Combinatorial clustering of distinct DNA motifs directs synergistic binding of Caenorhabditis elegans dosage compensation complex to X chromosomes.

Organisms that count X-chromosome number to determine sex utilize dosage compensation mechanisms to balance X-gene expression between sexes. Typically, a regulatory complex is recruited to X chromosomes of one sex to modulate gene expression. A major challenge is to determine the mechanisms that target regulatory complexes specifically to X. Here, we identify critical X-sequence motifs in Caenorhabditis elegans that act synergistically in hermaphrodites to direct X-specific recruitment of the dosage compensation complex (DCC), a condensin complex. We find two DNA motifs that collaborate with a previously defined 12-bp motif called MEX (motif enriched on X) to mediate binding: MEX II, a 26-bp X-enriched motif and Motif C, a 9-bp motif that lacks X enrichment. Inserting both MEX and MEX II into a new location on X creates a DCC binding site equivalent to an endogenous recruitment site, but inserting only MEX or MEX II alone does not. Moreover, mutating MEX, MEX II, or Motif C in endogenous recruitment sites with multiple different motifs dramatically reduces DCC binding in vivo to nearly the same extent as mutating all motifs. Changing the orientation or spacing of motifs also reduces DCC binding. Hence, synergy in DCC binding via combinatorial clustering of motifs triggers DCC assembly specifically on X chromosomes. Using an in vitro DNA binding assay, we refine the features of motifs and flanking sequences that are critical for DCC binding. Our work reveals general principles by which regulatory complexes can be recruited across an entire chromosome to control its gene expression.

Functional variants of DOG1 control seed chilling responses and variation in seasonal life-history strategies in Arabidopsis thaliana.

The seasonal timing of seed germination determines a plant's realized environmental niche, and is important for adaptation to climate. The timing of seasonal germination depends on patterns of seed dormancy release or induction by cold and interacts with flowering-time variation to construct different seasonal life histories. To characterize the genetic basis and climatic associations of natural variation in seed chilling responses and associated life-history syndromes, we selected 559 fully sequenced accessions of the model annual species Arabidopsis thaliana from across a wide climate range and scored each for seed germination across a range of 13 cold stratification treatments, as well as the timing of flowering and senescence. Germination strategies varied continuously along 2 major axes: 1) Overall germination fraction and 2) induction vs. release of dormancy by cold. Natural variation in seed responses to chilling was correlated with flowering time and senescence to create a range of seasonal life-history syndromes. Genome-wide association identified several loci associated with natural variation in seed chilling responses, including a known functional polymorphism in the self-binding domain of the candidate gene DOG1. A phylogeny of DOG1 haplotypes revealed ancient divergence of these functional variants associated with periods of Pleistocene climate change, and Gradient Forest analysis showed that allele turnover of candidate SNPs was significantly associated with climate gradients. These results provide evidence that A. thaliana's germination niche and correlated life-history syndromes are shaped by past climate cycles, as well as local adaptation to contemporary climate.

Treatment of Child with Four Lost Maxillary Incisors due to Traffic Injury.

Tooth injury is frequently encountered in pediatric clinical practice. A clinician may be requested to not only respond at the time of injury, but also properly diagnose how such injury will affect the teeth and dentition. Here, we present a case of a child who lost 4 bilateral maxillary incisors due to a traffic accident, and in whom marked mesial inclination of the bilateral maxillary canines and bilateral maxillary first premolars would subsequently occur. Dental management was provided over an extended period. The boy, aged 9 years 11 months, visited our department with the chief complaint of premature loss of 4 bilateral maxillary incisors and eating disturbance associated with the loss of these teeth. Initially, a denture was fitted. The bilateral maxillary canines subsequently demonstrated marked mesial inclination, however, and erupted from an area equivalent to that of the bilateral central incisors. The bilateral maxillary first premolars also showed mesial inclination and rotation. Taking esthetics into consideration, a Nance holding arch with resin buttons that extended to the maxillary incisors and attached to artificial teeth was used. This appliance was also equipped with a retracting hook, and the bilateral maxillary canines were moved downward and distally. Presently, the removable partial denture is worn for the purposes of retention, esthetics, and mastication. Long-term oral management will be necessary. Therefore, final prosthetic treatment for the missing maxillary incisors is planned for when the patient reaches adulthood.

