Enantiomeric cuparene-type sesquiterpenoids from Bazzania pompeana

Two enantiomeric cuparene-type sesquiterpenoids, (R)-(−)-cuparene (1) and (R)-(−)-δ-cuparenol (2), have been isolated from the liverwort, Bazzania pompeana. The structures and absolute configurations of the two compounds have been determined.     

RS-5645 attenuates inflammatory cytokine storm induced by SARS-CoV-2 spike protein and LPS by modulating pulmonary microbiota

An inflammatory cytokine storm is considered an important cause of death in severely and critically ill COVID-19 patients, however, the relationship between the SARS-CoV-2 spike (S) protein and the host''s inflammatory cytokine storm is not clear. Here, the qPCR results indicated that S protein induced a significantly elevated expression of multiple inflammatory factor mRNAs in peripheral blood mononuclear cells (PBMCs), whereas RS-5645 ((4-(thiophen-3-yl)-1-(p-tolyl)-1H-pyrrol-3-yl)(3,4,5-trimethoxyphenyl)methanone) attenuated the expression of the most inflammatory factor mRNAs. RS-5645 also significantly reduced the cellular ratios of CD45+/IFNγ+, CD3+/IFNγ+, CD11b+/IFNγ+, and CD56+/IFNγ+ in human PBMCs. In addition, RS-5645 effectively inhibited the activation of inflammatory cells and reduced inflammatory damage to lung tissue in mice. Sequencing results of 16S rRNA v3+v4 in mouse alveolar lavage fluid showed that there were 494 OTUs overlapping between the alveolar lavage fluid of mice that underwent S protein+ LPS-combined intervention (M) and RS-5645-treated mice (R), while R manifested 64 unique OTUs and M exhibited 610 unique OTUs. In the alveoli of group R mice, the relative abundances of microorganisms belonging to Porphyromonas, Rothia, Streptococcus, and Neisseria increased significantly, while the relative abundances of microorganisms belonging to Psychrobacter, Shimia, and Sporosarcina were significantly diminished. The results of KEGG analysis indicated that the alveolar microbiota of mice in the R group can increase translation and reduce the activity of amino acid metabolism pathways. COG analysis results indicated that the abundance of proteins involved in ribosomal structure and biogenesis related to metabolism was augmented in the alveolar microbiota of the mice in the R group, while the abundance of proteins involved in secondary metabolite biosynthesis was significantly reduced. Therefore, our research results showed that RS-5645 attenuated pulmonary inflammatory cell infiltration and the inflammatory storm induced by the S protein and LPS by modulating the pulmonary microbiota.     

Inhaltsstoffe von Pyxine coccifera

The red pigment pyxiferin from Pyxine coccifera (Fée) Nyl. is identical with chiodectonic acid, a naphthoquinone derivative. The lichen further contains 3β-hydroxy-25-acetoxy-20(S),24(R)-epoxy-dammarane, 25-acetoxy-20(S),24(R)-epoxy-3-oxo-dammarane, methyl pyxinate, atranorin, and chloroatranorin.     

Identification of novel genes for bitter taste receptors in sheep (Ovis aries)

Genetic studies on taste sensitivity, and bitter taste receptors (T2R) in particular, are an essential tool to understand ingestive behavior and its relation to variations of nutritional status occurring in ruminants. In the present study, we conducted a data-mining search to identify T2R candidates in sheep by comparison with the described T2R in cattle and using recently available ovine genome. In sheep, we identified eight orthologs of cattle genes: T2R16, T2R10B, T2R12, T2R3, T2R4, T2R67, T2R13 and T2R5. The in silico predicted genes were then confirmed by PCR and DNA sequencing. The sequencing results showed a 99% to 100% similarity with the in silico predicted sequence. Moreover, we address the chromosomal distribution and compare, in homology and phylogenetic terms, the obtained genes with the known T2R in human, mouse, dog, cattle, horse and pig. The eight novel genes identified map either to ovine chromosome 3 or 4. The phylogenetic data suggest a clustering by receptor type rather than by species for some of the receptors. From the species analyzed, we observed a clear proximity between the two ruminant species, sheep and cattle, in contrast with lower similarities obtained for the comparison of sheep with other mammals. Although further studies are needed to identify the complete T2R repertoire in domestic sheep, our data represent a first step for genetic studies on this field.     

