Endothelin‐1 increases the expression of VEGF‐R1/Flt‐1 receptors in rat cultured astrocytes through ETB receptors

Expressions of vascular endothelial growth factor (VEGF) receptors in astrocytes are increased in damaged brains. To clarify the regulatory mechanisms of VEGF receptors, the effects of endothelin‐1 (ET‐1) were examined in rat cultured astrocytes. Expressions of VEGF‐R1 and ‐R2 receptor mRNA were at similar levels, whereas the mRNA expressions of VEGF‐R3 and Tie‐2, a receptor for angiopoietins, were lower. Placenta growth factor, a selective agonist of the VEGF‐R1 receptor, induced phosphorylation of focal adhesion kinase (FAK) and extracellular signal regulated kinase 1/2 (ERK1/2). Phosphorylations of FAK and ERK 1/2 were also stimulated by VEGF‐E, a selective VEGF‐R2 agonist. Increased phosphorylations of FAK and ERK1/2 by VEGF₁₆₅ were reduced by selective antagonists for VEGF‐R1 and ‐R2. Treatment with ET‐1 increased VEGF‐R1 mRNA and protein levels. The effects of ET‐1 on VEGF‐R1 mRNA were mimicked by Ala^1,3,11,15‐ET‐1, a selective agonist for ET_B receptors, and inhibited by BQ788, an ET_B antagonist. ET‐1 did not affect the mRNA levels of VEGF‐R2, ‐R3, and Tie‐2. Pre‐treatment with ET‐1 potentiated the effects of placenta growth factor on phosphorylations of FAK and ERK1/2. These findings suggest that ET‐1 induces up‐regulation of VEGF‐R1 receptors in astrocytes, and potentiates VEGF signals in damaged nerve tissues.

     

Reproduction of an alien Ruditapes clam (Bivalvia: Veneridae) on recreational clam-gathering grounds in Tokyo Bay,Japan

The Manila clam Ruditapes philippinarum is an important fisheries resource and popular recreational native species in Japan. To counter the domestic shortage of Manila clams caused by depleted catches, similar clams were introduced into Japan from China and the Korean peninsula. The introduced clam is an unnamed morph closely related to R. philippinarum that originates from southern China. Its shell is morphologically different from that of the typical Manila clam and there are small but distinct genetic differences. There is evidence to suggest that the Chinese morph may hybridize with the Manila clam in the Ariake Sea, but not in Tokyo Bay. We carried out studies to determine the degree of overlap of the breeding seasons of both taxa by collecting samples of the Manila clam and the alien clam released onto recreational clam-gathering grounds in Tokyo Bay and their gonadal maturity was examined histologically. Spawning-stage individuals of the Manila clam appeared from April to August and those of the southern Chinese morph from July to August, indicating overlapping spawning periods and thus the potential for hybridization. We discussed the forming condition of the hybridized clam population in terms of reproduction and recruitment process of the clams.     

Exosomes from IL-1β stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes

Introduction

Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells.

Methods

Human synovial fibroblasts (SFB) and articular chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal articular chondrocytes were treated with exosomes from SFB, and Interleukin-1β (IL-1β) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis.

Results

Exosomes from IL-1β stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in articular chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1β stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1β, TNFα MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1β stimulated SFB compared to non-stimulated SFB.

Conclusions

Exosomes from IL-1β stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints.     

Alternative Mammalian Target of Rapamycin (mTOR) Signal Activation in Sorafenib-resistant Hepatocellular Carcinoma Cells Revealed by Array-based Pathway Profiling

