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In clinical studies, the next-generation anti-tumor necrosis factor-alpha (TNF-α) single domain antibody ozoralizumab showed high clinical efficacy shortly after the subcutaneous injection. To elucidate the mechanism underlying the rapid onset of the effects of ozoralizumab, we compared the biodistribution kinetics of ozoralizumab and adalimumab after subcutaneous injection in an animal model of arthritis. Alexa Fluor 680-labeled ozoralizumab and adalimumab were administered by subcutaneous injection once (2 mg/kg) at five weeks after induction of collagen-induced arthritis (CIA) in an animal arthritis model. The time-course of changes in the fluorescence intensities of the two compounds in the paws and serum were evaluated. The paws of the CIA mice were harvested at four and eight hours after the injection for fluorescence microscopy. Biofluorescence imaging revealed better distribution of ozoralizumab to the joint tissues than of adalimumab, as early as at four hours after the injection. Fluorescence microscopy revealed a greater fluorescence intensity of ozoralizumab in the joint tissues than that of adalimumab at eight hours after the injection. Ozoralizumab showed a significantly higher absorption rate constant as compared with adalimumab. These results indicate that ozoralizumab enters the systemic circulation more rapidly and is distributed to the target tissues earlier and at higher levels than conventional IgG antibodies. Our investigation provides new insight into the mechanism underlying the rapid onset of the effects of ozoralizumab in clinical practice.

We previously found that a single dose of B-type procyanidin mixture increase in skeletal muscle blood flow (BF). We compared BF changes following administration of (-)-epicatechin (EC, monomer) and the B-type procyanidins procyanidin B2 (B2, dimer), procyanidin C1 (C1, trimer), and cinnamtannin A2 (A2, tetramer). Each chemical was administered orally to rats, followed by BF measurement in cremaster arteriole for 60 min. About 10 and 100 µg/kg of B2 and C1 elicited BF increase, the effect was potent at 100 µg/kg. BF also increased significantly after administration of 10 µg/kg A2, but not with the administration at 100 µg/kg. EC yielded no BF changes. Co-treatment with the nonselective adrenaline blocker carvedilol attenuated the BF increase seen with 10 µg/kg A2 treatment. This outcome suggested the involvement of sympathetic nerve activation in the BF increase by this dose of A2. Co-treatment of 100 µg/kg A2 with the α2 blocker yohimbine exhibited an increase of BF significantly. The α2 adrenaline receptor in the vasomotor centre is an inhibitory receptor and it regulates hemodynamics. This result suggested that high doses of A2 did not alter BF because of activating the α2 adrenergic receptor. Phosphorylation of aortic endothelial nitric oxide synthase (eNOS) increased with 10 µg/kg A2 alone or co-treatment with 100 µg/kg A2 and yohimbine, but not with co-treatment of 10 µg/kg A2 and carvedilol or 100 µg/kg A2 alone. These results imply that A2 does not directly activate eNOS, but that shear stress from the increased BF might be associated with eNOS phosphorylation.

Muscle disuse induces atrophy through increased reactive oxygen species (ROS) released from damaged mitochondria. Mitophagy, the autophagic degradation of mitochondria, is associated with increased ROS production. However, the mitophagy activity status during disuse-induced muscle atrophy has been a subject of debate. Here, we developed a new mitophagy reporter mouse line to examine how disuse affected mitophagy activity in skeletal muscles. Mice expressing tandem mCherry-EGFP proteins on mitochondria were then used to monitor the dynamics of mitophagy activity. The reporter mice demonstrated enhanced mitophagy activity and increased ROS production in atrophic soleus muscles following a 14-day hindlimb immobilization. Results also showed an increased expression of multiple mitophagy genes, including Bnip3, Bnip3l, and Park2. Our findings thus conclude that disuse enhances mitophagy activity and ROS production in atrophic skeletal muscles and suggests that mitophagy is a potential therapeutic target for disuse-induced muscle atrophy.

