Functional analysis of chick ONT1 reveals distinguishable activities among olfactomedin-related signaling factors

The Olfactomedin family is a relatively new class of extracellular proteins. Two family members have been shown to play roles in the early development of ectodermal tissues: Noelin enhances neural crest generation in chick and Tiarin promotes dorsal neural specification in Xenopus. In this study, we introduce a novel member of the Olfactomedin family, ONT1. In the early chick embryo, ONT1 expression first appears at Hensen's node and subsequently in the axial and paraxial mesoderm. When the neural tube closes, strong expression of ONT1 is transiently found in the roof plate region from the rostral midbrain to the hindbrain. Overexpression of ONT1 in these regions prolongs the generation of neural crest cells in a manner similar to that of Noelin. Interestingly, ONT1 and Noelin have opposing effects on the expression of the migrating neural crest marker HNK-1 in the chick: they, respectively, cause suppression and ectopic induction of this marker. Differential activities among Olfactomedin-related factors are further examined in Xenopus. Microinjection of ONT1 mRNA into the Xenopus embryo expands the expression domain of the neural crest marker FoxD3 at the neurula stage whereas overexpression of Tiarin or Noelin suppresses FoxD3. ONT1 exhibits no dorsalizing effects on the Xenopus neural tube, which contrasts with the strong dorsalizing activity seen for Tiarin. Thus, distinct Olfactomedin-related factors evoke qualitatively different phenotypes even in the same experimental systems, suggesting that Olfactomedin family uses multiple response systems to mediate its signals in embryogenesis.     

Identification of 4-amino-2-cyclohexylaminoquinazolines as metabolically stable melanin-concentrating hormone receptor 1 antagonists

The optimization of the distance between two key pharmacophore features within our first hit compounds 1a and 2a led to the identification of a new class of potent non-peptidic antagonists for the MCH-R1, based around 4-amino-2-cyclohexylaminoquinazolines. In particular, ATC0065 (2c), N2-[cis-4-([2-[4-Bromo-2-(trifluoromethoxy)phenyl]ethyl]amino)cyclohexyl]-N4,N4-dimethylquinazoline-2,4-diamine dihydrochloride, bound with high affinity to the MCH-R1 (IC50 value of 16 nM) and showed good metabolic stability in liver microsomes from human and rat.     

A role for inflammatory mediators in the induction of immunoregulatory B cells

UV exposure suppresses the immune response to a variety of microbial, fungal, and viral Ags. In addition, UV radiation is a complete carcinogen and the immune suppression induced by UV radiation is a major risk factor for skin cancer induction. In this study, we examined the mechanisms underlying the induction of immune suppression and tolerance induction by UV radiation. Transferring lymph nodes cells from UV-irradiated, FITC-sensitized mice into normal recipients transferred immune tolerance. Contrary to expectations, the cell responsible was an FITC(+), IL-10-secreting, CD19(+), B220(+) B cell. Because the lipid mediator of inflammation, platelet-activating factor (PAF) is released by UV-irradiated keratinocytes and is essential for the induction of immune suppression, we determined its role in tolerance induction. When UV-irradiated mice were injected with PCA 4248, a selective PAF receptor (PAFR) antagonist, transfer of tolerance was suppressed. However, immune suppression was not transferred when FITC(+) cells from the draining lymph nodes of UV-irradiated, PAFR-deficient donor mice were injected into the recipients. Because PCA 4248 also blocks serotonin receptor binding, we measured the effect that blocking both serotonin and PAFR binding has on the transfer of immune suppression. Only when both PAF and serotonin binding were blocked could we inhibit tolerance induction. These data identify a novel function for PAF and serotonin in modulating immune function, the activation of immunoregulatory B cells.     

Comparative study on the relationship between photoperiodic host-seeking behavioral patterns and the eye parameters of mosquitoes

Relationships between the ommatidial structure and photoperiodic behavior of several mosquito species were investigated. Host-seeking behavioral patterns of mosquitoes were classified into four main groups based on previously compiled reports on field or laboratory biting activity. These groups were pattern I and I' (nocturnal), pattern II (crepuscular and nocturnal), pattern III (crepuscular and diurnal), and pattern IV (diurnal). Eye parameters (product of facet diameter and interommatidial angle) of mosquitoes that belong to the pattern I and I' group were higher (2.7-4.2) than those of mosquitoes that belong to the pattern IV group (0.8-2.3). Eye parameters of the mosquitoes categorized in the pattern II and III groups were intermediate (2.3-2.6). These results suggest that the crepuscular behavior of mosquitoes undergoes a transition in the course of evolution from nocturnal behavior to diurnal behavior. Large variations in the eye parameters were observed even within the same genus depending on their photoperiodic behavior. Therefore, the ommatidial structure of mosquitoes appears to be determined, not taxonomically, but evolutionarily by the photoenvironment in which the mosquitoes are most active.     

Ellagic acid in pomegranate suppresses resistin secretion by a novel regulatory mechanism involving the degradation of intracellular resistin protein in adipocytes

Resistin, an adipocytokine, is considered the link between obesity and type 2 diabetes. Pomegranate is a rich source of compounds used to treat metabolic diseases including type 2 diabetes. In this study, we found that consumption of pomegranate fruit extract (PFE) predominantly reduced the serum resistin levels in ovariectomized mice, an animal model with elevated resistin levels in serum and upregulated resistin mRNA expression in white adipose tissue. Moreover, the PFE significantly reduced the secretion and intracellular protein levels of resistin in differentiated murine 3T3-L1 adipocytes, but it did not alter resistin mRNA expression. When de novo protein synthesis was inhibited by the protein synthesis inhibitor cycloheximide, the intracellular resistin protein levels were drastically reduced by the PFE, suggesting that the PFE promoted the degradation of resistin at the protein level. We also found that ellagic acid (EA), a main component of pomegranate, had the same effects on the secretion and intracellular protein level of resistin. These results suggest that EA in pomegranate suppresses resistin secretion by a novel mechanism involving the degradation of intracellular resistin protein in adipocytes.     

