Preparation of a fluorescent derivative of benzoylecgonine, and preliminary studies of its application to the analysis of urine

A sensitive high-performance liquid chromatographic method using 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone (Br-DMEQ) as a fluorescent labeling reagent is described for the determination of benzoylecgonine (BE) and ecgonine (EC). The Br-DMEQ derivatives of BE and EC were separated on a C₁₈ column and detected at 455 nm with excitation at 370 nm. The detection limits of the proposed method were 18.7 fmol for BE and 12.5 pmol for EC at a signal-to-noise ratio of 3. Relative standard deviations of five replicate measurements were 1.94% (10 pmol) and 2.98% (50 pmol) for BE and 6.3% (250 pmol) and 5.62% (1.25 pmol) for EC. This method was applied to the determination of BE in human urine. BE was extracted from urine by solvent extraction with chloroform—isopropyl alcohol (9:1, v/v) solution. Levels of 2.5 · 10⁻⁸ M BE in urine (25 pmol/ml) could be determined.     

Liquid chromatographic determination of acetylcholine based on pre‐column alkaline cleavage reaction and post‐column tris(2,2′‐bipyridyl)ruthenium(III) chemiluminescence detection

A method for the determination of acetylcholine (ACh) has been developed using liquid chromatography with chemiluminescence detection. This method is based on the pre‐column alkaline cleavage of ACh to form trimethylamine (TMA) and the post‐column tris(2,2′‐bipyridyl)ruthenium(III) chemiluminescence detection of TMA. ACh was converted to TMA with high yield at 180°C in the presence of lithium hydroxide, and the produced TMA was separated on a cation‐exchange/reversed‐phase dual‐functional column using a mixture of 0.2 m potassium phosphate buffer (pH 5.9) and acetonitrile (20:1, v/v) as the mobile phase. The eluate was online mixed with acidic tris(2,2′‐bipyridyl)ruthenium(III) solution, and the generated chemiluminescence was detected. The detection limit (signal‐to‐noise ratio = 3) for ACh was 0.80 nmol/mL, which corresponded to 1.1 pmol TMA per injection volume of 5 µL. This is simple and robust method that does not need an expensive device and unstable enzymes, and was applied to the determination of ACh in pharmaceutical formulations. Copyright © 2009 John Wiley & Sons, Ltd.     

Describing size-related mortality and size distribution by nonparametric estimation and model selection using the Akaike Bayesian Information Criterion

When we calculate mortality along a gradient such as size, dividing into size classes and calculating rates for every class often involves a trade-off: fine class intervals produce fluctuating rates along the gradient, whereas broad ones may miss some trends within an interval. The same trade-off occurs when we want to illustrate size distribution by a histogram. This paper introduces nonparametric methods, published in a statistical journal, into forest ecology, in which the fine-class strategy is used in an extreme way: (1) a smoothly changing pattern is approximated by a fine step function, (2) the goodness-of-fit to the data and the smoothness along the gradient are formulated as a weighting sum within a Bayesian framework, (3) the Akaike Bayesian Information Criterion (ABIC) selects the weighting system that most appropriately balances the two demands, and (4) the values of the step function are optimized by the maximum likelihood method. The nonparametric estimates enable us to represent various patterns visually and, unlike parametric modeling, calculations do not demand the determination of a functional form. Mortality and size distribution analyses were conducted on 12-year forest tree monitoring data from a 4 ha permanent plot in an old-growth warm–temperate evergreen broad-leaved forest in Japan. From trees of 11 evergreen species with a diameter at breast height (DBH) greater than 5 cm, we found three types of trend with increasing DBH: decreasing, ladle-shaped and constant mortality. These patterns reflect variations in life history particular to each species.     

Free radical scavenging activity of propolis

We investigated the radical scavenging activity of propolis by ESR spectroscopy using spin trapping method. In addition, we examined the influence of a diet of 2% propolis on mice under oxidative stress. At low concentrations, the methanolic extract of propolis exhibited strong scavenging activity in vitro towards both the superoxide anion radical, generated by the hypoxanthine-xanthine oxidase reaction, and the NO radical, generated from the mixture of NOC-7 (NO generator) and carboxy-PTIO (spin trapping agent). An inhibitory effect of propolis on lipid peroxidation in vivo was observed, as determined by measurement of thiobarbituric acid-reactive substances in mouse liver homogenate. The level of vitamin C in the brain of mice under oxidative stress significantly increased compared with control mice under atmosphere, which was not observed in the mice given 2% propolis. The level of alpha-tocopherol in the brain of mice given 2% propolis significantly increased compared with control mice under atmosphere, which was not observed in mice under oxidative stress. SOD activity in the brain and plasma of mice given 2% propolis significantly decreased under atmosphere and oxidative stress compared with control mice. These results suggest that propolis possesses potent antioxidant activity in vitro and in vivo.     

Macaque monkeys show reversed ocular following responses to two-frame-motion stimulus presented with inter-stimulus intervals

Journal of Computational Neuroscience - When two-frame apparent motion stimuli are presented with an appropriate inter-stimulus interval (ISI), motion is perceived in the direction opposite to the...     