It is known that administering a gavage to rodents evokes a cardiac reflex, due to gastrointestinal stimulation. Consequently, it is difficult to evaluate changes in hemodynamics after a single oral dose of a pungent or astringent, which alters the circulation by increasing sympathetic activity. In the present study, we developed a method for administering a gavage without significantly affecting hemodynamics measurements. We marked a gastric tube at 10 cm from the tip, to mark the distance from the oral cavity to the stomach body of Wistar male rats. Rats were intubated under urethane anesthesia.After 10–15 min of stabilization, we measured the mean blood pressure (MBP), heart rate (HR), and blood flow (BF) in the cremaster arteriole under two different conditions; condition 1: a pointed gastric tube, room temperature distilled water, and injected at normal speed (approximately 3 ml/min); condition 2: a rounded gastric tube, 37°C distilled water, and injection at 1.0 ml/min. Under condition 1, we observed striking hemodynamic alterations, due to the somatic afferent reflex. In contrast, under condition 2, these hemodynamic changes were nearly eliminated. In addition, we could clearly detect hemodynamic changes in rats after a single gavage treatment of pungent (capsaicin) or astringent (cinnamtannin A2). We observed transient increases in the HR and MBP soon after treatment with capsaicin. Moreover, cremasteric BF was elevated with cinnamtannin A2. These results confirmed the utility of the gavage method developed in this study.

Craniometaphyseal dysplasia (CMD), a rare genetic bone disorder, is characterized by lifelong progressive thickening of craniofacial bones and metaphyseal flaring of long bones. The autosomal dominant form of CMD is caused by mutations in the progressive ankylosis gene ANKH (mouse ortholog Ank), encoding a pyrophosphate (PPi) transporter. We previously reported reduced formation and function of osteoblasts and osteoclasts in a knockin (KI) mouse model for CMD (AnkKI/KI) and in CMD patients. We also showed rapid protein degradation of mutant ANK/ANKH. Mutant ANK protein displays reduced PPi transport, which may alter the inorganic phosphate (Pi) and PPi ratio, an important regulatory mechanism for bone mineralization. Here we investigate whether reducing dietary Pi intake can ameliorate the CMD-like skeletal phenotype by comparing male and female Ank+/+ and AnkKI/KI mice exposed to a low (0.3%) and normal (0.7%) Pi diet for 13 weeks from birth. Serum Pi and calcium (Ca) levels were not significantly changed by diet, whereas PTH and 25-hydroxy vitamin D (25-OHD) were decreased by low Pi diet but only in male Ank+/+ mice. Importantly, the 0.3% Pi diet significantly ameliorated mandibular hyperostosis in both sexes of AnkKI/KI mice. A tendency of decreased femoral trabeculation was observed in male and female Ank+/+ mice as well as in male AnkKI/KI mice fed with the 0.3% Pi diet. In contrast, in female AnkKI/KI mice the 0.3% Pi diet resulted in increased metaphyseal trabeculation. This was also the only group that showed increased bone formation rate. Low Pi diet led to increased osteoclast numbers and increased bone resorption in all mice. We conclude that lowering but not depleting dietary Pi delays the development of craniofacial hyperostosis in CMD mice without severely compromising serum levels of Pi, Ca, PTH, and 25-OHD. These findings may have implications for better clinical care of patients with CMD. © 2020 American Society for Bone and Mineral Research.

Recent epidemiological and intervention studies have suggested that polyphenol-rich plant food consumption reduced the risk of cognitive decline. However, the findings were tentative and by no means definitive. In the present study, we examined the impact of short-term oral administration of cinnamtannin A2 (A2), an (−)-epicatechin tetramer, on adult hippocampal neurogenesis and cognitive function in mice. Mice received supplementation with vehicle (20% glycerol) or 100 μg/kg A2 for 10 days. Then, we conducted the open field test, the object location test, and the novel object test. In the open field test, the A2-treated group tended to spend more time in the center of the arena, compared to the vehicle-treated group. The A2-treated group spent significantly more time exploring objects placed in different locations, compared to the vehicle-treated group. There were no significant differences between groups in the object preference index or in the novel object test. In addition, A2 administration significantly increased the number of hippocampal bromodeoxyuridine-labeled cells in the dentate gyrus, but not in the CA1 or CA3 regions. These results suggested that short-term administration of A2 may impact spatial memory by enhancing neurogenesis in the dentate gyrus of adult mice.

