The diversity in sensitivity of TRPA1 and TRPV1 of various animals to polyphenols.

The perception of tastes is sensed by the receptors that stimulate sensory cells. We previously reported that TRPA1 and TRPV1 channels expressed in the oral cavity of mammals, are activated by the auto-oxidized product of epigallocatechin gallate (oxiEGCG), a major astringent catechin in green tea. Here, we investigated and compared the sensitivity of TRPA1 and TRPV1 from various animals to astringent polyphenols. We selected three polyphenols, oxiEGCG, tannic acid and myricetin. HEK293T cells expressing TRPA1 or TRPV1 from mammal, bird, reptile, amphibian, and fish, were analyzed for their activation by the Ca2+-imaging. We found the apparent diversity in the polyphenol-sensitivity among various animals. Mammalian TRPs showed relatively higher sensitivity to polyphenols, and especially, human TRPA1 and TRPV1 could be activated by all of three polyphenols at 20 µM. Reptile TRP channels, however, were insensitive to any polyphenols examined. Moreover, the polyphenol-sensitivity of zebrafish TRPA1 and TRPV1 was quite different from that of medaka TRP channels. Since many polyphenols are present in plants and the sensing of polyphenols using TRP channels in the oral cavity might cause astringent taste, the observed diversity of the polyphenol-sensitivity of TRP channels might be involved in the divergence in the food habit of various animals.

Chemical sensing properties of Takifugu TRPA1.

Transient receptor potential ankyrin 1 (TRPA1) is one of the main sensors for noxious stimuli in animals. Recent studies on the cloning and characterization of TRPA1 channels from several organisms showed the functional diversity of TRPA1 in sensing chemicals and temperature. Nociceptive receptors have been suggested to play important roles in adaptation to the environment by and survival strategies of animals; therefore, the sensitivity of various vertebrate TRPA1s needs to be examined in more detail. Here, we focused on fish TRPA1s and investigated the chemical sensing properties of pufferfish (Takifugu) TRPA1 (pfTRPA1). We determined how mammalian TRPA1 ligands activated pfTRPA1 using a Ca-imaging technique. The results obtained indicated that the sensitivity of pfTRPA1 to known TRPA1 ligands was lower than that of mammalian TRPA1s, except for the response ability to allyl isothiocyanate. We also investigated the effects of tannic acid, a type of polyphenol, by measuring ionic currents in Xenopus oocytes in a two-electrode voltage clamp. Although mouse TRPA1 was inhibited by tannic acid, pfTRPA1 channels were enhanced by the treatment with tannic acid. Taken together, these results suggest that pfTRPA1 is not a simple sensor with a lower sensitivity to chemical stimulation, but is actually a specialized sensor with unique properties.

Green tea polyphenol epigallocatechin gallate activates TRPA1 in an intestinal enteroendocrine cell line, STC-1.

A characteristic astringent taste is elicited by polyphenols. Among the polyphenols, catechins and their polymers are the most abundant polyphenols in wine and tea. A typical green tea polyphenol is epigallocatechin gallate (EGCG). Currently, the mechanism underlying the sensation of astringent taste is not well understood. We observed by calcium imaging that the mouse intestinal endocrine cell line STC-1 responds to the astringent compound, EGCG. Among major catechins of green tea, EGCG was most effective at eliciting a response in this cell line. This cellular response was not observed in HEK293T or 3T3 cells. Further analyses demonstrated that the 67-kDa laminin receptor, a known EGCG receptor, is not directly involved. The Ca(2+) response to EGCG in STC-1 cells was decreased by inhibitors of the transient receptor potential A1 (TRPA1) channel. HEK293T cells transfected with the mouse TRPA1 (mTRPA1) cDNA showed a Ca(2+) response upon application of EGCG, and their response properties were similar to those observed in STC-1 cells. These results indicate that an astringent compound, EGCG, activates the mTRPA1 in intestinal STC-1 cells. TRPA1 might play an important role in the astringency taste on the tongue.

Variants of the RELA gene are associated with schizophrenia and their startle responses.

