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Soybean Trypsin Inhibitor (soybean + trypsin_inhibitor)
Selected AbstractsImmobilized Metal Affinity Chromatography without Chelating Ligands: Purification of Soybean Trypsin Inhibitor on Zinc Alginate BeadsBIOTECHNOLOGY PROGRESS, Issue 1 2002Munishwar N. Gupta Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL -1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. [source] Effect of addition of soybean trypsin inhibitor to colostrum on immunological status in goat kidsJOURNAL OF ANIMAL PHYSIOLOGY AND NUTRITION, Issue 1 2010J. J. Ramos Summary The aim of the study was to evaluate the influence of soybean trypsin inhibitor (TI) on immunoglobulin G (IgG) serum levels and growth in neonatal goat kids. Twenty-four newborn kids were fed with natural colostrum (group A), and 24 kids received the same colostrum with 1 g of TI per litre (group B). Blood samples were obtained at birth and on days 1, 2 and 4 of life to analyze serum proteins, IgG and haematological parameters. There were no clinical signs of disease and no significant differences in body weight between the groups. Haematological parameters were not affected by treatment. The peak of serum IgG was reached at 24 h of life, but no effects of soybean TI was observed on serum IgG levels. The apparent efficiency of absorption of IgG was similar in both groups (group A 24.5% vs. group B 25.2%, p > 0.05). The addition of TI to colostrum did not change the concentration of serum proteins and their fractions in goat kids. The correlation between serum IgG and ,-globulin was positive and significant (p < 0.01, r = 0.64) in group A, but not in group B (p > 0.05, r = 0.08), suggesting a negative influence of soybean TI on ,-globulin absorption. These results show that addition of soybean TI to colostrum did not improve the performance or immunological status in goat kids. [source] PROTEINASES IN HYBRID CATFISH VISCERA: CHARACTERIZATION AND EFFECT OF EXTRACTION MEDIAJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2010SAPPASITH KLOMKLAO ABSTRACT Proteolytic activity from viscera extract of hybrid catfish (Clarias macrocephalus × Clarias gariepinus) was investigated. Optimal pH and temperature for casein hydrolysis were 9.0 and 50C, respectively. The enzyme was stable to heat treatment up to 40C and over a pH range of 7,11 for 30,120 min. The proteolytic activity was effectively inhibited by soybean trypsin inhibitor, benzamidine, phenylmethylsulfonyl fluoride and N -p-tosyl-L-lysine chloromethyl ketone. Activities of the viscera extract continuously decreased as NaCl concentration increased, while activities increased as CaCl2 concentration increased. Based on the proteinase activity of zones separated by electrophoresis, the molecular mass of the major proteinases in hybrid catfish viscera was 23 and 20 kDa. The effect of extraction media on recovery of proteinases was also studied. Extraction of the viscera powder with 50 mM Tris-HCl, pH 7.0 containing 0.5 M NaCl and 0.2% (v/v) Brij 35 rendered a higher recovery of proteinase activity than other extractants tested (P < 0.05). The results suggested that major proteinases in hybrid catfish viscera were heat-activated alkaline proteinases, most likely trypsin-like serine proteinases. PRACTICAL APPLICATIONS Hybrid catfish viscera is an abundant and underutilized resource that can be used as a unique proteinase source. Proteinase from various sources catalyzes the hydrolysis of peptide bonds. Thus, it is expected that like other proteinases, hybrid catfish proteinase would be useful in biomedical, food and beverage application. Moreover, the presented extraction media could be adopted to recover the trypsin-like serine proteinase from hybrid catfish viscera, which is currently a solid waste of Pa-duk-ra industry. [source] COMPARATIVE STUDIES ON PROTEOLYTIC ACTIVITY OF SPLENIC EXTRACT FROM THREE TUNA SPECIES COMMONLY USED IN THAILANDJOURNAL OF FOOD BIOCHEMISTRY, Issue 5 2004SUPPASITH KLOMKLAO ABSTRACT Proteolytic activities of splenic extract from three tuna species including skipjack tuna (Katsuwonus pelamis), yellowfin tuna (Thunnus albacores) and tongol tuna (Thunnus tonggol) were studied. Optimal activity of splenic extract from all tuna species was at pH 9.0 and 55C when casein was used as a substrate. Among all species tested, yellowfin tuna showed the highest activity, followed by skipjack tuna and tongol tuna. The proteolytic activity was strongly inhibited by soybean trypsin inhibitor, TLCK and partially inhibited by ethylenediaminetetraacetic acid. E-64, N-ethylmaleimide, iodoacetic acid, TPCK and pepstatin A showed no inhibition. The effect of NaCl and CaCl2 on proteolytic activity was also investigated. Activities continuously decreased as NaCl concentration increased, and no activity remained in the presence of 