Characterization of coumarin-specific prenyltransferase activities in Citrus limon peel

Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-geranyltransferase activity revealed its unique properties: apparent K(m) values for GPP (9 μM) and bergaptol (140 μM) and a broad divalent cation requirement. These findings provide information towards the discovery of a yet unidentified coumarin-specific prenyltransferase gene.     

Evaluation of extra capsular lymph node involvement in patients with extra-hepatic bile duct cancer

ABSTRACT: BACKGROUND: Lymph node metastasis (LNM) is one of the most important prognostic factors for extra-hepatic bile duct carcinoma (ExHBDC). Extra capsular lymph node involvement (ExCLNI) is the extension of cancer cells through the nodal capsule into the perinodal fatty tissue. The prognostic impact of ExCLNI has been shown to be mainly significant in head and neck malignancies. Recently, the prognostic impacts of ExCLNI have been evaluated in gastrointestinal malignancies. However, no data are available regarding the incidence and prognostic significance of extra-capsular lymph node involvement (ExCLNI) in resectable ExHBDCs. The aim of the present study is first to evaluate the incidence of ExCLNI in surgically-treated ExHBDCs and, second, to determine the prognostic impact of ExCLNI in patients with surgically-treated ExHBDCs. METHODS: A total of 228 patients, (110 cases of hilar cholangiocarcinoma and 118 cases of distal cholangiocarcinoma), with surgically-treated ExHBDCs were included in this retrospective study. ExCLNI was defined as the extension of cancer cells through the nodal capsule into the perinodal fatty tissue. The existence of ExCLNI and its prognostic value were analyzed as a subgroup of lymph node metastasis. RESULTS: ExCLNI was detected in only 22% of patients with lymph node metastasis of surgically-treated ExHBDC. The presence of ExCLNI correlated with distal cholangiocarcinoma (P = 0.002). On univariate analysis for survival, perineural invasion, vascular invasion, histological grade, and lymph node metastasis were statistically significant factors. On multivariate analysis, only lymph node metastasis was identified as a significant independent prognostic factor in patients with resectable ExHBDC. Subgroups of lymph node metastasis including the presence of ExCLNI, location of lymph node metastasis, and the number of lymph node metastasis had no statistically significant impact on survival. CONCLUSION: ExCLNI was present in only 22% of the LNM (7% of overall patients) in patients with surgically-treated ExHBDCs. And ExCLNI would have no impact on the survival of patients with surgically-treated ExHBDCs.     

The Rab21-GEF activity of Varp, but not its Rab32/38 effector function, is required for dendrite formation in melanocytes

Vacuolar protein sorting 9 (VPS9)-ankyrin-repeat protein (Varp) has recently been identified as an effector molecule for two small GTPases-Rab32 and Rab38-in the transport of a melanogenic enzyme tyrosinase-related protein 1 (Tyrp1) to melanosomes in melanocytes. Although Varp contains a Rab21-guanine nucleotide exchange factor (GEF) domain (i.e., VPS9 domain), since Rab21-GEF activity is not required for Tyrp1 transport, nothing is known about the physiological significance of the Rab21-GEF activity in melanocytes. Here we show by knockdown-rescue experiments that the Rab21-GEF activity of Varp, but not its Rab32/38 effector function, is required for forskolin-induced dendrite formation of cultured melanocytes. We found that Varp-deficient cells are unable to extend dendrites in response to forskolin stimulation and that reexpression of wild-type Varp or a Rab32/38-binding-deficient mutant Varp(Q509A/Y550A) in Varp-deficient cells completely restores their ability to form dendrites. By contrast, VPS9 mutants (D310A and Y350A) and a vesicle-associated membrane protein 7 (VAMP7)-binding-deficient mutant were unable to support forskolin-induced dendrite formation in Varp-deficient cells. These findings indicate that the Rab21-GEF activity and Rab32/38 binding activity of Varp are required for different melanocyte functions, that is, Rab21 activation by the VPS9 domain is required for dendrite formation, and the Rab32/38 effector function of the ankyrin repeat 1 domain is required for Tyrp1 transport to melanosomes, although VAMP7-binding ability is required for both functions.     