Neolignans from Ocotea catharinensis

The trunk bark of Ocotea catharinensis yielded, besides the known bicyclo(3.2.1)octanoid neolignans canellin-C and 5′-methoxycanellin-C, two epimers rel-(1R,4S and 4R,5S,6R,7S,8R)-1-allyl-4,8-dihydroxy-3,5-dimethoxy-7-methyl-6-piperonyl-bicyclo(3.2.1)oct-2-enes and rel-(1R,5S,6R,7S,8R)-1-allyl-3,8-dihydroxy-5-methoxy-7-methyl-6-piperonyl-4-oxobicyclo(3.2.1)oct-2-ene. The hydrobenzofuranoid neolignans are represented by the equally novel (2S,3S,5R)-5-allyl-5,7-dimethoxy-3-methyl-2-piperonyl-2,3,5,6-tetrahydro-6-oxobenzofuran and (2R,3S,3aS)-3a-allyl-5,7-dimethoxy-3-methyl-2-piperonyl-2,3,3a,6-tetrahydro-6-oxobenzofuran.     

Evidence for an anisotropic magnetic interaction between the (bacteriopheophytin) intermediary acceptor and the first quinone acceptor in bacterial photosynthesis

We have investigated electron spin polarization effects occurring in protonated and perdeuterated reaction centers of Rhodospirillum rubrum with electron spin resonance at 9 and 35 GHz (X- and Q-band). As for Rhodopseudomonas sphaeroides strains 2.4.1 and R-26 (Gast, P. and Hoff, A.J. (1979) Biochim. Biophys. Acta 548, 520–535; Gast, P., Mushlin, R.A. and Hoff, A.J. (1982) J. Phys. Chem. 86, 2886–2891), electron spin polarization effects of the prereduced first quinone acceptor Q^?_A in R. rubrum are strongly nonuniform. This nonuniformity is due to an anisotropic magnetic coupling between the intermediary bacteriopheophytin acceptor (I^?) and Q^?_A. It is argued that the anisotropy is too strong to arise solely from an anisotropy in the exchange interaction between I^? and Q^?_A and that dipolar contributions to the magnetic coupling between I^? and Q^?_A are important. The anisotropy in the magnetic coupling for reaction centers of Rps. sphaeroides strains 2.4.1 and R-26 is different from that of R. rubrum wild type. The combination of the 4-fold higher resolution at Q-band and the line narrowing upon deuteration has enabled us to obtain the principal g values and two hyperfine interaction constants of the reduced first quinone acceptor Q^?_A. The principal g values are g_x = 2.0067, g_y = 2.0056 and g_z = 2.0024; the hyperfine constant of the CH₂ group at position 1 is 1.6 G and that of the CH₃ group at position 2 is 2.1 G. These values are close to those found for ubisemiquinone in vitro (Okamura, M.Y., Debus, R.J., Isaacson, R.A. and Feher, G. (1980) Fed. Proc. 39, 1802; Hales, B.J. (1975) J. Am. Chem. Soc. 97, 5993–5997).     

Molecular Genetics of Pre-B Acute Lymphoblastic Leukemia Sister Cell Lines during Disease Progression

For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors (CDKN2A, CDKN2B), expression of fusion genes (ETV6-RUNX1, BCR-ABL1, MEF2D-BCL9) or of genes targeted by point mutations (KRAS A146T, NRAS G12C, PAX5 R38H). MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.     

Antitumor activities and interaction with DNA of oxaliplatin-type platinum complexes with linear or branched alkoxyacetates as leaving groups

Five oxaliplatin-typed platinum complexes containing trans-1R, 2R-diaminocyclohexane chelating platinum cores, characteristic of linear or branched alkoxycarboxylates as leaving groups, were biologically evaluated. These compounds showed higher antitumor activity, lower toxicity in vivo than cisplatin or oxaliplatin. And the results revealed that the antitumor activity and interaction with DNA of these compounds were highly related to the nature of leaving groups. Among these complexes, 5a, cis-(trans-1R, 2R-diaminocyclohexane) bis (2-tert-butoxyacetate) platinum(II), showed the highest antitumor activity and the lowest toxicity.     

