Biomedical subjects
Publications and source records attributed to Masato Kobori.
We describe the prospects of bioinformatics for drug discovery and discuss the current status, problems, and future direction of the interface between bioinformatics and docking studies. We also describe our recent work on sequence and structure analysis using the guanidino-modifying enzymes superfamily as a good example.
Explore the source record for details and available documents.
Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1- and PKR2-gene-disrupted mice (Pkr1(-/-) and Pkr2(-/-), respectively). Phenotypic analysis indicated that not Pkr1(-/-)but Pkr2(-/-)mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2(-/-) mice. In addition, the Pkr2(-/-) mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2(-/-) mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2(-/-) mice. The phenotype of the Pkr2(-/-) mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.
Several previous linkage scans in type 2 diabetes (T2D) families indicated a putative susceptibility locus on chromosome 12q15-q22, while the underlying gene for T2D has not yet been identified. We performed a region-wide association analysis on 12q15-q22, using a dense set of >500 single-nucleotide polymorphisms (SNPs), in 1492 unrelated Japanese individuals enrolled in this study. We identified an association between T2D and a haplotype block spanning 13.6 kb of genomic DNA that includes the entire SOCS2 gene. Evolutionary-based haplotype analysis of haplotype-tagging SNPs followed by a "sliding window" haplotypic analysis indicated SNPs that mapped to the 5' region of the SOCS2gene to be associated with T2D with high statistical significance. The SOCS2 gene was expressed ubiquitously in human and murine tissues, including pancreatic beta-cell lines. Adenovirus-mediated expression of the SOCS2 gene in MIN6 cells or isolated rat islets significantly suppressed glucose-stimulated insulin secretion. Our data indicate that SOCS2 may play a role in susceptibility to T2D in the Japanese.
We present a simple method for neural cell fate specification directly from mouse embryonic stem cells (ES cells) in serum-free conditions in the absence of embryoid body formation. Dissociated ES cells were cultured in serum-free media supplemented with vitamin B12 and heparin, but without any expensive cytokines. After 14 days in culture, beta-tubulin type III (TuJ1) and tyrosine hydroxylase (TH)-positive colonies were detected by immunocytochemical examinations. In addition, specific gene analyses by RT-PCR demonstrated expression of an early central nerve system, mature neuron, and midbrain dopaminergic neuron-specific molecules (i.e., nestin, middle molecular mass neurofilament protein, Nurr1, and TH, respectively). Dopamine was also detected in the culture media by reverse-phase HPLC analysis. These facts indicate that addition of vitamin B12/heparin to serum-free culture media induced neurons from ES cells, which included cells that released dopamine. Other supplements, such as putrescine, biotin, and Fe2+, could not induce neurons from ES cells by themselves, but produced synergistic effects with vitamin B12/heparin. The rate of TuJ1+/TH+ colony formation was increased threefold and the amounts of dopamine released increased 1.5-fold by the addition of a mixture of putrescine, biotin, and Fe2+ to vitamin B12/heparin culture media. Our method is a simple tool to differentiate ES cells to dopaminergic neurons for the preparation of dopamine-releasing cells for the cell transplantation therapy of Parkinson's disease. In addition, this method can facilitate the discovery of soluble factors and genes that can aid in the induction of the ES cell to its neural fate.
Histamine H(4) receptor is considered as a novel therapeutic target for allergic diseases. To enhance the knowledge about species difference, which is essential for drug discovery research, monkey H(4) receptor was identified. Monkey H(4) receptor was characterized to have comparable similarity with its human counterpart. Discovery of monkey H(4) receptor will contribute to a better interpretation of effective drug discovery.
G-protein-coupled receptors (GPCRs) are important mediators of signal transduction and are therefore potential targets for pharmacological therapeutics. Here, we report the identification and characterization of an orphan GPCR, termed GPRg1, which was found in the GenBank database following searches with GPCR query sequences. Quantitative PCR analysis revealed that GPRg1 transcripts are expressed almost exclusively in the brain. Moreover, in situ hybridization experiments in brain demonstrated that GPRg1 is abundantly expressed in the ventrolateral region of caudate putamen, the habenular nucleus, the zona incerta, and the medial mammillary nucleus. In addition, overexpression of GPRg1 in 293-EBNA cells activates serum response factor mediated transcription, which was completely inhibited by the Gq/11 selective inhibitor YM-254890, indicating the coupling of GPRg1 with Gq/11. These findings suggest that GPRg1 is a candidate receptor for novel physiologically bioactive substrates and that it plays important roles in the central nervous system.