25-hydroxycholesterol enhances cytokine release and toll-like receptor 3 response in airway epithelial cells

Background

25-hydroxycholesterol (25-HC) is one of the oxysterols, which are oxidized derivatives of cholesterol, and has been reported to be involved in the pathogenesis of atherosclerosis and Alzheimer’s disease. In lung, the possible involvement of 25-HC in airway diseases has been revealed. In the present study, we examined whether 25-HC affects the release of cytokines and also modulates the responses of toll-like receptor 3 (TLR3) in airway epithelial cells.

Methods

The effect of 25-HC on the release of cytokines from primary human bronchial epithelial cells after stimulation with or without polyinosine-polycytidylic acid [poly(I:C)], a ligand for TLR3, and the signal transduction were examined.

Results

25-HC significantly potentiated the release of interleukin-8 (IL-8) and IL-6 from the cells. This effect was more potent compared with that of other oxysterols, 22-HC and 27-HC. GW3965 and TO901317, synthetic agonists of liver X receptors that are receptors for oxysterols, did not augment the IL-8 release. 25-HC enhanced the nuclear factor-kappa B (NF-κB) DNA binding activity and translocation of phosphorylated c-Jun into the nucleus. The release of IL-8 was inhibited by the NF-κB inhibitor, caffeic acid phenethyl ester (CAPE), an inhibitor of nuclear factor kappa-B alpha (IκBα) inhibitor, BAY 11–7085, and an inhibitor of nuclear factor kappa-B kinase-2 (IKK-2) inhibitor, SC-514, but not by a c-Jun N-terminal kinase (JNK) inhibitory peptide, L-JNKi1. 25-HC significantly potentiated IL-8 release in poly(I:C)-treated cells and the augmentation was inhibited by CAPE, BAY 11–7085, and SC-514. Furthermore, 25-HC potentiated the translocation of interferon regulatory factor 3 into the nucleus and the release of interferon-beta (IFN-β) in poly(I:C)-treated cells.

Conclusions

These data demonstrated that 25-HC augments the release of IL-8 and IL-6 via NF-κB signalling pathway and enhances the release of IL-8 and IFN-β after stimulation of TLR3 in airway epithelial cells. 25-HC may be involved in the neutrophilic airway inflammation through the stimulant effect of IL-8 and IL-6 release and also potentiate the TLR3-mediated innate immunity in airway diseases.     

Peroxynitrite augments fibroblast-mediated tissue remodeling via myofibroblast differentiation

Irreversible airflow limitation in asthma is associated with airway remodeling in which the differentiation of fibroblasts to myofibroblasts plays a pivotal role. In asthmatic airways, excessive production of reactive nitrogen species (RNS) has been observed. The aim of this study is to evaluate whether peroxynitrite, one of the RNS, can affect the differentiation of fibroblasts to myofibroblasts. Human fetal lung fibroblasts were treated with various concentrations of authentic peroxynitrite or a peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1), and the expressions of alpha-smooth muscle actin (alpha-SMA) and desmin, markers of myofibroblast differentiation, were evaluated. The releases of transforming growth factor-beta(1) (TGF-beta(1)) and ECM proteins including fibronectin and collagen I were assessed. To clarify the mechanism in this differentiation, the effect of anti-TGF-beta antibody or NF-kappaB inhibitors on the alpha-SMA expression and ECM production was assessed. Peroxynitrite and SIN-1 significantly augmented the alpha-SMA expression compared with control in a concentration-dependent manner (P < 0.01 and P < 0.05, respectively). Peroxynitrite significantly increased desmin and TGF-beta(1) production (P < 0.01). Peroxynitrite enhanced the translocation of NF-kappaB into the nucleus confirmed by immunocytostaining and immunoblotting. Peroxynitrite-augmented alpha-SMA expression was blocked by NF-kappaB inhibitors, MG132 and caffeic acid phenethyl ester (CAPE), and anti-TGF-beta antibody. CAPE completely inhibited the peroxynitrite-augmented TGF-beta(1) release. The production of fibronectin and collagen I was significantly increased by peroxynitrite (P < 0.01) and inhibited by anti-TGF-beta antibody. These results suggest that RNS can affect the differentiation to myofibroblasts and excessive ECM production via a NF-kappaB-TGF-beta(1)-dependent pathway.     

Protein phosphatase 2A regulatory subunit Bbeta promotes MAP kinase-mediated migration of A431 cells.

