瓠瓜胰蛋白酶抑制剂的提取

采用水提法从瓠瓜中提取胰蛋白酶抑制剂,以瓠瓜提取液对胰蛋白酶的抑制率为指标,研究了提取温度、液料比、提取时间、pH四个主要影响因素,优化选出最佳提取方案。从瓠瓜中提取胰蛋白酶抑制剂的最佳条件为：温度70℃、pH=8、液料比4∶1（mL/g）、提取时间1h。     

Inhibitory effects of lactoferrin on pulmonary inflammatory processes induced by lipopolysaccharide by modulating the TLR4-related pathway

This study tested the ability of lactoferrin to modulate pulmonary inflammation. To construct in vitro and in vivo inflammatory lung models, cells from the human lung adenocarcinoma cell line (A549) were exposed to lipopolysaccharide (LPS, 1 µg/mL), and mice (CD-1) were intratracheally administered LPS [10 mg/kg of body weight (BW), tracheal lumen injection], respectively. The A549 cells were preincubated with lactoferrin (10 mg/mL), and the mice were intraperitoneally injected with lactoferrin (100 mg/kg of BW), followed by LPS treatment. The concentrations of proinflammatory cytokines (IL-1β and TNF-α) in culture medium of A549 cells and in bronchoalveolar lavage fluid of the mice were determined using enzyme-linked immunosorbent assays. The toll-like receptor 4–related pathway (TLR4/MyD88/IRAK1/TRAF6/NFκB) was determined at gene and protein expression levels in A549 cells and mouse lung tissue. Results showed that LPS treatment significantly elevated the concentrations of IL-1β and TNF-α in the A549 cell culture medium and in bronchoalveolar lavage fluid of the mice; it also elevated both the mRNA and protein expressions of TLR4 and the TLR4 downstream factors in A549 cells and mouse lung tissue. Nevertheless, lactoferrin apparently depressed the releases of IL-1β and TNF-α from A549 cells and lung tissues stimulated by LPS, and significantly suppressed the TLR4 signaling pathway. Lactoferrin also promoted the enhancement of miR-146a expression in A549 cells and mouse lung tissue. Moreover, 100°C heating for 3 min caused total loss of the previously listed bioactivity of lactoferrin. Collectively, we proved that lactoferrin intervened in LPS-induced inflammation in the pulmonary cell model and in the mouse model, through inhibiting the TLR4-related pathway.     

不同还原剂对大豆胰蛋白酶抑制剂的钝化研究

研究了二巯基苏糖醇(DTT)、亚硫酸钠(Na2SO3)和半胱氨酸(Cys)对大豆胰蛋白酶抑制剂活性的影响。实验在温度80℃,pH7.5条件下反应,并以BAPNA为底物,采用改进的方法测定DTT对大豆蛋白酶抑制剂的钝化作用,最后用SDS-PAGE方法和凝胶排阻色谱法研究其蛋白酶钝化敏感性。通过改进的BAPNA法得出还原剂钝化大豆胰蛋白酶抑制剂由大到小顺序依次为:DTT>Na2SO3>Cys,并通过SDS-PAGE进一步证实了比色法得出的结论。而凝胶排阻色谱法说明了还原剂对大豆胰蛋白酶抑制剂作用使得抑制剂中二硫键被打断,抑制剂结构发生改变,有新的物质生成。所以,得出大豆胰蛋白酶抑制剂的稳定性与二硫键的存在有关。     

Ruminal lipopolysaccharide concentration and inflammatory response during grain-induced subacute ruminal acidosis in dairy cows

The effects of grain-induced subacute ruminal acidosis (SARA) in lactating dairy cows on free ruminal lipopolysaccharide (LPS) and indicators of inflammation were determined. Four mid lactation dairy cows were divided into 2 groups of 2 cows and used in a repeated switchover design. During each period, SARA was induced in 2 animals for 5 subsequent days by replacing 25% of their total mixed ration (dry matter basis) with a concentrate made of 50% wheat and 50% barley. The other 2 cows acted as controls and were fed a total mixed ration diet in which 44% of dry matter was concentrate. On average, inducing SARA did not affect milk composition, increased the duration of rumen pH below 5.6 from 187 to 309 min/d, and increased free ruminal LPS concentration from 24,547 endotoxin units (EU)/mL to 128,825 EU/mL. Averaged across treatments, milk fat yield and milk protein yield were 0.66 and 1.00 kg/d, respectively. Rumen pH and milk fat data suggest that control cows also experienced ruminal acidosis, albeit a milder form of this disease than SARA cows. Serum LPS concentration in both control and SARA cows was less than the detection limit of <0.01 EU/mL for the assay. Induction of SARA elevated serum amyloid A concentration from 286.8 to 498.8 μg/mL, but did not affect other markers of inflammation including haptoglobin, fibrinogen, serum copper, or white blood cells. These results suggest that grain-induced SARA in mid lactation dairy cows increases the lysis of gram-negative bacteria and activates an inflammatory response.     