The pathogenesis of schizophrenia is thought to involve aberrant immune and inflammatory responses. Nuclear factor kappa B (NF-κB) has important roles in the immune and inflammatory responses. The v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) gene encodes the major component of the NF-κB complex. We genotyped four single-nucleotide polymorphisms (SNPs) in the RELA gene and performed a gene-based association analysis using 1224 patients with schizophrenia and 1663 controls. We found significant associations of three SNPs (rs11820062: p=0.00011, rs2306365: p=0.0031, and rs7119750: p=0.0080) with schizophrenia and stronger evidence for association in a multi-marker sliding window haplotype analysis (the lowest p=0.00006). The association between this gene and schizophrenia was evident in male subjects but not in female subjects, when separately analyzed by gender. In silico genotype-gene expression analysis using web database and the WGAViewer software revealed that these three schizophrenia-associated SNPs might be related to RELA mRNA expression in immortalized B-lymphocytes. In silico analysis also suggested the putative promoter SNP, rs11820062, might disrupt the consensus transcription factor binding sequence of the androgen receptor. The impact of four RELA polymorphisms on pre-pulse inhibition (PPI) was investigated in 53 patients with schizophrenia. We provided evidence that at risk genotypes of three SNPs were associated with deficits in PPI; however, there was no effect of the one non-risk SNP on PPI. These findings suggest that variants of the RELA gene are associated with risk for schizophrenia and PPI deficits in a Japanese population.

Establishment of clonal cell lines of taste buds from a p53(-/-) mouse tongue.

A taste bud is a sensory organ and consists of 50-100 spindle-shaped cells. The cells function as taste acceptors. They have characteristics of both epithelial and neuronal cells. A taste bud contains four types of cells, type I, type II, type III cells, and basal cells. Taste buds were isolated from a tongue of a p53-deficient mouse at day 12, and 11 clonal taste bud (TBD) cell lines were established. In immunochemical analysis, all cell lines expressed cytokeratin 18, gustducin, T1R3, and neural cellular adhesion molecule, but not GLAST. In RT-PCR analysis, shh was not expressed in any of the cell lines. Further analysis with RT-PCR was conducted on four cell lines. They expressed G protein-coupled taste receptors; T1R3, T2R8 for sweet, bitter, umami. And they also expressed α-ENaC for salty taste. While, a candidate for sour receptor HCN4 was expressed in TBD-a1 and TBD-a7 lines. And another candidate for sour receptor PKD1L3 was slightly expressed in TBD-a1 and TBD-c1.

Possible association between the pituitary adenylate cyclase-activating polypeptide (PACAP) gene and major depressive disorder.

Pituitary adenylate cyclase-activating polypeptide (PACAP, ADCYAP1: adenylate cyclase-activating polypeptide 1) is a neuropeptide with neurotransmission modulating activity. The associations of the PACAP gene with schizophrenia and hippocampal volume have been reported. We recently reported depression-like behavior in the forced swimming test in PACAP deficient mice. Here we examined a possible association between the PACAP gene and major depressive disorder (MDD) in 637 patients and 967 controls and found that a genetic variant in the gene was associated with MDD. The present results suggest that PACAP signaling might contribute to the pathogenesis of MDD.

Antipsychotic medication and cognitive function in schizophrenia.

Antipsychotic polypharmacy and excessive dosing still prevail worldwide in the treatment of schizophrenia, while their possible association with cognitive function has not well been examined. We examined whether the "non-standard" use of antipsychotics (defined as antipsychotic polypharmacy or dosage >1,000 mg/day of chlorpromazine equivalents) is associated with cognitive function. Furthermore, we compared cognitive function between patients taking only atypical antipsychotics and those taking only conventionals. Neurocognitive functions were assessed in 67 patients with chronic schizophrenia and 92 controls using the Wechsler Memory Scale-Revised (WMS-R), the Wechsler Adult Intelligence Scale-Revised (WAIS-R), the Wisconsin Card Sorting Test (WCST), and the Advanced Trail Making Test (ATMT). Patients showed markedly poorer performance than controls on all these tests. Patients on non-standard antipsychotic medication demonstrated poorer performance than those on standard medication on visual memory, delayed recall, performance IQ, and executive function. Patients taking atypical antipsychotics showed better performance than those taking conventionals on visual memory, delayed recall, and executive function. Clinical characteristics such as duration of medication, number of hospitalizations, and concomitant antiparkinsonian drugs were different between the treatment groups (both dichotomies of standard/non-standard and conventional/atypical). These results provide evidence for an association between antipsychotic medication and cognitive function. This association between antipsychotic medication and cognitive function may be due to differential illness severity (e.g., non-standard treatment for severely ill patients who have severe cognitive impairment). Alternatively, poorer cognitive function may be due in part to polypharmacy or excessive dosing. Further investigations are required to draw any conclusions.

