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1.
Intern Med ; 61(7): 1033-1037, 2022 Apr 01.
Article in English | MEDLINE | ID: mdl-35110484

ABSTRACT

A 28-year-old woman experienced gross hematuria after the administration of the second dose of an messenger ribonucleic acid (mRNA) vaccine (BNT162b2). She was diagnosed with Immunogloblin A nephropathy (IgAN) by a renal biopsy two weeks after vaccination, which revealed a mild increase in mesangial cells and a matrix with co-depositions of galactose-deficient IgA1 and C3 in the mesangial region. The gross hematuria and proteinuria gradually improved without any medication, suggesting that immune activation by the mRNA vaccine may not elicit continuous disease progression of IgAN. Thus, further studies investigating the relationship between mRNA vaccines against COVID-19 and the progression of IgAN should be conducted.


Subject(s)
COVID-19 , Glomerulonephritis, IGA , Adult , BNT162 Vaccine , COVID-19 Vaccines/adverse effects , Female , Glomerulonephritis, IGA/diagnosis , Hematuria/etiology , Humans , Immunoglobulin A , RNA, Messenger , Vaccination , Vaccines, Synthetic , mRNA Vaccines
2.
PLoS One ; 14(8): e0217329, 2019.
Article in English | MEDLINE | ID: mdl-31419225

ABSTRACT

Silene latifolia is a dioecious flowering plant with sex chromosomes in the family Caryophyllaceae. Development of a gynoecium and stamens are suppressed in the male and female flowers of S. latifolia, respectively. Microbotryum lychnidis-dioicae promotes stamen development when it infects the female flower. If suppression of the stamen and gynoecium development is regulated by the same mechanism, suppression of gynoecium and stamen development is released simultaneously with the infection by M. lychnidis-dioicae. To assess this hypothesis, an asexual mutant without a gynoecium or stamen was infected with M. lychnidis-dioicae. A filament of the stamen in the infected asexual mutant was elongated at stages 11 and 12 of flower bud development as well as in the male, but the gynoecium did not form. Instead of the gynoecium, a filamentous structure was suppressed as in the male flower. Developmental suppression of the stamen was released by M. lychnidis-dioicae, but that of gynoecium development was not released. M. lychnidis-dioicae would have a function similar to stamen-promoting factor (SPF), since the elongation of the stamen that is not observed in the healthy asexual mutant was observed after stage 8 of flower bud development. An infection experiment also revealed that a deletion on the Y chromosome of the asexual mutant eliminated genes for maturation of tapetal cells because the tapetal cells did not mature in the asexual mutant infected with M. lychnidis-dioicae.


Subject(s)
Basidiomycota/pathogenicity , Flowers/microbiology , Silene/microbiology , Basidiomycota/physiology , Chromosome Deletion , Crosses, Genetic , Flowers/growth & development , Flowers/physiology , Genes, Plant , Host Microbial Interactions/genetics , Host Microbial Interactions/physiology , Microscopy, Electron, Scanning , Mutation , Plant Diseases/genetics , Plant Diseases/microbiology , Reproduction/genetics , Reproduction/physiology , Reproduction, Asexual/genetics , Reproduction, Asexual/physiology , Silene/genetics , Silene/physiology
3.
Plant Cell Physiol ; 58(2): 320-328, 2017 02 01.
Article in English | MEDLINE | ID: mdl-28011871

ABSTRACT

Mechanisms of suppression of pistil primordia in male flowers and of stamen primordia in female flowers differ in diclinous plants. In this study, we investigated how cell death and cell cycle arrest are related to flower organ formation in Silene latifolia. Using in situ hybridization and a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, we detected both cell cycle arrest and cell death in suppressed stamens of female flowers and suppressed pistils of male flowers in S. latifolia. In female flowers infected with Microbotryum lychnidis-dioicae, developmental suppression of stamens is released, and cell cycle arrest and cell death do not occur. Smut spores are formed in S. latifolia anthers infected with M. lychnidis-dioicae, followed by cell death in the endothelium, middle layer, tapetal cells and pollen mother cells. Cell death is difficult to detect using a fluorescein isothiocyanate-labeled TUNEL assay due to strong autofluorescence in the anther. We therefore combined a TUNEL assay in an infrared region with transmission electron microscopy to detect cell death in anthers. We show that following infection by M. lychnidis-dioicae, a TUNEL signal was not detected in the endothelium, middle layer or pollen mother cells, and cell death with outflow of cell contents, including the nucleoplast, was observed in tapetal cells.


