Positive Charges on the Surface of Thaumatin Are Crucial for the Multi-Point Interaction with the Sweet Receptor.

Thaumatin, an intensely sweet-tasting protein, elicits sweet taste with a threshold of only 50 nM. Previous studies from our laboratory suggested that the complex model between the T1R2-T1R3 sweet receptor and thaumatin depends critically on the complementarity of electrostatic potentials. In order to further validate this model, we focused on three lysine residues (Lys78, Lys106, and Lys137), which were expected to be part of the interaction sites.

Molecular Analysis of the Polymeric Glutenins with Gliadin-Like Characteristics That Were Produced by Acid Dispersion of Wheat Gluten.

We had earlier shown that the dispersion of wheat gluten in acetic acid solution conferred gliadin-like characteristics to the polymeric glutenins. To elucidate the molecular behavior of its polymeric glutenins, the characteristics of gluten powder prepared from dispersions with various types of acid were investigated in this study. Mixograph measurements showed that the acid-treated gluten powders, regardless of the type of acid, had dough properties markedly weakened in both resistance and elasticity properties, as though gliadin was supplemented.

A Hypersweet Protein: Removal of The Specific Negative Charge at Asp21 Enhances Thaumatin Sweetness.

Thaumatin is an intensely sweet-tasting protein that elicits sweet taste at a concentration of 50 nM, a value 100,000 times larger than that of sucrose on a molar basis. Here we attempted to produce a protein with enhanced sweetness by removing negative charges on the interacting side of thaumatin with the taste receptor. We obtained a D21N mutant which, with a threshold value 31 nM is much sweeter than wild type thaumatin and, together with the Y65R mutant of single chain monellin, one of the two sweetest proteins known so far.

Dispersion in the presence of acetic acid or ammonia confers gliadin-like characteristics to the glutenin in wheat gluten.

Spray-dried gluten has unique properties and is commercially available in the food industry worldwide. In this study, we examined the viscoelastic properties of gluten powder prepared by dispersion in the presence of acetic acid or an ammonia solvent and then followed by lyophilization instead of a spray drying. Mixograph measurements showed that the acid- and ammonia-treated gluten powders had marked decreases in the time to peak dough resistance when compared with the control gluten powder.

Isolation of lactic acid-tolerant Saccharomyces cerevisiae from Cameroonian alcoholic beverage.

We investigated yeast strains used in Cameroonian microbreweries, and identified a Saccharomyces cerevisiae strain (OCY3) with an excellent capacity for alcoholic fermentation. OCY3 showed higher tolerance to lactic acid and better fermentation performance under acidic conditions than a representative Japanese sake yeast, Kyokai No. 7, and a wine yeast, EC1118.

Five amino acid residues in cysteine-rich domain of human T1R3 were involved in the response for sweet-tasting protein, thaumatin.

Thaumatin, a sweet-tasting plant protein, elicits a sweet taste sensation at 50 nM in humans but not rodents. Although it was shown that the cysteine-rich domain (CRD) of human T1R3 (hT1R3) is important for the response to thaumatin, the amino acid residues within CRD critical for response are still unknown. A comparison of the amino acid sequence (69 amino acid residues) of CRD between hT1R3 and mouse T1R3 (mT1R3) revealed sixteen amino acids that differ.

Changes in textural properties of Japanese tenobe somen noodles during storage.

Tenobe somen (TS) noodles are traditional Japanese wheat-based noodles that are produced manually using a sophisticated method called tenobe (literally, "hand stretched"). In the tension test, both the tensile strength and extensibility of TS noodles were greater than those of machine-made (MS) noodles. In the biting test, the chewy texture of TS noodles was realized in the analysis of the force-deformation curves of each type of somen noodles.

Atomic structure of the sweet-tasting protein thaumatin I at pH 8.0 reveals the large disulfide-rich region in domain II to be sensitive to a pH change.

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at 50 nM. Although the sweetness remains when thaumatin is heated at 80 °C for 4h under acid conditions, it rapidly declines when heating at a pH above 6.5.