We previously found increases in uncoupling protein (Ucp)-1 transcription in brown adipose tissue (BAT) of mice following a single oral dose of flavan 3-ol (FL)s, a fraction of catechins and procyanidins. It was confirmed that these changes were totally reduced by co-treatment of adrenaline blockers. According to these previous results, FLs possibly activate sympathetic nervous system (SNS). In this study, we confirmed the marked increase in urinary catecholamine (CA) s projecting SNS activity following a single dose of 50 mg/kg FLs. In addition, we examined the impact of the repeated administration of 50 mg/kg FLs for 14 days on adipose tissues in mice. In BAT, FLs tended to increase the level of Ucp-1 along with significant increase of thermogenic transcriptome factors expressions, such as peroxisome proliferator-activated receptor γ coactivator (PGC)-1α and PR domain-containing (PRDM)1. Expression of browning markers, CD137 and transmembrane protein (TMEM) 26, in addition to PGC-1α were increased in epididymal adipose (eWAT) by FLs. A multilocular morphology with cell size reduction was shown in the inguinal adipose (iWAT), together with increasing the level of Ucp-1 by FLs. These results exert that FLs induce browning in adipose, and this change is possibly produced by the activation of the SNS.

Human dental pulp stem cells (DPSCs) have high clonogenic and proliferative potential. We previously reported that a helioxanthin derivative (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2&ndash, b]pyridine-2-carboxamide (TH)) enhances osteogenic differentiation of DPSCs derived from young patients. However, in the clinical field, elderly patients more frequently require bone regenerative therapy than young patients. In this study, we examined and compared the osteogenic differentiation potential of TH-induced DPSCs from elderly patients and young patients to explore the potential clinical use of DPSCs for elderly patients. DPSCs were obtained from young and elderly patients and cultured in osteogenic medium with or without TH. We assessed the characteristics and osteogenic differentiation by means of specific staining and gene expression analyses. Moreover, DPSC sheets were transplanted into mouse calvarial defects to investigate osteogenesis of TH-induced DPSCs by performing micro-computed tomography (micro-CT). We demonstrated that osteogenic conditions with TH enhance the osteogenic differentiation marker of DPSCs from elderly patients as well as young patients in vitro. In vivo examination showed increased osteogenesis of DPSCs treated with TH from both elderly patients and young patients. Our results suggest that the osteogenic differentiation potential of DPSCs from elderly patients is as high as that of DPSCs from young patients. Moreover, TH-induced DPSCs showed increased osteogenic differentiation potential, and are thus a potentially useful cell source for bone regenerative therapy for elderly patients.

Flavan-3-ols (FLs), specifically catechin and its oligomer B-type procyanidins, are suggested to potently bind to bovine serum albumin (BSA). We examined the interaction between BSA and FLs by fluorescence quenching and found the following order of binding activities to BSA: cinnamtannin A2 (A2, tetramer) &gt, procyanidin C1 (C1, trimer) &asymp, procyanidin B2 (B2, dimer) &gt, (&minus, )epicatechin (EC, monomer). Docking simulations between BSA and each compound at the binding site showed that the calculated binding energies were consistent with the results of our experimental assay. FLs exerted cytotoxicity at 1000 &mu, g/mL in F11 cell culture with fetal bovine serum containing BSA. In culture containing serum-free medium, FLs exhibited significant cell proliferation at 10&minus, 4 &mu, g/mL and cytotoxicity was observed at concentrations greater than 10 &mu, g/mL. Results of this study suggest that interactions between polyphenols and BSA should be taken into account when evaluating procyanidin in an in vitro cell culture system.