Auditory imagery mismatch negativity elicited in musicians.

A mismatch between auditory sensation and expectant imagery of syllables elicited a possible equivalent of mismatch negativity in a previous study. The purpose of this study was to verify whether auditory imagery from musical notation could also mediate such imagery-based mismatch negativity. Neuromagnetic recording was obtained from eight musicians, who were instructed to identify unpredictably occurring pitch mismatches between a random tone sequence and a visually presented musical score. The difference between incongruent and congruent responses showed a magnetic distribution consistent with two frontal-negative current dipoles bilaterally located in the vicinity of Heschl's gyrus, peaking at approximately 150 ms in latency. This imagery-based mismatch negativity may represent an early neural process of deviance detection between the sensory input and expectant imagery.

Audiovisual phonological mismatch produces early negativity in auditory cortex.

During silent reading, visual information provided by letters is converted to auditory information in the mind. The purpose of this study was to identify the primary locus for auditory verbal imagery in the brain. Neuromagnetic recording was obtained from 10 right-handed study participants, who were instructed to identify infrequently occurring phonological mismatches between a random-ordered sequence of syllable sounds and a visually presented syllabogram sequence. The activity difference in early latency, calculated by subtracting the averaged responses to matched syllables from the averaged responses to mismatched syllables, showed a spatiotemporal profile strikingly similar to that of mismatch negativity. Auditory imagery of forthcoming verbal sounds may establish a memory trace as a template for imagery-based mismatch negativity generation in the auditory cortex.

Functional expression of single-chain heterodimeric G-protein-coupled receptors for adenosine and dopamine.

The direct homo- and heteromeric association between G-protein-coupled receptors (GPCRs), adenosine A2A receptor (A(2A)R) and dopamine D2 receptor (D2R), occurs although little is known about the selectivity of their formation (A(2A)R/A(2A)R vs. A(2A)R/D2R). In order to stimulate the heteromerization of A(2A)R and D2R, we have designed a single-polypeptide-chain heterodimeric A(2A)R/D2R complex by fusing the C-terminus of the A(2A)R via transmembrane (TM) of a type II TM protein with the N-terminus of D2R in tandem. This was successfully expressed on the cell surface as a full-length protein with specific binding to the respective ligands and functional coupling to G-proteins comparable to wild-type receptors, suggesting the possible creation of physiologically relevant heteromeric A(2A)R/D2R. This expression system would be useful to exclusively clarify the properties of heteromeric GPCRs irrespective of homomeric receptors.

Characterization of bitter taste responses of intestinal STC-1 cells.

Cellular responses of STC-1 cells to two bitter tastants (denatonium and caffeine) were investigated using a calcium-imaging technique and compared with the response to bombesin. Caffeine is known to stimulate taste receptor cells, but the properties of its signaling have not been well studied. STC-1 cells responded to all three molecules in a dose-dependent manner, and when a reverse transcriptase-polymerase chain reaction (RT-PCR) for denatonium receptor was performed, the product of predicted size was detected in STC-1 cells. Furthermore, all three signaling pathways were blocked by a phospholipase C (PLC) inhibitor, demonstrating the essential involvement of PLC in cellular responses. To study the regulatory system of G protein signaling in STC-1 cells, we searched G protein-coupled receptor kinases (GRKs) by the degenerate-primer PCR method and found that GRK2 is expressed. We also demonstrated that three GRKs (GRK2, GRK3 and GRK5) are differentially distributed in the circumvallate papilla while only GRK2 is present in taste bud cells. Finally, we overexpressed GRK2 in SCT-1 cells and found that bombesin-induced response was strongly inhibited by GRK2 but denatonium-activated signaling was not affected. In the case of caffeine, response was decreased by expression of GRK2 only when cells were activated by 1 mM caffeine. Thus, we showed that STC-1 cells emerge as a cell model for studying the molecular mechanism of bitter taste signaling, and could indicate properties of caffeine-induced signaling in comparison with other signaling.