Subject(s)
Basidiomycota/physiology , Flowers/metabolism , Silene/metabolism , Silene/microbiology , Cell Cycle Checkpoints/physiology , Cell Death/physiology , Flowers/microbiology , Pollen/metabolism , Pollen/microbiology
4.
Sci Rep ; 6: 18917, 2016 Jan 08.
Article in English | MEDLINE | ID: mdl-26742857

ABSTRACT

Sex chromosomes are particularly interesting regions of the genome for both molecular genetics and evolutionary studies; yet, for most species, we lack basic information, such as the gene order along the chromosome. Because they lack recombination, Y-linked genes cannot be mapped genetically, leaving physical mapping as the only option for establishing the extent of synteny and homology with the X chromosome. Here, we developed a novel and general method for deletion mapping of non-recombining regions by solving "the travelling salesman problem", and evaluate its accuracy using simulated datasets. Unlike the existing radiation hybrid approach, this method allows us to combine deletion mutants from different experiments and sources. We applied our method to a set of newly generated deletion mutants in the dioecious plant Silene latifolia and refined the locations of the sex-determining loci on its Y chromosome map.


Subject(s)
Base Sequence , Chromosomes, Plant/chemistry , Flowers/genetics , Physical Chromosome Mapping/methods , Sequence Deletion , Silene/genetics , Biological Evolution , Genetic Loci , Sex Determination Processes , Software
5.
PLoS One ; 7(4): e35380, 2012.
Article in English | MEDLINE | ID: mdl-22536376

ABSTRACT

One of the most distinctive features of human sweet taste perception is its broad tuning to chemically diverse compounds ranging from low-molecular-weight sweeteners to sweet-tasting proteins. Many reports suggest that the human sweet taste receptor (hT1R2-hT1R3), a heteromeric complex composed of T1R2 and T1R3 subunits belonging to the class C G protein-coupled receptor family, has multiple binding sites for these sweeteners. However, it remains unclear how the same receptor recognizes such diverse structures. Here we aim to characterize the modes of binding between hT1R2-hT1R3 and low-molecular-weight sweet compounds by functional analysis of a series of site-directed mutants and by molecular modeling-based docking simulation at the binding pocket formed on the large extracellular amino-terminal domain (ATD) of hT1R2. We successfully determined the amino acid residues responsible for binding to sweeteners in the cleft of hT1R2 ATD. Our results suggest that individual ligands have sets of specific residues for binding in correspondence with the chemical structures and other residues responsible for interacting with multiple ligands.


Subject(s)
Receptors, G-Protein-Coupled/agonists , Sweetening Agents/chemistry , Amino Acid Motifs , Amino Acid Substitution , Aspartame/chemistry , Binding Sites , Computer Simulation , Cyclamates/chemistry , Cyclamates/pharmacology , HEK293 Cells , Humans , Models, Molecular , Molecular Weight , Mutagenesis, Site-Directed , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/genetics , Saccharin/chemistry , Saccharin/pharmacology , Sucrose/analogs & derivatives , Sucrose/chemistry , Sucrose/pharmacology , Sweetening Agents/pharmacology , Thiazines/chemistry , Thiazines/pharmacology , Tryptophan/chemistry , Tryptophan/pharmacology
6.
Proc Natl Acad Sci U S A ; 108(40): 16819-24, 2011 Oct 04.
Article in English | MEDLINE | ID: mdl-21949380

