Soy ß-Conglycinin Peptide Attenuates Obesity and Lipid Abnormalities in Obese Model OLETF Rats.

We previously reported that soy ß-conglycinin (ßCG) improves obesity-induced metabolic abnormalities, but not obesity, in obese model Otsuka Long-Evans Tokushima fatty (OLETF) rats. In the present study, we aimed to investigate the effects of ßCG-derived peptide consumption on obesity and lipid abnormality in OLETF rats. To this end, wild-type Long-Evans Tokushima Otsuka and OLETF rats were provided a normal diet containing 20% casein for four weeks as a control. In addition, we prepared ßCG peptide by enzymatic hydrolysis, and OLETF rats were fed a diet in which half of the casein was replaced by ßCG peptide (ßCG peptide group). Consequently, rats in the ßCG peptide group showed decreased abdominal white adipose tissue weight and lipid content (serum and liver triglycerides, and serum and liver cholesterol) compared to control OLETF rats. Further analysis demonstrated that ßCG peptide consumption decreased lipogenic enzyme activity and increased lipolytic enzyme activity in the liver of OLETF rats. In addition, suppressive effects on both synthesis and absorption of cholesterol were observed in ßCG peptide-fed OLETF rats. These findings suggest that peptidization of ßCG enhanced the anti-obese and hypolipidemic effects of ßCG.

Narrow-leafed lupin (Lupinus angustifolius L.) seed ß-conglutins reverse the induced insulin resistance in pancreatic cells.

Insulin resistance (IR) is the main contributor to the development of type 2 diabetes. In this study, we have purified recombinant ß-conglutin proteins (rß1 to rß4, and rß6) from narrow-leafed lupin (NLL) by using affinity chromatography. The objective of this study was to evaluate the capacity of these ß-conglutins to improve the IR state using ex vivo and in vitro systems. rß1, rß3, and rß6 produced lower levels of pro-inflammatory mediator nitric oxide (about -7-fold in all cases), up-regulated mRNA expression levels of IRS-1 (+201, +173, +192%) and Glut-4 (+286, +121, +147%), increased levels of p85-PI3K (+188, +187, +137-fold) and Glut-4 (+503, +548, +515-fold) proteins, higher phosphorylation levels of the insulin signalling pathway activator p-IRS-1 and downstream mediators such as p-Akt, p-Cbl, and p-caveolin, and improved glucose uptake in insulin resistant (IR-C) culture cells. ß-conglutin proteins were able to suppress the oxidative stress produced by insulin-induced resistance on PANC-1 control (C) cells by strongly reducing the protein oxidative carbonylation induced by ROS and balancing the metabolic homeostasis in IR-C cells through regulation of mRNA expression. At the same time, ß-conglutins are able to reduce the levels of the pro-inflammatory mediator nitric oxide and promote the anti-oxidative capacity of cells by increasing the levels of reduced glutathione. These results suggest NLL ß-conglutins might play a fundamental role as functional food components, since ß-conglutins' nutraceutical properties could enhance the effectiveness of dietary improvement of type 2 diabetes complications.

Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

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.

Amino acid profiles and quality from lotus seed proteins.

BACKGROUND: Protein composition, amino acid profile and nutritional value of the lotus seed and its Osborne fractions were investigated. The seed was rich in protein with 19.85%, and showed well balanced amino acid composition compared with FAO/WHO pattern, Its nutritive properties were similar to those observed in the reference soybean protein. Phenylalanine, tyrosine, leucine and lysine were the limiting amino acids in the seed proteins. The albumin and globulin were the main protein fraction, the amino acid profile and nutritional value were close to the seed protein. RESULTS: Changes in transition temperature and thermal stability were observed through different solvent extractions. Albumin possessed the predominant thermal stability (81.4 °C) followed by globulin (74.49 °C), prolamin (69 °C) and glutelin (65.6 °C). So, solvent compositions influence the profile of AAs and their nutritive value, and aqueous solvent with 0.1 mol L⁻¹ NaCl was an efficient protein solubiliser. CONCLUSION: The results indicated that the extraction processes influenced the lotus seed protein quality and thermal stability. Overall, the study revealed that the lotus seed protein was nutritionally well-balanced protein and might be of significant importance in the formulation of diets for humans.