Sorafenib is a multi-kinase inhibitor that has been proven effective for the treatment of unresectable hepatocellular carcinoma (HCC). However, its precise mechanisms of action and resistance have not been well established. We have developed high-density fluorescence reverse-phase protein arrays and used them to determine the status of 180 phosphorylation sites of signaling molecules in the 120 pathways registered in the NCI-Nature curated database in 23 HCC cell lines. Among the 180 signaling nodes, we found that the level of ribosomal protein S6 phosphorylated at serine residue 235/236 (p-RPS6 S235/236) was most significantly correlated with the resistance of HCC cells to sorafenib. The high expression of p-RPS6 S235/236 was confirmed immunohistochemically in biopsy samples obtained from HCC patients who responded poorly to sorafenib. Sorafenib-resistant HCC cells showed constitutive activation of the mammalian target of rapamycin (mTOR) pathway, but whole-exon sequencing of kinase genes revealed no evident alteration in the pathway. p-RPS6 S235/236 is a potential biomarker that predicts unresponsiveness of HCC to sorafenib. The use of mTOR inhibitors may be considered for the treatment of such tumors.Hepatocellular carcinoma (HCC)¹ is the third most common cause of cancer-related death worldwide (1). Advanced HCC often cannot be managed with local treatments (surgical resection, ethanol injection, radiofrequency ablation, chemoembolization), but no systemic chemotherapy with conventional cytotoxic agents had been shown to be effective until a landmark phase III clinical trial (the Sorafenib HCC Assessment Randomized Protocol) revealed significant survival prolongation in patients treated with sorafenib (Nexavar; Bayer Healthcare Pharmaceuticals Inc. Berlin, Germany) (2). Furthermore, it has been reported that some patients show remarkable tumor shrinkage after short-term administration of sorafenib (3). Based on these results, sorafenib monotherapy has been employed as the current standard first-line treatment for unresectable HCC. However, not all HCC patients show the desired therapeutic benefits of sorafenib. The overall survival prolongation of unselected patients in the SHARP trial was limited to 2.8 months (2), and an objective tumor response was observed only in a small proportion of patients (0.6% to 2%) (2, 4). Given the relatively high cost and occasional severe adverse events (diarrhea, hand-foot skin reaction, hypertension, and others) (2, 4), there is an urgent need to identify a predictive biomarker that could exclude advanced HCC patients who are unlikely to benefit from sorafenib therapy.Sorafenib is a multi-kinase inhibitor that blocks tumor cell proliferation and angiogenesis through the inhibition of c-RAF and b-RAF, as well as many receptor tyrosine kinases, including vascular endothelial growth factor receptors 2 and 3, platelet-derived growth factor receptor-α, Fms-related tyrosine kinase 3, RET, and c-KIT (5). In view of this broad inhibitory spectrum, the precise mechanisms underlying the anti-tumor activity remain elusive. To date, factors that have been identified as correlated with the efficacy of sorafenib include phosphorylated extracellular signal-regulated kinase 1 (p-ERK) (6), serum des-γ-carboxyprothrombin level (7), phosphorylated c-Jun protein (8), and fibroblast growth factor-3/4 gene amplification (3), but their clinical utility as predictive biomarkers has not been established.In the present study, we developed a new technique, high-density fluorescence reverse-phase protein array (RPPA), and used it to search for a biomarker that would identify patients in whom sorafenib would be effective, employing a large library of phosphorylation-site-specific antibodies. RPPA represents an emerging technology for proteomics, and it is well suited for the profiling of phosphorylated proteins. It involves micro-format dot immunoblotting of lysates from tissues or cells (9), allowing simultaneous monitoring of the expression of a particular phosphoprotein in hundreds to thousands of samples under identical conditions in a highly quantitative manner (10). In this study we profiled the activation status of 180 key signaling nodes across a panel of 23 HCC cell lines and identified de novo activation of mTOR signaling in sorafenib-resistant HCC cells.     

A high-throughput screening utilizing intramolecular fluorescence resonance energy transfer for the discovery of the molecules that bind HIV-1 TAR RNA specifically

A 16-residue peptide, including the Tat(49-57) sequence was labeled with a fluorescein and a tetramethylrhodamine at its N- and C-terminus, respectively. This double dye-labeled peptide was prepared as a tracer for high-throughput screening utilizing intramolecular fluorescence resonance energy transfer (FRET). The binding of the competitor molecules for HIV-1 TAR RNA were monitored and dissociation constants of those molecule were determined by using this tracer. This novel screening system might be useful to discover the drug for HIV-1 TAR RNA.     

A novel l-isoleucine hydroxylating enzyme, l-isoleucine dioxygenase from Bacillus thuringiensis, produces (2S,3R,4S)-4-hydroxyisoleucine

The unique function of 4-hydroxyisoleucine (4-HIL) is to stimulate glucose-induced insulin secretion in a glucose-dependent manner. 4-HIL is distributed only in certain kinds of plants and mushrooms, but the biosynthetic mechanism of 4-HIL has not been elucidated. Moreover, 4-HIL-producing microorganisms have not been reported. l-isoleucine (l-Ile) hydroxylating activity producing 4-HIL was detected in a cell lysate of Bacillus thuringiensis strain 2e2 AKU 0251 obtained from the mid-late exponential phase of growth. Properties of the purified hydroxylase demonstrated that it is a α-ketoglutaric acid (α-KG) dependent l-Ile dioxygenase (IDO) and requires α-KG, ferric ion, and ascorbic acid for its maximum activity. IDO showed high stereoselectivity in l-Ile hydroxylation producing only (2S,3R,4S)-4-HIL. The N-terminal 22 amino acids sequence revealed high homology to a hypothetical protein (GenBank ID: RBTH_06809) in B. thuringiensis serovar israelensis ATCC 35646. The histidine motif, which is conserved in α-KG dependent dioxygenases, is found in RBTH_06809.     