BACKGROUND/AIMS: Phosphatases are involved in regulation of MAP kinase (MAPK). A431 cells migrate on collagen after EGF stimulation using MAPK. To clarify the involvement of PP2A in this MAPK-dependent migration, the expression of an isoform of the B regulatory subunit was inhibited. METHODS: An antisense sequence corresponding to Bbeta cDNA was transfected into A431 cells. Their migratory activity on collagen was examined using Transwell, and MAPK phosphorylation and phosphatase activity were measured, and the results were compared with those obtained with mock-transfected cells. RESULTS: Antisense-transfected cells showed less Bbeta protein and phosphatase activity than mock-transfected controls. Migration of antisense-transfected cells showed a low response to EGF. The response of MAPK phosphorylation of antisense-transfected cells to EGF stimulation and adhesion to collagen in the presence or absence of EGF were markedly decreased. Phosphatase activity of PP2A-Bbeta also did not respond to EGF, collagen or EGF plus collagen, and remained at low levels. CONCLUSION: These results suggested that PP2A-Bbeta promotes cell migration through the MAPK cascade.     

Intraluteal release of angiotensin II and progesterone in vivo during corpora lutea development in the cow: effect of vasoactive peptides.

The newly formed corpus luteum (CL) develops rapidly and has the features of active vascularization and mitosis of steroidogenic cells. Such local mechanisms must be strictly regulated by the complex relationship between angiogenic growth factors and vasoactive peptides such as angiotensin (Ang) II, atrial natriuretic peptide (ANP), and endothelin (ET)-1. Thus, the objective of the present study was to determine 1) the changes in vasoactive peptides and progesterone (P) concentrations within the developing CL, along with the changes in concentration in ovarian venous plasma (OVP) and jugular venous plasma (JVP) in the cow, 2) the effects of CL exposure to vasoactive peptides on Ang II and P secretion, and 3) the expression of mRNA for ANP type C receptor in the bovine CL and endothelial cells (ETC) from bovine developing CL. A microdialysis system (MDS) was surgically implanted into multiple CL of six cows on Day 3 after a GnRH injection that induced superovulation, and a catheter was simultaneously inserted into the ovarian vein. The Ang II concentration in OVP was higher than that in JVP throughout the experiment, while the intraluteal release of Ang II was stable. During the experimental period, the concentrations of other vasoactive peptides (ANP and ET-1) showed no clear changes in plasma and were below detectable levels in the MDS perfusate. Exposure of CL to Ang II using the MDS stimulated P release, while exposure to ANP enhanced Ang II release within the developing CL. However, ET-1 had no effect on either P or Ang II release. The expression of mRNA for ANP type C receptor was mainly observed in early CL and ETC. The results suggest that the ET-Ang-ANP system in the preovulatory follicle switches to an Ang-ANP system to enhance both the angiogenesis and steroidogenesis that are actively occurring in developing CL.     

Effects of LPA1 and LPA6 on the regulation of colony formation activity in colon cancer cells treated with anticancer drugs

Lysophosphatidic acid (LPA) is a simple physiological lipid and exhibits a variety of cellular responses via the activation of G protein-coupled transmembrane LPA receptors (LPA receptor-1 (LPA₁) to LPA₆). The aim of our study was to investigate effects of LPA receptors on soft agar colony formation in colon cancer cells treated with anticancer drugs. DLD1 cells were treated with fluorouracil (5-FU) or cisplatin (CDDP) for at least six months (DLD-5FU and DLD-CDDP cells, respectively). LPAR1 gene expression was markedly elevated in DLD-5FU cells. In contrast, DLD-CDDP cells showed the high expression of LPAR6 gene. In colony formation assay, DLD-5FU cells formed markedly large-sized colonies, while no colony formation was observed in DLD1 and DLD-CDDP cells. The large-sized colonies formed in DLD-5FU cells were suppressed by LPA₁ knockdown. In contrast, LPA₆ knockdown increased the size of colonies. In addition, DLD-5FU cells were further treated with CDDP for three months (DLD-C-F cells). DLD-CDDP cells were also treated with 5-FU (DLD-F-C cells). DLD-C-F cells formed large-sized colonies, but not DLD-F-C cells, correlating with LPAR1 and LPAR6 gene expression levels. These results suggest that LPA₁ and LPA₆ may regulate the colony formation activity in DLD1 cells treated with anticancer drugs.