The potential protective and therapeutic effects of cannabidiol oil on experimental Leukemia induced by DMBA in male rats.

BACKGROUND: 7,12-Dimethylbenzanthracene (DMBA) is a member of the polycyclic aromatic hydrocarbon family. It is a member of the polycyclic aromatic hydrocarbon family. It is a mutagenic, carcinogenic, and immunosuppressor agent. Cannabidiol (CBD) is a phytocannabinoid. It has anticonvulsant, anti-inflammatory, anti-anxiety, antioxidant, and anti-cancer properties. The purpose of this study was to investigate the possible protective and therapeutic benefits of CBD oil in DMBA-induced leukemia in rats. METHOD: Experimental animals were divided into six groups of five rats each. Group 1 (normal control) included healthy rats. Group 2 included normal rats that received olive oil. Group 3 included normal rats that received CBD. Group 4 included the DMBA-induced leukemic group. Group 5 (prophylactic group) included rats that received CBD as a prophylaxis before IV injection with DMBA. Group 6 (treated group) included DMBA-induced leukemic rats that received CBD as treatment. Liver functions (total, direct and indirect bilirubin, alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), albumin, globulin, and albumin globulin ratio) were measured. Superoxide dismutase (SOD) and catalase (CAT) were also measured. Total RNA extraction followed by-real time qRT-PCR gene expression of LC3-II, Beclin, mTOR, and P62 was performed. Histopathological examination of liver and spleen tissues was performed. RESULTS: Administration of CBD in groups 5 and 6 resulted in a significant improvement of the levels of liver functions compared to the leukemic untreated rats. Also, the levels of catalase and SOD significantly increased after treatment with CBD compared to the leukemic group. After treatment with CBD in groups 5 and 6, there were downregulations in the expression of all studied genes compared to leukemic untreated rats. Treatment with CBD was more statistically effective than prophylactic use. CONCLUSION: Administration of CBD resulted in a significant improvement in the biochemical, antioxidant status, morphological, and molecular measures in DMBA-induced leukemia in adult male rats. The therapeutic use was more effective than the prophylactic one.

Soybean protein hydrolysate stimulated cholecystokinin secretion and inhibited feed intake through calcium-sensing receptors and intracellular calcium signalling in pigs.

Although soybean protein is the major component in livestock feeds, its effect on pigs' appetites is largely unknown. Recently, the importance of gut nutrient-sensing for appetite modulation by regulating anorectic gut hormone release has been recognised. This study investigates the roles of soybean proteins in appetite regulation, anorectic gut hormone secretion, and underlying mechanisms. The duodenal-cannulated piglets were used to evaluate the effects of soybean protein hydrolysate (SPH) on feed intake and anorectic hormone release, including cholecystokinin (CCK), peptide YY (PYY), glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) in the hepatic vein by infusing SPH. Identifying which nutrient-sensing receptor in pig duodenum response to SPH stimulation for gut hormone release was conducted. Using its antagonist, the role of the identified receptor in feed intake and anorectic hormone release was also investigated. Combination with an ex vivo perfusion system, the possible mechanism by which SPH exerts the effects in porcine duodenum was further illustrated. Results in vivo showed that intraduodenal infusion of SPH inhibited short-term feed intake in pigs and promoted CCK, PYY, and GIP secretion in the hepatic vein. SPH also increased duodenum calcium-sensing receptor (CaSR) expression. Pre-treated with CaSR antagonist NPS 2143, the feed intake of pigs tended to be attenuated by SPH (P = 0.09), and CCK release was also suppressed (P < 0.05), indicating that CaSR was involved in SPH-stimulated CCK release and inhibited feed intake in pigs. The ex vivo perfused duodenum tissues revealed that SPH-triggered CCK secretion was likeliest due to the activation of the intracellular Ca2+/TRPM5 pathway. Overall, this study's result illustrates that the diet soybean protein might decrease appetite in pigs by triggering duodenum CCK secretion by activating CaSR and the intracellular Ca2+/TRPM5 pathway.