ABSTRACT

Miraculin (MCL) is a homodimeric protein isolated from the red berries of Richadella dulcifica. MCL, although flat in taste at neutral pH, has taste-modifying activity to convert sour stimuli to sweetness. Once MCL is held on the tongue, strong sweetness is sensed over 1 h each time we taste a sour solution. Nevertheless, no molecular mechanism underlying the taste-modifying activity has been clarified. In this study, we succeeded in quantitatively evaluating the acid-induced sweetness of MCL using a cell-based assay system and found that MCL activated hT1R2-hT1R3 pH-dependently as the pH decreased from 6.5 to 4.8, and that the receptor activation occurred every time an acid solution was applied. Although MCL per se is sensory-inactive at pH 6.7 or higher, it suppressed the response of hT1R2-hT1R3 to other sweeteners at neutral pH and enhanced the response at weakly acidic pH. Using human/mouse chimeric receptors and molecular modeling, we revealed that the amino-terminal domain of hT1R2 is required for the response to MCL. Our data suggest that MCL binds hT1R2-hT1R3 as an antagonist at neutral pH and functionally changes into an agonist at acidic pH, and we conclude this may cause its taste-modifying activity.


Subject(s)
Glycoproteins/metabolism , Models, Molecular , Protein Conformation , Receptors, G-Protein-Coupled/metabolism , Taste Buds/metabolism , Animals , Cell Line , Fluorescence , Glycoproteins/chemistry , Humans , Hydrogen-Ion Concentration , Mice , Receptors, G-Protein-Coupled/chemistry
7.
Biosci Biotechnol Biochem ; 75(8): 1600-2, 2011.
Article in English | MEDLINE | ID: mdl-21821940

ABSTRACT

Neoculin, a sweet protein found in the fruit of Curculigo latifolia, has the ability to change sourness into sweetness. Neoculin turns drinking water sweet, indicating that non-acidic compounds may induce the sweetness. We report that ammonium chloride and certain amino acids elicit the intense sweetness of neoculin. Neoculin can thus sweeten amino acid-enriched foods.


Subject(s)
Curculigo/chemistry , Food Technology , Fruit/chemistry , Plant Proteins , Sweetening Agents/metabolism , Amino Acids/metabolism , Amino Acids/pharmacology , Circular Dichroism , Curculigo/metabolism , Fruit/metabolism , Hydrogen-Ion Concentration , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/metabolism , Spectrometry, Fluorescence , Sweetening Agents/chemistry , Taste/drug effects , Taste Perception/drug effects
8.
PLoS One ; 6(4): e19448, 2011 Apr 29.
Article in English | MEDLINE | ID: mdl-21559382

ABSTRACT

Neoculin occurring in the tropical fruit of Curculigo latifolia is currently the only protein that possesses both a sweet taste and a taste-modifying activity of converting sourness into sweetness. Structurally, this protein is a heterodimer consisting of a neoculin acidic subunit (NAS) and a neoculin basic subunit (NBS). Recently, we found that a neoculin variant in which all five histidine residues are replaced with alanine elicits intense sweetness at both neutral and acidic pH but has no taste-modifying activity. To identify the critical histidine residue(s) responsible for this activity, we produced a series of His-to-Ala neoculin variants and evaluated their sweetness levels using cell-based calcium imaging and a human sensory test. Our results suggest that NBS His11 functions as a primary pH sensor for neoculin to elicit taste modification. Neoculin variants with substitutions other than His-to-Ala were further analyzed to clarify the role of the NBS position 11 in the taste-modifying activity. We found that the aromatic character of the amino acid side chain is necessary to elicit the pH-dependent sweetness. Interestingly, since the His-to-Tyr variant is a novel taste-modifying protein with alternative pH sensitivity, the position 11 in NBS can be critical to modulate the pH-dependent activity of neoculin. These findings are important for understanding the pH-sensitive functional changes in proteinaceous ligands in general and the interaction of taste receptor-taste substance in particular.