The role of climate adaptation in colonization success in Arabidopsis thaliana.

Understanding the genetic mechanisms that contribute to range expansion and colonization success within novel environments is important for both invasion biology and predicting species-level responses to changing environments. If populations are adapted to local climates across a species' native range, then climate matching may predict which genotypes will successfully establish in novel environments. We examine evidence for climate adaptation and its role in colonization of novel environments in the model species, Arabidopsis thaliana. We review phenotypic and genomic evidence for climate adaptation within the native range and describe new analyses of fitness data from European accessions introduced to Rhode Island, USA, in spring and fall plantings. Accessions from climates similar to the Rhode Island site had higher fitness indicating a potential role for climate pre-adaptation in colonization success. A genomewide association study (GWAS), and genotypic mean correlations of fitness across plantings suggest the genetic basis of fitness in Rhode Island differs between spring and autumn cohorts, and from previous fitness measurements in European field sites. In general, these observations suggest a scenario of conditional neutrality for loci contributing to colonization success, although there was evidence of a fitness trade-off between fall plantings in Norwich, UK, and Rhode Island. GWAS suggested that antagonistic pleiotropy at a few specific loci may contribute to this trade-off, but this conclusion depended upon the accessions included in the analysis. Increased genomic information and phenotypic information make A. thaliana a model system to test for the genetic basis of colonization success in novel environments.

De novo genome assembly of the economically important weed horseweed using integrated data from multiple sequencing platforms.

Horseweed (Conyza canadensis), a member of the Compositae (Asteraceae) family, was the first broadleaf weed to evolve resistance to glyphosate. Horseweed, one of the most problematic weeds in the world, is a true diploid (2n = 2x = 18), with the smallest genome of any known agricultural weed (335 Mb). Thus, it is an appropriate candidate to help us understand the genetic and genomic bases of weediness. We undertook a draft de novo genome assembly of horseweed by combining data from multiple sequencing platforms (454 GS-FLX, Illumina HiSeq 2000, and PacBio RS) using various libraries with different insertion sizes (approximately 350 bp, 600 bp, 3 kb, and 10 kb) of a Tennessee-accessed, glyphosate-resistant horseweed biotype. From 116.3 Gb (approximately 350× coverage) of data, the genome was assembled into 13,966 scaffolds with 50% of the assembly = 33,561 bp. The assembly covered 92.3% of the genome, including the complete chloroplast genome (approximately 153 kb) and a nearly complete mitochondrial genome (approximately 450 kb in 120 scaffolds). The nuclear genome is composed of 44,592 protein-coding genes. Genome resequencing of seven additional horseweed biotypes was performed. These sequence data were assembled and used to analyze genome variation. Simple sequence repeat and single-nucleotide polymorphisms were surveyed. Genomic patterns were detected that associated with glyphosate-resistant or -susceptible biotypes. The draft genome will be useful to better understand weediness and the evolution of herbicide resistance and to devise new management strategies. The genome will also be useful as another reference genome in the Compositae. To our knowledge, this article represents the first published draft genome of an agricultural weed.

Acquisition and evolution of enhanced mutualism-an underappreciated mechanism for invasive success?