Identification, Characterization and Antihypertensive Effect In Vivo of a Novel ACE-Inhibitory Heptapeptide from Defatted Areca Nut Kernel Globulin Hydrolysates.

The areca (Areca catechu L.) nut kernel (ANK) is a good potential protein source for its high protein content of 9.89-14.62 g/100 g and a high yield of around 300,000 tons per year in China. However, utilization of the areca nut kernel is limited. To expand the usage of ANK in pharmaceutical or foods industries, areca nut kernel globulin was extracted and angiotensin-I converting enzyme (ACE) inhibition peptides were prepared and identified using gel chromatography, reversed phase HPLC separation, UPLC-ESI-MS/MS analysis and in silico screening. Finally, a novel ACE-inhibitory heptapeptide (Ala-Pro-Lys-Ile-Glu-Glu-Val) was identified and chemically synthesized. The combination pattern between APKIEEV and ACE, and the inhibition kinetics, antihypertensive effect and endothlein-1 inhibition activity of APKIEEV were studied. The results of the molecular docking demonstrated that APKIEEV could bind to four active sites (not the key active sites) of ACE via short hydrogen bonds and demonstrated high ACE-inhibitory activity (IC50: 550.41 µmol/L). Moreover, APKIEEV exhibited a significantly lowering effect on both the systolic blood pressure and diastolic blood pressure of spontaneously hypertensive rats, and had considerable suppression ability on intracellular endothelin-1. These results highlight the potential usage of APKIEEV as ingredients of antihypertensive drugs or functional foods.

Distribution and effects of natural selenium in soybean proteins and its protective role in soybean ß-conglycinin (7S globulins) under AAPH-induced oxidative stress.

The effects of selenium (Se) on the protein content, amino acid profile, secondary structure and subunit composition of soy proteins and its distribution were evaluated, as was the effect of peroxyl radicals produced by thermal decomposition of AAPH on the conformational changes of Se-enriched ß-conglycinin (S-7S). The Se biofortification ability of soy was very strong, 7S had strongest ability to incorporate Se, and lower amounts of inorganic Se existed in Se-enriched beans. Se could promote protein synthesis and thus improve the protein content, increase the total amino acid content with a decrease in cysteine, combine into low-molecular-weight proteins, and influence the secondary structure of soybean proteins. Se was involved in the relevant protein changes in surface hydrophobicity, intrinsic fluorescence, infrared absorption and solubility and played an antioxidant role as an effectual "protector" to reduce the influence of peroxyl radical oxidation on S-7S, thereby maintaining the structural rearrangement between aggregation and protein unfolding.

Tarin stimulates granulocyte growth in bone marrow cell cultures and minimizes immunosuppression by cyclo-phosphamide in mice.

Chemotherapeutic drugs, such as cyclophosphamide, cause severe immunosuppression and patients become susceptible to infections. Based on this, the immunomodulatory potential of tarin, a lectin from Colocasia esculenta, was evaluated in bone marrow cell cultures and in cyclophosphamide-immunosuppressed mice. Tarin promoted maintenance of hematopoietic progenitors and repopulation of Gr1 cells in vitro which was supported by in vivo results. In immunosuppressed mice, tarin increased bone marrow cell numbers and altered cell profile distribution by enhancing the frequency of Gr1+ progenitors, including Ly6-CintLy6-Glo, and anticipating their proliferation/differentiation in mature cells, especially Ly6-CloLy6-Ghi. Bone marrow cells harvested from tarin-treated immunosuppressed mice proliferated in response to GM-CSF or G-CSF in vitro and, the low numbers of bone marrow cells in the G0 phase, combined with a high number cells undergoing apoptosis confirmed that tarin promoted a faster and intense proliferation/differentiation, even in the presence of CY-induced toxicity. As a result, tarin minimized leukopenia in immunosuppressed mice promoting a faster recovery of peripheral leucocytes and protected erythroid bone marrow cells from CY-cytotoxicity in a dose-dependent manner. Data suggest that tarin could be considered a potential adjuvant to decrease leukopenia and possibly ameliorate anemia, if carefully evaluated in human cancer cell lineages and in clinical trials.

