0.05) the mRNA abundance of genes associated with de novo fatty acid synthesis (FASN, ACACA, SCD1) and triacylglycerol (TAG) synthesis (DGAT1, DGAT2, GPAM, and AGPAT6). Genes responsible for lipid droplet formation and secretion (PLIN2 and PLIN3) and fatty acid oxidation (ATGL, HSL, ACOX, and CPT1A) all decreased (p < 0.05) after ACSS2 and ACLY knockdown. Total cellular TAG content and lipid droplet formation also decreased. Use of a luciferase reporter assay revealed a direct regulation of ACSS2 by SREBP‐1. Furthermore, SREBP‐1 interacted with an SRE (SREBP response element) spanning at −475 to −483 bp on the ACSS2 promoter. Taken together, our results revealed a novel pathway that SREBP‐1 may regulate fatty acid and TAG synthesis by regulating the expression of ACSS2.

This paper is focused on the effects of radio frequency (RF) heating on the relative activity of polyphenol oxidase (PPO), weight loss, texture, color, and microstructure of potatoes. The results showed that pure mushroom PPO was almost completely inactivated at 80 °C by RF heating. The relative activity of potato PPO reduced to less than 10% with increasing temperature (25-85 °C). Enzyme extract showed the lowest PPO relative activity at 85 °C after RF treatment, followed by the potato cuboids and mashed potato, about 0.19 ± 0.017%, 3.24 ± 0.19%, and 3.54 ± 0.04%, respectively. Circular dichroism analysis indicated that RF heating changed the secondary structure of PPO, as α-helix content decreased. Both electrode gap and temperature had significant effect (P < .05) on weight loss, color, and texture of the potato cuboids. Microstructure analysis showed the changes of potato cell and starch during RF heating.