Subject(s)
Amino Acids/chemistry , Plant Proteins/chemistry , Taste , Animals , Aspergillus oryzae/metabolism , Calcium/chemistry , Cell Line , Curculigo , Dose-Response Relationship, Drug , Genetic Variation , Humans , Hydrogen-Ion Concentration , Models, Molecular , Plant Proteins/metabolism , Protein Folding , Protein Structure, Tertiary
9.
Biotechnol Lett ; 33(1): 103-7, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20936326

ABSTRACT

PURPOSE OF WORK: Soluble protein expression is an important first step during various types of protein studies. Here, we present the screening strategy of secretable mutant. The strategy aimed to identify those cysteine residues that provoke protein misfolding in the heterologous expression system. Intentional mutagenesis studies should consider the size of the library and the time required for expression screening. Here, we proposed a cysteine-to-serine shuffling mutation strategy (CS shuffling) using a Saccharomyces cerevisiae expression system. This strategy of site-directed shuffling mutagenesis of cysteine-to-serine residues aims to identify the cysteine residues that cause protein misfolding in heterologous expression. In the case of a nonglycosylated mutant of the taste-modifying protein miraculin (MCL), which was used here as a model protein, 25% of all constructs obtained from CS shuffling expressed MCL mutant, and serine mutations were found at Cys47 or Cys92, which are involved in the formation of the disulfide bond. This indicates that these residues had the potential to provoke protein misfolding via incorrect disulfide bonding. The CS shuffling can be performed using a small library and within one week, and is an effective screening strategy of soluble protein expression.


Subject(s)
Cysteine/genetics , Glycoproteins/metabolism , Mutagenesis, Site-Directed/methods , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Serine/genetics , Amino Acid Sequence , Genetic Vectors , Glycoproteins/genetics , Molecular Sequence Data , Protein Folding , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sweetening Agents/metabolism
10.
Biochim Biophys Acta ; 1800(9): 986-92, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20542090

ABSTRACT

BACKGROUND: Miraculin (MCL) is a taste-modifying protein that converts sourness into sweetness. The molecular mechanism underlying the taste-modifying action of MCL is unknown. METHODS: Here, a yeast expression system for MCL was constructed to accelerate analysis of its structure-function relationships. The Saccharomyces cerevisiae expression system has advantages as a high-throughput analysis system, but compared to other hosts it is characterized by a relatively low level of recombinant protein expression. To alleviate this weakness, in this study we optimized the codon usage and signal-sequence as the first step. Recombinant MCL (rMCL) was expressed and purified, and the sensory taste was analyzed. RESULTS: As a result, a 2 mg/l yield of rMCL was successfully obtained. Although sensory taste evaluation showed that rMCL was flat in taste under all the pH conditions employed, taste-modifying activity similar to that of native MCL was recovered after deglycosylation. Mutagenetic analysis revealed that the N-glycan attached to Asn42 was bulky in rMCL. CONCLUSIONS: The high-mannose-type N-glycan attached in yeast blocks the taste-modifying activity of rMCL. GENERAL SIGNIFICANCE: The bulky N-glycan attached to Asn42 may cause steric hindrance in the interaction between active residues and the sweet taste receptor hT1R2/hT1R3.


Subject(s)
Glycoproteins , Polysaccharides , Receptors, G-Protein-Coupled/agonists , Recombinant Proteins , Taste/drug effects , Glycoproteins/biosynthesis , Glycoproteins/genetics , Glycoproteins/pharmacology , Humans , Polysaccharides/biosynthesis , Polysaccharides/genetics , Polysaccharides/pharmacology , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Saccharomyces cerevisiae/genetics , Taste/physiology
11.
Plant Cell Physiol ; 51(2): 282-93, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20064843

ABSTRACT

Carpel suppression is a trigger for sexual dimorphism in the dioecious plant Silene latifolia. To clarify what kind of genes are involved in carpel suppression in this species, we generated a bisexual mutant, R025, by C-ion beam irradiation. R025 produces bisexual flowers with a mature gynoecium and mature stamens. Genetic analysis of R025 attributed the bisexual trait to mutations on the Y chromosome. Scanning electron microscopy (SEM) analysis of early floral development revealed that the carpel size of R025 was different from that of wild-type males in spite of the male background in R025. We also identified an S. latifolia CLAVATA1-like gene (SlCLV1) as a candidate of the CLAVATA-WUSCHEL (CLV-WUS) pathway. Two separate pathways, the CLV-WUS pathway and the CUP-SHAPED COTYLEDON (CUC)-SHOOT MERISTEMLESS (STM) pathway, contribute to carpel development in the Arabidopsis floral meristem. SlSTM1 and SlSTM2 (orthologs of STM) and SlCUC (an ortholog of CUC1 and CUC2) have already been identified in S. latifolia. We therefore examined the expression patterns of SlCLV1, SlSTM (SlSTM1 and SlSTM2) and SlCUC in young flowers of R025 and wild-type males and females. The expression patterns of the three genes in the two pathways differ between the wild-type male and the bisexual mutant, and the differences in expression patterns of the three genes occur at the same stage. These results suggest that in addition to SlSTM1, SlSTM2 and SlCUC, SlCLV1 is also involved in carpel suppression in S. latifolia. They also suggest that a gynoecium-suppressing factor (GSF), which is lost in the R025 Y chromosome, acts on an upstream gene that is common to the two pathways, triggering sexual dimorphism in S. latifolia.