Bacterial heat shock protein 60, GroEL, can induce the conversion of naïve T cells into a CD4 CD25(+) Foxp3-expressing phenotype.

Recent publications report that heat shock proteins (HSPs) can endow regulatory responses to the systemic immune system when administered via the mucosal route, leading to an amelioration of atherosclerosis and allergy. However, it remains poorly understood if HSP antigens exist in the luminal contents of the gastrointestinal tract and which types of HSP induce regulatory responses. Here we addressed these problems, considering that numerous gut microflora and foods are natural sources of HSPs.

Introduction of a negative charge at Arg82 in thaumatin abolished responses to human T1R2-T1R3 sweet receptors.

Thaumatin, an intensely sweet-tasting protein, elicits a sweet-taste sensation at a level as low as 50 nM. Although previous sensory analyses have suggested that Lys67 and Arg82 are important to the sweetness of thaumatin, the exact effects of each residue on sweet receptors are still unknown. In the present study, various mutants of thaumatin altered at Arg82 as well as Lys67 were prepared and their sweetness levels were quantitatively evaluated by cell-based assays using HEK293 cells expressing human sweet receptors.

Crystal structure of the sweet-tasting protein thaumatin II at 1.27Å.

Thaumatin, an intensely sweet-tasting protein, elicits a sweet taste sensation at 50 nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27 Å.

High-resolution structure of the recombinant sweet-tasting protein thaumatin I.

Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at a concentration of 50 nM. The crystal structure of a recombinant form of thaumatin I produced in the yeast Pichia pastoris has been determined to a resolution of 1.1 Å.

Changes in lamina propria dendritic cells on the oral administration of exogenous protein antigens during weaning.

Two critical periods of maximum exposure to antigens occur in young mammals, immediately after birth and at weaning, as a result of colonization by commensal bacteria and the ingestion of new diets. At weaning, active immune responses of antibody production against dietary proteins are known to occur, but simultaneously, oral tolerance is acquired for harmless food proteins. However, regulated mechanisms of the immune system at weaning remain to be elucidated although its immune responses may be somewhat similar to those in adulthood.

The cysteine-rich domain of human T1R3 is necessary for the interaction between human T1R2-T1R3 sweet receptors and a sweet-tasting protein, thaumatin.

Thaumatin is an intensely sweet-tasting protein perceived by humans but not rodents. Its threshold value of sweetness in humans is 50nM, the lowest of any sweet-tasting protein. In the present study, the sites where sweet receptors interact with thaumatin were investigated using human embryonic kidney 293 (HEK293) cells expressing the sweet receptors T1R2-T1R3.

Lymphoid neoplastic P388D1 cells express membrane protein candidates that discriminate among the C-terminal phylogenetic diversity in heat shock protein 70 sequences.

We previously found that mouse inducible Hsp72 bound more extensively to lymphoblast-like lymphoid neoplastic P388D1 cells than to RAW264.7 monocyte-macrophages. In the present study, we analyzed the characteristics of the binding to P388D1 cells of recombinant HSP70 derived from different species.

Rheological properties of somen noodles--a traditional Japanese wheat product.

We investigated the rheological properties of the Japanese wheat product tenobe somen noodles manufactured using a unique traditional process-"Te-nobe (hand-stretched)." In an extension test, the maximum resistance to extension (R(max)) and extensibility until rupture (Erup) of boiled somen noodles were measured on a Texture Analyzer in the tension test mode and compared with those of machine-made somen noodles. The R(max) and Erup values per unit cross-sectional area were significantly higher for boiled tenobe somen noodles than for machine-made somen noodles, clearly indicating the higher resistance to extension and extensibility of the former.

Interactions of the sweet-tasting proteins thaumatin and lysozyme with the human sweet-taste receptor.