30% NaCl. On the other hand, activities increased as CaCl2 concentration increased. The highest activity was obtained in the presence of 1 mM CaCl2. SDS-substrate gel electrophoresis revealed that major proteinases in splenic extract from different tuna species were different in apparent molecular weights and sensitivity to TLCK. Although the major activity bands of all species were strongly inhibited by soybean trypsin inhibitor, varying sensitivity to TLCK probably implied the differences in binding characteristic of enzyme to substrate and/or inhibitors. The results suggest that major proteinases in spleen of all tuna species were trypsin-like serine proteinases. [source] Tuna Pepsin: Characteristics and Its Use for Collagen Extraction from the Skin of Threadfin Bream (Nemipterus spp.)JOURNAL OF FOOD SCIENCE, Issue 5 2008S. Nalinanon ABSTRACT:, Pepsin from the stomach of albacore tuna, skipjack tuna, and tongol tuna was characterized. Pepsin from all tuna species showed maximal activity at pH 2.0 and 50 °C when hemoglobin was used as a substrate. Among the stomach extract of all species tested, that of albacore tuna showed the highest activity (40.55 units/g tissue) (P < 0.05). Substrate-Native-PAGE revealed that pepsin from albacore tuna and tongol tuna consisted of 2 isoforms, whereas pepsin from skipjack tuna had only 1 form. The activity was completely inhibited by pepstatin A, while EDTA (ethylenediaminetetraacetic acid), SBTI (soybean trypsin inhibitor), and E-64 (1-(L -trans-epoxysuccinyl-leucylamino)-4-guanidinobutane) exhibited negligible effect. The activity was strongly inhibited by SDS (sodium dodecyl sulfate) (0.05% to 0.1%, w/v). Cysteine (5 to 50 mM) also showed an inhibitory effect in a concentration dependent manner. ATP, molybdate, NaCl, MgCl2, and CaCl2 had no impact on the activity. When tuna pepsin (10 units/g defatted skin) was used for collagen extraction from the skin of threadfin bream for 12 h, the yield of collagen increased by 1.84- to 2.32-fold and albacore pepsin showed the comparable extraction efficacy to porcine pepsin. The yield generally increased with increasing extraction time (P < 0.05). All collagen obtained with the aid of tuna pepsin showed similar protein patterns compared with those found in acid-solubilized collagen. Nevertheless, pepsin from skipjack tuna caused the degradation of , and , components. All collagens were classified as type I with large portion of ,-chain. However, proteins with molecular weight (MW) greater than 200 kDa were abundant in acid-solubilized collagen. [source] Soy-Derived Immunoglobulin Production Stimulating Factor Enhances IgM Production of Mouse Spleen LymphocytesJOURNAL OF FOOD SCIENCE, Issue 7 2006N. Maeda ABSTRACT:, We have reported previously that some proteins such as lactoferrin and lysozyme control the immune system via immunoglobulin (Ig) production. In the course of the study about the function of dietary proteins and peptides, Ig production-stimulating activity of an unknown protein contained in soybean trypsin inhibitor (STI) preparation was found. Thus, we examined the activity of the unknown activator and the mechanism of the activity. The factor significantly elevated IgM production by mouse spleen lymphocytes in a dose-dependent manner. In addition, the unknown activator up-regulated the expression of interleukin (IL)-6 and IL-10 mRNA. And IgM production enhancing activity of STI was significantly suppressed by neutralization of IL-6. Then, to clarify whether STI itself is the active compound or not, STI was fractionized by gel filtration chromatography. We found that the activity the content of STI did not correlate with and the active fractions contained some proteins whose molecular weight is more than 20 kDa. These suggest that an unknown activator exists in STI preparation. [source] Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activityJOURNAL OF MOLECULAR RECOGNITION, Issue 4 2005Yasuhiro Nishiyama Abstract Covalent nucleophile,electrophile interactions have been established to be important for recognition of substrates by several enzymes. Here, we employed an electrophilic amidino phosphonate ester (EP1) to study the nucleophilic reactivity of the following proteins: albumin, soluble epidermal growth factor receptor (sEGFR), soluble CD4 (sCD4), calmodulin, casein, ,-lactalbumin, ovalbumin, soybean trypsin inhibitor and HIV-1 gp120. Except for soybean trypsin inhibitor and ,-lactalbumin, these proteins formed adducts with EP1 that were not dissociated by denaturing treatments. Despite their negligible proteolytic activity, gp120, sEGFR and albumin reacted irreversibly with EP1 at rates comparable to the serine protease trypsin. The neutral counterpart of EP1 reacted marginally with the proteins, indicating the requirement for a positive charge close to the electrophilic group. Prior