Anti-Melanogenic Properties of Velutin and Its Analogs.

Velutin, one of the flavones contained in natural plants, has various beneficial activities, such as skin whitening, as well as anti-inflammatory, anti-allergic, antioxidant, and antimicrobial activities. However, the relationship between the structure of velutin and its anti-melanogenesis activity is not yet investigated. In this study, we obtained 12 velutin derivatives substituted at C5, C7, C3', and C4' of the flavone backbone with hydrogen, hydroxyl, and methoxy functionalities by chemical synthesis, to perform SAR analysis of velutin structural analogues. The SAR study revealed that the substitution of functional groups at C5, C7, C3', and C4' of the flavone backbone affects biological activities related to melanin synthesis. The coexistence of hydroxyl and methoxy at the C5 and C7 position is essential for inhibiting tyrosinase activity. However, 1,2-diol compounds substituted at C3' and C4' of flavone backbone induce apoptosis of melanoma cells. Further, substitution at C3' and C4' with methoxy or hydrogen is essential for inhibiting melanogenesis. Thus, this study would be helpful for the development of natural-derived functional materials to regulate melanin synthesis.

Activity-expression profiling of glucose-6-phosphate dehydrogenase in tissues of normal and diabetic mice.

Glucose-6-phosphate dehydrogenase (G6PD) plays a principal role in the regulation of oxidative stress by modulating the nicotinamide adenine dinucleotide phosphate pool and is expected to be associated with metabolic diseases such as diabetes mellitus (DM). However, it is unclear whether hyperglycemia increases G6PD activity levels in DM because suitable assays for quantifying the activity in a high-throughput manner are lacking. Using liquid droplet arrays tailored to analyze tissue lysates, we performed G6PD activity profiling in eight tissues of normal and diabetic mice: brain, heart, kidney, liver, lung, muscle, spleen, and thyroid. Diabetic mice exhibited significantly higher G6PD activities in the kidney, liver, spleen, and thyroid than normal mice; no significant difference was found in the brain, heart, lung, or muscle. We also performed G6PD expression profiling in the eight tissues using Western blot analysis. Diabetic mice showed significantly elevated G6PD expression levels in the kidney, lung, spleen, and thyroid compared with normal mice; no significant difference was found in the brain, heart, liver, or muscle. An analysis of G6PD activity-expression profiles demonstrated tissue-specific changes in response to hyperglycemia. Thus, our approach would be helpful for understanding the role of G6PD in tissue-based pathogenesis of diabetic complications.

The benzodiazepine anesthetic midazolam prevents hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

We investigated the beneficial effects of midazolam against vascular endothelial growth factor (VEGF)-induced vascular leakage and its molecular mechanism of action in human retinal endothelial cells (HRECs) and the retinas of diabetic mice. Midazolam inhibited VEGF-induced elevation of intracellular Ca2+, generation of reactive oxygen species (ROS), and transglutaminase activation in HRECs; these effects were reversed by the GABA, type A (GABAA) receptor antagonist flumazenil but not by the translocator protein antagonist PK11195. Midazolam also prevented VEGF-induced disassembly of adherens junctions and in vitro permeability. Intravitreal injection of midazolam prevented hyperglycemia-induced ROS generation, transglutaminase activation, and subsequent vascular leakage in the retinas of diabetic mice, and those effects were reversed by flumazenil. The roles of flumazenil were further supported by identifying GABAA receptors in mouse retinas. Thus, midazolam prevents hyperglycemia-induced vascular leakage by inhibiting VEGF-induced intracellular events in the retinas of diabetic mice.-Lee, Y.-J., Kim, M., Lee, J.-Y., Jung, S.-H., Jeon, H.-Y., Lee, S.-A., Kang, S., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The benzodiazepine anesthetic midazolam prevents hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

Velutin, an Aglycone Extracted from Korean Mistletoe, with Improved Inhibitory Activity against Melanin Biosynthesis.