Molecular docking of glucosamine-6-phosphate synthase in Rhizopus oryzae

Recent expansion of immunocompromised population has led to significant rise in zygomycosis caused by filamentous fungus Rhizopus oryzae. Due to emergence of fungal resistance and side-effects of antifungal drugs, there is increased demand for novel drug targets. The current study elucidates molecular interactions of peptide drugs with G-6-P synthase (catalyzing the rate-limiting step of fungal cell wall biosynthetic pathway) of R.oryzae by molecular docking studies. The PDB structures of enzyme in R.oryzae are not known which were predicted using I-TASSER server and validated with PROCHECK. Peptide inhibitors, FMDP and ADGP previously used against enzyme of E.coli (PDBid: 1XFF), were used for docking studies of enzyme in R.oryzae by SchrödingerMaestro v9.1. To investigate binding between enzyme and inhibitors, Glide and Induced Fit docking were performed. IFD results of 1XFF with FMDP yielded C1, R73, W74, T76, G99 and D123 as the binding sites. C379 and Q427 appear to be vital for binding of R.oryzae enzymes to inhibitors. The comparison results of IFD scores of enzyme in R.oryzae and E.coli (PDBid: 2BPL) yield appreciable score, hinting at the probable effectiveness of inhibitors FMDP and ADGP against R.oryzae, with ADGP showing an improved enzyme affinity. Moreover, the two copies of gene G-6-P synthase due to extensive fungal gene duplication, in R. oryzae eliminating the problem of drug ineffectiveness could act as a potential antifungal drug target in R. oryzae with the application of peptide ligands.     

An H-2 linked difference in the binding of dexamethasone to murine hepatic cytosol receptor a possible role of endogenous modifier(s)

The binding of dexamethasone to its receptor in hepatic cytosol preparations from pregnant mice of four congenic and recombinant strains, C57BL/10, B10.A, B10.A(2R) and B10.A(5R), which have almost identical genetic backgrounds other than the H-2 complex, on day 12 of gestation was analyzed by plotting the binding of ligand against cytosol concentration. The plots of C57BL/10 and B10.A(5R) mice were straight lines, but those of the strains B10.A and B10.A(2R) were upward concave curves. The curvature probably did not result from denaturation of the receptor, as indicated by the time course of the dexamethasone binding and by the fact that at a lower concentration of the ligand, at which the receptor would be less stable, there was less curvature than at a higher concentration of the ligand. The curvature can be explained by the presence of endogenous modifier(s) using an analogy from enzymology. Mathematical analysis, partial removal of the modifier(s) by gel filtration, and mixing of the cytosols from the two types of strains indicated the presence of an unsaturated amount of a modifier(s) in the cytosol of the B10.A and B10.A(2R) strains, and the presence of a saturated amount in the cytosol of the c57BL/10 and B10.A(5R) strains. Thus, the H-2 complex contains a gene(s) which regulates the level of a modifier(s) in hepatic cytosol which affects the binding of glucocorticoid to its hepatic cytosolic receptor.     

miR-223 contributes to the AGE-promoted apoptosis via down-regulating insulin-like growth factor 1 receptor in osteoblasts