Subject(s)
Flowers/growth & development , Plant Proteins/metabolism , Silene/genetics , Amino Acid Sequence , Arabidopsis Proteins/metabolism , Cloning, Molecular , Gene Expression Profiling , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genes, Plant , Homeodomain Proteins/metabolism , Meristem/genetics , Meristem/growth & development , Molecular Sequence Data , Mutation , Plant Proteins/genetics , Protein Serine-Threonine Kinases , RNA, Plant/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Sequence Alignment , Silene/growth & development , Silene/metabolism
12.
Bioorg Med Chem Lett ; 20(3): 1081-3, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-20031409

ABSTRACT

Some D-amino acids such as d-tryptophan and D-phenylalanine are well known as naturally-occurring sweeteners. Photoreactive D-phenylalanine derivatives containing trifluoromethyldiazirinyl moiety at 3- or 4-position of phenylalanine, were designed as sweeteners for functional analysis with photoaffinity labeling. The trifluoromethyldiazirinyl D-phenylalanine derivatives were prepared effectively with chemo-enzymatic methods using L-amino acid oxidase and were found to have potent activity toward the human sweet taste receptor.


Subject(s)
Phenylalanine/chemical synthesis , Phenylalanine/metabolism , Photoaffinity Labels/chemical synthesis , Photoaffinity Labels/metabolism , Receptors, G-Protein-Coupled/metabolism , Taste Buds/metabolism , Cell Line , Crystallography, X-Ray , Humans , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/chemistry , Sweetening Agents/metabolism , Taste Buds/chemistry
13.
Biosci Biotechnol Biochem ; 73(11): 2552-5, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19897888

ABSTRACT

Neoculin has pH-dependent taste-modifying activity. This study found that neoculin changed pH-dependently in its tryptophan- and ANS-derived fluorescence spectra, while no such change occurred in a neoculin variant whose histidine residues were replaced with alanine. These results suggest that the sweetness of neoculin depends on structural change accompanying the pH change, with the histidine residues playing a key role.


Subject(s)
Plant Proteins/chemistry , Plant Proteins/pharmacology , Taste/drug effects , Humans , Hydrogen-Ion Concentration , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Taste/physiology , Tryptophan
15.
J Plant Physiol ; 166(16): 1832-8, 2009 Nov 01.
Article in English | MEDLINE | ID: mdl-19457581

ABSTRACT

The genes SlSTM1 and SlSTM2 (orthologs of Arabidopsis SHOOT MERISTEMLESS) and SlCUC (an ortholog of CUP-SHAPED COTYLEDON1 and CUC2) of the dioecious species Silene latifolia have been proposed to control the gynoecium suppression pathway in developing flowers. In a mutant of S. latifolia (K034) that produces no males but only asexual and imperfect female (female-like) flowers, both on the same individual, gynoecia are completely suppressed in asexual flowers and partially suppressed in female-like flowers. To determine whether these two epigenetic phenotypes in gynoecium development are caused by changes in SlSTM and SlCUC expression, we performed in situ hybridization with probes of SlSTM and SlCUC. We found two different pattern of gene expression in flower buds prior to the onset of phenotypic differentiation, which were similar to the reciprocal expression of the two genes described in male and female wild-type plants. In young K034 flower buds, 14.3% of developing structures showed female and the rest male determination. This ratio corresponds to the ratio of female-like to asexual flowers eventually produced by the K034 plants. The same ratio (7-16%) was not only found in the original mutants but also in the first and second backcross generations and in vegetative clones of the original mutant line. Hence, the switch-like and reciprocal SlSTM and SlCUC expression patterns in K034 correspond to the gynoecium suppression patterns in the wild type, suggesting that the mutation(s) responsible for the two mutant genotypes acts upstream of SlSTM and SlCUC.