Bone defects affect patients functionally and psychologically and can decrease quality of life. To resolve these problems, a simple and efficient method of bone regeneration is required. Human dental pulp stem cells (DPSCs) have high proliferative ability and multilineage differentiation potential. In our previous study, we reported a highly efficient method to induce osteogenic differentiation using DPSC sheets treated with a helioxanthin derivative (4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH)) in a mouse calvarial defect model. However, the localization of the DPSCs after transplantation remains unknown. Therefore, in this study, we investigated the localization of transplanted DPSCs in a mouse fracture model. DPSCs were collected from six healthy patients aged 18&ndash, 29 years, cultured in normal medium (NM), osteogenic medium (OM), or OM with TH, and fabricated them into cell sheets. To evaluate the efficacy of fracture healing using DPSCs treated with OM+TH, and to clarify the localization of the transplanted DPSC sheets in vivo, we transplanted OM+TH-treated DPSC sheets labeled with PKH26 into mouse tibiae fractures. We demonstrated that transplanted OM+TH-treated DPSCs sheets were localized to the fracture site and facilitated bone formation. These results indicated that transplanted OM+TH-treated DPSCs were localized at fracture sites and directly promoted fracture healing.

Acylated anthocyanins are more stable than monomeric anthocyanins, but little is known about their physiological effects. We evaluated the hemodynamic effects of single intragastric doses of purple carrot (Daucus carota L.) anthocyanin (PCA) and two monomeric anthocyanins, cyanidin 3-O-glycoside (C3G) and delphinidin 3-O-ruthenoside (D3R). PCA, C3G, or D3R was administered orally to rat and blood flow in the cremaster artery was measured for 60 min using a laser Doppler blood flow meter. After measurements, the aorta of the animal was removed and the extent of phosphorylation of aortic epithelial nitric oxide synthase (eNOS) and Akt were determined by western blotting. PCA (10 mg kg-1) or C3G (1 mg kg-1) significantly increased rat cremaster arteriole blood flow and phosphorylation of eNOS and Akt; D3G (1 mg kg-1) only slightly altered cremaster arteriole blood flow and did not affect the phosphorylation of eNOS and Akt in the aorta. These results suggest that hemodynamic alterations depend more on the chemical structure of anthocyanins, particularly the aglycon, than on the glycoside. In addition, increase of blood flow by a single oral dose of PCA was practically reduced with treatment of carvedilol (CR), a non-specific adrenaline blocker. Blood concentrations of cyanidin or its glycoside 15, 30, or 60 min after the administration of 10 mg kg-1 PCA were below the limit of detection. These hemodynamic changes may have been associated with an indirect adrenergic action induced following a single dose of PCA.

We previously confirmed that postprandial alterations in the circulation and metabolism after a single oral dose of flavan 3-ols (mixture of catechin and catechin oligomers) were involved in an increase in sympathetic nervous activity. However, it is well known that, in response to various stresses, activation of the hypothalamic-pituitary-adrenal (HPA) axis occurs together with sympathetic nerve activity, which is associated with activation of the sympathetic-adrenal-medullary (SAM) axis. In this study, we examined whether the HPA axis was activated after a single dose of flavan 3-ols. We administered an oral dose of 10 or 50 mg/kg flavan 3-ols to male ICR mice, removed the brains, and fixed them in paraformaldehyde-phosphate buffer. Other animals that were treated similarly were decapitated, and blood was collected. In the paraventricular nucleus (PVN), c-fos mRNA expression increased significantly at 15 min after administration of either 10 or 50 mg/kg flavan 3-ols. Corticotropin-releasing hormone (CRH) mRNA expression levels significantly increased at 240 min after administration of 10 mg/kg flavan 3-ols, and at 60 min after administration of 50 mg/kg flavan 3-ols. Plasma corticosterone levels were also significantly increased at 240 min after ingestion of 50 mg/kg flavan 3-ols. In this experiment, we confirmed that the ingestion of flavan 3-ols acted as a stressor in mammals with activation both the SAM and HPA axes.