Advanced glycation end products (AGEs) have been confirmed to induce bone quality deterioration in diabetes mellitus (DM), and to associate with abnormal expression of miRNAs in DM patients or in vitro. Recently, miRNAs have been recognized to mediate the onset or progression of DM. In the present study, we investigated the regulation on miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells, with real-time quantitative PCR assay. And then we examined the inhibition of insulin-like growth factor 1 receptor (IGF-1R) expression by miR-223, via targeting of the 3′ UTR of IGF-1R with real-time quantitative PCR, western blotting and luciferase reporter assay. Then we explored the regulation of miR-223 and IGF-1R levels, via the lentivirus-mediated miR-223 inhibition and IGF-1R overexpression in the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It was demonstrated that AGE-BSA treatment with more than 100 μg/ml significantly up-regulated miR-223 level, whereas down-regulated IGF-1R level in MC3T3-E1 cells. And the up-regulated miR-223 down-regulated IGF-1R expression in both mRNA and protein levels, via targeting the 3′ UTR of IGF-1R. Moreover, though the AGE-BSA treatment promoted apoptosis in MC3T3-E1 cells, the IGF-1R overexpression or the miR-223 inhibition significantly attenuated the AGE-BSA-promoted apoptosis in MC3T3-E1 cells. In summary, our study recognized the promotion of miR-223 level by AGE-BSA treatment in osteoblast-like MC3T3-E1 cells. The promoted miR-223 targeted IGF-1R and mediated the AGE-BSA-induced apoptosis in MC3T3-E1 cells. It implies that miR-223 might be an effective therapeutic target to antagonize the AGE-induced damage to osteoblasts in DM.     

Asymmetric hybridization in Rhododendron agastum: a hybrid taxon comprising mainly F1s in Yunnan, China

Background and Aims

Rhododendron (Ericaceae) is a large woody genus in which hybridization is thought to play an important role in evolution and speciation, particularly in the Sino-Himalaya region where many interfertile species often occur sympatrically. Rhododendron agastum, a putative hybrid species, occurs in China, western Yunnan Province, in mixed populations with R. irroratum and R. delavayi.

Methods

Material of these taxa from two sites 400 km apart (ZhuJianYuan, ZJY and HuaDianBa, HDB) was examined using cpDNA and internal transcribed spacer (ITS) sequences, and amplified fragment length polymorphism (AFLP) loci, to test the possibility that R. agastum was in fact a hybrid between two of the other species. Chloroplast trnL-F and trnS-trnG sequences together distinguished R. irroratum, R. delavayi and some material of R. decorum, which is also considered a putative parent of R. agastum.

Key Results

All 14 R. agastum plants from the HDB site had the delavayi cpDNA haplotype, whereas at the ZJY site 17 R. agastum plants had this haplotype and four had the R. irroratum haplotype. R. irroratum and R. delavayi are distinguished by five unequivocal point mutations in their ITS sequences; every R. agastum accession had an additive pattern (double peaks) at each of these sites. Data from AFLP loci were acquired for between ten and 21 plants of each taxon from each site, and were analysed using a Bayesian approach implemented by the program NewHybrids. The program confirmed the identity of all accessions of R. delavayi, and all R. irroratum except one, which was probably a backcross. All R. agastum from HDB and 19 of 21 from ZJY were classified as F₁ hybrids; the other two could not be assigned a class.

Conclusions

Rhododendron agastum represents populations of hybrids between R. irroratum and R. delavayi, which comprise mostly or only F₁s, at the two sites examined. The sites differ in that at HDB there was no detected variation in cpDNA type or hybrid class, whereas at ZJY there was variation in both.     

Synthesis and in vitro antitumor activity of novel iridium(III) complexes with enantiopure C2-symmetrical vicinal diamine ligands

Four novel iridium(III) complexes with enantiopure C₂-symmetrical vicinal diamine ligands were designed, synthesized, and characterized by FT-IR, NMR, and MS. The cytotoxicities of all of the complexes against the human solid tumor cell lines A2780, A549, KB, and MDA-MB-231 were evaluated. Both R,R-configured complexes (R,R)-5a and (R,R)-5b exhibited more potent or similar activity compared with oxaliplatin, whereas their corresponding (S,S)-isomers (S,S)-5a and (S,S)-5b were found to be mostly inactive. As indicated by the activation of caspase-3, the cleavage of PARP, and the upregulation of p53, the preliminary mechanism studies revealed that the mode of cell death initiated by (R,R)-5a in A2780 cells was predominantly p53-mediated apoptosis. In addition, the structure of (R,R)-5a was unambiguously confirmed through single crystal X-ray structure determination.     