Soil biota can determine plant invasiveness, yet biogeographical comparisons of microbial community composition and function across ranges are rare. We compared interactions between Conyza canadensis, a global plant invader, and arbuscular mycorrhizal (AM) fungi in 17 plant populations in each native and non-native range spanning similar climate and soil fertility gradients. We then grew seedlings in the greenhouse inoculated with AM fungi from the native range. In the field, Conyza plants were larger, more fecund, and associated with a richer community of more closely related AM fungal taxa in the non-native range. Fungal taxa that were more abundant in the non-native range also correlated positively with plant biomass, whereas taxa that were more abundant in the native range appeared parasitic. These patterns persisted when populations from both ranges were grown together in a greenhouse; non-native populations cultured a richer and more diverse AM fungal community and selected AM fungi that appeared to be more mutualistic. Our results provide experimental support for evolution toward enhanced mutualism in non-native ranges. Such novel relationships and the rapid evolution of mutualisms may contribute to the disproportionate abundance and impact of some non-native plant species.

Effects of lifelong intake of lemon polyphenols on aging and intestinal microbiome in the senescence-accelerated mouse prone 1 (SAMP1).

Polyphenols have been examined for their beneficial effects on health, particularly in rodents, but their lifelong effects are unclear. Lemons (Citrus limon), containing lemon polyphenols (LPP), are widely consumed but the effects of LPP on aging are unknown. Therefore, we examined the effects of LPP on aging such as aging-related scores, locomotor activity, cognitive functions, and intestinal microbiome using senescence-accelerated mouse prone 1 (SAMP1) and senescence-accelerated resistant mouse 1 (SAMR1). All mice had ad libitum access to water (P1_water group, SAMR1) or 0.1% LPP (P1_LPP group). In the P1_LPP group, LPP intake prolonged the lifespan by approximately 3 weeks and delayed increases in aging-related scores (e.g., periophthalmic lesions) and locomotor atrophy. The P1_water group showed large changes in the intestinal microbiome structure, while the R1 and P1_LPP groups did not. The phylum Bacteroidetes/Firmicutes, which is associated with obesity, in the P1_water group was significantly lower and higher than that in the P1_LPP and R1 groups, respectively. Although the relative abundance of Lactobacillus significantly increased in both P1 groups with aging, the P1_LPP group showed a significantly lower increase than the P1_water group. Thus, lifelong intake of LPP may have anti-aging effects on both phenotypes and the intestinal environment.

Lemon Polyphenols Suppress Diet-induced Obesity by Up-Regulation of mRNA Levels of the Enzymes Involved in beta-Oxidation in Mouse White Adipose Tissue.

The aim of this study was to investigate the effect of dietary lemon polyphenols on high-fat diet-induced obesity in mice, and on the regulation of the expression of the genes involved in lipid metabolism to elucidate the mechanisms. Mice were divided into three groups and fed either a low fat diet (LF) or a high fat diet (HF) or a high fat diet supplemented with 0.5% w/w lemon polyphenols (LP) extracted from lemon peel for 12 weeks. Body weight gain, fat pad accumulation, the development of hyperlipidemia, hyperglycemia, and insulin resistance were significantly suppressed by lemon polyphenols. Supplementation with lemon polyphenols also significantly up-regulated the mRNA level of the peroxisome proliferator activated receptor-alpha (PPARalpha) compared to the LF and HF groups in the liver. Furthermore, the mRNA level of acyl-CoA oxidase (ACO) was up-regulated in the LP group compared to the LF group, but not HF group in the liver, and was also significantly increased in the epididymal white adipose tissue. Thus, feeding with lemon polyphenols suppressed body weight gain and body fat accumulation by increasing peroxisomal beta-oxidation through up-regulation of the mRNA level of ACO in the liver and white adipose tissue, which was likely mediated via up-regulation of the mRNA levels of PPARalpha.

Antioxidative catechol lignans converted from sesamin and sesaminol triglucoside by culturing with Aspergillus.