heating resulted in altered rates of formation of the EP1,protein adducts accompanied by discrete changes in the fluorescence emission spectra of the proteins, suggesting that the three-dimensional protein structure governs the nucleophilic reactivity. sCD4 and vasoactive intestinal peptide (VIP) containing phosphonate groups (EP3 and EP4, respectively) reacted with their cognate high-affinity binding proteins gp120 and calmodulin, respectively, at rates exceeding the corresponding reactions with EP1. Reduced formation of EP3,gp120 adducts and EP4,calmodulin adducts in the presence of sCD4 and VIP devoid of the phosphonate groups was evident, suggesting that the nucleophilic reactivity is expressed in coordination with non-covalent recognition of peptide determinants. These observations suggest the potential of EPs for specific and covalent targeting of proteins, and raise the possibility of nucleophile,electrophile pairing as a novel mechanism stabilizing protein,protein complexes. Copyright © 2005 John Wiley & Sons, Ltd. [source] Preparation of trypsin-immobilised chitosan beads and their application to the purification of soybean trypsin inhibitorJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 13 2008Li Zhang Abstract BACKGROUND: Trypsin inhibitors are among the most important antinutritional factors in legumes. Recent research has shown that soybean trypsin inhibitor (SBTI) exhibits multiple bioactivities, but very few studies on the purification of SBTI are available. Enzymes are commonly used as biospecific ligands in affinity purification of their substrates or inhibitors. The aim of the present study was to prepare trypsin (EC 3.4.21.4)-immobilised chitosan beads and use them to purify trypsin inhibitor from soybean whey. RESULTS: Compared with free trypsin, the immobilised trypsin had higher thermal and pH stability. The adsorption ratio of SBTI from crude SBTI aqueous solution by trypsin-immobilised chitosan beads was 33.3%. The purified SBTI obtained by affinity chromatography was characterised by sodium dodecyl sulfate polyacrylamide gel electrophoresis as a single polypeptide band with an Mr of 8.3 kDa belonging to the Bowman,Birk family. CONCLUSION: Trypsin-immobilised chitosan beads were effectively used in the affinity separation of trypsin inhibitor from soybean seeds, thus indicating that immobilised trypsin may have practical application in the soybean-processing industry. The results of this study provide a background for further investigation of potential applications of soybean bioactive constituents in the areas of agriculture and food. Copyright © 2008 Society of Chemical Industry [source] Comparative study on proteolysis of two species of bigeye snapper, Priacanthus macracanthus and Priacanthus tayenusJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 9 2003Soottawat Benjakul Abstract Proteolytic activity in muscle from two species of bigeye snapper (Priacanthus macracanthus and Priacanthus tayenus) was studied. Autolysis of mince and washed mince at 50 and 60 °C was compared. Higher degradation of myosin heavy chain was observed in both mince and washed mince from P macracanthus than in those from P tayenus, especially when the incubation time was increased. Autolysis of washed mince from both species was inhibited by soybean trypsin inhibitor, suggesting that myofibril-associated proteases were serine proteases. When sarcoplasmic proteolytic activity in P macracanthus muscle was studied, two activity peaks with an optimum temperature of 60 °C were observed at pH 6.5 and 8.5. The activities of both peaks were mostly inhibited by soybean trypsin inhibitor, suggesting that the major protease was a serine protease. Major sarcoplasmic proteolytic activity in P macracanthus muscle was found at Mr 62 000 on sodium dodecyl sulphate substrate gel. For P tayenus sarcoplasmic proteolytic activity, two activity peaks with an optimum temperature of 60 °C were found at pH 5.0 and 8.5. The pH 5.0 peak activity was effectively inhibited by pepstatin A, while the pH 8.5 peak activity was inhibited by several inhibitors. The results indicated that various sarcoplasmic proteases were present in P tayenus muscle. The two species contained different sarcoplasmic proteases in terms of composition and activity level. P macracanthus muscle generally had higher sarcoplasmic proteolytic activities than P tayenus muscle. Copyright © 2003 Society of Chemical Industry [source] Effect of diets containing a purified soybean trypsin inhibitor on growth performance, digestive proteases and intestinal histology in juvenile sea bream (Sparus aurata L.)AQUACULTURE RESEARCH, Issue 9 2010Ester Santigosa Abstract Juvenile sea bream were fed on diets containing 0.0, 2.0 or 4.0 g kg,1 of a soybean trypsin inhibitor (SBTI) for 30 days. The growth performance, total protease