Synthesis and evaluation of substituted hexahydronaphthalenes as novel inhibitors of the Mcl-1/BimBH3 interaction

Mcl-1, an anti-apoptotic member of the Bcl-2 protein family, is overexpressed in a broad range of human cancers and plays a critical role in conferring resistance to chemotherapy. In the course of screening a natural product-like library of sesquiterpenoid analogs, we identified substituted hexahydronaphthalenes that showed activity against the Mcl-1/BimBH3 interaction in vitro. Here, we describe the synthesis of a small library of analogs and their biological evaluation. The most potent inhibitor in the series (19) exhibits an IC(50) of 8.3μM by ELISA and disrupts the interaction between endogenously expressed Mcl-1 and Bim in cultured MDA-MB-468 breast cancer cells.     

Toll‐like receptors 2 and 3 enhance melanogenesis and melanosome transport in human melanocytes

Because little is known about how the innate immune response influences skin pigmentation, we examined whether Toll‐like receptor (TLR) agonists participate in melanogenesis and melanosome transportation. We observed that TLR2/2 agonist HKLM and TLR3 agonist Poly(I:C) increased the amount of extracellular melanin from primary human epidermal melanocytes. HKLM, but not Poly(I:C), increased the melanogenic genes such as tyrosinase and dopachrome tautomerase. Poly(I:C) increased the expression of Rab27A, a molecule that facilitates melanosome transport to perimembranous actin filament. UVB irradiation induced Rab27A and melanosome transportation in a similar manner of Poly(I:C). SiRNA for TLR3 or Rab27A suppressed the perimembranous accumulation of Gp100‐positive vesicles in melanocytes and decreased melanin transfer to neighboring keratinocytes induced by both Poly(I:C) and UVB. These results suggest that the microenvironment in the epidermis and innate immune stimuli, such as microbiome and ultraviolet represented here by TLR2 and TLR3 agonists, could affect the melanogenesis in human melanocytes.     

Increased Serum LIGHT Levels Correlate with Disease Progression and Severity of Interstitial Pneumonia in Patients with Dermatomyositis: A Case Control Study

BackgroundActivated CD8+ T cells play an important role in the pathogenesis of dermatomyositis (DM) with interstitial pneumonia (IP). Serum CD8+ T-cell activator, LIGHT, and Th1/Th2/Th17 cytokines were measured in DM-IP patients and compared with clinical parameters to investigate their usefulness.MethodsThe correlations between the clinical findings and serum LIGHT and Th1/Th2/Th17 cytokine levels were investigated in 21 patients with DM-IP (14 with rapidly progressive IP [RPIP] and 7 with chronic IP [CIP], including 4 fatal cases of IP).ResultsThe median serum LIGHT level was 119 (16–335.4) pg/ml, which was higher than that in healthy control subjects and DM patients without IP. The median serum IL–6 level was 14.7 (2.4–154.5) pg/ml (n = 13). The other cytokines were detected in only a few patients. The median serum LIGHT level in DM-RPIP patients (156 [49.6–335.4] pg/ml) was significantly higher than that in DM-CIP patients (94.3 [16–164.2] pg/ml) (P = 0.02). The serum IL–6 level did not correlate with either progression or outcome of DM-IP. ROC curve analysis determined a serum LIGHT level of ≥120 pg/ml to be the cut-off value for the rapid progression of DM-IP. Serum LIGHT levels correlated significantly with %DLco (R = 0.55, P = 0.04) and total ground-glass opacity scores (R = 0.72, P = 0.0002). The serum LIGHT level significantly decreased to 100.5 (12.4–259.3) pg/ml 4 weeks after treatment initiation (P = 0.04).ConclusionsThe serum LIGHT level may be a promising marker of disease progression and severity in patients with DM-IP.     

Heterogenous induction of carcinoma-associated fibroblast-like differentiation in normal human prostatic fibroblasts by co-culturing with prostate cancer cells

In the tumor microenvironment, carcinoma-associated fibroblasts (CAFs) are considered to play a critical role in the promotion of tumorigenesis. However, the mechanisms that generate CAFs are not well elucidated. To understand how CAFs are generated during primary cancer progression, we investigated the biochemical characteristics of normal human prostate stromal cells (PrSC) co-cultured with human prostate cancer (PCa) cells in vitro. In primary cultures of human PCa-derived stromal cells (PCaSC-8 and PCaSC-9), expression of TNC, ACTA2, EGF, FGF7, and IGF1 mRNA was generally higher than PrSC but gene expression patterns were not uniform between PCaSC-8 and PCaSC-9 cells. Transforming growth factor β (TGFβ) and vascular endothelial growth factor (VEGF) protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than PrSC but levels of both secreted proteins were not same. When PrSCs were co-cultured with androgen-sensitive LNCaP cells or its sublines, androgen-low-sensitive E9 cells and androgen-insensitive AIDL cells, mRNA expression of IGF1 was significantly increased in all combinations. In contrast, expression of COL1A1, TNC, and ACTA2 mRNA was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Protein production of VEGF was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Increase of TGFβ protein was observed only in E9 + PrSC co-cultures. These biochemical characteristics of PrSC were partially recapitulated in TGFβ-treated PrSC. We have demonstrated that normal fibroblasts co-cultured with cancer cells become activated and exhibit biochemical characteristics of CAFs in a heterogenous manner. Our results suggest that heterogenous induction of CAF-like differentiation might be strongly dependent on biochemical characteristics of adjacent cancer cells.