Sesamin and sesaminol triglucoside in sesame seeds are major lignans that display an abundance of biological activities. Although their antioxidative activity in vitro is weak, they have been reported to suppress oxidative stress in vivo. We investigated the production of new antioxidative lignans from sesame lignans by culturing with the genus Aspergillus to enhance the function of food materials. Media containing sesamin or sesaminol triglucoside increased antioxidative activity for DPPH radical scavenging by culturing with Aspergillus usamii mut. shirousamii RIB2503. The antioxidative lignans in sesamin medium were identified as sesamin 2,6-dicatechol and episesamin 2,6-dicatechol. Those in sesaminol triglucoside medium were identified as sesaminol 6-catechol and episesaminol 6-catechol, which are novel antioxidative lignans. It is suggested that they may exhibit higher antioxidative activity than sesamin and sesaminol triglucoside because they have the catechol functional moiety.

Hsp90 is a direct target of the anti-allergic drugs disodium cromoglycate and amlexanox.

Hsp90 (heat-shock protein 90) alone can act to prevent protein aggregation and promote refolding in vitro, but in vivo it operates as a part of a multichaperone complex, which includes Hsp70 and cohort proteins. Since the physiological function of Hsp90 is not yet fully understood, the development of specific antagonists might open new lines of investigation on the role of Hsp90. In an effort to discover Hsp90 antagonists, we screened many drugs and found that the anti-allergic drugs DSCG (disodium cromoglycate) and amlexanox target Hsp90. Both drugs were found to bind directly wild-type Hsp90 via the N- and C-terminal domains. Both drugs strongly suppressed the in vitro chaperone activity of native Hsp90 towards citrate synthase at 1.5-3.0 microM. Amlexanox suppressed C-terminal chaperone activity in vitro, but not N-terminal chaperone activity, and inhibited the association of cohort proteins, such as cyclophilin 40 and Hsp-organizing protein, to the C-terminal domain of Hsp90. These data suggest that amlexanox might disrupt the multichaperone complex, including Hsp70 and cohort proteins, both in vitro and in vivo. Although DSCG inhibited the in vitro chaperone activity of the N-terminal domain, the drug had no effect either on the C-terminal chaperone activity or on the association of the cohort proteins with the C-terminus of Hsp90. The physiological significance of these interactions in vivo remains to be investigated further, but undoubtedly must be taken into account when considering the pharmacology of anti-allergic drugs. DSCG and amlexanox may serve as useful tools for evaluating the physiological significance of Hsp90.

Neuronal calcium sensor proteins are direct targets of the insulinotropic agent repaglinide.

The NCS (neuronal calcium sensor) proteins, including neurocalcins, recoverins and visinin-like proteins are members of a family of Ca2+-sensitive regulators, each with three Ca2+-binding EF-hand motifs. In plants, lily CCaMK [chimaeric Ca2+/CaM (calmodulin)-dependent protein kinase] and its PpCaMK ( Physcomitrella patens CCaMK) homologue are characterized by a visinin-like domain with three EF-hands. In the present study, in an effort to discover NCS antagonists, we screened a total of 43 compounds using Ca2+-dependent drug affinity chromatography and found that the insulinotropic agent repaglinide targets the NCS protein family. Repaglinide was found to bind to NCS proteins, but not to CaM or S100 proteins, in a Ca2+-dependent manner. Furthermore, the drug antagonized the inhibitory action of recoverin in a rhodopsin kinase assay with IC50 values of 400 microM. Moreover, repaglinide tightly bound to the visinin-like domain of CCaMK and PpCaMK in a Ca2+-dependent manner and antagonized the regulatory function of the domain with IC50 values of 55 and 4 microM for CCaMK and PpCaMK respectively. Although both repaglinide and a potent insulin secretagogue, namely glibenclamide, blocked K(ATP) channels with similar potency, glibenclamide had no antagonizing effect on the Ca2+-stimulated CCaMK and PpCaMK autophosphorylation, mediated by their visinin-like domain. In addition, a typical CaM antagonist, trifluoperazine, had no effect on the CCaMK and PpCaMK autophosphorylation. Repaglinide appears to be the first antagonist of NCS proteins and visinin-like domain-bearing enzymes. It may serve as a useful tool for evaluating the physiological functions of the NCS protein family. In addition, since repaglinide selectively targets NCS proteins among the EF-hand Ca2+-binding proteins, it is a potential lead compound for the development of more potent NCS antagonists.