Subacute ruminal acidosis induces ruminal lipopolysaccharide endotoxin release and triggers an inflammatory response

Subacute ruminal acidosis (SARA) was induced in 3 rumen fistulated Jersey steers by offering them different combinations of wheat-barley pellets and chopped alfalfa hay. Steers were offered 4, 5, and 6 kg/d of pelleted concentrate and 6, 5, and 4 kg/d of chopped alfalfa hay for diets 1, 2, and 3, respectively, during 5-d treatment periods and were fed chopped alfalfa hay between treatment periods. Inducing SARA increased blood concentrations of haptoglobin and serum amyloid-A. Dry matter intake of concentrate and hay decreased from d 1 to 5 in each period. Subacute ruminal acidosis was induced in all steers during d 4 and 5 when concentrate was fed, with ruminal pH remaining below 5.6 for an average of 187 and 174 min/d on these days. Lipopolysaccharide concentration increased significantly during periods of grain feeding compared with times when only hay was fed. Inducing SARA by feeding wheat-barley pellets activated a systemic inflammatory response in the steers.     

The determination of trypsin inhibitor levels in foodstuffs

The trypsin inhibitor activity of processed foods can be determined by measuring the loss of activity of added trypsin under standard conditions. Observed values are not usually independent of the degree of inhibition, and averaging over a range of inhibition levels or extrapolation to zero inhibition may not produce a more reliable value. A somewhat modified method is described which has been tested in two laboratories and used for large numbers of different samples on a routine basis; its application and limitations are discussed.     

Buckwheat trypsin inhibitor enters Hep G2 cells by clathrin-dependent endocytosis

Recombinant buckwheat trypsin inhibitor (rBTI) was studied to evaluate if it could enter cancer cells and to determine the mechanism. Fluorescein isothiocyanate-labelled buckwheat trypsin inhibitor (FITC-BTI) entered Hep G2 cells in a concentration-dependent manner. FITC-BTI colocalised with labelled transferrin (Tf) in the punctate structure, implying that rBTI enters Hep G2 cells by clathrin-dependent endocytosis. Incubation of Hep G2 cells with different chemical inhibitors abolished diffuse, but not punctate fluorescence, thus indicating that membrane potential plays a critical role in this process. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a theory of a single route of endocytosis. Consistent with our working hypothesis, Hep G2 cells which were arrested in the M phase did not show any vesicular or diffuse FITC-BTI. We conclude from these results that both endocytosis and membrane potential are required for rBTI entry into Hep G2 cells.     

Changes of trypsin in activity and secondary structure induced by complex with trypsin inhibitors and tea polyphenol

To reveal the relationships between the activity of trypsin and its structural change, changes of trypsin in biological activity induced by complex with Bowman-Birk trypsin inhibitor (BBTI), Kunitz soybean trypsin inhibitor (KSTI, type I-S) and tea polyphenol (TP) were detected and their relationship with the secondary structure changes were studied by far-UV circular dichroism (CD) spectra measurement. BBTI and KSTI were also irradiated by ultrasonic to compare the effects on trypsin. The rank was found as KSTI > BBTI > TP according to their inhibitory activities against trypsin. Yet BBTI exhibited much stronger resistance against ultrasonic irradiation than KSTI. BBTI, KSTI and TP were found inactivate trypsin by modifying the secondary structures and far-UV spectrum of trypsin. Complex of trypsin with ultrasonic-treated BBTI and native BBTI and KSTI exhibited the similar modified effects in secondary structures, decrease of α-helix and β-turn content, increase of β-sheet content and unchanged random coil content basically. But complex of trypsin with ultrasonic-treated KSTI exhibited less modified effects because of inactivation by ultrasonic irradiation. The changes of trypsin in secondary structure induced by complex with TP showed different from those induced by complex with BBTI and KSTI, increase of α-helix content, decrease of random coil content and unchanged β-sheet and β-turn content basically.     

