Rapid screening of lignans from Phyllanthus myrtifolius and stilbenoids from Syagrus romanzoffiana by HPLC-SPE-NMR

Introduction – Application of on‐line solid‐phase extraction (SPE) as an interface between HPLC and NMR has gained great improvement in solving sensitivity problems and signal interferences by the eluents. Objective – Rapid analysis and characterisation by HPLC‐SPE‐NMR and LC/MS of the arylnaphthalene‐type lignans present in Phyllanthus myrtifolius and the minor stilbenoids present in the polyphenol‐rich fraction from the ethanol extract of the seeds of Syagrus romanzoffiana. Methodology – Pretreatment of fractions by liquid–liquid partitioning, followed by Sephadex LH‐20 fractionation, was found very useful to facilitate the focusing and analysis of the polyphenolic fraction. HPLC‐DAD‐SPE‐NMR (400 MHz and 600 MHz) analysis was carried out using an Agilent 1100 liquid chromatography, followed by a Prospekt 2 automated solid‐phase extraction unit, containing 96 HySphere‐Resin GP cartridges (10 × 2 mm, 10–12 µm), which was connected to a 120 or 60 µL LC probe. Results – Seven arylnaphthalene‐type lignans from the chloroform‐soluble fraction of P. myrtifolius and nine stilbenoids from a polyphenol‐rich butanol‐soluble fraction of the seeds of S. romanzoffiana were characterised. Conclusion – HPLC‐SPE‐NMR associated with HR‐ESI/MS, which consumed only analytical amounts of partially purified mixtures, was demonstrated to be a good tool for rapid screening of both known and new natural products. Copyright © 2011 John Wiley & Sons, Ltd.     

An amplified surface plasmon resonance "turn-on" sensor for mercury ion using gold nanoparticles

Inorganic mercury ion (Hg²⁺) has been shown to coordinate to DNA duplexes that feature thymine–thymine (T–T) base pair mismatches. This observation suggests that an Hg²⁺-induced conformational change in a single-stranded DNA molecule can be used to detect aqueous Hg²⁺. Here, we have developed an analytical method using surface plasmon resonance (SPR) to develop a highly selective and sensitive detection technique for Hg²⁺ that takes advantage of T–Hg²⁺–T coordination chemistry. The general concept used in this approach is that the “turn-on” reaction of a hairpin probe via coordination of Hg²⁺ by the T–T base pair results in a substantial increase in the SPR response, followed by specific hybridization with a gold nanoparticle probe to amplify the sensor performance. Meanwhile, the limit of detection is 1 nM, which is lower than other recently developed techniques. A linear correlation is observed between the measured SPR reflectivity and the logarithm of the Hg²⁺ concentration over the concentration range of 5–5000 nM. Additionally, the SPR system provides high selectivity for Hg²⁺ in the presence of other divalent metal ions up to micromolar concentration levels. The proposed approach is also successfully utilized for the determination of Hg²⁺ in water samples.     

Endothelin‐1 Suppresses Long‐Chain Fatty Acid Uptake and Glucose Uptake Via Distinct Mechanisms in 3T3‐L1 Adipocytes

Endothelin‐1 (ET‐1) has been demonstrated to induce insulin resistance (IR) and lipolysis, raising the possibility that ET‐1 may also contribute to the elevated fatty acid levels in IR‐associated comorbidities. We attempted to evaluate whether ET‐1 also affects the long‐chain fatty acid (LCFA) utilization in 3T3‐L1 adipocytes. The effects of chronic ET‐1 exposure on basal and insulin‐stimulated LCFA uptake, and LCFA uptake kinetics were examined in 3T3‐L1 adipocytes. Chronic exposure to ET‐1 induced IR and suppressed basal and insulin‐stimulated LCFA uptake. Given that insulin acutely stimulates LCFA uptake, there was dramatically similar trend of dose‐response curves for ET‐1‐suppressed LCFA uptake, and also similar corresponding IC₅₀ values, between basal and insulin‐stimulated states, reflecting that ET‐1 predominantly suppresses basal LCFA uptake. Results of LCFA kinetics, western blots, and CD36 inhibition using sulfosuccinimidyl oleate (SSO) revealed that suppression of LCFA uptake by ET‐1 is associated with downregulation of CD36. ET type A receptor (ET_AR) antagonist BQ‐610 reversed the IR induction and the ET‐1‐suppressed LCFA uptake. Exogenous replenishment of phosphatidylinositol (PI) 4, 5‐bisphosphate (PIP₂) prevented IR induction, but not the suppression of LCFA uptake by ET‐1. Pharmacological inhibition of the activation of mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) completely blocked the ET‐1‐suppressed LCFA uptake. Serving as an inducer of IR, ET‐1 also chronically suppresses LCFA uptake via PIP₂‐independent and ERK‐dependent pathway. The interplay between impaired glucose disposal and diminished LCFA utilization, induced by ET‐1, could worsen the dysregulation of adipose metabolism and energy homeostasis in insulin‐resistant states.     