FGF21 Alleviates Hepatic Endoplasmic Reticulum Stress under Physiological Conditions.

Fibroblast growth factor 21 (FGF21), mainly synthesized and secreted from the liver, is an endocrine FGF that regulates glucose and fatty acid metabolism to maintain whole body energy homeostasis. Gene expression of FGF21 was previously reported to be induced by endoplasmic reticulum (ER) stress through activating transcription factor 4 (ATF4). It has been reported that drug-induced ER stress is reduced by overexpression of FGF21. However, the function of endogenous FGF21 under physiological conditions such as the postprandial state remains unknown. Here, we examined the effects of both endogenous and exogenous FGF21 on postprandial hepatic ER stress. In mice, postprandial and tunicamycin-induced ER stress was significantly reduced by overexpression of FGF21 using a recombinant adenovirus. FGF21-deficient mice exhibited a more considerable increase in drug-induced ER stress target gene expression than wild-type mice. Following refeeding after fasting, FGF21 deficiency caused severe ER stress in the liver. The postprandial ER stress response was significantly reduced when hepatic FGF21 gene expression was increased by feeding a diet containing the soy protein ß-conglycinin which activates ATF4. Together, these results demonstrate that FGF21 reduces the increased expression of a subset of genes in the liver in response to ER stress and may correct metabolic disorders caused by ER stress.

Characterization and Immunomodulatory Activity of a Novel Peptide, ECFSTA, from Wheat Germ Globulin.

A novel peptide was extracted from wheat germ globulin and purified using ion-exchange chromatography, gel filtration chromatography, and semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The sequence of the peptide was found to be Glu-Cys-Phe-Ser-Thr-Ala (ECFSTA). Its immunomodulatory effects were evaluated, and the results showed that ECFSTA could enhance phagocytosis of RAW 264.7 cells and significantly increase their secretion of nitric oxide (NO), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and reactive oxygen species (ROS). ECFSTA activated macrophages mainly through the pattern recognition receptors (PRRs) of toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4), and the production of ROS simultaneously stimulated macrophages to produce TNF-α. Thus, ECFSTA could be used as an immunomodulator and might be a promising component of functional foods.

Antibacterial Actions of Glycinin Basic Peptide against Escherichia coli.

Glycinin basic peptide (GBP) is an antibacterial ingredient that occurs naturally in the basic parts of soybean glycinin. The antibacterial actions of GBP against Escherichia coli ATCC 8739 were investigated in this study. The minimum inhibitory concentration of GBP against E. coli was 200 µg/mL. The exposure of E. coli cells to GBP induced significant cell damage and inactivated intracellular esterases (stressed and dead cells, 70.9% ± 0.04 for 200 µg/mL of GBP and 91.9% ± 0.06 for 400 µg/mL of GBP), as determined through dual staining in flow cytometry. GBP resulted in the exposure of phosphatidylserine in E. coli cells. The analyses of flow cytometry-manifested GBP treatment led to the shrinkage of the cell surface and the complication of cell granularity. The observations in transmission electron microscopy demonstrated that 400 µg/mL of GBP severely disrupted the membrane integrity, resulting in ruptures or pores in the membrane, outflows of intracellular contents, or aggregation of the cytoplasm. Release of alkaline phosphatase, lipopolysaccharide, and reducing sugar further verified that the membrane damage was due to GBP. In addition, GBP treatment changed the helicity and base staking of DNA, as determined by circular dichroism spectroscopy. These results showed that GBP had strong antibacterial activity against E. coli via membrane damage and DNA perturbation. Additionally, GBP exhibited no cytotoxicity on the viability of human embryonic kidney cells. Thus, GBP may be a promising candidate as a natural antibacterial agent.

Two Peptides from Soy ß-Conglycinin Induce a Hypocholesterolemic Effect in HepG2 Cells by a Statin-Like Mechanism: Comparative in Vitro and in Silico Modeling Studies.