Volunteer study and serum protein profiling to understand inflammatory response induced by Satsuma mandarin

It has been observed that consumption of a certain amount of Satsuma, lychee, and longan often caused a symptom characterized by dry or sore throat, gum swelling and even mouth ulcer, which significantly impaired the life quality of a large population. We define the adverse reaction to Satsuma as Satsuma-induced syndrome (SIS). Volunteers were assigned to oral Satsuma challenge in an open manner. The results showed that SIS was characterized with symptoms affecting the throat, oral cavity, face, gastrointestinal system and eye either individually or in combination. A comparative proteomic study was performed to investigate the differences of serum proteins in the Post-SC (after Satsuma challenge) and Pre-SC (before Satsuma challenge) serum samples of 15 volunteers with severe SIS. Ten proteins were identified to be differentially expressed (P < 0.05). Of these, levels of complement component C9 precursor were elevated significantly in the Post-SC serum samples and were further verified by enzyme-linked immunosorbent assay, indicating that the complement system may be activated and plays a significant role in inflammatory response. Meanwhile, serum samples were subjected to immobilized metal affinity capture (IMAC3) protein chip surfaces and tested by surface-enhanced laser desorption/ionization-time of flight-mass spectrometry. The data were analyzed by Ciphergen ProteinChip Software. A diagnostic model was constructed to discriminate the SIS from normal samples, using principal component analysis. A total of 50 detected biomarkers were found to be different with statistical significance (P < 0.05). The multivariate logistic analysis demonstrates a complete distinction between the two groups. Our findings suggest that these assays may provide potential biomarkers for the diagnosis of SIS.     

Inactivation of trypsin inhibitor,lectin and urease in soybean by hydrothermal treatment

The effects of hydrothermal treating parameters on trypsin inhibitor (TI), lectin and urease activities in whole soybean seeds were investigated by Response Surface Analysis (RSA). Independent variables with equidistant levels examined in this study were the following: treating temperature (levels: 70, 85 and 100°C), treating time (levels: 10, 30 and 50 min), and soaking time (2, 4 and 6 h). The functions between treating parameters and responses values of TI, lectin and urease activities in treated soybean were calculated by multiple, non-linear regression analysis and analysis of variance. Mathematical models were developed in this study to predict TI, lectin and urease activities in soybean during hydrothermal processing and they have been found to be significant (P[%] = 0.1, 1.0, and 0.1, respectively). Differences between the effects of processing parameters on the inactivation of TI, lectin and urease in soybean were observed and they can be seen either from the mathematical models or the typical figures. The modeling of the effects should help in selection of optimal parameters of hydrothermal treatment for destruction of TI. lectin and urease in soybean. Based on the modeling, lectin and urease can be fully inactivated in soybean treated at proper conditions, while remaining TI activity can be expected in soybean treated at any conditions examined in this study.     

Soybean trypsin inhibitor neutralization of its inhibitory effect upon the casein precipitating reaction of trypsin by rabbit antiserum

Summary Antiserum produced against Soybean Trypsin Inhibitor (SBTI) was shown to neutralize the SBTI inhibition of the Casein precipitating activity of trypsin thus indicating that the precipitating interactions observed in agar gel double diffusion analysis were at least partly due to reaction between SBTI and specific antibody.On this basis the use of antiserum for the detection of SBTI in various soy proteins is discussed.

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Zusammenfassung Es konnte gezeigt werden, daß Antiserum, welches gegen die Soja-Trypsin-Hemmer hergestellt worden war, die SBTI Hemmung der caseinfällenden Trypsinaktivität neutralisieren kann; daraus wird gefolgert, daß die bei der Ausfällung mit dem Agar-Gel-Diffusions-test beobachteten Reaktionen zumindest teilweise auf eine Reaktion zwischen SBTI und den spezifischen Antikörpern beruhen.Aufgrund dieser Ergebnisse wird die Anwendung von Antiserum zur Bestimmung von SBTI in verschiedenen Soja-Proteinen beschrieben.

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This work was supported by a grant from the Agricultural Research Council of Norway.     