Proadrenomedullin N-terminal 20 peptide (PAMP[1-20]/PAMP-20) and its truncated analog, PAMP[9-20]/PAMP-12, are endogenous peptides that elicit hypotension through inhibiting catecholamine secretion from sympathetic nerve endings and adrenal chromaffin cells. Although the binding sites for PAMP are widely distributed, the nature of its receptor has been elusive. In an effort to identify potential PAMP receptor(s), we found that a human G-protein-coupled receptor, MrgX2, was specifically activated by PAMP. Although a previous study revealed that MrgX2 was a receptor for cortistatin, a neuropeptide involved in sleep regulation and locomotor activity, our present data indicated that the rank order of the agonistic effect against MrgX2 was "PAMP-12> or =cortistatin>PAMP-20". These activities were confirmed by the inhibition of the forskolin-elevated cAMP accumulation, Ca(2+) mobilization, and [(35)S]guanosine 5'-(gamma-thio)triphosphate binding assays. These findings suggest that MrgX2 couples with not only G(alpha q) but also G(alpha i), consistent with previous reports on the pharmacological profile of PAMP signaling. Furthermore, by immunostaining, we found that MrgX2 was expressed in the adrenal chromaffin cells as well as the dorsal root ganglia. From these results, we concluded that MrgX2 is a potential human PAMP-12 receptor that regulates catecholamine secretion from adrenal glands. The present discovery will eventually lead to a better understanding of the pathophysiological role of proadrenomedullin peptides.
A lysophospholipid series, such as lysophosphatidic acid, lysophosphatidylserine, and lysophosphatidylcholine (LPC), is a bioactive lipid mediator with diverse physiological and pathological functions. LPC has been reported to induce insulin secretion from pancreatic beta-cells, however, the precise mechanism has remained elusive to date. Here we show that an orphan G-protein-coupled receptor GPR119 plays a pivotal role in this event. LPC potently enhances insulin secretion in response to high concentrations of glucose in the perfused rat pancreas via stimulation of adenylate cyclase, and dose-dependently induces intracellular cAMP accumulation and insulin secretion in a mouse pancreatic beta-cell line, NIT-1 cells. The Gs-protein-coupled receptor for LPC was identified as GPR119, which is predominantly expressed in the pancreas. GPR119-specific siRNA significantly blocked LPC-induced insulin secretion from NIT-1 cells. Our findings suggest that GPR119, which is a novel endogenous receptor for LPC, is involved in insulin secretion from beta-cells, and is a potential target for anti-diabetic drug development.
Adipose tissue is a source of adult multipotent stem cells that can differentiate along mesenchymal lineage. When mature fat cells obtained from human subcutaneous adipose tissue were maintained with attachment to the ceiling surface of culture flasks filled with medium, two fibroblastic cell populations appeared at the ceiling and the bottom surface. Both populations were positive to CD13, CD90, and CD105, moderately positive to CD9, CD166, and CD54, negative to CD31. CD34, CD66b, CD106, and CD117, exhibited potential of unlimited proliferation, and differentiated along mesenchymal lineage to produce adipocytes, osteoblasts, and chondrocytes. The population that appeared at the ceiling surface showed higher potential of adipogenic differentiation. These observations showed that the cells tightly attached to mature fat cells can generate two fibroblastic cell populations with multiple but distinct potential of differentiation. Since enough number of both populations for clinical transplantation can be easily obtained by maintaining fat cells from a small amount of subcutaneous adipose tissue, this method has an advantage in preparing autologous cells for patients needing repair of damaged tissues by reconstructive therapy.
YM-254890, which was isolated from the culture broth of Chromobacterium sp., inhibits ADP-induced platelet aggregation and has antithrombotic and thrombolytic effects. YM-254890 blocks Galpha(q/11)-coupled ADP receptor P2Y1-mediated Ca(2+) mobilization. Here we report that YM-254890 is a selective Galpha(q/11) inhibitor. YM-254890 blocked Ca(2+) mobilization mediated by several Galpha(q/11)-coupled receptors but not by Galpha(i)- or Galpha(15)-coupled receptor, indicating that phospholipase Cbeta activation and subsequent signaling molecules are not the target of YM-254890. YM-254890 completely prevented the serum response factor (SRF)-mediated gene transcription induced by Galpha(q)R183C, which is constitutively active in a receptor-dependent manner because of its reduced k(cat) of GTP hydrolysis. Conversely, YM-254890 had only a modest effect on the SRF-mediated gene transcription by Galpha(q)Q209L, which is GTPase-deficient (activated) Galpha(q). These suggested that the acting point of YM-254890 is receptor-Galpha(q) interaction or the subsequent guanine nucleotide exchange step. The fact that YM-254890 (i) inhibited the SRF-mediated gene transcription by Galpha(qi5), which interacts with Galpha(i)-coupled receptor and possesses the effector function of Galpha(q), and (ii) had no effect on the K(d) value of high affinity [(3)H]2MeSADP binding to P2Y1, which reflects the agonist-receptor-Galpha ternary complex, suggested that receptor-Galpha(q/11) interaction is not the target of YM-254890. On the other hand, specific [(35)S]GTPgammaS binding to Galpha(q/11) stimulated by the M1 muscarinic acetylcholine receptor and P2Y1 were inhibited by YM-254890. These data indicate that YM-254890 blocks the exchange of GDP for GTP in Galpha(q/11) activation. This novel Galpha(q/11)-selective inhibitor is a promising and powerful tool for studying Galpha(q/11) protein activation, Galpha(q/11) -coupled receptor signaling, and Galpha(q/11)-mediated biological events.