Two peptides from soybean ß-conglycinin, i.e., YVVNPDNDEN (peptide 2) and YVVNPDNNEN (peptide 3), are known to be absorbed by human enterocytes. The former is a fragment of LRVPAGTTFYVVNPDNDENLRMIA (peptide 1), previously shown to increase the low-density lipoprotein (LDL) uptake and degradation in hepatocytes. Research carried out in silico on their interactions with the catalytic site of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR) demonstrated that they behave as competitive inhibitors of HMGCoAR activity with a statin-like mechanism, confirmed by direct inhibition experiments. Research in HepG2 cells aimed at investigating the effects of these peptides on cholesterol metabolism showed that compared to mock treatment peptide 2 at 350 µM up-regulates the mature SREBP2 protein level by 134.0 ± 10.5%, increases the LDLR protein level by 152.0 ± 20.0%, and enhances the HMGCoAR protein production by 171 ± 29.9%, whereas peptide 3 up-regulates the mature SREBP2 protein level by 158.0 ± 9.2%, increases the LDL level 164.0 ± 17.9%, and induces a HMGCoAR protein increase by 170 ± 50.0%.

Soy ß-conglycinin retards progression of diabetic nephropathy via modulating the insulin sensitivity and angiotensin-converting enzyme activity in rats fed with high salt diet.

Soy protein was known to have renal-protective effects. The aim of this study was to investigate the effects of different doses of soybean ß-conglycinin, one of the main storage proteins in soy, on diabetic nephropathy in the rat. We used 40 Wistar rats with eight rats in each group. Diabetes mellitus was induced in rats by an intravenous injection of streptozotocin. The groups included a control group (Ctrl) fed with the standard AIN93-M diet, while other groups were fed with AIN-93M with the addition of NaCl to induce diabetic nephropathy (DN). DN rats were divided into the DN control (DN) group, the soy protein (DN + SP) group, the low-dose ß-conglycinin (DN + B) group, and the high-dose ß-conglycinin (DN + 2B) group. After a 27 weeks experimental period, we found that soy protein and ß-conglycinin decreased blood glucose via increasing the insulin sensitivity, with an enhanced cholesterol-lowering effect of ß-conglycinin-mediated hepatic LDL receptor protein expression. Otherwise, there were beneficial effects of soy protein and ß-conglycinin on renal function markers. Through the inhibition of angiotensin-converting enzyme (ACE), soy protein and ß-conglycinin retarded the progression of diabetic nephropathy by decreasing the blood pressure and histological changes. In conclusion, soy protein and ß-conglycinin may retard the progression of diabetic nephropathy by increasing insulin sensitivity, regulating lipid metabolism, improving renal function markers, and inhibiting ACE activity.

Production of the growth factors GM-CSF, G-CSF, and VEGF by human peripheral blood cells induced with metal complexes of human serum γ -globulin formed with copper or zinc ions.

As it was established in our previous studies, the proteins of human serum γ-globulin fraction could interact with copper or zinc ions distributed in the periglobular space, form metal complexes, and become able to perform effector functions differing due to the conformational shifts from those mediated by them in native conformation of their Fc regions. In the present work we have evaluated ability of the γ-globulin metal complexes formed with copper or zinc ions in the conditions like to the physiological ones to induce production or to regulate induction in the culture of freshly isolated human peripheral blood cells (PBC) of granulocyte (G) and granulocyte-macrophage (GM) colony-stimulating factors (CSF) as well as of vascular endothelial growth factor (VEGF). The γ-globulin metal complexes formed with both copper and zinc ions were found to similarly reduce production of GM-CSF, G-CSF, and VEGF induced in normal human PBC cultures by the control γ-globulins or by copper and zinc ions used alone. In context of theory and practice of inflammation the properties of the γ-globulin metal complexes might impact the basic knowledge in search of novel approaches to anti-inflammatory drugs development.

Polyclonal rabbit anti-human ovarian cancer globulins inhibit tumor growth through apoptosis involving the caspase signaling.