Distribution and diversity of rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in Northwest China

A total of 155 nodule isolates that originated from seven sites in Northwest China were characterized by PCR-RFLP of the 16S rRNA gene and sequence analysis of multiple core genes (16S rRNA, recA, atpD, and glnII) in order to investigate the diversity and biogeography of Glycine soja-nodulating rhizobia. Among the isolates, 80 were Ensifer fredii, 19 were Ensifer morelense, 49 were Rhizobium radiobacter, and 7 were putative novel Rhizobium species. The phylogenies of E. fredii and E. morelense isolates in a concatenate tree (assembly of all housekeeping genes) were generally consistent with those in individual gene trees. However, incongruence was found in the phylogenies of the different genes of Rhizobium isolates, indicating that lateral transfer or recombination possibly occurred in these gene loci. Despite their species identity, all the isolates in this study formed a single lineage related to E. fredii in nodAand nifH gene phylogenies, which also indicated that the symbiotic genes were laterally transferred between different species. Biogeographic patterns were found at the species and strain genomic type levels, as revealed by BOXA1R fingerprinting, demonstrating that the evolution of rhizobial populations in different geographic locations was related to soil types, altitude and spatial effects. This study is the first to report that E. morelense, R. radiobacter, and Rhizobium sp. are microsymbionts of G. soja, as well as showing that the diversity of G. soja rhizobia is enhanced and new rhizobia have evolved in Northwest China.     

The glucagon-like peptide 2 receptor is expressed in enteric neurons and not in the epithelium of the intestine

Glucagon-like peptide 2 (GLP-2) is a potent intestinotrophic growth factor with therapeutic potential in the treatment of intestinal deficiencies. It has recently been approved for the treatment of short bowel syndrome. The effects of GLP-2 are mediated by specific binding of the hormone to the GLP-2 receptor (GLP-2R) which was cloned in 1999. However, consensus about the exact receptor localization in the intestine has never been established.By physical, chemical and enzymatic tissue fragmentation, we were able to divide rat jejunum into different compartments consisting of: (1) epithelium alone, (2) mucosa with lamina propria and epithelium, (3) the external muscle coat including myenteric plexus, (4) a compartment enriched for the myenteric plexus and (5) intestine without epithelium. Expression of Glp2r; chromogranin A; tubulin, beta 3; actin, gamma 2, smooth muscle, enteric and glial fibrillary acidic protein in these isolated tissue fractions was quantified with qRT-PCR. Expression of the Glp2r was confined to compartments containing enteric neurons and receptor expression was absent in the epithelium.Our findings provide evidence for the expression of the GLP-2R in intestinal compartments rich in enteric neurons and, importantly they exclude significant expression in the epithelium of rat jejunal mucosa.     

Identification and Characterization of a Mycobacterial (2R,3R)-2,3-Butanediol Dehydrogenase

Bacterial strain B-009, capable of using racemic 1,2-propanediol (PD), was identified as a rapid-growing member of the genus Mycobacterium. The strain is phylogenetically related to M. gilvum, but has slightly different physiological characteristics. An NAD⁺-dependent enantioselective alcohol dehydrogenase, which acts on R-PD, was purified from the strain. The enzyme was a homodimer of a peptide coded by a 1047-bp gene (mbd1). A highly conserved sequence for medium-chain dehydrogenase/reductases with a preference for secondary alcohols was found in the gene. Hydroxyacetone was produced from R-PD by an enzymatic reaction, indicating that position 2 of the substrate was oxidized. The enzyme activity was highest for (2R,3R)-2,3-butanediol (R,R-BD), enabling the enzyme to be identified as (2R,3R)-2,3-butanediol dehydrogenase (R,R-BD-DH). A homology search revealed M. gilvum, M. vanbaalenii, and M. semegmatis to have ORFs similar to mbd1, suggesting the widespread distribution of genes encoding R,R-BD-DH among mycobacterial strains.     