Acoustic adaptations to anthropogenic noise in the cicada Cryptotympana takasagona Kato (Hemiptera: Cicadidae)

Anthropogenic noise produced by human activities affects acoustic communication in animals living in urban habitats. We recorded the calling songs of the cicada Cryptotympana takasagona in the Kaohsiung metropolitan areas of southern Taiwan to investigate possible acoustic adaptations to anthropogenic noise. C. takasagona did not call more in noise gaps. Acoustic features (peak frequency, quartile 25%, quartile 50%, and quartile 75%) of calling songs significantly increased with ambient noise levels. C. takasagona shifted the energy distribution of calling songs to higher frequencies in the presence of higher noise levels. We suggest that the acoustic adaptation by which song frequencies increase with levels of anthropogenic noise in C. takasagona may result from a size-dependent calling strategy in which small-sized males call more in noise conditions or large-sized males adjust their song frequency by changing their abdominal cavities.     

The indispensable role of CCR5 for in vivo suppressor function of tumor-derived CD103+ effector/memory regulatory T cells

CD103 is a marker for identification of effector/memory regulatory T cells (Tregs). CD103(+) Tregs are potent suppressors of tissue inflammation in several infectious diseases, autoimmune diseases, and cancers. However, the underlying mechanisms for this potent suppression ability remain unclear. The current study was designed to clarify this issue. Unexpectedly, we found both CD103(+) and CD103(-) Tregs had similar suppression capacity in vitro. We then chose a murine tumor model for investigation of the in vivo behavior of these Tregs. The suppression ability in vivo against the anti-tumor ability of CD8(+) T cells was restricted to CD103(+) Tregs although both Tregs had equal in vitro suppression ability. In addition, CD103(+) Tregs expressed significantly higher levels of CCR5 than those of CD103(-) Tregs and accumulated more in tumors than did CD103(-) Tregs. Furthermore, blockade of CCR5 signaling, either by CCR5(-/-)CD103(+) Tregs or by CCL5 knockdown tumor, could reduce the migration of CD103(+) Tregs into tumors and impair their in vivo suppression ability. In conclusion, these results indicate that the potent in vivo suppression ability of CD103(+) Tregs is due to the tissue-migration ability through CCR5 expression.     

Signal Regulatory Protein α Negatively Regulates β2 Integrin-Mediated Monocyte Adhesion,Transendothelial Migration and Phagocytosis

Background

Signal regulate protein α (SIRPα) is involved in many functional aspects of monocytes. Here we investigate the role of SIRPα in regulating β₂ integrin-mediated monocyte adhesion, transendothelial migration (TEM) and phagocytosis.

Methodology/Principal Findings

THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs) resulted in a decrease of SIRPα expression but an increase of β₂ integrin cell surface expression and β₂ integrin-mediated adhesion to tumor necrosis factor-α (TNFα)–stimulated human microvascular endothelial cell (HMEC-1) monolayers. In contrast, SIRPα overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1)–triggered cell surface expression of β₂ integrins, in particular CD11b/CD18. SIRPα overexpression reduced β₂ integrin-mediated firm adhesion of THP-1 cells to either TNFα–stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1). SIRPα overexpression also reduced MCP-1–initiated migration of THP-1 cells across TNFα–stimulated HMEC-1 monolayers. Furthermore, β₂ integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPα overexpression.

Conclusions/Significance

SIRPα negatively regulates β₂ integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis.     

Inactivation by UDP-glucuronosyltransferase enzymes: the end of androgen signaling

Conjugation by UDP-Glucuronosyltransferase (UGT) is the major pathway of androgen metabolism and elimination in the human. High concentrations of glucuronide conjugates of androsterone (ADT) and androstane-3alpha,17beta-diol (3alpha-diol) are present in circulation and several studies over the last 30 years have concluded that the serum levels of these metabolites might reflect the androgen metabolism in several tissues, including the liver and androgen target tissues. Three UGT2B enzymes are responsible for the conjugation of DHT and its metabolites ADT and 3alpha-diol: UGT2B7, B15 and B17. UGT2B7 is expressed in the liver and skin whereas UGT2B15 and B17 were found in the liver, prostate and skin. Very specific antibodies against each UGT2B enzyme have been obtained and used for immunohistochemical studies in the human prostate. It was shown that UGT2B17 is expressed in basal cells whereas UGT2B15 is only localized in luminal cells, where it inactivates DHT. By using LNCaP cells, we have also demonstrated that the expression and activity of UGT2B15 and B17 are modulated by several endogenous prostate factors including androgen. Finally, to study the physiological role of UGT2B enzymes, transgenic mice bearing the human UGT2B15 gene were recently obtained. A decrease in reproductive tissue weight from transgenic animals compared to those from control animals was observed. In conclusion, the conjugation by UGT2B7, B15 and B17, which represents a non-reversible step in androgen metabolism, is an important means by which androgens are regulated locally. It is also postulated that UGT enzymes protect the tissue from deleteriously high concentrations of active androgen.