Most women with ovarian cancer are diagnosed at an advanced stage and there are few therapeutic options. Recently, monoclonal antibody therapies have had limited success, thus more effective antibodies are needed to improve long-term survival. In this report, we prepared polyclonal rabbit anti-ovarian cancer antibody (Poly Ab) by immunizing rabbits with the human ovarian cancer cell line SKOV3. The Poly Ab bound to SKOV3 and inhibited the cancer cells proliferation. Western blot analysis was conducted, which indicated that Poly Ab inhibited cancer cells through apoptosis involving the caspase signaling pathway including caspase-3 and caspase-9. Finally, compared with the control antibody, administration of Poly Ab reached 64% and 72% tumor inhibition in the subcutaneous and intraperitoneal xenograft mouse model, respectively. Our findings suggest that Poly Ab is an effective agent for apoptosis induction and may be useful as a safe anticancer agent for ovarian cancer therapy.

Soy ß-conglycinin improves glucose uptake in skeletal muscle and ameliorates hepatic insulin resistance in Goto-Kakizaki rats.

Although the underlying mechanism is unclear, ß-conglycinin (ßCG), the major component of soy proteins, regulates blood glucose levels. Here, we hypothesized that consumption of ßCG would normalize blood glucose levels by ameliorating insulin resistance and stimulating glucose uptake in skeletal muscles. To test our hypothesis, we investigated the antidiabetic action of ßCG in spontaneously diabetic Goto-Kakizaki (GK) rats. Our results revealed that plasma adiponectin levels and adiponectin receptor 1 messenger RNA expression in skeletal muscle were higher in ßCG-fed rats than in casein-fed rats. Phosphorylation of adenosine monophosphate-activated protein kinase (AMP kinase) but not phosphatidylinositol-3 kinase was activated in ßCG-fed GK rats. Subsequently, ßCG increased translocation of glucose transporter 4 to the plasma membrane. Unlike the results in skeletal muscle, the increase in adiponectin receptor 1 did not lead to AMP kinase activation in the liver of ßCG-fed rats. The down-regulation of sterol regulatory element-binding factor 1, which is induced by low insulin levels, promoted the increase in hepatic insulin receptor substrate 2 expression. Based on these findings, we concluded that consumption of soy ßCG improves glucose uptake in skeletal muscle via AMP kinase activation and ameliorates hepatic insulin resistance and that these actions may help normalize blood glucose levels in GK rats.

Identification of peptides from soybean protein, glycinin, possessing suppression of intracellular Ca2+ concentration in vascular smooth muscle cells.

In this study, we challenged to identify vasoactive peptides in soybean 11S glycinin hydrolysate by thermolysin to regulate intracellular Ca(2+) concentration ([Ca(2+)]i) that can induce constrictive vascular tension. As a function of the inhibition of elevated [Ca(2+)]i by 10 µM angiotensin (Ang) II in vascular smooth muscle cells (VSMCs), eleven peptides were successfully identified from the hydrolysate, among which His-Gly-Lys exhibited the most potent inhibition against [Ca(2+)]i elevation in Ang II-stimulated VSMCs (inhibition at 300 µM: 46.5±8.0% vs. control). The biological capacity of His-Gly-Lys analogues as an [Ca(2+)]i inhibitor was also proven when His-Lys and His-Gly-Arg elicited a significant reduction in [Ca(2+)]i. In contrast, less reduction of [Ca(2+)]i by His-Gly-Ile and His-(3-methyl)-Gly-Lys indicated the importance of the imino proton in His, along with basic amino acids positioned at C-terminal for the effect.

Purification and characterization of the lectin from taro (Colocasia esculenta) and its effect on mouse splenocyte proliferation in vitro and in vivo.