Acid-labile handles for Fmoc solid-phase synthesis of peptide N-alkylamides

Summary By analogy to established methodology for the preparation of C-terminal peptide amides by 9-fluorenylmethyl-oxycarbonyl (Fmoc) chemistry, in conjunction with the acidolyzable 5-(4-Fmoc-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL, 1) handle, the present paper reports on 5-(4-(N-Fmoc-N-alkyl)aminomethyl-3,5-dimethoxyphenoxy)valeric acid [(R)PAL, 2] handles that can be used for synthesis of peptide N-alkylamides. The key step in the preparation of these handles was the NaBH₃CN-mediated reductive amination (60 to 85% yields; R=CH₃, CH₃CH₂, C₆H₅CH₂CH₂, 4-NO₂C₆H₅) of 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid (4), an aldehyde precursor to PAL. The (R)PAL handles (2a and b) were applied to the preparation of LHRH analogues. After anchoring of handles to PEG-PS supports, peptide chain assemblies were carried out, and treatments with TFA-thioanisolephenol-1,2-ethanedithiol (87:5:5:3) for 90 min at 25 °C, followed by aqueous workups, provided the expected products in excellent yields and purities as supported by HPLC and mass spectrometric characterization.Taken in part from the Ph.D. Thesis of M.F. Songster, University of Minnesota, 1996. Preliminary reports of this work were presented at the 14th American Peptide Symposium, Columbus, OH, June 18–23, 1995 (poster P047), and at the Fourth International Symposium on Solid Phase Synthesis and Combinatorial Chemical Libraries, Edinburgh, Scotland, UK, September 12–16, 1995.     

Enzymatic synthesis of an orlistat intermediate using a mutant short-chain dehydrogenase from Novosphingobium aromaticivorans

Methyl (R)-3-hydroxytetradeconoate ((R)-MHOT) is a crucial chiral intermediate for the chemical synthesis of the anti-obesity drug, orlistat. Here, (R)-MHOT was prepared from methyl 3-oxotetradecanoate (MOT) using a mutant of the short-chain dehydrogenase/reductase (SDR) from Novosphingobium aromaticivorans (NaSDR). Mutant NaSDR-G145A/I199L had a 3.23 times greater k_cat value than that of wild type toward MOT. The conditions for the expression of recombinant NaSDR-G145A/I199L were further investigated and obtained cells were used for gram-scale preparation of (R)-MHOT with 50 g/L of MOT. The target product was extracted and confirmed by gas chromatography; the enantiomeric excess value of (R)-MHOT was 99.0 %. Molecular docking analysis was used to reveal the molecular basis of the enhanced catalytic activity of NaSDR-G145A/I199L; NaSDR-G145A/I199L presented a more effective docking posture than NaSDR. This is the first reported use of SDR for preparing (R)-MHOT via the reduction of MOT. Our study provides a foundation for greener preparation of (R)-MHOT.     

Key Amino Acid Residues Involved in Multi-Point Binding Interactions between Brazzein, a Sweet Protein, and the T1R2-T1R3 Human Sweet Receptor

The sweet protein brazzein [recombinant protein with sequence identical with the native protein lacking the N-terminal pyroglutamate (the numbering system used has Asp2 as the N-terminal residue)] activates the human sweet receptor, a heterodimeric G-protein-coupled receptor composed of subunits Taste type 1 Receptor 2 (T1R2) and Taste type 1 Receptor 3 (T1R3). In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: site 1 (Loop43), site 2 (N- and C-termini and adjacent Glu36, Loop33), and site 3 (Loop9-19). Basic residues in site 1 and acidic residues in site 2 were essential for positive responses from each assay. Mutation of Y39A (site 1) greatly reduced positive responses. A bulky side chain at position 54 (site 2), rather than a side chain with hydrogen-bonding potential, was required for positive responses, as was the presence of the native disulfide bond in Loop9-19 (site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus flytrap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in brazzein response. The exception, hT1R2 (human T1R2 subunit of the sweet receptor):R217A/hT1R3 (human T1R3 subunit of the sweet receptor), which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site is involved in subunit-subunit interaction rather than in direct brazzein binding. Results from this study support a multi-point interaction between brazzein and the sweet receptor by some mechanism other than the proposed wedge models.