Eucalcemia during lipopolysaccharide challenge in postpartum dairy cows: I. Clinical,inflammatory, and metabolic response

Hypocalcemia induced by immune activation is a conserved response across mammalian species; however, administration of Ca is discouraged in other species as it is associated with increased morbidity and mortality. Early postpartum cows experience a decrease in circulating Ca concentration following acute inflammation. Corrective Ca therapy during the transition period, particularly in dairy cows experiencing acute disease, is common practice. However, the effect of Ca administration on the inflammatory response during acute immune activation is unknown. Our objective was to compare the clinical, inflammatory, and metabolic response to an intravenous (IV) lipopolysaccharide (LPS) challenge between postpartum cows infused, or not, with IV Ca to maintain eucalcemia. Cows (n = 14, 8 ± 1 d in milk) were enrolled in a matched-pair randomized controlled design to receive IV Ca (IVCa) or sterile 0.9% NaCl (CTRL) during an IV LPS challenge (0.040 or 0.045 µg of LPS/kg of body weight over 1 h). Ionized Ca (iCa) was monitored cow-side, and IV Ca infusion was adjusted in a eucalcemic clamp for 12 h following the start of LPS infusion. Cows were monitored during the 24 h following challenge and serial blood samples were collected to quantify concentrations of glucose, β-hydroxybutyrate, nonesterified fatty acids, urea nitrogen, cytokines, acute-phase proteins, and cortisol. Blood iCa concentration decreased to 0.87 ± 0.03 mM in CTRL during challenge, and by design, iCa concentration was maintained within 3% of baseline in IVCa. Body temperature, heart rate, and respiratory rate were monitored for 24 h following the start of challenge and did not differ between groups. A treatment × time interaction was identified such that serum cortisol concentrations increased in both groups at 2 h but decreased to a greater extent at 6 h in IVCa compared with CTRL. Rumination time (min/h) over the first 12 h following challenge was greater in IVCa, but total rumination time in the 24 h following challenge did not differ from CTRL. Serum glucose and nonesterified fatty acid concentrations decreased, and β-hydroxybutyrate and urea nitrogen concentrations increased over time, but did not differ between groups. Acute leukopenia occurred in both groups at 4 h before leukocytosis was observed at 24 h with total white blood cell counts returning to baseline within 72 h. Plasma concentrations of tumor necrosis factor (TNF) and interleukin-10 (IL-10) increased within 1 h following the start of challenge and did not differ between groups. Serum haptoglobin and serum amyloid A concentrations increased within the 24 h following challenge and were elevated through 72 h but did not differ between groups. Eucalcemia during the acute systemic inflammatory response did not alter the TNF or IL-10 cytokine response, or the acute-phase protein SAA and haptoglobin response in this LPS challenge model; however, eucalcemia was associated with a more rapid decline in cortisol response and greater rumination time in the first 12 h following challenge. We did not find evidence that eucalcemia exacerbated the inflammatory response in early postpartum cows, but Ca administration may alter the clinical response to acute systemic inflammation.     

虫草素对脂多糖诱导巨噬细胞过度活化的抑制作用研究

目的:研究虫草素对脂多糖（LPS）诱导巨噬细胞过度活化的抑制作用。方法:培养RAW264.7小鼠巨噬细胞,采用LPS（1μg/m L）激活巨噬细胞,同时给予0.1¹0μmol/L的虫草素处理细胞,采用四甲基偶氮唑盐（MTT）法测定细胞存活率,Griess法检测细胞一氧化氮（NO）释放量,酶联免疫吸附分析实验（ELISA）分别检测三种细胞炎症因子肿瘤坏死因子α（TNF-α）、白介素-1β（IL-1β）和白介素-6（IL-6）的释放水平,采用Fluo-3/AM染色法以流式细胞仪测定胞浆游离钙离子浓度([Ca²⁺]i)的变化,采用水杨酸法考察虫草素对羟自由基（·OH）清除作用,L-酪氨酸法检测其对过氧亚硝酸根离子自由基（ONOO-）清除作用,邻苯三酚自氧化法检测其对超氧阴离子自由基（O₂^-·）清除作用。结果:虫草素在0.1¹0μmol/L浓度范围内可显著抑制LPS激活的RAW264.7巨噬细胞释放NO及3种炎症因子（IL-1β、IL-6和TNF-α）,同时对细胞存活率无显著影响;虫草素（10μmol/L）可显著抑制LPS诱导的巨噬细胞内[Ca²⁺]i水平异常升高;此外,虫草素对·OH、ONOO-和O₂^-·自由基具有显著清除作用。结论:虫草素可抑制LPS激活的RAW264.7巨噬细胞释放NO及炎症因子TNF-α、IL-1β和IL-6,这可能与其抑制了细胞内[Ca²⁺]i的增高及清除·OH、ONOO-和O₂^-·自由基有关。虫草素可能对巨噬细胞过度激活引起的炎症相关疾病具有一定的防治作用。      

Relationships between rumen lipopolysaccharide and mediators of inflammatory response with milk fat production and efficiency in dairy cows