activity and intestinal histology were studied after 0, 15 and 30 days of dietary treatment. No significant differences were found in the weight gain, specific growth rate (SGR) and feed conversion rate in fish fed on inhibitor-supplemented diets when compared with those fed on an inhibitor-free diet. Only the SGR at day 15 decreased significantly with protease inhibitor inclusion, although this effect was not observed at day 30. In relation to proteolytic activity at day 15, the total protease activity in the distal intestine decreased in fish fed on inhibitor-supplemented diets. Zymograms of these extracts showed that the SBTI reduced the intensity of some proteolytic fractions in the distal intestine. A noticeable reduction in the protease activity of the intestinal content in fish fed on the highest level of soybean inhibitor (4.0 g kg,1) was also observed. However, at day 30, the inhibition effect on these active bands was not detected, and the total protease activity was similar to that in fish fed on an inhibitor-free diet. Histological examination revealed no perceptible differences in the intestinal structure between any of the diet groups. In addition, all fish were maintained under experimentation for 10 more days and fed on an inhibitor-free diet to determine whether the possible effects caused by the protease inhibitor could be reverted. The administration of SBTI-supplemented diets did not affect sea bream growth performance or intestine histology after 30 days, and only a decrease in the total alkaline protease activity was found at day 15. [source] Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet, Brassica species, and protease inhibitor,ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2010Martin A. Erlandson Abstract The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one-dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55,kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100,kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24,h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease-encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin-like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin-like gene McSP34. The expression of the trypsin-like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources. Published 2010 Wiley Periodicals, Inc. [source] Exogenous and endogenous protease inhibitors in the gut of the fall armyworm larvae, Spodoptera frugiperdaARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2010Digali Lwalaba Abstract A dose-dependent inhibition of endogenous trypsin and aminopeptidase occurs in the lumen of Spodoptera frugiperda after feeding L6 larvae exogenous inhibitors soybean trypsin inhibitor (SBTI), tosyl-L-lysine chloromethyl ketone-HCl (TLCK), or bestatin, respectively, for 3 days. TLCK inhibits trypsin in tissue extracts and in secretions more strongly than SBTI. The aminopeptidase released into the lumen (containing the peritrophic membrane) is strongly inhibited by bestatin, but the membrane-bound enzyme is not. A bound enzyme may be more resistant to an inhibitor than unbound. A cross-class elevation of aminopeptidase activity occurs in response to ingested trypsin inhibitor, but there was no cross-class effect of aminopeptidase inhibitor (bestatin) on trypsin activity. An endogenous trypsin and aminopeptidase inhibitor is present in the lumen and ventricular cells. The strength of the endogenous trypsin inhibition seems to be in the same range as that resulting from ingestion of the exogenous inhibitor SBTI. In some insect species, considerable trypsin secretion occurs in unfed as well as in fed animals, and endogenous protease inhibitors might function to protect the ventricular epithelium by inactivation of trypsin when less food is available. © 2010 Wiley Periodicals, Inc. [source] Immobilized Metal Affinity Chromatography without Chelating Ligands: Purification of Soybean Trypsin Inhibitor on Zinc Alginate BeadsBIOTECHNOLOGY PROGRESS, Issue 1 2002Munishwar N. Gupta Immobilized metal affinity chromatography (IMAC) is a widely used technique for bioseparation of proteins in general and recombinant proteins with polyhistidine fusion tags in particular. An expensive and critical step in this process is coupling of a chelating ligand to the chromatographic matrix. This chelating ligand coordinates metal ions such as Cu2+, Zn2+, and Ni2+, which in turn bind proteins. The toxicity of chemicals required for coupling and their slow release during the separation process are of considerable concern. This is an important issue in the context of purification of proteins/enzymes which are used in food processing or pharmaceutical purposes. In this work, a simpler IMAC design is described which should lead to a paradigm shift in the application of IMAC in separation. It is shown that zinc alginate beads (formed by chelating alginate with Zn2+ directly) can be used for IMAC. As "proof of concept", soybean trypsin inhibitor was purified 18-fold from its crude extract with 90% recovery of biological activity. The dynamic binding capacity of the packed bed was 3919 U mL -1, as determined by frontal analysis. The media could be regenerated with 8 M urea and reused five times without any appreciable loss in its binding capacity. [source] AT2 receptor-dependent vasodilation is mediated by activation of vascular kinin generation under flow conditionsBRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2002Jun Katada Physiological roles of angiotensin II type 2 receptor (AT2) are not well defined. This study was designed to investigate the mechanisms of AT2 -dependent vascular relaxation by studying vasodilation in pressurized and perfused rat mesenteric arterial segments. Perfusion of angiotensin II in the presence of AT1 antagonist elicited vascular relaxation, which was completely dependent on AT2 receptors on endothelium. FR173657 (>1 ,M), a bradykinin (BK) B2 -specific antagonist, significantly suppressed AT2 -dependent vasodilation (maximum inhibition: 68.5% at 10 ,M). Kininogen-deficient Brown Norway Katholiek rats showed a significant reduction in AT2 -mediated vasodilatory response compared with normal wild-type Brown Norway rats. Indomethacin (>1 ,M), aprotinin (10 ,M) and soybean trypsin inhibitor (10 ,M) also reduced AT2 -dependent vasodilation. Our results demonstrated that stimulation of AT2 receptors caused a significant vasodilation through local production of BK in resistant arteries of rat mesentery in a flow-dependent manner. Such vasodilation counterbalances AT1 -dependent vasoconstriction to regulate the vascular tone. British Journal of Pharmacology (2002) 136, 484,491; doi:10.1038/sj.bjp.0704731 [source] Changes in inhibitory activity and secondary conformation of soybean trypsin inhibitors induced by tea polyphenol complexationJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 14 2009Huihua Huang Abstract BACKGROUND: Tea polyphenol (TP) is a new food additive for antioxidant application, while soybean is an important resource for food and feed processing. It is therefore of rational and practical significance to investigate the influence of TP on soybean trypsin inhibitors (TIs). The aim of this study was to determine the effects of TP on the inhibitory activity of Kunitz (KTI) and Bowman,Birk (BBTI) TIs and to reveal the relationship between the inhibitory activity and conformation of KTI and BBTI by measurement of circular dichroism (CD) spectra. RESULTS: KTI and BBTI were found to be partially deactivated by TP. BBTI exhibited stronger resistance than KTI to TP deactivation. The unchanged KM value of trypsin for benzoyl- DL -arginine- p -nitroanilide hydrolysis indicated that KTI and BBTI inhibited trypsin in a non-competitive pattern when complexed with TP. As the TP/TI ratio was increased and the inhibitory activity of KTI and BBTI decreased, the conformation of KTI and BBTI showed relevant changes and the major CD negative bands shifted progressively towards the near-UV region. CONCLUSION: These results show the deactivation effects of TP on KTI and BBTI and reveal preliminarily the relationship between the inhibitory activity and secondary structure of KTI and BBTI. Copyright © 2009 Society of Chemical Industry [source] Effects of tea polyphenols on the activities of soybean trypsin inhibitors and trypsinJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 2 2004Huihua Huang Abstract Tea polyphenols (TPs) and other materials were extracted from Chinese green tea, and their effects on trypsin inhibitors and trypsin were analysed. TPs were found to have a deactivation effect on both Kunitz trypsin inhibitor (KTI) and Bowman,Birk trypsin inhibitor (BBTI). KTI was more easily deactivated than BBTI by complexing with TPs. The deactivation effect of TPs on KTI and BBTI reached a maximum at a TP/KTI ratio of 25 and a TP/BBTI ratio of 16. However, the deactivation effect of TPs on KTI and BBTI was reduced dramatically when KTI and BBTI were already complexed with trypsin. TPs were also found to inhibit trypsin. The inhibitory activity of TPs, KTI and BBTI on trypsin was found to decrease in the order BBTI > gtTI > gtPs. Complete inhibition of trypsin by TPs could not be achieved. When the TP concentration was increased to about 17 µg ml,1, the residual activity of trypsin was maintained at 400 TU mg,1, equivalent to 32% of the initial trypsin activity. In TP inhibition the KM value for trypsin remained unchanged at 5.88 × 10,4 mol l,1 and Vmax decreased when benzoyl- DL -arginine- p -nitroanilide (BAPNA) was used as substrate. The pattern of trypsin inhibition by TPs is non-competitive. Copyright © 2004 Society of Chemical Industry [source] | |||||||||||||