Background Human dental pulp stem cells (DPSCs), which have the ability to differentiate into multiple lineages, were recently identified. DPSCs can be collected readily from extracted teeth and are now considered to be a type of mesenchymal stem cell with higher clonogenic and proliferative potential than bone marrow stem cells (BMSCs). Meanwhile, the treatment of severe bone defects, such as fractures, cancers, and congenital abnormalities, remains a great challenge, and novel bone regenerative techniques are highly anticipated. Several studies have previously shown that 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin derivative, induces osteogenic differentiation of preosteoblastic and mesenchymal cells. However, the osteogenic differentiation activities of TH have only been confirmed in some mouse cell lines. Therefore, in this study, toward the clinical use of TH in humans, we analyzed the effect of TH on the osteogenic differentiation of DPSCs, and the in-vivo osteogenesis ability of TH-induced DPSCs, taking advantage of the simple transplantation system using cell-sheet technology. Methods DPSCs were obtained from dental pulp of the wisdom teeth of five healthy patients (18–22 years old) and cultured in regular medium and osteogenic medium with or without TH. To evaluate osteogenesis of TH-induced DPSCs in vivo, we transplanted DPSC sheets into mouse calvaria defects. Results We demonstrated that osteogenic conditions with TH induce the osteogenic differentiation of DPSCs more efficiently than those without TH and those with bone morphogenetic protein-2. However, regular medium with TH did not induce the osteogenic differentiation of DPSCs. TH induced osteogenesis in both DPSCs and BMSCs, although the gene expression pattern in DPSCs differed from that in BMSCs up to 14 days after induction with TH. Furthermore, we succeeded in bone regeneration in vivo using DPSC sheets with TH treatment, without using any scaffolds or growth factors. Conclusions Our results demonstrate that TH-induced DPSCs are a useful cell source for bone regenerative medicine, and the transplantation of DPSC sheets treated with TH is a convenient scaffold-free method of bone healing. Electronic supplementary material The online version of this article (10.1186/s13287-018-0783-7) contains supplementary material, which is available to authorized users.

Objectives We previously found that elevated energy expenditure following a single oral dose of flavan 3-ols (FL), a mixture of catechins and B type procyanidins, is caused by sympathetic nerve activation. In the present study, we compared the activity of the FL components (-)-epicatechin (EC; monomer), procyanidin B2 (B2; dimer), procyanidin C1 (C1; trimer), cinnamtannin A2 (A2; tetramer), and more than pentamer fraction (P5). Methods Male ICR mice were treated with a single oral dose of FL, EC, B2, C1, A2, or P5. The animals were sacrificed and blood and brown adipose tissue (BAT) sampled. The plasma catecholamine (CA) levels and BAT uncoupling protein (UCP)-1 mRNA expression were determined. Results A single dose of 10 mg/kg FL significantly increased plasma CA and UCP-1 mRNA levels. B2, C1, and A2, but not EC and P5 (all at 1 mg/kg), significantly increased plasma adrenaline levels. Plasma noradrenaline was significantly elevated by B2 and A2, but not by EC, C1, or P5. UCP-1 mRNA levels were significantly increased by C1 and P5. In the dose response study of A2, 10−3 mg/kg A2 increased UCP-1 mRNA levels significantly, but not 10−2 and 10−1 mg/kg A2. In addition, combination treatment with 10−1 mg/kg A2 and yohimbine, an α2 adrenalin blocker, remarkably increased UCP-1 mRNA levels. Conclusion These results suggest that FL and its components, except EC, increase UCP-1 mRNA and plasma CA with varying efficacy.