Lectins are proteins found in a wide range of organisms, with the ability to bind reversibly to specific carbohydrates. They can display important biological activities, such as the activation of the cell cycle in lymphocytes. Storage proteins with lectin activity have been reported in tuberous plant species, such as Colocasia esculenta, popularly known as taro. A simple strategy based on Cibacron Blue chromatography was used to purify a 12 kDa polypeptide 1.3-fold, with a recovery of 30 %. The purified protein was identified as tarin by mass spectrometry, which indicated that it was present in G1a/G1d isoforms. Tarin exhibited both agglutinating activity against hamster erythrocytes and mitogenic activity in vitro and in vivo toward mouse splenocytes. Optimum cellular proliferation in vitro was achieved by 625 ng of the crude extract or 500 ng of the purified tarin. Total mouse splenocyte proliferation measured after 5 days of intraperitoneal inoculation of purified tarin was increased 3.3-fold in comparison to the control group. Half of the proliferating cells were identified as B lymphocytes by flow cytometry. These results show that this is an efficient and simple strategy to purify tarin and aid in establishing this protein as a new therapeutic drug, able to promote cell proliferation in a murine model.

Use of phytochemomics to evaluate the bioavailability and bioactivity of antioxidant peptides of soybean ß-conglycinin.

This research investigates how in vitro digestion contributes to the release of antioxidant peptides crypted in soybean ß-conglycinin (7S) and its deglycosylated form (D7S). It also investigates the uptake of the bioactive peptides by human intestinal Caco-2 cells using a bicameral system, and their effect on the antioxidant cell defense. Phytochemomics is used as a tool for achieving this goal. The peptides are obtained by mimicking human physiological gastrointestinal digestion conditions. The antioxidant capacity of the peptides is tested by ABTS•(+) radical cation decolorization (2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)) and oxygen radical absorbance capacity assays. The antioxidant power of the peptides recovered from the basolateral chamber is also evaluated by an analysis of biomarkers of cellular oxidative stress such as cell proliferation, alkaline phosphatase, and secretion of nitric oxide, lipid peroxidation, superoxide dismutase and catalase. Peptides from D7S were more active than those of 7S in the modulation of the cell proliferation, oxidative status and differentiation of Caco-2 cells treated with H2 O2 . Differences in the bioactivity of the peptides of both proteins can be explained by analysis of the structural data obtained by mass spectrophotometry. Our findings support the bioavailability of antioxidant peptides of 7S. The antioxidant properties of 7S soy protein were influenced by events such as glycosylation, digestion, and absorption. Deglycosylation seems to be an innovative strategy for improving the properties of 7S. Deglycosylation might enhance 7S antioxidant power and reduce its immunoreactivity. The combined use of advanced analytical techniques and biochemical analyses (phytochemomics) has been a key part of this study.

Protective and reparative effects of peptides from soybean ß-conglycinin on mice intestinal mucosa injury.

Peptides derived from alcalase digestion of soybean ß-conglycinin, containing 8.52% carbohydrate, exhibits an inhibition effect on pathogen adhesion or translocation to intestinal cells in vitro. In this study, the protective and reparative effects of ß-conglycinin peptides on intestinal mucosa injury in vivo were studied using mice with dextran sulfate sodium (DSS)-induced intestinal mucosa injury. The results showed that ß-conglycinin peptides contained approximately 21.77% glutamic acid (Glu), and significantly reduced the histological injury in mice both in the protective and reparative experiments. The myeloperoxidase activity of mice treated with ß-conglycinin peptides decreased compared with those treated DSS in the positive control group. Immunohistochemical analysis also showed that ß-conglycinin peptides inhibited the expression of inflammatory factor NF-κB/p65. These results suggested that peptides derived from soybean ß-conglycinin exhibited protective and reparative effects on mice intestinal mucosa injury.

Soybean ß-conglycinin induces inflammation and oxidation and causes dysfunction of intestinal digestion and absorption in fish.