Molecular evidence for hybridization in Colias (Lepidoptera: Pieridae): are Colias hybrids really hybrids?

Gene flow and hybridization among species dramatically affect our understanding of the species as a biological unit, species relationships, and species adaptations. In North American Colias eurytheme and Colias eriphyle, there has been historical debate over the extent of hybridization occurring and the identity of phenotypically intermediate individuals as genetic hybrids. This study assesses the population structure of these two species to measure the extent of hybridization and the genetic identity of phenotypic intermediates as hybrids. Amplified fragment length polymorphism (AFLP) marker analysis was performed on 378 specimens collected from northern California and Nevada. Population structure was inferred using a Bayesian/Markov chain Monte Carlo method, which probabilistically assigns individuals to genetic clusters. Three genetic clusters provided the best fit for the data. C. eurytheme individuals were primarily assigned to two closely related clusters, and C. eriphyle individuals were mostly assigned to a third, more distantly related cluster. There appeared to be significant hybridization between the two species. Individuals of intermediate phenotype (putative hybrids) were found to be genetically indistinguishable from C. eriphyle, indicating that previous work based on the assumption that these intermediate forms are hybrids may warrant reconsideration.

High-Density Single Nucleotide Polymorphism Linkage Maps of Lowland Switchgrass using Genotyping-by-Sequencing.

Switchgrass (Panicum virgatum L.) is a warm-season perennial grass with promising potential as a bioenergy crop in the United States. However, the lack of genomic resources has slowed the development of plant lines with optimal characteristics for sustainable feedstock production. We generated high-density single nucleotide polymorphism (SNP) linkage maps using a reduced-representation sequencing approach by genotyping 231 F1 progeny of a cross between two parents of lowland ecotype from the cultivars Kanlow and Alamo. Over 350 million reads were generated and aligned, which enabled identification and ordering of 4611 high-quality SNPs. The total lengths of the resulting framework maps were 1770 cM for the Kanlow parent and 2059 cM for the Alamo parent. These maps show collinearity with maps generated with polymerase chain reaction (PCR)-based simple-sequence repeat (SSR) markers, and new SNP markers were identified in previously unpopulated regions of the genome. Transmission segregation distortion affected all linkage groups (LGs) to differing degrees, and ordering of distorted markers highlighted several regions of unequal inheritance. Framework maps were adversely affected by the addition of distorted markers with varying severity, but distorted maps were of higher marker density and provided additional information for analysis. Alignment of these linkage maps with a draft version of the switchgrass genome assembly demonstrated high levels of collinearity and provides greater confidence in the validity of both resources. This methodology has proven to be a rapid and cost-effective way to generate high-quality linkage maps of an outcrossing species.

Evolution and spread of glyphosate resistance in Conyza canadensis in California.

Recent increases in glyphosate use in perennial crops of California, USA, are hypothesized to have led to an increase in selection and evolution of resistance to the herbicide in Conyza canadensis populations. To gain insight into the evolutionary origins and spread of resistance and to inform glyphosate resistance management strategies, we investigated the geographical distribution of glyphosate resistance in C. canadensis across and surrounding the Central Valley, its spatial relationship to groundwater protection areas (GWPA), and the genetic diversity and population structure and history using microsatellite markers. Frequencies of resistant individuals in 42 sampled populations were positively correlated with the size of GWPA within counties. Analyses of population genetic structure also supported spread of resistance in these areas. Bayesian clustering and approximate Bayesian computation (ABC) analyses revealed multiple independent origins of resistance within the Central Valley. Based on parameter estimation in the ABC analyses, resistant genotypes underwent expansion after glyphosate use began in agriculture, but many years before it was detected. Thus, diversity in weed control practices prior to herbicide regulation in GWPA probably kept resistance frequencies low. Regionally coordinated efforts to reduce seed dispersal and selection pressure are needed to manage glyphosate resistance in C. canadensis.