The abnormal regulation of melanin synthesis leads to a wide range of pigmentary disorders. Although various melanin biosynthesis inhibitors have been developed, their efficacy and long-term safety needs to be further improved, and thus the goal of this study is to develop promising natural compound inhibitors of melanin biosynthesis. Here, we obtained aglycone flavonoid extract through the microwave-assisted hydrolysis of glycoside extract from Korean mistletoe in acidic condition. The aglycone extract inhibited tyrosinase activity more efficiently with better antioxidant activity than glycoside extract in vitro. The microwave-assisted aglycone extract of mistletoe was further analyzed for in vivo activity, and the results showed the aglycone extract inhibited both early melanocyte development and melanin synthesis more efficiently in zebrafish embryo in a dose-dependent manner. Our in vivo toxicity assay quantitatively measured cell death in zebrafish embryos and showed that the microwave-assisted aglycone extract of mistletoe had no significant effect on cell death (p < 0.001), indicating that aglycone extract is more biocompatible than glycoside extract. Furthermore, our in vitro and in vivo analyses successfully identified and characterized velutin, an aglycone of a homoflavoyadorinin B glycoside, as a major inhibitory component in the microwave-assisted mistletoe extract. Ultimately, this study showed that the novel natural compound inhibitor velutin, which was generated through microwave-assisted extraction from mistletoe, improved the efficacy of melanin biosynthesis inhibition with little toxicity.

High-throughput investigation of transglutaminase 2 kinase regulation using a novel cysteine-modified peptide array.

Transglutaminase 2 (TGase2) kinase has emerged as an important regulator of apoptosis as well as chromatin structure and function; however, details about the pathophysiological functions of TGase2 kinase have been limited because of the lack of a suitable activity assay for systematic investigation of TGase2 kinase regulation in a high-throughput manner. Thus, we developed a novel on-chip TGase2 kinase activity assay using a cysteine-modified insulin-like growth factor-binding protein-3-derived peptide (CMI peptide) on an array platform. This peptide array-based activity assay was reproducible, with a detection limit of 2.127 µg/ml. We successfully applied this assay to investigate the effects of thiol-reactive compounds and divalent cations on TGase2 kinase by determining the half maximal inhibitory concentrations (IC50). Thiol-reactive compounds inhibited TGase2 kinase activity in a concentration-dependent manner, with IC50 values ranging from 0.125 to 5.550 mM. Divalent metal cations also showed a concentration-dependent inhibition, with IC50 values ranging from 0.005 to 1.937 mM; however, Ca2+ had no effect on TGase2 kinase activity. Thus, this novel kinase activity assay using the CMI peptide array described here is suitable for systematic investigation of TGase2 kinase regulation and may be useful for investigating the roles of TGase2 kinase in pathogenesis of kinase-mediated diseases.

Systematic investigation of protein kinase A substrate proteins using on-chip protein kinase kinetic profiling.

Protein kinases play a critical role in a wide variety of cellular processes through post-translational protein phosphorylation and identification of their substrate proteins is important for understanding the enzymes' mechanism of action and elucidating their functions in physiological and disease processes. However, there have been few systematic investigations of protein kinase substrates due to a lack of high-throughput kinetic assays. Thus, we designed an on-chip protein kinase assay for profiling kinase kinetic parameters by introducing the phosphorylation rate (Vp) under physiological conditions, instead of the maximal velocity (Vmax), in a high-throughput manner. We applied this method to the kinetic analysis of protein kinase A (PKA) for 28 nuclear, cytosolic, plasma membrane, and extracellular target proteins to determine the substrate affinity (Km), Vp (FIATP10/s), and substrate preference (Vp/Km). We then constructed a map of PKA's kinetic parameters with respect to the 28 proteins based on subcellular localization. Thus, the on-chip protein kinase kinetic profiling is an ideal approach for systematically investigating substrate proteins of protein kinases and fully characterizing the enzymes' physiological functions.

Array-Based High-Throughput Analysis of Silk-Elastinlike Protein Polymer Degradation and C-Peptide Release by Proteases.

The objective of this study was to utilize an on-chip degradation assay to evaluate polymer depots and the predicted drug release from the depots. We conjugated four silk-elastinlike protein (SELP) polymers including SELP-815K, SELP-815K-RS1, SELP-815K-RS2, and SELP-815K-RS5 with a Cy5-NHS ester and fabricated SELP arrays by immobilizing the conjugated polymers onto well-type amine arrays. SELP polymer degradation rates were investigated by calculating the half-maximal effective concentration (EC50). Eight cleavage enzymes were applied, all of which exhibited distinctive EC50 values for SELP-815K and its three analogues. We successfully utilized this assay to study the in vitro release of the Cy5-conjugated C-peptide from SELP-815K hydrogel arrays. Additionally, cumulative C-peptide release from the SELP-815K depots was also demonstrated using repetitive elastase treatments. Therefore, this array-based on-chip degradation assay could potentially be used for evaluating depot degradation and controlled drug release from polymer depots at the molecular level.