The main objective of this study was to evaluate correlative relationships between rumen lipopolysaccharide (LPS) and mediators of acute phase response with milk fat yield and efficiency in dairy cows challenged with graded amounts of barley grain in the diet. An additional aim of the study was to quantify the intercow variation in relation to milk fat production and acute phase response in cows fed graded amounts of grain. Eight primiparous, lactating Holstein cows (60 d in milk) were assigned to 1 of the 4 total mixed rations containing barley grain at 0, 15, 30, and 45% (dry matter basis) in a replicated 4 × 4 Latin square design. Free rumen LPS, plasma acute phase proteins, and milk fat content were quantified in multiple samples collected on d 5 and 7 of the measurement periods shortly before the morning feeding. Results showed markedly greater concentrations of rumen LPS with increasing dietary grain level. The correlative analysis revealed strong negative relationships between rumen LPS and milk fat content and yield. The predictor variable of rumen LPS explained 69% of the variation during the milk fat reduction of the cows. The stronger depression in milk fat percentage was obtained when rumen LPS exceeded a threshold of 5,564 ng/mL, corresponding to a milk fat content of 3.39%. The increase in concentration of rumen LPS was also associated with declines in milk fat yield and 3.5% fat-corrected milk (R² = 0.50), as well as milk energy efficiency (R² = 0.43). The correlative analysis also indicated that the increase of plasma C-reactive protein (CRP) in response to higher grain feeding was associated with a linear decrease of milk fat content and yield (R² = 0.28 to 0.46). Furthermore, the statistical analysis revealed high percentages of intercow variation related to milk fat variables, as well as the responses of rumen LPS and plasma CRP. Taken together, the current results implicate rumen LPS and the host CRP response in the lowering of milk fat content and milk energy efficiency in dairy cows fed high-grain diets. Further research is warranted to understand the mechanism(s) by which rumen LPS and inflammatory responses to LPS lower milk fat synthesis and milk energy efficiency and to develop novel strategies for their prevention.     

Mechanisms involved in milk endogenous proteolysis induced by a lipopolysaccharide experimental mastitis

Experimental mastitis has been induced by the lipopolysaccharide (LPS) of Escherichia coli on six dairy cows in order to study the mechanisms involved in milk endogenous proteolysis during the inflammatory process. Variations in somatic cell count (SCC), plasmin activity, and total casein (CN) content were measured, and proteose-peptone content and the percentage of pH 4.6 insoluble peptides including gamma-CN have been considered as indicators of endogenous proteolysis. Furthermore, polymorphonuclear neutrophils (PMN) maturity has been evaluated by optical microscopy, and proteolysis by PMN proteinases has been studied at neutral and acidic pH in order to establish a link between caseinolysis, proteinase class, and PMN maturation. Two peaks of proteose-peptones content have been noticed for the six cows. First peak could be explained by both plasmin activity and SCC, while second peak was concomitant with a low plasmin activity but a SCC remaining high. The second peak of proteose-peptones content confirmed the role of cellular proteases in milk caseinolysis. Casein breakdown by cellular proteases was confirmed by SDS-PAGE electrophoresis, and a link between neutral proteinases activity and immature PMN recruitment was shown. Acidic proteinases activity was effective with mature PMN and during the recovery phase.     

Kunitz trypsin inhibitor in addition to Bowman-Birk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis

Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor (BBI) and Kunitz trypsin inhibitor (KTI) concentrations on the stability of lunasin against pepsin-pancreatin hydrolysis (PPH). In addition, the effect of KTI mutation on lunasin stability against PPH was determined. LEP were produced by calcium and pH precipitation methods of 30% aqueous ethanol extract from defatted soybean flour. LEP, lunasin-enriched commercially available products and KTI control and mutant flours underwent PPH and samples were taken after pepsin and pepsin-pancreatin hydrolysis. The concentrations of BBI, KTI, and lunasin all decreased after hydrolysis, but they had varying results. BBI concentration ranged from 167.5 to 655.8 μg/g pre-hydrolysis and 171.5 to 250.1 μg/g after hydrolysis. KTI concentrations ranged from 0.3 to 122.3 μg/g pre-hydrolysis and 9.0 to 18.7 μg/g after hydrolysis. Lunasin concentrations ranged from 8.5 to 71.0 μg/g pre-hydrolysis and 4.0 to 13.2 μg/g after hydrolysis. In all products tested, lunasin concentration after PPH significantly correlated with BBI and KTI concentrations. Mutation in two KTI isoforms led to a lower concentration of lunasin after PPH. This is the first report on the potential role of KTI in lunasin stability against PPH and must be considered in designing lunasin-enriched products that could potentially survive digestion after oral ingestion.