Subject(s)
Flowers , Mutation , Silene/growth & development , Base Sequence , DNA Primers , Gene Expression Regulation, Plant , Genes, Plant , In Situ Hybridization , Silene/genetics
16.
Plant Cell Physiol ; 50(6): 1127-41, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19406862

ABSTRACT

To elucidate the mechanism(s) underlying dioecious flower development, the present study analyzed a SUPERMAN (SUP) homolog, SlSUP, which was identified in Silene latifolia. The sex of this plant is determined by heteromorphic X and Y sex chromosomes. It was revealed that SlSUP is a single-copy autosomal gene expressed exclusively in female flowers. Introduction of a genomic copy of SlSUP into the Arabidopsis thaliana sup (sup-2) mutant complemented the excess-stamen and infertile phenotypes of sup-2, and the overexpression of SlSUP in transgenic Arabidopsis plants resulted in reduced stamen numbers as well as the suppression of petal elongation. During the development of the female flower in S. latifolia, the expression of SlSUP is first detectable in whorls 2 and 3 when the normal expression pattern of the B-class flowering genes was already established and persisted in the stamen primordia until the ovule had matured completely. In addition, significant expression of SlSUP was detected in the ovules, suggestive of the involvement of this gene in ovule development. Furthermore, it was revealed that the de-suppression of stamen development by infection of the S. latifolia female flower with Microbotryum violaceum was accompanied by a significant reduction in SlSUP transcript levels in the induced organs. Taken together, these results demonstrate that SlSUP is a female flower-specific gene and suggest that SlSUP has a positive role in the female flower developmental pathways of S. latifolia.


Subject(s)
Flowers/growth & development , Plant Proteins/genetics , Silene/genetics , Transcription Factors/genetics , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/growth & development , Flowers/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Genes, Plant , Genetic Complementation Test , Molecular Sequence Data , Phylogeny , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , RNA, Plant/genetics , Sequence Alignment , Silene/growth & development
17.
FASEB J ; 22(7): 2323-30, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18263698

ABSTRACT

Neoculin (NCL) is a sweet protein that also has taste-modifying activity to convert sourness to sweetness. However, it has been unclear how NCL induces this unique sensation. Here we quantitatively evaluated the pH-dependent acid-induced sweetness of NCL using a cell-based assay system. The human sweet taste receptor, hT1R2-hT1R3, was functionally expressed in HEK293T cells together with G alpha protein. When NCL was applied to the cells under different pH conditions, it activated hT1R2-hT1R3 in a pH-dependent manner as the condition changed from pH 8 to 5. The pH-response sigmoidal curve resembled the imidazole titration curve, suggesting that His residues were involved in the taste-modifying activity. We then constructed an NCL variant in which all His residues were replaced with Ala and found that the variant elicited strong sweetness at neutral pH as well as at acidic pH. Since NCL and the variant elicited weak and strong sweetness at the same neutral pH, respectively, we applied different proportions of NCL-variant mixtures to the cells at this pH. As a result, NCL competitively inhibits the variant-induced receptor activation. All these data suggest that NCL acts as an hT1R2-hT1R3 agonist at acidic pH but functionally changes into its antagonist at neutral pH.


Subject(s)
Curculigo , Hydrogen-Ion Concentration , Plant Proteins/chemistry , Taste/physiology , Aspartame , Humans , Sweetening Agents , Taste Buds/physiology
18.
Plant Cell Physiol ; 48(10): 1450-61, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17720717