Chanarin–Dorfman syndrome: Deficiency in CGI-58, a lipid droplet-bound coactivator of lipase

Chanarin–Dorfman syndrome (CDS) is a rare autosomal recessive disease of lipid metabolism; it is associated with congenital ichthyosis typed as non-bullous congenital ichthyosiform erythroderma (NCIE). CDS is characterized by the presence of an abnormally large number of cytosolic lipid droplets containing triacylglycerol (TG) in various tissues such as the skin, liver, and leukocytes. Mutations in the CGI-58 (also called ABHD5) gene encoding a 39-kDa protein of the α/β hydrolase domain subfamily have been shown to be responsible for this disorder. In adipocytes, CGI-58 is involved in TG degradation on lipid droplets; in doing so, it coordinates with several lipolytic factors including perilipin, a member of the PAT protein family, and ATGL, a putative rate-limiting lipase in adipocytes. In quiescent adipocytes, CGI-58 interacts with perilipin on the surfaces of lipid droplets. Upon hormonal stimulation, CGI-58 facilitates massive lipolysis by activating ATGL. Some CGI-58 mutations found in CDS patients cancel the ability to interact with perilipin or activate ATGL, indicating that the loss of these interactions is physiologically important. However, based on the tissue distributions of these lipolytic factors, there are likely multiple molecular targets of CGI-58 actions. This in turn gives rise to the multiple phenotypes of CDS, such as ichthyosis, liver steatosis, or neurosensory diseases.     

Chemokine receptor expression and functional effects of chemokines on B cells: implication in the pathogenesis of rheumatoid arthritis

Introduction  

Accumulation of B cells in the rheumatoid arthritis (RA) synovium has been reported, and it has been thought that these cells might contribute to the pathogenesis of RA by antigen presentation, autoantibody production, and/or inflammatory cytokine production. Chemokines could enhance the accumulation of B cells in the synovium. The aims of this study were to determine chemokine receptor expression by B cells both in the peripheral blood of normal donors and subjects with RA, and at the inflammatory site in RA, and the effects of chemokines on B cell activation.     

Association of the insulin-like growth factor1 gene with myocardial infarction in Japanese subjects

During adult life, the insulin/insulin-like growth factor1 (IGF1) signaling pathway plays an important role in cardiovascular function. Several reports have suggested that low baseline levels of IGF1 increase the risk of fatal ischemic heart disease. Thus, IGF1 may be involved in cardiovascular disease. The aim of the present study was to investigate the relationship between the human IGF1 gene and myocardial infarction (MI) in the Japanese population via the use of single nucleotide polymorphisms (SNPs). After selecting six SNPs in the human IGF1 gene (rs2162679, rs7956547, rs2288378, rs2072592, rs978458 and rs6218), we performed a case-control study using each of the SNPs and haplotypes in 320 MI patients and 307 non-MI controls. Multiple logistic regression analysis demonstrated that the GG+GA variant of rs2162679 (p=0.009) and the AA+GA variant of rs2072592 (p=0.026) exhibited a resistant effect for MI. The haplotype-based case-control study revealed that the frequency of the A-T-G-G haplotype for rs2162679-rs7956547-rs2072592-rs978458 was significantly higher in the MI group (47.3%) as compared to the non-MI group (41.4%) (p=0.037, odds ratio=1.270). The frequency of the A-T-G-T haplotype for rs2162679-rs7956547-rs978458-rs6218 was also significantly higher in the MI group (47.3%) as compared to the non-MI group (41.3%) (p=0.033, odds ratio=1.276). The current results suggest that specific SNPs and haplotypes can be utilized as genetic markers for MI risk or MI resistance. In addition, IGF1 or a neighboring gene might be associated with increased or decreased susceptibility to MI.     

Halophilic β-lactamase as a new solubility- and folding-enhancing tag protein: production of native human interleukin 1α and human neutrophil α-defensin

The amino acid composition of halophilic enzymes is characterized by an abundant content of acidic amino acid, which confers to the halophilic enzymes extensive negative charges at neutral pH and high aqueous solubility. This negative charge prevents protein aggregation when denatured and thereby leads to highly efficient protein refolding. β-Lactamase from periplasmic space of moderate halophile (BLA), a typical halophilic enzyme, can be readily expressed as a native, active form in Escherichia coli cytoplasm. Similar to other halophilic enzymes, BLA is soluble upon denaturation by heat or urea treatments and, hence, can be efficiently refolded. Such high solubility and refolding efficiency make BLA a potential fusion partner for expression of aggregation-prone heterologous proteins to be expressed in E. coli. Here, we succeeded in the soluble expression of several “difficult-to-express” proteins as a BLA fusion protein and verified biological activities of human interleukin 1α and human neutrophil α-defensin, HNP-1.