Identification of adenosine receptor subtypes expressed in the human endothelial-like ECV304 cells.

Adenosine, acting through its receptors, is a potent endogenous regulator of endothelial cells. The cultured endothelial cells expressing adenosine receptors are thus important for elucidation of molecular mechanism of adenosine functions in these cell systems. Therefore, identification of adenosine receptors in the human ECV304 cell line derived from a human umbilical vein endothelial cell culture was performed. RT-PCR experiments revealed that ECV304 cells express mRNAs for A1 and A2B adenosine receptors. The expression of mRNA for A2A adenosine receptor was not in a significant level and that for A3 adenosine receptor was not detected. The binding study of ECV304 cell membrane fractions using various radiolabeled ligands for adenosine receptors indicated the presence of A1 adenosine receptors 245 fmol/mg of membrane proteins, but the specific binding for A2A and for A3 adenosine receptors were found to be negligible. The functional expression of A1 and A2B adenosine receptors in ECV304 cells was detected by assays for adenosine-3',5'-cyclic monophosphate and for extracellular signal-regulated kinase, but that of A2A adenosine receptors was not confirmed under the assay conditions employed. In conclusion, this study presented evidence for functional A1 and A2B adenosine receptors in human endothelial-like ECV304 cells, indicating that ECV304 cells can be a good model for the study of adenosine receptors, especially for A2B adenosine receptor, in endothelial cells.

Oligomerization of adenosine A2A and dopamine D2 receptors in living cells.

We investigated whether oligomerization of adenosine A(2A) receptor (A(2A)R) and dopamine D(2) receptor (D(2)R) exists in living cells using modified bioluminescence resonance energy transfer (BRET(2)) technology. Fusion of these receptors to a donor, Renilla luciferase (Rluc), and to an acceptor, modified green fluorescent protein (GFP(2)), did not affect the ligand binding affinity, subcellular distribution, and coimmunoprecipitation of the receptors. BRET was detected not only between Myc-D(2)R-Rluc and A(2A)R-GFP(2) but also between HA-tagged A(2A)R-Rluc and A(2A)R-GFP(2). These results indicate A(2A)R, either homomeric or heteromeric with D(2)R, exists as an oligomer in living cells.

Agonist-promoted heteromeric oligomerization between adenosine A(1) and P2Y(1) receptors in living cells.

We have explored the process of oligomerization of G protein-coupled purinergic receptors, adenosine A(1) receptor (A(1)R) and P2Y(1) receptor (P2Y(1)R), in intact HEK293T cells by means of modified bioluminescence resonance energy transfer technology (BRET(2)) that offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared to traditional BRET. This approach identified both constitutive and agonist-promoted heteromeric oligomerization between Myc-tagged P2Y(1)R fused to a donor, Renilla luciferase (Myc-P2Y(1)R-Rluc) and HA-tagged A(1)R fused to an acceptor, a different form of green fluorescent protein (HA-A(1)R-GFP(2)). The BRET(2) signal increased in a time-dependent manner in the cells expressing HA-A(1)R-GFP(2)/Myc-P2Y(1)R-Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y(1)R antagonist MRS2179. A high degree of HA-A(1)R-GFP(2) and Myc-P2Y(1)R-Rluc co-localization in the co-transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A(1)R and P2Y(1)R can form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous activation of both receptors.

Alternative splicing of RGS8 gene determines inhibitory function of receptor type-specific Gq signaling.

The regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling. RGS8 is a brain-specific RGS protein of 180 aa. Here we identified a short isoform of RGS8, RGS8S, that arises by alternative splicing. RGS8S cDNA encodes a N terminus of 7 aa instead of amino acids 1-9 of RGS8 and 10-180 of RGS8. The subcellular distribution of RGS8 and RGS8S did not differ significantly in transfected cells. RGS8S accelerated, not as efficiently as RGS8, the turning on and off of Gi/o-mediated modulation of G protein-gated inwardly rectifying K(+) channels in Xenopus oocytes. We next examined the effects of RGS8 and RGS8S on Gq-mediated signaling. RGS8 decreased the amplitude of the response upon activation of m1 muscarinic or substance P receptors, but did not remarkably inhibit signaling from m3 muscarinic receptors. In contrast, RGS8S showed much less inhibition of the response of either of these Gq-coupled receptors. By quantitative analysis of the inhibitory effect and the protein expression level, we confirmed that the difference of inhibitory effect is caused by both the qualitative difference between RGS8 and RGS8S and the quantitative difference of the protein expression level. We also confirmed that the receptor-type specificity of inhibition is not caused by the difference of the expression level of the receptors. In summary, we showed that 9 aa in the N terminus of RGS8 contribute to the function to inhibit Gq-coupled signaling in a receptor type-specific manner and that the regulatory function of RGS8S is especially diminished on Gq-coupled responses.