Direct observation of interactions of silk-elastinlike protein polymer with adenoviruses and elastase.

Silk-elastinlike protein polymer (SELP) hydrogels have been investigated for sustained local delivery of adenoviral gene carriers to solid tumors. These polymers degrade in the presence of proteases such as elastase. A detailed understanding of the interaction of SELPs with viruses and their degradation in the presence of elastase can provide useful information about mechanisms of sustained gene delivery from these systems. In this work, we investigated the interactions of SELPs with adenoviruses (Ads) and elastase using atomic force microscopy. We observed that viral particles interacted strongly with SELP networks formed by cross-linking of nanofibers. The presence of viruses contributed to enhanced network formation. Incubation of Ad with SELPs in the liquid state induced close packing of the viral colony. Morphological changes of SELP networks cleaved by enzymatic interaction with elastase were investigated. SELP-415K fiber networks were more responsive to temperature changes and were slowly degraded by elastases compared to SELP-47K, a SELP analogue with shorter elastin units in the monomer repeat. These studies provide insight into the influence of SELP structure on degradation and potential mechanisms of increased viral stability.

A peptide array-based serological protein kinase A activity assay and its application in cancer diagnosis.

Protein kinase A (PKA) plays a crucial role in several biological processes; however, there is no assay with sufficient sensitivity and specificity to determine serological PKA (sPKA) activity. Here we present an on-chip activity assay that employs cysteine-modified kemptide arrays to determine specific sPKA activity in human sera that eliminates the potential contributions of other kinases with a protein kinase peptide inhibitor. The sensitivity of the on-chip sPKA activity assay was greatly enhanced by Triton X-100, with a 0.01 U mL(-1) detection limit. sPKA activity was determined by subtracting nonspecific sPK activity from total sPK activity. Our assay provided greater sensitivity and specificity and more accurate area under the curve values for gastric cancer compared to the total sPK activity assay. sPKA activities in human sera from patients with hepatic (n = 30), gastric (n = 30), lung (n = 30), and colorectal (n = 30) cancers were significantly higher than those in controls (n = 30, p < 10(-4)), but no significant difference in sPKA activities between normal and inflammation groups was observed. These results demonstrate that the on-chip assay accurately measures sPKA activity in human sera and that the sPKA activity may be a potential biomarker for cancer diagnosis.

Dammarenediol-II Prevents VEGF-Mediated Microvascular Permeability in Diabetic Mice.

Diabetic retinopathy is a major diabetic complication predominantly caused by vascular endothelial growth factor (VEGF)-induced vascular permeability in the retina; however, treatments targeting glycemic control have not been successful. Here, we investigated the protective effect of dammarenediol-II, a precursor of triterpenoid saponin biosynthesis, on VEGF-induced vascular leakage using human umbilical vein endothelial cells (HUVECs) and diabetic mice. We overproduced the compound in transgenic tobacco expressing Panax ginseng dammarenediol-II synthase gene and purified using column chromatography. Analysis of the purified compound using a gas chromatography-mass spectrometry system revealed identical retention time and fragmentation pattern to those of authentic standard dammarenediol-II. Dammarenediol-II inhibited VEGF-induced intracellular reactive oxygen species generation, but it had no effect on the levels of intracellular Ca(2+) in HUVECs. We also found that dammarenediol-II inhibited VEGF-induced stress fiber formation and vascular endothelial-cadherin disruption, both of which play critical roles in modulating endothelial permeability. Notably, microvascular leakage in the retina of diabetic mice was successfully inhibited by intravitreal dammarenediol-II injection. Our results suggest that the natural drug dammarenediol-II may have the ability to prevent diabetic microvascular complications, including diabetic retinopathy.

Sustained local delivery of oncolytic short hairpin RNA adenoviruses for treatment of head and neck cancer.