Complete switchgrass genetic maps reveal subgenome collinearity, preferential pairing and multilocus interactions.

Polyploidy is an important aspect of the evolution of flowering plants. The potential of gene copies to diverge and evolve new functions is influenced by meiotic behavior of chromosomes leading to segregation as a single locus or duplicated loci. Switchgrass (Panicum virgatum) linkage maps were constructed using a full-sib population of 238 plants and SSR and STS markers to access the degree of preferential pairing and the structure of the tetraploid genome and as a step toward identification of loci underlying biomass feedstock quality and yield. The male and female framework map lengths were 1645 and 1376 cM with 97% of the genome estimated to be within 10 cM of a mapped marker in both maps. Each map coalesced into 18 linkage groups arranged into nine homeologous pairs. Comparative analysis of each homology group to the diploid sorghum genome identified clear syntenic relationships and collinear tracts. The number of markers with PCR amplicons that mapped across subgenomes was significantly fewer than expected, suggesting substantial subgenome divergence, while both the ratio of coupling to repulsion phase linkages and pattern of marker segregation indicated complete or near complete disomic inheritance. The proportion of transmission ratio distorted markers was relatively low, but the male map was more extensively affected by distorted transmission ratios and multilocus interactions, associated with spurious linkages.

Microsatellite variation points to local landscape plantings as sources of invasive pampas grass (Cortaderia selloana) in California.

International trade in horticultural plants is a major pathway of introduction of invasive species. Pampas grass (Cortaderia selloana) is an invasive species of horticultural origin that is native to South America but cultivated as an ornamental in regions with Mediterranean climates worldwide. To gain insight into the introduction history of invasive populations in California, we analysed microsatellite marker variation in cultivated and invasive C. selloana. We sampled 275 cultivated plants from diverse sources and 698 invasive plants from 33 populations in four geographical regions of California. A model-based Bayesian clustering analysis identified seven distinct gene pools in cultivated C. selloana. Probabilities of assignment of invasive individuals to cultivated gene pools indicated that two gene pools accounted for the genomic origin of 78% of the invasive C. selloana sampled. Extensive admixture between cultivated source gene pools was detected within invasive individuals. Sources of admixed invasive individuals are probably landscape plantings. Consistent with the Bayesian assignment results indicating that multiple cultivated gene pools and landscape plantings are probable sources of invasive populations, F(ST) and neighbour-joining clustering analyses indicated multiple escapes from shared sources in each geographical region. No isolation by distance or geographical trend in reduction of genetic diversity was evident. Furthermore, a generally random and discontinuous distribution of proportional assignments of invasive populations to cultivated gene pools suggests that introductions occurred recurrently within each geographical region. Our results strongly suggest that dispersal through local landscape plantings has contributed to the range expansion of invasive C. selloana in California.

Isolation of antioxidative phenolic glucosides from lemon juice and their suppressive effect on the expression of blood adhesion molecules.

Phenolic glucosides having radical scavenging activity were examined from the fraction eluted with 20% methanol on Amberlite XAD-2 resin applied to lemon (Citrus limon) juice by using reversed phase chromatography. Four phenolic glucosides were identified as 1-feruloyl-beta-D-glucopyranoside, 1-sinapoyl-beta-D-glucopyranoside, 6,8-di-C-glucosylapigenin and 6,8-di-C-glucosyldiosmetin by (1)H-NMR, (13)C-NMR, and MS analyses. They exhibited radical scavenging activity for 1,1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide, although the activity was low in comparison with eriocitrin, a potent antioxidant in lemon fruit, and the eriodictyol of its aglycone. The phenolic compounds in lemon juice were examined for their suppressive effect on the expression of blood adhesion molecules by measuring the expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVECs) induced by necrosis factor-alpha (TNF-alpha). 6,8-Di-C-glucosylapigenin, apigenin, and diosmentin of the flavones were found to significantly suppress the expression of ICAM-1 at 10 muM (P<0.05). The phenolic glucosides isolated in this study were contained in comparative abundance in daidai (Citrus aurantium) and niihime (Citrus unshiu x Citrus tachibana) among the sour citrus juices.