Previous reports have shown that osteoblasts are mechano-sensitive. Low-intensity pulsed ultrasound (LIPUS) induces osteoblast differentiation and is an established therapy for bone fracture. Here we have examined how LIPUS affects inflammatory responses of osteoblasts to LPS. LPS rapidly induced mRNA expression of several chemokines including CCL2, CXCL1, and CXCL10 in both mouse osteoblast cell line and calvaria-derived osteoblasts. Simultaneous treatment by LIPUS significantly inhibited mRNA induction of CXCL1 and CXCL10 by LPS. LPS-induced phosphorylation of ERKs, p38 kinases, MEK1/2, MKK3/6, IKKs, TBK1, and Akt was decreased in LIPUS-treated osteoblasts. Furthermore, LIPUS inhibited the transcriptional activation of NF-κB responsive element and Interferon-sensitive response element (ISRE) by LPS. In a transient transfection experiment, LIPUS significantly inhibited TLR4-MyD88 complex formation. Thus LIPUS exerts anti-inflammatory effects on LPS-stimulated osteoblasts by inhibiting TLR4 signal transduction.

A novel series of 2-aminothiazole-oxazoles was designed and synthesized as part of efforts to develop potent phosphoinositide 3-kinase γ (PI3Kγ) inhibitors. The modification of a high-throughput screening hit, compound 1, resulted in the identification of compounds 10 and 15, which displayed potent inhibitory activities in enzyme-based and cell-based assays.

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P(1)) and participates in many pathological conditions. We developed a novel type S1P(1)-selective antagonist, TASP0251078, which is structurally unrelated to S1P. This competitive antagonist inhibited binding of S1P to S1P(1) resulting in reduced signaling downstream of S1P(1), including GTPγS-binding and cAMP formation. TASP0251078 also inhibited S1P-induced cellular responses such as chemotaxis and receptor-internalization. Furthermore, when administered in vivo, TASP0251078 induced lymphopenia in blood, which is different from previously reported effects of other S1P(1)-antagonists. In a mouse contact hypersensitivity model, TASP0251078 effectively suppressed ear swelling, leukocyte infiltration, and hyperplasia. These findings provide the chemical evidence that S1P(1) antagonism is responsible for lymphocyte sequestration from the blood, and suggest that the effect of S1P(1) agonists on lymphocyte sequestration results from their functional antagonism.

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking through the type 1 sphingosine 1-phosphate receptor (S1P1) and participates in many pathological conditions, including autoimmune diseases. We developed a novel S1P1-selective antagonist, TASP0277308, which is structurally unrelated to S1P. This antagonist competitively inhibited S1P-induced cellular responses, such as chemotaxis and receptor internalization. Furthermore, differing from previously reported S1P1 antagonists, TASP0277308 demonstrated in vivo activities to induce lymphopenia, a block in T cell egress from the thymus, displacement of marginal zone B cells, and upregulation of CD69 expression on both T and B cells, all of which recapitulate phenotypes of S1P1-deficient lymphocytes. In a mouse collagen-induced arthritis model, TASP0277308 significantly suppressed the development of arthritis, even after the onset of disease. These findings provide the first chemical evidence to our knowledge that S1P1 antagonism is responsible for immunosuppression in the treatment of autoimmune diseases and also resolve the discrepancies between genetic and chemical studies on the functions of S1P1 in lymphocytes.