BACKGROUND: Oncolytic adenovirus (Ad)-mediated gene therapy is a promising approach for suppression of primary tumors. Therapeutic efficacy of Ad-mediated gene therapy has been limited by immunogenicity, rapid dissemination of viral progenies into systemic circulation and short duration of biological activity. Polymeric sustained local delivery can overcome many of these challenges to produce a viable therapy with improved outcomes. METHODS: Silk-elastinlike protein polymer (SELP) hydrogels were used for matrix-mediated delivery of oncolytic Ad, containing short hairpin RNA (shRNA) targeted to C-Met (sh-C-Met), to solid tumors in a nude mouse model of human head and neck cancer. The biological activity of Ad released from SELP hydrogels was examined as a function of time to investigate protective effects on viral activity. Antitumor efficacy and viral distribution were investigated for 3 weeks in tumor-bearing mice. RESULTS: The encapsulation of Ad with SELP hydrogels sustained biological activity longer than Ad alone. Ad in SELP matrix showed 1.5-fold greater antitumor efficacy compared to that of naked Ad in human xenograft tumor models. Histological analysis demonstrated that treatment with Ad in a SELP matrix resulted in apoptosis in a wider area of tumor tissue and higher density of Ad infection compared to Ad administered alone. CONCLUSIONS: Matrix-mediated delivery of Ad-containing shRNA with SELP hydrogels enhances therapeutic efficacy by tumor-selective infection, spatiotemporal control and preservation of biologic activity.

Integrative proteomic profiling of protein activity and interactions using protein arrays.

Proteomic studies based on abundance, activity, or interactions have been used to investigate protein functions in normal and pathological processes, but their combinatory approach has not been attempted. We present an integrative proteomic profiling method to measure protein activity and interaction using fluorescence-based protein arrays. We used an on-chip assay to simultaneously monitor the transamidating activity and binding affinity of transglutaminase 2 (TG2) for 16 TG2-related proteins. The results of this assay were compared with confidential scores provided by the STRING database to analyze the functional interactions of TG2 with these proteins. We further created a quantitative activity-interaction map of TG2 with these 16 proteins, categorizing them into seven groups based upon TG2 activity and interaction. This integrative proteomic profiling method can be applied to quantitative validation of previously known protein interactions, and in understanding the functions and regulation of target proteins in biological processes of interest.

Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays.

Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

Extracellular ultrathin fibers sensitive to intracellular reactive oxygen species: formation of intercellular membrane bridges.

Membrane bridges are key cellular structures involved in intercellular communication; however, dynamics for their formation are not well understood. We demonstrated the formation and regulation of novel extracellular ultrathin fibers in NIH3T3 cells using confocal and atomic force microscopy. At adjacent regions of neighboring cells, phorbol 12-myristate 13-acetate (PMA) and glucose oxidase induced ultrathin fiber formation, which was prevented by Trolox, a reactive oxygen species (ROS) scavenger. The height of ROS-sensitive ultrathin fibers ranged from 2 to 4 nm. PMA-induced formation of ultrathin fibers was inhibited by cytochalasin D, but not by Taxol or colchicine, indicating that ultrathin fibers mainly comprise microfilaments. PMA-induced ultrathin fibers underwent dynamic structural changes, resulting in formation of intercellular membrane bridges. Thus, these fibers are formed by a mechanism(s) involving ROS and involved in formation of intercellular membrane bridges. Furthermore, ultrastructural imaging of ultrathin fibers may contribute to understanding the diverse mechanisms of cell-to-cell communication and the intercellular transfer of biomolecules, including proteins and cell organelles.

Simultaneous activity assay of two transglutaminase isozymes, blood coagulation factor XIII and transglutaminase 2, by use of fibrinogen arrays.