ß-Conglycinin has been identified as one of the major feed allergens. However, studies of ß-conglycinin on fish are scarce. This study investigated the effects of ß-conglycinin on the growth, digestive and absorptive ability, inflammatory response, oxidative status and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian) in vivo and their enterocytes in vitro. The results indicated that the specific growth rate (SGR), feed intake, and feed efficiency were reduced by ß-conglycinin. In addition, activities of trypsin, chymotrypsin, lipase, creatine kinase, Na(+),K(+)-ATPase and alkaline phosphatase in the intestine showed similar tendencies. The protein content of the hepatopancreas and intestines, and the weight and length of the intestines were all reduced by ß-conglycinin. ß-Conglycinin increased lipid and protein oxidation in the detected tissues and cells. However, ß-conglycinin decreased superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and glutathione (GSH) content in the intestine and enterocytes. Similar antioxidant activity in the hepatopancreas was observed, except for GST. The expression of target of rapamycin (TOR) gene was reduced by ß-conglycinin. Furthermore, mRNA levels of interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and transforming growth factor-ß (TGF-ß) genes were increased by ß-conglycinin. However, ß-conglycinin increased CuZnSOD, MnSOD, CAT, and GPx1b gene expression. In conclusion, this study indicates that ß-conglycinin induces inflammation and oxidation, and causes dysfunction of intestinal digestion and absorption in fish, and finally reduces fish growth. The results of this study provide some information to the mechanism of ß-conglycinin-induced negative effects.

Purification and biophysical characterization of an 11S globulin from Wrightia tinctoria exhibiting hemagglutinating activity.

Wrightia tinctoria globulin (WTG), one of the major seed storage proteins, was isolated for the first time from seeds of the medicinal plant. WTG was extracted and purified to homogeneity in two steps using anion-exchange and size-exclusion chromatographies. On an SDS-PAGE gel under non-reducing conditions, a major band of ~56 kDa was observed; under reducing conditions, however, two major polypeptides, one with molecular weight ~32-34 kDa and the other with molecular weight ~22-26 kDa were observed. Intact mass determination by MALDI-TOF supported this observation. The N-terminal amino acid sequence of WTG matched in NCBI database with an expressed sequence tag obtained from the c-DNA of developing embryo m-RNA of Wrightia tinctoria. The EST sequence was further substantiated by partial de novo internal sequencing using MALDI-TOF/TOF. The high sequence homology with seed storage protein 11S globulin confirmed that WTG is a type of 11S globulin. Circular dichroism analysis showed that the secondary structure of WTG consists predominantly of ß-sheets (44.2%) and moderate content of α-helices (10.3%). WTG showed hemagglutinating property indicating that the protein may possess lectin-like activity. WTG was crystallized at 20 Å°C by the vapour diffusion method using PEG 400 as precipitant. The crystals belonged to the orthorhombic space group P212121 with cell dimensions of a=109.9Å, b=113.2Å and c=202.2Å with six molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.2Å under cryocondition. Preliminary structure solution of WTG indicated the possibility of a hexameric assembly in its asymmetric unit.

Heterologous expression and purification of the soybean 7S globulin α' subunit extension region: in vitro evidence of its involvement in cell cholesterol homeostasis.

In a previous paper, the biological activity of a 216-amino acid recombinant truncated form of the soybean 7S globulin α' subunit, known to control cholesterol and triglyceride homeostasis, was described. In this work, a shorter version of the polypeptide chain, spanning 142 amino acid residues from the N-terminus and thus exclusively including the so-called extension region, was cloned and overexpressed in Pichia pastoris. The yield of the recombinant polypeptide, which was termed α'E, was 8-fold greater than the previous truncated version. The α'E polypeptide was purified by simple conventional biochemical techniques to make it available for biological assays. Human hepatoma cell lines (Hep G2) were used to monitor the uptake and degradation of labeled low-density lipoproteins (LDL), according to an established procedure. The LDL uptake (+86%) and degradation (+94%) by cells tested at the highest α'E dose (2 µM) were similar to those found in cells incubated with 1 µM simvastatin, a potent inhibitor of cholesterol biosynthesis. Additionally, the cell response to α'E was found to be dose-dependent. The present findings strongly suggest that this recombinant polypeptide, or a fragment thereof, is the molecular determinant for cholesterol homeostasis and open new prospects for understanding the mechanism involved in this biological response, as a gateway to its utilization in lipid-lowering therapies.