Interictal schizophrenia-like psychosis in a patient with double cortex syndrome.

The authors report a female patient with sporadic double cortex syndrome who manifested recurrent interictal schizophrenia-like psychoses. She had no mutations in the doublecortin gene but a pericentric inversion of chromosome 9. Neurodevelopmental disturbances and seizures may be associated with her mental dysfunction.

A simple method of detecting K-ras point mutations in stool samples for colorectal cancer screening using one-step polymerase chain reaction/restriction fragment length polymorphism analysis.

BACKGROUND: We examined a technique for detecting point mutations of K-ras codon 12 in stool samples using one-step polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis, in order to determine whether it could be used to screen for colorectal cancer. METHODS: DNA was extracted from 200-mg stool specimens of 5 healthy controls and 31 colorectal cancer patients. A 107-base-pair fragment of exon 1 of K-ras was amplified by PCR using mismatched primers. PCR products were digested with Bst NI and analyzed by gel electrophoresis followed by silver staining. Specificity of one-step PCR/RFLP was examined by using synthetic oligonucleotides. The detection limit of K-ras codon 12 mutations was determined by using SW480 and HT29 cells. RESULTS: The K-ras gene was successfully amplified from all healthy controls and colorectal cancer patients studied. Mutations of K-ras codon 12 were not detected in any of the healthy controls, but were identified in 13 (41.9%) of the 31 patients with colorectal cancer. Mutations were detectable in all six synthetic mutant DNAs, while none were detected among the wild type. The detection limit of this method was > or = 0.1%. CONCLUSIONS: PCR/RFLP analysis could be used in mass screening for colorectal cancer, because it is highly specific, has a low detection limit, and is simpler than conventional methods for detecting genetic abnormalities.

Lactoferrin in whole gut lavage fluid as a marker for disease activity in inflammatory bowel disease: comparison with other neutrophil-derived proteins.

OBJECTIVES: We investigated which neutrophil-derived proteins in whole gut lavage fluid (WGLF) most accurately reflect disease activity in inflammatory bowel disease. METHODS: WGLF was obtained from patients undergoing whole gut lavage as a bowel preparation for colonoscopy. Twenty-seven patients with ulcerative colitis (UC), 23 patients with Crohn's disease (CD), and 35 control subjects were examined. The concentrations of lactoferrin, polymorphonuclear neutrophil elastase (PMN-E), myeloperoxidase, and lysozyme in WGLF were measured by ELISA. For the assessment of stability, WGLF samples were stored at 37 degrees C for various periods. RESULTS: In UC, the concentrations of lactoferrin, myeloperoxidase, and lysozyme in WGLF had good correlations with colonoscopic grading. Zero, 12, five, and 10 of 28 samples from active UC patients showed normal concentrations of lactoferrin, PMN-E, myeloperoxidase, and lysozyme, respectively. In CD, the concentrations of lactoferrin and myeloperoxidase had good correlations with the Crohn's disease activity index. Thirteen and seven of 36 samples from inactive CD patients (Crohn's disease activity index < or = 150) showed high concentrations of lactoferrin and myeloperoxidase, respectively. Most of them (11/13, 6/7) were found to have ulceration by colonoscopy or small bowel x-ray. The ratio of the lactoferrin concentration in the WGLF supernatant to that in total WGLF was highest among these proteins in all disease groups and control subjects. Lactoferrin and myeloperoxidase showed good stability in WGLF, whereas PMN-E and lysozyme did not. CONCLUSION: Lactoferrin is the most suitable of these proteins for use as a neutrophil-derived WGLF marker of intestinal inflammation.