We developed an on-chip activity assay system to simultaneously determine the transamidating activities of blood coagulation factor XIII (FXIII) and transglutaminase 2 (TG2) by use of fibrinogen arrays. FXIII and TG2 are transglutaminase family members that are involved in various physiological functions, including vascular pathophysiology, bone development, and cancer progression. However, investigation of their differential functions is limited by the lack of high-throughput and isozyme-specific activity assays. For the on-chip activity assay, we fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane surface of well-type arrays, and we determined transamidating activity by probing biotinylated fibrinogen with Cy3-conjugated streptavidin on arrays. We optimized assay conditions, such as buffer pH, concentrations of dithiothreitol and 5-(biotinamido)pentylamine, and incubation time, and we created equations to determine specific FXIII and TG2 activities in samples. We successfully applied this assay system to monitor changes in FXIII and TG2 activities in THP-1 monocytic cells differentiated with phorbol 12-myristate13-acetate and interleukin-4. This activity assay is sensitive and suitable for high-throughput determination of FXIII and TG2 activities and thus has a strong potential for investigating the differential functions of these isozymes in cell signaling and cardiovascular pathophysiology research.

Rapid determination of blood coagulation factor XIII activity using protein arrays for serodiagnosis of human plasma.

We developed a novel on-chip assay using protein arrays for quantitative and rapid analysis of blood coagulation factor XIII (FXIII) activity in human plasma. FXIII is activated by concerted action of thrombin and Ca(2+) and plays essential roles in hemostasis, angiogenesis, and wound healing. We fabricated protein arrays by immobilizing fibrinogen onto the 3-aminopropyltrimethoxysilane layer of well-type arrays and determined FXIII activity by analyzing biotinylated fibrinogen with Cy3-conjugated streptavidin. We determined optimal concentrations of Ca(2+), thrombin, and 5-(biotinamido)pentylamine (BAPA) for the on-chip activity assay, and the detection limit was 0.01 Lowey U/mL (9.9 pM). Using the on-chip activity assay, hepatocellular carcinoma patients (n = 24), but not hepatitis (n = 24) or liver cirrhosis patients (n = 41), had significantly lower FXIII activities (p < 0.001) than normal individuals (n = 41), indicating that FXIII activity is a possible diagnostic marker for hepatocellular carcinoma. In addition, we have successfully used this activity assay to reveal individual variations (37-57%, n = 65) in the inhibition rate of FXIII activity by isoniazid, the first-line antituberculosis agent. Thus, our optimized on-chip FXIII activity assay provides a quantitative and high-throughput approach to investigating the role(s) of FXIII in human diseases. Moreover, it has a strong potential to be applied toward FXIII-related personalized medicines.

Characterization of Phenethyl Cinnamamide Compounds from Hemp Seed and Determination of Their Melanogenesis Inhibitory Activity.

Hyperpigmentation is induced by the overactivation of tyrosinase, which is a rate-limiting enzyme in melanogenesis. The defatted extract of hemp (Cannabis sativa L.) seed is known to have inhibitory effects on melanogenesis; however, effective compounds in the extract have not been identified yet. In this study, three phenethyl cinnamamides present in hemp seed extract were prepared by purification and chemical synthesis and were assessed for their inhibitory effect on melanogenesis in B16F10 melanoma cells. A comparison of the anti-melanogenesis and anti-tyrosinase activity of hemp seed solvent fractions revealed that the ethyl acetate fraction possessed the greatest potential for suppressing melanogenesis in melanoma cells by decreasing tyrosinase activity. We tentatively identified 26 compounds in the ethyl acetate fraction by comparing spectroscopic data with the literature. Three phenethyl cinnamamides such as N-trans-caffeoyltyramine, N-trans-coumaroyltyramine, and N-trans-feruloyltyramine present abundantly in the ethyl acetate fraction were prepared and their anti-melanogenesis and anti-tyrosinase activities in melanoma cells were evaluated. We found that N-trans-caffeoyltyramine and N-trans-feruloyltyramine inhibited alpha melanocyte stimulating hormone (α-MSH)-induced melanogenesis without cytotoxicity, while N-trans-coumaroyltyramine inhibited melanogenesis with cytotoxicity. IC50 values of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and N-trans-coumaroyltyramine for inhibition of α-MSH-mediated tyrosinase activation were 0.8, 20.2, and 6.3 µM, respectively. Overall, N-trans-caffeoyltyramine possessed the strongest anti-melanogenesis activity among the three phenethyl cinnamamides evaluated. The inhibitory effect of N-trans-caffeoyltyramine was verified by determining the melanin content and tyrosinase activity in melanoma after treating the cells with synthetic compounds. Thus, N-trans-caffeoyltyramine isolated from hemp seed extract could be useful in cosmetics as a skin-whitening agent.

