GPR34 is a receptor for lysophosphatidylserine with a fatty acid at the sn-2 position

GPR34 is a G protein-coupled receptor belonging to the P2Y family. Here, we attempted to resolve conflicting reports about whether it is a functional lysophosphatidylserine (LysoPS) receptor. In HEK293 cells expressing human, mouse or rat GPR34 and Gα chimera between Gαq and Gαi1(Gq/i1), LysoPS quickly elevated intracellular Ca(2+) ion levels ([Ca(2+)](i)). LysoPS also stimulated alkaline phosphatase (AP)-tagged TGFα (AP-TGFα) release in GPR34-expressing HEK293 cells and induced the migration of CHO-K1 cells expressing GPR34. Other lysophospholipids did not induce these actions. Replacement of the serine residue of LysoPS abolished the reactivity of LysoPS with GPR34, indicating that GPR34 strictly recognizes the serine head group of LysoPS. Recombinant phosphatidylserine-specific phospholipase A(1) (PS-PLA(1)) that deacylates fatty acid at the sn-1 position of PS and produces 2-acyl-LysoPS, but not catalytically inactive mutant PS-PLA(1), stimulated the release of AP-TGFα from GPR34-expressing cells. Consistent with the result, LysoPS was detected in the cells treated with wild-type PS-PLA(1) but not with the mutant PS-PLA(1). PS treated with PLA(1) was much more effective at stimulating AP-TGFα release than PS treated with PLA(2). In addition, migration-resistant 2-acyl-1-deoxy-LysoPS, a 2-acyl-LysoPS analogue, was much more potent than 1-acyl-2-deoxy-LysoPS. The present studies confirm that GPR34 is a cellular receptor for LysoPS, especially with a fatty acid at the sn-2 position.     

Lead optimization of a dihydropyrrolopyrimidine inhibitor against phosphoinositide 3-kinase (PI3K) to improve the phenol glucuronic acid conjugation

Our lead compound for a phosphoinositide 3-kinase (PI3K) inhibitor (1) was metabolically unstable because of rapid glucuronidation of the phenol moiety. Based on structure–activity relationship (SAR) information and a FlexSIS docking simulation score, aminopyrimidine was identified as a bioisostere of phenol. An X-ray structure study revealed a hydrogen bonding pattern of aminopyrimidine derivatives. Finally, aminopyrimidine derivatives 33 showed strong tumor growth inhibition against a KPL-4 breast cancer xenograft model in vivo.     

Dehydroabietic acid derivatives as a novel scaffold for large-conductance calcium-activated K+ channel openers

We found that the dehydroabietic acid structure is a new scaffold for chemical modulators of large-conductance calcium-activated K+ channels (BK channels). Structure-activity relationship (SAR) studies of the dehydroabietic acid derivatives and related non-aromatic compounds such as pimaric acid revealed the importance of the carboxyl functionality and an appropriate hydrophobic moiety of the molecules for BK channel-opening ability.     

Design and synthesis of a highly selective, orally active and potent anaplastic lymphoma kinase inhibitor (CH5424802)

Anaplastic lymphoma kinase (ALK) receptor tyrosine kinase is considered an attractive therapeutic target for human cancers, especially non-small cell lung cancer (NSCLC). Our previous study revealed that 8,9-side-chains of 6,6-dimethyl-11-oxo-6,11-dihydro-5H-benzo[b]carbazole scaffold crucially affected kinase selectivity, cellular activity, and metabolic stability. In this work, we optimized the side-chains and identified highly selective, orally active and potent ALK inhibitor CH5424802 (18a) as the clinical candidate.     

Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork

DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNA methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNA expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNA expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNA expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNA methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNA methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNA expression and the consequent dynamic changes in global DNA methylation.     