ABSTRACT

Sexual dimorphism is controlled by genes on the Y chromosome in the dioecious plant Silene latifolia. K034 is the first mutant with female flowers and asexual flowers in one individual. Its stamens are suppressed completely, and its gynoecium exhibits two suppression patterns. One gynoecium resembles a thin rod, as in wild-type males (asexual flower); the other is imperfectly suppressed, having 1-3 carpels (female-like flower). The ratio of these patterns was 9 : 1. To exclude the possibility of chimerism in K034, we crossed a female-like flower of K034 with a wild-type male. Progeny obtained from this crossing had asexual and female-like flowers in one individual. This two-flower-type phenotype was inherited without separating. To examine the identity of flower organs in K034, we analyzed the development of asexual and female-like flowers using scanning electron microscopy and in situ hybridization with SLM1 and SLM2 (orthologs of AGAMOUS and PISTILLATA, respectively) as probes. Mitotic spreads of root tip chromosomes from hairy root cultures showed that K034 had 25 chromosomes. Fluorescent in situ hybridization analysis, using a subtelomeric repetitive sequence (KpnI subfamily) as a probe, indicated that K034 possessed two X chromosomes and one Y chromosome (Y(d)), of which Y(d) had been rearranged to lose the pseudoautosomal region (PAR). PCR analysis using Y-specific sequence-tagged site (STS) markers clarified that Y(d) of K034 had two other deletions in gynoecium-suppressing and stamen-promoting regions. It is reasonable to suggest that these sex chromosomal abnormalities resulted in two abnormal sexual phenotypes: the asexual and imperfect female (female-like) flowers in K034.


Subject(s)
Flowers/anatomy & histology , Gene Expression Regulation, Plant , Mutation , Plant Proteins/genetics , Silene/genetics , Flowers/genetics , Reproduction
19.
Biochem Biophys Res Commun ; 360(2): 407-11, 2007 Aug 24.
Article in English | MEDLINE | ID: mdl-17592723

ABSTRACT

Miraculin (MCL), a tropical fruit protein, is unique in that it has taste-modifying activity to convert sourness to sweetness, though flat in taste at neutral pH. To obtain a sufficient amount of MCL to examine the mechanism involved in this sensory event at the molecular level, we transformed Aspergillus oryzae by introducing the MCL gene. Transformants were expressed and secreted a sensory-active form of MCL yielding 2 mg/L. Recombinant MCL resembled native MCL in the secondary structure and the taste-modifying activity to generate sweetness at acidic pH. Since the observed pH-sweetness relation seemed to reflect the imidazole titration curve, suggesting that histidine residues might be involved in the taste-modifying activity. H30A and H30,60A mutants were generated using the A. oryzae-mediated expression system. Both mutants found to have lost the taste-modifying activity. The result suggests that the histidine-30 residue is important for the taste-modifying activity of MCL.


Subject(s)
Aspergillus oryzae/metabolism , Glycoproteins/biosynthesis , Glycoproteins/chemistry , Sweetening Agents/chemistry , Sweetening Agents/metabolism , Aspergillus oryzae/genetics , Dose-Response Relationship, Drug , Glycoproteins/administration & dosage , Humans , Hydrogen-Ion Concentration , Protein Engineering/methods , Recombinant Proteins/administration & dosage , Recombinant Proteins/biosynthesis , Sweetening Agents/administration & dosage , Taste/drug effects
20.
Biochem Biophys Res Commun ; 358(2): 585-9, 2007 Jun 29.
Article in English | MEDLINE | ID: mdl-17499612

ABSTRACT

This study examines taste reception of neoculin, a Curculigo latifolia sweet protein with taste-modifying activity which converts sourness to sweetness. Neoculin tastes sweet to humans, but not to mice, and is received by the human sweet taste receptor hT1R2-hT1R3. In the present study with calcium imaging analysis of HEK cells expressing human and mouse T1Rs, we demonstrated that hT1R3 is required for the reception of neoculin. Further experiments using human/mouse chimeric T1R3s revealed that the extracellular amino terminal domain (ATD) of hT1R3 is essential for the reception of neoculin. Although T1R2-T1R3 is known to have multiple potential ligand-binding sites to receive a wide variety of sweeteners, the present study is apparently the first to identify the ATD of hT1R3 as a new sweetener-binding region.


Subject(s)
Kidney/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Amino Acid Sequence , Binding Sites , Cell Line , Humans , Molecular Sequence Data , Protein Binding , Sweetening Agents/chemistry , Sweetening Agents/metabolism
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