Purification and partial characterization of low molecular weight vicilin-like glycoprotein from the seeds of Citrullus lanatus.

The watermelon (Citrullus lanatus) seeds are highly nutritive and contain large amount of proteins and many beneficial minerals such as magnesium, calcium, potassium, iron, phosphorous, zinc etc. In various parts of the world, C. lanatus seed extracts are used to cure cancer, cardiovascular diseases, hypertension, and blood pressure. C. lanatus seed extracts are also used as home remedy for edema and urinary tract problems. In this study, we isolated protein fraction of C. lanatus seeds using various protein separation methods. We successfully purified a low molecular weight vicilin-like glycoprotein using chromatographic methods followed by SDS-PAGE and MALDI-TOF/MS identification. This is the first report of purification of a vicilin like polypeptide from C. lanatus seeds. In next step, we extracted mRNA from immature seeds and reverse transcribed it using suitable forward and reverse primers for purified glycoprotein. The PCR product was analysed on 1% agarose gel and was subsequently sequenced by Dideoxy DNA sequencing method. An amino acid translation of the gene is in agreement with amino acid sequences of the identified peptides.

Process improvement for semipurified oleosomes on a pilot-plant scale.

Semipurified oleosomes were isolated on a pilot-plant scale using improved-process extraction conditions. The improved process consisted of continuous centrifugation in a three-phase decanter with recirculation of slurry until most of the oleosomes were recovered. Oleosome fractionation, oleosin identification, and isoflavone and saponin mass distributions and recoveries were investigated. The improved pilot-plant oleosome extraction process was achieved in 8 h. A total of 91%± 1% of soybean oil was recovered as intact oleosomes. The oil content of the aqueous supernatant and the residue fractions were low at 2% and 3%, respectively. The aqueous supernatant fraction contained 40% total soybean protein. About 76% of the proteins present in the oleosome fraction were soybean storage proteins. Washing the semipurified oleosomes with a 0.1 M Tris-HCl, pH 8.6 containing 0.4 M sucrose, and 0.5 M NaCl resulted in the recovery of the associated storage proteins. The recovery of these proteins in addition to the protein in aqueous supernatant accounted for 79% of the total soybean storage proteins fractionated by this process. Oleosins were detected at 17 and 18 kDa. Isoflavones and saponins partitioned into the oleosome, aqueous supernatant, and residue fractions at different ratios with the majority, about 82 and 63 mole%, respectively, in oleosome and aqueous supernatant fractions, making these fractions an attractive source for phytochemicals.

Effect of protein hydrolysates from germinated soybean on cancerous cells of the human cervix: an in vitro study.

Consumption of soybeans can reduce the risk of different types of cancer. Little is known about the effect of germination on the anticancer properties of soya. This study was done to determine if germination improves the anticancer properties of soybean protein through generation of amino acids or bioactive peptides. Soybean was germinated for 0, 2, 3, 4, 5, and 6 days and proteins were isolated from the seeds. Isolates with and without ethanol-soluble phytochemicals were hydrolyzed with digestive enzymes and their effect on growth in HeLa and C-33 (epidermoid cervical carcinoma) and HaCaT (non-cancerous human keratinocytes) cells were evaluated with the Alamar Blue method. Germination induced degradation of the alpha and alpha' fractions of beta-conglycinin and acid fraction of glycinin, generating low molecular weight peptides. Degrees of hydrolysis ranged from 73-77%. Hydrolysates inhibited the growth of HeLa cells and C-33 at concentrations exceeding 1.25 mg/ml. Major inhibition was observed with the hydrolysate germinated for 2 days and containing ethanolsoluble phytochemicals (IC(50) 2.15 and 2.27 mg/ml for HeLa and C-33, respectively). Interestingly, hydrolysate cytoxicity for normal cells was minimal in comparison to cancer cells.