High-dose ascorbic acid induces carcinostatic effects through hydrogen peroxide and superoxide anion radical generation-induced cell death and growth arrest in human tongue carcinoma cells

High-dose ascorbic acid (AsA) treatment, known as pharmacological AsA, has been shown to exert carcinostatic effects in many types of cancer cells and in vivo tumour models. Although pharmacological AsA has potential as a complementary and alternative medicine for anticancer treatment, its effects on human tongue carcinoma have not yet been elucidated. In this study, we investigated the effect of AsA treatment on human tongue carcinoma HSC-4 cells compared with non-tumourigenic tongue epithelial dysplastic oral keratinocyte (DOK) cells. Our results show that treatment with 1 and 3?mM of AsA for 60?min preferentially inhibits the growth of human tongue carcinoma HSC-4 over DOK cells. Furthermore, AsA-induced effects were accompanied by increased intracellular oxidative stress and were repressed by treatment with a hydrogen peroxide (H₂O₂) scavenger catalase and a superoxide anion radical (O₂^?) scavenger, tempol. Time-lapse observation and thymidine analog EdU incorporation revealed that AsA treatment induces not only cell death but also suppression of DNA synthesis and cell growth. Moreover, the growth arrest was accompanied by abnormal cellular morphologies whereby cells extended dendrite-like pseudopodia. Taken together, our results demonstrate that AsA treatment can induce carcinostatic effects through induction of cell death, growth arrest, and morphological changes mediated by H₂O₂ and O₂^? generation. These findings suggest that high-dose AsA treatment represents an effective treatment for tongue cancer as well as for other types of cancer cells.     

Pathologic Findings in Rabbit Models of Hereditary Hypertriglyceridemia and Hereditary Postprandial Hypertriglyceridemia

In recent years, the association between hyperlipidemia and the development of arteriosclerosis has been addressed in several studies. Rabbit models of hypertriglyceridemia (TGH) and postprandial hypertriglyceridemia (PHT) have been developed at the authors'' institute. TGH rabbits manifest pathology similar to that of humans with TGH, such as xanthoma, in addition to atherosclerosis of arterioles. Furthermore, PHT rabbits show visceral obesity, insulin resistance, and impaired glucose tolerance, with pathologic features similar to those of the metabolic syndrome assumed to be the cause of human ischemic heart disease. This study was designed to investigate the histopathologic features of TGH and PHT rabbits. TGH rabbits showed advanced aortic atherosclerosis, accompanied by intimal thickening of coronary and renal arteries, fatty liver changes, and xanthoma. PHT rabbits demonstrated aortic intimal thickening and hepatic fatty degeneration. The results of this study suggest that TGH and PHT rabbits are useful animal models for studying human hyperlipidemia and metabolic syndrome and the cardiovascular diseases that result from these conditions.Abbreviations: LDL, low-density lipoprotein; PHT, postprandial hypertriglyceridemia; TGH, high triglycerideThe 3 leading diseases causing death in humans currently are cancer, cardiac disease (ischemic heart disease), and cerebrovascular accidents. Because the total number of deaths attributed to ischemic heart disease and cerebrovascular accidents exceeds those due to cancer, seeking a remedy for cardiovascular disease is an important task in current medical research.Cardiac disease and cerebrovascular accidents are caused by arteriosclerosis, and high cholesterol levels play an important role in the onset of this disease.^44,46 However, not all patients affected by ischemic heart disease have high cholesterol levels, and hypertriglyceridemia recently has been reported to be associated with the onset of ischemic heart disease.^2,12,18,26 Patients with visceral fat accumulation, hypertriglyceridemia, glucose intolerance, and hypertension have high risk for cardiovascular disease.^32,41,47 These relationships indicate the importance of determining the mechanism that underlie of the onset of hypertriglyceridemia and to establish prophylactic and therapeutic modalities.Many animal models, including WHHL rabbits,^1,45 KHC rabbits,²³ and mice deficient in receptors for low-density lipoproteins (LDL),⁴ used to study hyperlipemia have innately high cholesterol levels. Among rodent models of hyperlipemia, apoE-deficient mice²⁹ and LDL receptor-deficient mice⁴ are well known. Rodents produce apoB48-containig lipoproteins in liver, they have high levels of high-density lipoprotein–cholesterol. However, unlike humans (who produce apoB100-containing very-low–density lipoproteins), rodents lack cholesteryl ester transfer protein, which plays a key role in the onset of atherosclerosis, and the underlying mechanism therefore differs from that of human lipoprotein metabolism.⁵ Therefore the usefulness of comparisons between humans and rodents in regard to lipid metabolism is limited.In contrast to rodents, rabbits and humans are similar with regard to the chemical composition and content of lipoproteins (including apoB), production of lipoproteins (including apoB100) in the liver, and the presence of cholesteryl ester transfer protein.⁵ WHHL and KHC rabbits are well known as hypercholesterolemic rabbit models. WHHL rabbits also are prone to developing marked coronary atherosclerosis,³⁷ and WHHLMI rabbits derived from WHHL have symptoms of spontaneous myocardial infarction.³⁸ These models are used in various medical fields such as in the study of the mechanism of lesion onset and development of curative therapy.^36,38 Two groups^3,25 have developed the SMHL rabbit as a hypertriglyceridemia model. However, neither the triglyceride concentration nor the cholesterol level in the blood of these rabbits exceeded 300 mg/dl even if fatty foods were supplied, and both triglyceride and cholesterol undergo lipolysis by complex mechanisms.⁶ Therefore, the SMHL rabbit is unsuitable for confirming the influence of triglyceride in cardiac disease and atherosclerosis.Scientists at the Yamagata University Faculty of Medicine have developed inbred rabbit models of both high hereditary triglyceridemia (the TGH model) and hereditary postprandial hypertriglyceridemic (the PHT rabbit) by selectively mating WHHL rabbits and normal Japanese White (JW) rabbits and screening blood triglycerides values^14,15,43 (Figure 1).The hereditary type of TGH shows autosomal recessive inheritance,^14,43 and TGH rabbits show blood cholesterol values in excess of 1000 to 1500 mg/dl and triglyceride values greater than 500 mg/dl. In addition, physical examination and necropsy of TGH rabbits often reveal xanthomas and lesions of aortic atherosclerosis similar to those in human disease.^9,11Open in a separate windowFigure 1.Breeding schema for TGH and PHT rabbits. TGH rabbits were bred with the progeny of a cross between JW and WHHL. TGH rabbits were generated by selectively crossbreeding animals with plasma triglyceride values of 500 mg/dl or more. In addition, progeny rabbits that showed plasma triglyceride levels of 50 to 100 mg/dl after 24 h of feed deprivation and 500 to 3000 mg/dl after 24 of continuous food supply obtained as a result of breeding TGH (F1) × TGH (F1) were separated into the PHT subpopulation.The PHT rabbit model was established from the F2 generation of TGH × JW crosses and shows increased postprandial blood levels of cholesterol (100 to 150 mg/dl) and triglyceride (500 to 3000 mg/dl), despite fasting levels similar to those in JW rabbits (cholesterol, 50 to 100 mg/dl; triglyceride, 50 to 100 mg/dl). In addition, the PHT rabbit model shows visceral fat accumulation,^14,19 glucose intolerance,^13-15,19 insulin resistance,^13,14,16,19 and a syndrome very similar to the metabolic syndrome^8,47 that is considered to be the origin of human ischemic heart disease. Therefore, the TGH and PHT rabbit models are considered to be extremely useful in studies of lifestyle-related diseases such as hyperlipidemia, obesity, glucose intolerance, and insulin resistance and to identify prophylactic treatments as well as remedies for such conditions.Thus far, studies of the PHT and TGH rabbit models have been conducted mainly from the viewpoint of clinical and blood biochemistry.^13-17,19 This study therefore was designed to investigate the histopathologic findings in the main internal organs of TGH and PHT rabbits.     

Differentiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage

M cells are a kind of intestinal epithelial cell in the follicle-associated epithelium of Peyer's patches. These cells can transport antigens and microorganisms into underlying lymphoid tissues. Despite the important role of M cells in mucosal immune responses, the origin and mechanisms of differentiation as well as cell death of M cells remain unclear. To clarify the mechanism of M cell differentiation, we established a novel murine intestinal epithelial cell line (MIE) from the C57BL/6 mouse. MIE cells grow rapidly and have a cobblestone morphology, which is a typical feature of intestinal epithelial cells. Additionally, they express cytokeratin, villin, cell-cell junctional proteins, and alkaline phosphatase activity and can form microvilli. Their expression of Musashi-1 antigen indicates that they may be close to intestinal stem cells or transit-amplifying cells. MIE cells are able to differentiate into the M cell lineage following coculture with intestinal lymphocytes, but not with Peyer's patch lymphocytes (PPL). However, PPL costimulated with anti-CD3/CD28 MAbs caused MIE cells to display typical features of M cells, such as transcytosis activity, the disorganization of microvilli, and the expression of M cell markers. This transcytosis activity of MIE cells was not induced by T cells isolated from PPL costimulated with the same MAbs and was reduced by the depletion of the T cell population from PPL. A mixture of T cells treated with MAbs and B cells both from PPL led MIE cells to differentiate into M cells. We report here that MIE cells have the potential ability to differentiate into M cells and that this differentiation required activated T cells and B cells.