We sequenced the genome of Theileria orientalis, a tick-borne apicomplexan protozoan parasite of cattle. The focus of this study was a comparative genome analysis of T. orientalis relative to other highly pathogenic Theileria species, T. parva and T. annulata. T. parva and T. annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T. orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within infected erythrocytes. While synteny across homologous chromosomes of the three Theileria species was found to be well conserved overall, subtelomeric structures were found to differ substantially, as T. orientalis lacks the large tandemly arrayed subtelomere-encoded variable secreted protein-encoding gene family. Moreover, expansion of particular gene families by gene duplication was found in the genomes of the two transforming Theileria species, most notably, the TashAT/TpHN and Tar/Tpr gene families. Gene families that are present only in T. parva and T. annulata and not in T. orientalis, Babesia bovis, or Plasmodium were also identified. Identification of differences between the genome sequences of Theileria species with different abilities to transform and immortalize bovine leukocytes will provide insight into proteins and mechanisms that have evolved to induce and regulate this process. The T. orientalis genome database is available at http://totdb.czc.hokudai.ac.jp/., IMPORTANCE Cancer-like growth of leukocytes infected with malignant Theileria parasites is a unique cellular event, as it involves the transformation and immortalization of one eukaryotic cell by another. In this study, we sequenced the whole genome of a nontransforming Theileria species, Theileria orientalis, and compared it to the published sequences representative of two malignant, transforming species, T. parva and T. annulata. The genome-wide comparison of these parasite species highlights significant genetic diversity that may be associated with evolution of the mechanism(s) deployed by an intracellular eukaryotic parasite to transform its host cell.

Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Currently, with the rapid growth of transcriptome data of various species, more reliable orthology information is prerequisite for further studies. However, detection of orthologs could be erroneous if pairwise distance-based methods, such as reciprocal BLAST searches, are utilized. Thus, as a sub-database of H-InvDB, an integrated database of annotated human genes (http://h-invitational.jp/), we constructed a fully curated database of evolutionary features of human genes, called 'Evola'. In the process of the ortholog detection, computational analysis based on conserved genome synteny and transcript sequence similarity was followed by manual curation by researchers examining phylogenetic trees. In total, 18 968 human genes have orthologs among 11 vertebrates (chimpanzee, mouse, cow, chicken, zebrafish, etc.), either computationally detected or manually curated orthologs. Evola provides amino acid sequence alignments and phylogenetic trees of orthologs and homologs. In 'd(N)/d(S) view', natural selection on genes can be analyzed between human and other species. In 'Locus maps', all transcript variants and their exon/intron structures can be compared among orthologous gene loci. We expect the Evola to serve as a comprehensive and reliable database to be utilized in comparative analyses for obtaining new knowledge about human genes. Evola is available at http://www.h-invitational.jp/evola/.

In order to assist the progression of comparative genomics, we have developed a new web-based tool, named G-compass, for browsing and analysis of genome alignments. G-compass utilizes 829,311 pieces of genome alignments between human and mouse that were originally produced for this tool. The quality of the genome alignment set was evaluated by using several statistics. As a result, the alignment set is found to cover approximately 17% of the human genome and 82% of the annotated exons. The averages of nucleotide sequence identity and sequence length are 71.2% and 673.6 bp, respectively. In comparison with public data, it appeared that our data is more expansive and possesses greater genome coverage. G-compass incorporates unique functions such as window analysis of individual alignments. Furthermore, with G-compass and the joint help of H-InvDB, we were able to find highly conserved genomic segments and a human specific antisense transcript candidate, demonstrating that G-compass is useful for facilitating biological discoveries. G-compass is publicly accessible on the WWW at http://www.jbirc.aist.go.jp/g-compass/.

MIST (mast cell immunoreceptor signal transducer; also termed Clnk) is an adaptor protein structurally related to SLP-76-family hematopoietic cell-specific adaptor proteins. We demonstrate here that two major MIST-associated phosphoproteins expressed in mast cell lines are SLAP-130 and SKAP55, adaptors known to interact with the Src-homology (SH) 2 domain of Src-family protein tyrosine kinases (PTKs). MIST directly associated with SLAP-130 via its SH2 domain, and collaboration of SLAP-130 with SKAP55 was required for the recruitment of MIST to Lyn. Furthermore, MIST was preferentially recruited to Fyn rather than Lyn, which is regulated by higher affinity binding of SLAP-130 and SKAP55 with the Fyn-SH2 domain than the Lyn-SH2 domain. Our results suggest that the MIST-SLAP-130-SKAP55 adaptor complex functions downstream of high-affinity IgE receptor-associated Src-PTKs in mast cells.