Rapid analysis of matrix metalloproteinase-3 activity by gelatin arrays using a spectral surface plasmon resonance biosensor.

We developed a novel assay system using an array-based spectral surface plasmon resonance (SPR) biosensor for a high-throughput analysis of matrix metalloproteinase (MMP)-3 activity. Gelatin arrays were fabricated by immobilizing gelatin, a MMP-3 substrate, on amine-modified gold arrays. MMP-3 activity was determined by monitoring the shift of SPR wavelength caused by gelatin proteolysis. The gelatinolytic activity of MMP-3, which caused a decrease of the SPR wavelength, was verified by SPR spectroscopy, atomic force microscopy, and fluorescence-based protein arrays. MMP-3 activity increased by three non-ionic detergents in a dose-dependent manner, and Brij-35 was most effective. The array-based SPR biosensor was successfully applied to the rapid analysis of dose-dependent MMP-3 activity and its inhibition with tissue inhibitors of metalloproteinase 1 and GM6001, MMP inhibitors. Therefore, this new assay system using a spectral SPR biosensor is simple, label-free, and high-throughput, and is likely to have a strong potential for inhibitor screening.

Characterization of Caffeoylquinic Acids from Lepisorus thunbergianus and Their Melanogenesis Inhibitory Activity.

Hyperpigmentation resulting from the overactivation of tyrosinase leads to darker spots or patches on the human skin. Although these phenomena are harmless, there is still great demand for melanogenesis inhibitors to prevent hyperpigmentation by inhibiting the tyrosinase, a rate-limiting enzyme in melanogenesis. Although Lepisorus thunbergianus has been used in folk remedies as a diuretic and hemostatic agent, its effect on melanogenesis has not yet been reported. In this study, we prepared an L. thunbergianus extract and its solvent fractions and evaluated their biological activity against free radical and melanin synthesis. The extract of L. thunbergianus inhibited mushroom tyrosinase activity more efficiently than, and with similar antioxidant activity to, arbutin in vitro. Comparative evaluation of the anti-melanogenesis and anti-tyrosinase activity of L. thunbergianus solvent fractions demonstrated that, by inhibiting tyrosinase activity, the butanol fraction has the highest potential for the inhibition of melanogenesis in melanoma cells. We found by structural analysis using high-performance liquid chromatography (HPLC) and NMR spectroscopy that the major compounds in butanol fraction were three caffeoylquinic acid derivatives. The three derivatives had similar radical scavenging and anti-tyrosinase activities in vitro, while only 5-caffeoylquinic acid had an inhibitory effect on α-MSH-induced melanogenesis. The inhibitory effect of 5-caffeoylquinic acid was verified by the determination of the melanin content and tyrosinase activity in melanoma after treating the cells with a commercial compound. Further, we revealed that 5-caffeoylquinic acid inhibited melanogenesis by chelating a copper cation from a copper-tyrosinase complex. Thus, 5-caffeoylquinic acid or butanol fraction isolated from L. thunbergianus might be useful in cosmetics as a skin-whitening agent.

A novel array-based assay of in situ tissue transglutaminase activity in human umbilical vein endothelial cells.

Transglutaminases (TGs), a family of calcium-dependent transamidating enzymes, are involved in functions such as apoptosis andinflammation and play a role in autoimmune diseases and neurodegenerative disorders. In this study, we describe a novel array-based approach to rapidly determine in situ TG activity in human umbilical vein endothelial cells and J82 human bladder carcinoma cells. Amine arrays were fabricated by immobilizing 3-aminopropyltrimethoxysilane on glass slides. The assay was specific and highly reproducible. The average coefficient of variation between spots was 2.6% (n=3 arrays), and the average correlation coefficients between arrays and between arrays/reactions were 0.998 and 0.976, respectively (n=3 arrays). The assay was successfully applied to detect changes in TG activity induced by maitotoxin and to analyze inhibition of the TG activation with cystamine and monodansyl cadaverine. In addition, the assay demonstrated that intracellular reactive oxygen species regulate the maitotoxin-induced activation of TG. Thus, the array-based in situ TG activity assay constitutes a rapid and high-throughput approach to investigating the roles of TGs in cell signaling.