Formation of Nepsilon-(succinyl)lysine in vivo: a novel marker for docosahexaenoic acid-derived protein modification.

Free radical-catalyzed peroxidation of docosahexaenoic acid (DHA, C22:6/omega-3) generates various lipid peroxidation products that covalently modify biomolecules such as proteins. Under a free radical-generating system, DHA significantly modified lysine residues in bovine serum albumin. Upon incubation of oxidized DHA with an amino-compound pyridoxamine or a lysine-containing peptide, N-propanoyl and N-succinyl adducts were determined to be the major modification products. The hydroperoxide levels in the oxidized DHA closely reflected the formation of the N(epsilon)-(succinyl)lysine (SUL) upon reaction with the peptide, indicating that the hydroperoxides of DHA represent a potential pathway for the formation of SUL. To detect the DHA-derived protein modification in vivo, we developed a monoclonal antibody (mAb2B12) specific to SUL and found that the antibody specifically reacts with the SUL moiety. The formation of SUL was then immunochemically demonstrated in the liver of mice fed with DHA followed by intraperitoneal injection of carbon tetrachloride (CCl(4)), a hepatic lipid peroxidation model. Immunoreactive materials with mAb2B12 were observed in the DHA + CCl(4) group, but were not significant in the control, DHA-alone, and CCl(4)-alone groups. These data suggest that the formation of DHA-derived adducts such as SUL may be implicated in the oxidative damage observed in DHA-enriched tissues.

Interaction of S100 proteins with the antiallergic drugs, olopatadine, amlexanox, and cromolyn: identification of putative drug binding sites on S100A1 protein.

S100 proteins are a multigenic family of low-molecular-weight Ca(2+)-binding proteins comprising 19 members. These proteins undergo a conformational change by Ca(2+)-binding and consequently interact with their target proteins. Recently, we reported that two antiallergic drugs, Amlexanox and Cromolyn, bind to S100A12 and S100A13 of the S100 protein family. In the present study, we used a newly developed antiallergic drug, Olopatadine, as a ligand for affinity chromatography and examined binding specificity of the drug to S100 protein family. Olopatadine binds specifically to S100 proteins, such as S100A1, S100B, S100L, S100A12, and S100A13, in a Ca(2+)-dependent manner but not to calmodulin. Mutagenesis study showed that amino acid residues 76-85 in S100A1 are necessary for its binding to Olopatadine. In contrast, residues 89-94 were identified as an Amlexanox-binding site in S100A1. Moreover, Olopatadine did not competitively inhibit S100A1-binding site of Amlexanox. Furthermore, we showed that Olopatadine inhibited the binding of S100A1 target protein's binding site peptides to S100A1. These results indicate that C-terminal region of S100A1 is important for antiallergic drug binding, although the drug binding sites are different according to each antiallergic drug. Differences in the binding sites of S100A1 to antiallergic drugs suggest that the regulatory functions of S100 proteins may exist in several regions. Therefore, these drugs may serve as useful tools for evaluating the physiological significance of S100 protein family.

Identification of intracellular target proteins of the calcium-signaling protein S100A12.

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+-dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+-dependent isocitrate dehydrogenase (IDH), fructose-1,6-bisphosphate aldolase A (aldolase), glyceraldehyde-3-phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12-binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+-dependent, whereas that of GAPDH and IDH was only weakly Ca2+-dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C-terminal truncation mutants to the S100A12-immobilized sensor chip. The results indicated that the S100A12-binding site on S100A12 itself is located at the C-terminus (residues 87-92). However, cross-linking experiments with the truncation mutants indicated that residues 87-92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo- or heterodimerization model. Ca2+-dependent affinity chromatography revealed that C-terminal residues 75-92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone-like function which is Ca2+-dependent.