This study investigated the sweetness of the sweet-tasting protein thaumatin and lysozyme by both an in vitro cell-based assay and an in vivo sensory analysis to elucidate the differences between in vitro and in vivo response profiles. Hek293 cells were constructed that stably expressed the human T1R2+T1R3 sweet-taste receptor, and their responses to thaumatin and lysozyme were analyzed by monitoring the levels of intracellular cAMP. The results indicated that thaumatin and lysozyme as well as aspartame induced a decrease in the intracellular cAMP accumulation of the T1R2+T1R3-transfected cells and that EC(50) values of thaumatin and lysozyme determined by cell-based assay are well-consistent with the results of the sweetness threshold value determined by sensory analysis in the presence of 140 mM NaCl.

Surface expression of a C-terminal alpha-helix region in heat shock protein 72 on murine LL/2 lung carcinoma can be recognized by innate immune sentinels.

Surface expression of Hsp70 members has been previously reported on human tumor cell lines. Here we examined how the inducible mouse Hsp72 can be expressed on the surface of two types of murine tumor cell lines in response to non-lethal heat shock. Exposure to 42 degrees C for 2h led to the intracellular production of Hsp72 for both murine LL/2 lung carcinoma and B16 melanoma cells.

Oligoclonal Enzyme-linked Immunosorbent Assay Capable of Determining the Major Food Allergen, Ovomucoid, Irrespective of the Degree of Heat Denaturation.

We have recently established an enzyme-linked immunosorbent assay (ELISA) for total ovomucoid determination, irrespective of the degree of its heat denaturation, by using a monoclonal antibody (mAb) 7D specific to the carbohydrate moiety of ovomucoid (Biosci Biothechnol Biochem, 68, 2490-2497, 2004). Two novel methods have been developed to improve the ELISA. First, its sensitivity was enhanced 100 times by using an oligoclonal cocktail of mAb 7D and two other mAbs with different epitopes as a second antibody.

Effects of CpG-oligodeoxynucleotides on dendritic cell development.

Dendritic cell (DC) development begins in the bone marrow and immature progenitors reach their sites of residence in lymphoid organs. The mechanism of DC development in the bone marrow and in peripheral lymphoid organs is poorly understood. Here, we examined the effects of synthetic oligodeoxynucleotides containing a CpG motif (CpG-ODNs) on the development of DC from the bone marrow cells.

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I.

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness.

Effects of pre- and pro-sequence of thaumatin on the secretion by Pichia pastoris.

Thaumatin is a 22-kDa sweet-tasting protein containing eight disulfide bonds. When thaumatin is expressed in Pichia pastoris using the thaumatin cDNA fused with both the alpha-factor signal sequence and the Kex2 protease cleavage site from Saccharomyces cerevisiae, the N-terminal sequence of the secreted thaumatin molecule is not processed correctly. To examine the role of the thaumatin cDNA-encoded N-terminal pre-sequence and C-terminal pro-sequence on the processing of thaumatin and efficiency of thaumatin production in P.

Cloning of the thaumatin I cDNA and characterization of recombinant thaumatin I secreted by Pichia pastoris.

Thaumatin is a sweet-tasting protein comprising a mixture of some variants. The major variants are thaumatins I and II. Although the amino acid sequence of thaumatin I was known and the nucleotide sequence of cDNA of thaumatin II was elucidated, the nucleotide sequence of thaumatin I has been controversial.

Developments in biotechnological production of sweet proteins.

Most proteins are tasteless and flavorless, while some proteins elicit a sweet-taste response on the human palate. Six proteins, thaumatin, monellin, mabinlin, brazzein, egg lysozyme, and neoculin (previously considered as curculin) have been identified as sweet-tasting proteins. However, no common features among them have been observed.

Functional region of mouse heat shock protein 72 for its binding to lymphoid neoplastic P388D1 cells.

Extracellular heat shock proteins have been reported to participate in both innate and adaptive immune responses. We have found that recombinant mouse inducible heat shock protein 72 (Hsp72) bound to lymphoid neoplastic P388D1 cells. In the present study, we examined which region of mouse Hsp72 interacted with this cell line by using truncated variants that are sequentially lacking sections of the C-terminal region.