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.     

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.     

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.     

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.     

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.     

Cryptorchidism induced in normal rats by the relaxin-like factor inhibitor

Cryptorchidism is a serious problem, which affects 2-5% of the male population. Failure of the testes to descend into the scrotal region impairs germ cell development and is associated with a greater incidence of testicular cancer. The relaxin-like factor (RLF or insulin-like-3) has been shown to be critically important for the timely descent of the testicles in mice. We have discovered that the signal initiation site of the RLF can be eliminated without measurable effects on hormone binding to its receptor and that the resulting RLF derivative is a competitive inhibitor of RLF called RLFi. RLFi administered to pregnant rats causes dose-dependent gonadal retention in the offspring. The ability to control the severity of the syndrome by altering the concentration of RLFi and the timing of administration enables us to study in detail the structural changes that are associated with the action of RLF during critical stages of development. Targeted inhibition of the physiological migration pattern of testicles by RLFi lets one dissect the physiological process such as to find a window for clinical application of RLF and to search for ancillary factors that might play a role during normal development.     

大豆中植酸和胰蛋白酶抑制剂的抗营养和抗癌效应

大豆是重要的油料作物和植物蛋白来源,富含植物化学物质,如植酸和胰蛋白酶抑制荆等。传统营养理论认为这些植物化学物质是抗营养因子,然而近来研究显示这些化学成分具有天然抗癌作用。以植酸和STI为例主要介绍了它们的结构、分布、生理活性、抗营养效应及抗癌效应。     

Cleavage of a trypsin chymotrypsin inhibitor from Phaseolus vulgaris in active fragments

Summary Two peptide fragments can be separated from one of thePhaseolus vulgaris trypsin chymotrypsin inhibitors (PVI 3) after modifying with trypsin and cleaving the disulfide bonds by S-sulfonation. Reogidation of the larger C-terminal fragment yields a polypeptide with chymotrypsin-inhibitory activity. — After incubation with chymotrypsin two different behaving fragments are obtained from PVI 3 by treatment as described above. The now larger N-terminal fragment inhibits after reogidation trypsin and chymotrypsin, and this reogidation gives chymotrypsin-inhibitory activity for the C-terminal fragment, too.

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Zusammenfassung Nach der Modifizierung mit Trypsin und Spaltung der Disulfidbindungen durch S-Sulfonierung kann einer der Trypsin-Chymotrypsin-Inhibitoren ausPhaseolus vulgaris (PVI 3) in zwei Peptidfragmente aufgetrennt werden. Reogidation des größeren, C-terminalen Bruchstücks führt zu einem Polypeptid mit Chymotrypsin-Inhibitoraktivitiit. - Analoge Behandlung von PVI 3 nach der Incubation mit Chymotrypsin ergab ein größeres, C-terminalen Fragment mit Trypsin- und Chymotrypsin-Inhibitoraktivität und ein kleineres, C-terminales Peptid mit Aktivität gegen Chymotrypsin.

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Dedicated to Prof. Dr. Josef Schormüller on his 70th birthday.     

The nutrition evaluation of kidney beans (Phaseolus vulgaris). The effect of nutritional value of seed germination and changes in trypsin inhibitor content

The total N content of kidney beans (Phaseolus vulgaris) was found to increase on germination. The overall amino acid composition however changed very little. Rats fed on a diet containing raw beans lost more weight than the protein-free controls (negative n.p.u. value). Germination was found to bring about a gradual improvement in nutritive value probably through the elimination of some of the toxic constituents of the seed. The near doubling of the amount of trypsin inhibitors by the 8th day of germination taken together with the substantial improvement in the nutritive value of the bean appeared to rule out trypsin inhibitors as the main toxic components of raw beans.     

Costunolide inhibits interferon regulatory factor 3 activation induced by lipopolysaccharide and polyinosinic-polycytidylic acid

Inflammation can be initiated by invading microbial pathogens. Toll-like receptors (TLRs) recognize molecular structures derived from microbial pathogens and regulate the activation of innate immunity. In general, TLRs have 2 major downstream signaling pathways, the myeloid differentiation primary response protein 88 (MyD88)- and Toll/interleukin 1 receptor (TIR) domaincontaining adaptor protein (TIRAP) inducing interferon (TRIF)-dependent pathways, which lead to the activation of nuclear factor (NF)-κB and interferon regulatory factor 3 (IRF3). Costunolide, one of the active ingredients in Aucklandiae Radix (Saussurea lappa), has been used to treat many chronic inflammatory diseases. To evaluate the therapeutic potential of costunolide, its effect on signal transduction via the TLR signaling pathways was examined. Costunolide inhibited lipopolysaccharide or polyinosinicpolycytidylic acid-induced NF-κB and IRF3 activation and IRF3 phosphorylation, as well as interferon-inducible genes such as interferon inducible protein-10. The results suggest that costunolide can modulate immune responses regulated by TLR signaling pathways and may be the basis of effective therapeutics for chronic inflammatory diseases.     

Therapeutic effect of ginsenoside Rg1 on mastitis experimentally induced by lipopolysaccharide in lactating goats

Escherichia coli is a cause of subclinical and clinical mastitis in dairy cattle and goats, and sometimes causes severe clinical disease that may result in death of the animal. Previous investigation showed that ginsenoside Rg1 extracted from Panax ginseng C.A. Meyer (Araliaceae) has an anti-inflammatory effect on the sepsis induced by E. coli lipopolysaccharide via competitive binding to toll-like receptor 4. We hypothesized that intravenous injection of Rg1 had therapeutic effect on mastitis experimentally induced by intramammary infusion of lipopolysaccharide in lactating goats. In this study, 9 lactating goats were randomly assigned to 1 of the 3 groups: (1) lipopolysaccharide intramammary infusion + saline intravenous injection, (2) lipopolysaccharide intramammary infusion + Rg1 intravenous injection, and (3) saline intramammary administration + saline intravenous injection. Because no adverse clinical signs were observed after intramammary infusion of saline and intravenous injection of Rg1 in a preliminary experiment, and available qualified goats were limited in this study, this treatment was not included in this study. One udder half of each goat received intramammary infusion of lipopolysaccharide (50 μg/kg of body weight; groups 1 and 2) or saline solution (group 3), and the other half was infused with 2 mL of saline solution at h 0. Afterward, intravenous injections of saline solution (groups 1 and 3) or Rg1 (2.5 mg/kg of body weight; group 2) were administered at h 2 and 4 post-lipopolysaccharide challenge. Blood and milk samples were collected 3, 6, 9, 12, 15, 18, 21, 24, 48, and 72 h post-lipopolysaccharide challenge, and clinical signs were monitored hourly after lipopolysaccharide challenge within the first 10 h and at the same time points as blood samples. The results showed that Rg1 treatment downregulated rectal temperature, udder skin temperature, udder girth, milk somatic cell count, and N-acetyl-β-d-glucosaminidase and upregulated milk production, lactose, and recovered blood components, such as white blood cells, neutrophils, lymphocytes, total proteins, albumin, and globulin. Considering the positive therapeutic effect on lipopolysaccharide-induced mastitis in goats presented in this study as well as the anti-inflammatory activity found previously, the botanical Rg1 deserves further study as a therapeutic agent in the treatment of E. coli mastitis in dairy animals.