Decalpenic acid induces early osteoblastic markers in pluripotent mesenchymal cells via activation of retinoic acid receptor γ

Decalpenic acid is a natural small molecule previously isolated from the fermentation broth of fungi that induces early osteoblastic markers in pluripotent mesenchymal cells. Treatment of mouse pluripotent mesenchymal C3H10T1/2 cells with decalpenic acid gave rise to a morphological change similar to that induced by the treatment with retinoic acid, i.e. the cells adopted a more elongated spindle shape. Using a retinoic acid response element reporter and receptor activity assays, we show that decalpenic acid is a new retinoid with selectivity towards retinoic acid receptors γ and α. The induction of early osteoblastic markers by decalpenic acid was significantly inhibited by treatment with the retinoid antagonist, LE540, or with small interfering RNA-mediated knockdown of retinoic acid receptor γ. These results demonstrated that decalpenic acid induces early osteoblastic markers in pluripotent mesenchymal cells through activation of retinoic acid receptor γ.     

Serpin B1 protects colonic epithelial cell via blockage of neutrophil elastase activity and its expression is enhanced in patients with ulcerative colitis

Serpin B1 is a monocyte neutrophil elastase (NE) inhibitor and is one of the most efficient inhibitors of NE. In the present study, we investigated the role of serpin B1 in the pathogenesis of ulcerative colitis by using clinical samples and an experimental model. The colonic expression of serpin B1 was determined by real-time polymerase chain reaction (PCR), Western blot analysis, and immunohistological studies in both normal and inflamed mucosa from patients with ulcerative colitis. Serpin B1 mRNA expression was determined by real-time PCR in the mouse dextran sodium sulfate (DSS)-induced colitis model. Young adult mouse colonic epithelial (YAMC) cells were used to determine the role of serpin B1. Serpin B1 gene transfected YAMC cells were treated with H(2)O(2) to measure cell viability. The expression of NE was determined in YAMC cells treated with H(2)O(2). NE-silenced YAMC cells were also treated with H(2)O(2) and then measured for viability. Upregulated expression of serpin B1 in colonic mucosa was confirmed from patients with active ulcerative colitis. Immunohistochemical studies showed that serpin B1 expression was localized not only in inflammatory infiltration cells but also in epithelial cells. Serpin B1 mRNA expression was also increased in colonic mucosa of mouse DSS-induced colitis. Serpin B1-transfected YAMC cells were resistant against the treatment of H(2)O(2). H(2)O(2) treatment significantly induced NE in YAMC cells, and NE-silenced YAMC cells were also resistant against the treatment of H(2)O(2). These results suggest that serpin B1 may be a novel marker of active ulcerative colitis and may play an important role in the pathogenesis of inflammatory bowel disease.     

Multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study

Biomarkers are most frequently proteins that are measured in the blood. Their development largely relies on antibody creation to test the protein candidate performance in blood samples of diseased versus nondiseased patients. The creation of such antibody assays has been a bottleneck in biomarker progress due to the cost, extensive time, and effort required to complete the task. Targeted proteomics is an emerging technology that is playing an increasingly important role to facilitate disease biomarker development. In this study, we applied a SRM-based targeted proteomics platform to directly detect candidate biomarker proteins in plasma to evaluate their clinical utility for pancreatic cancer detection. The characterization of these protein candidates used a clinically well-characterized cohort that included plasma samples from patients with pancreatic cancer, chronic pancreatitis, and healthy age-matched controls. Three of the five candidate proteins, including gelsolin, lumican, and tissue inhibitor of metalloproteinase 1, demonstrated an AUC value greater than 0.75 in distinguishing pancreatic cancer from the controls. In addition, we provide an analysis of the reproducibility, accuracy, and robustness of the SRM-based proteomics platform. This information addresses important technical issues that could aid in the adoption of the targeted proteomics platform for practical clinical utility.     

Non-targeted metabolite profiling in activated macrophage secretion

Periodontal diseases are inflammatory infectious diseases that affect the periodontal tissue. Macrophages play a central role in inflammatory conditions, leading to the destruction of tissues. Identifying the signaling molecules secreted by macrophages would be valuable to the study of these diseases. Here, we present non-targeted analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) for the profiling of extracellular metabolites released during macrophage activation. Lipopolysaccharide (LPS)-induced activation of a mouse macrophage-like cell line RAW264.7 was used as a model system. Cells were treated without (control) or with LPS for 22?h and, after washing, were incubated for 1?h in phosphate-buffered saline. The accumulation of metabolites in the culture supernatant was monitored. LPS treatment significantly enhanced the accumulation of prostaglandins, tumor necrosis factor-??, nitric oxide and citrulline in the culture medium. RAW264.7 cells produced 46 metabolites and 66% of these showed significant changes (P?<?0.05) following cell activation. In particular, the production of leucine, hypoxanthine, choline, putrecine, N ₈-acetylspermidine, succinate, itaconate, and 4-methyl-2-oxopentanoate was significantly increased by cell activation (P?<?0.001). Significantly elevated production of lactate and glycine was also observed. Here, we present the first catalog of the up and down-regulation of the various metabolites secreted by macrophages following inflammatory activation.