Discovery and functional characterization of flavone O-glycosyltransferases in Scutellaria baicalensis / 药学学报

Huang-Qin is a traditional Chinese medicine with antiviral, antioxidant, and anti-inflammatory activities. Its major bioactive compounds are diverse flavone O-glucuronides and glucosides. Although three flavonoid O-glycosyltransferases have been identified from S. baicalensis, this information is not sufficient to elucidate the structural diversity of flavonoid glycosides. In this study, nine glycosyltransferase candidate genes were discovered from S. baicalensis by BLAST analysis and their functions were characterized after heterologous expression. Three new flavone O-glycosyltransferases were able to catalyze the formation of major compounds in S. baicalensis, including baicalin and wogonoside. These enzymes could also utilize exogenous flavones as sugar acceptors. This work further elucidates biosynthetic pathways for Scutellaria flavonoid O-glycosides.

Association between Genetic Variations of MERTK and Chronic Obstructive Pulmonary Disease in Koreans

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease. To date, a large number of clinical studies have been conducted to investigate the association between genetic variations and COPD. However, little is known regarding the genetic susceptibility of Koreans to this disease. MER receptor tyrosine kinase (MERTK) plays important roles in the inhibition of inflammation and in the clearance of apoptotic cells. Here, we investigated the association between genetic variations in MERTK and the development of COPD in Koreans. METHODS: We conducted genetic analysis of MERTK using genomic DNA samples from 87 patients with COPD and 88 healthy controls and compared the frequency of each variation or haplotype between the patient and control groups. Subsequently, the effect of each variation was evaluated using in vitro assays. RESULTS: Ten variations were identified in this study, four of them for the first time. In addition, we found that the frequency of each variation or haplotype was comparable between the patient and control groups. However, we observed that the frequency for the wild-type haplotype was higher in the control group, compared to that in the group of patients with COPD, in the subgroup analysis of current smokers, although the difference was not statistically significant (P = 0.080). In in vitro assays, we observed that none of the variations affected the activity of the promoter or the expression of MERTK. CONCLUSION: Our findings indicate that the susceptibility to COPD is not related to the genetic variations or haplotypes of MERTK in Koreans.

Functional characterization of human equilibrative nucleoside transporter 1

Equilibrative nucleoside transporters (ENTs), which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer chemotherapy, and treatment for virus infections. Functional characterization of ENTs at the molecular level remains technically challenging and hence scant. In this study, we report successful purification and biochemical characterization of human equilibrative nucleoside transporter 1 (hENT1) in vitro. The HEK293F-derived, recombinant hENT1 is homogenous and functionally active in proteoliposome-based counter flow assays. hENT1 transports the substrate adenosine with a K of 215 ± 34 µmol/L and a V of 578 ± 23.4 nmol mg min. Adenosine uptake by hENT1 is competitively inhibited by nitrobenzylmercaptopurine ribonucleoside (NBMPR), nucleosides, deoxynucleosides, and nucleoside-derived anti-cancer and anti-viral drugs. Binding of hENT1 to adenosine, deoxyadenosine, and adenine by isothermal titration calorimetry is in general agreement with results of the competitive inhibition assays. These results validate hENT1 as a bona fide target for potential drug target and serve as a useful basis for future biophysical and structural studies.

Functional Characterization of Resting and Adenovirus-Induced Reactive Astrocytes in Three-Dimensional Culture

Brain is a rich environment where neurons and glia interact with neighboring cells as well as extracellular matrix in three-dimensional (3D) space. Astrocytes, which are the most abundant cells in the mammalian brain, reside in 3D space and extend highly branched processes that form microdomains and contact synapses. It has been suggested that astrocytes cultured in 3D might be maintained in a less reactive state as compared to those growing in a traditional, two-dimensional (2D) monolayer culture. However, the functional characterization of the astrocytes in 3D culture has been lacking. Here we cocultured neurons and astrocytes in 3D and examined the morphological, molecular biological, and electrophysiological properties of the 3D-cultured hippocampal astrocytes. In our 3D neuron-astrocyte coculture, astrocytes showed a typical morphology of a small soma with many branches and exhibited a unique membrane property of passive conductance, more closely resembling their native in vivo counterparts. Moreover, we also induced reactive astrocytosis in culture by infecting with high-titer adenovirus to mimic pathophysiological conditions in vivo. Adenoviral infection induced morphological changes in astrocytes, increased passive conductance, and increased GABA content as well as tonic GABA release, which are characteristics of reactive gliosis. Together, our study presents a powerful in vitro model resembling both physiological and pathophysiological conditions in vivo, and thereby provides a versatile experimental tool for studying various neurological diseases that accompany reactive astrocytes.

Functional Characterization of ABCB4 Mutations Found in Low Phospholipid-Associated Cholelithiasis (LPAC)

Multidrug resistance 3 (MDR3) is expressed on the canalicular membrane of the hepatocytes and plays an important role in protecting the liver from bile acids. Altered ABCB4 gene expression can lead to a rare hepatic disease, low phospholipid-associated cholelithiasis (LPAC). In this study, we characterized 3 ABCB4 mutations in LPAC patients using various in vitro assay systems. We first measured the ability of each mutant to transport paclitaxel and then the mechanisms by which these mutations might change MDR3 transport activity were determined using immunoblotting, cell surface protein biotinylation, and immunofluorescence. Through a membrane vesicular transport assay, we observed that the uptake of paclitaxel was significantly reduced in membrane vesicles expressing 2 ABCB4 mutations, F165I and S320F. Both mutants showed significantly decreased total and cell surface MDR3 expression. These data suggest two missense mutations of ABCB4 may alter function of MDR3 and ultimately can be determined as LPAC-causing mutations.

Caracterização funcional da interação entre as proteínas CTSP-1 e CTCF / Functional characterization of the interaction between the proteins CTSP-1 and CTCF

Os antígenos cancer-testis (CT) são proteínas imunogênicas expressas em tecido gametogênico e em diferentes tipos de tumor, sendo considerados candidatos promissores para a imunoterapia do câncer. Entretanto, pouco se sabe sobre a função desses antígenos na tumorigênese. Em 2006, identificamos CTSP-1 como um novo antígeno CT, frequentemente expresso em vários tumores. Nesse trabalho, investigamos a função de CTSP-1 por meio da identificação de proteínas expressas em tumores de próstata e que são capazes de interagir fisicamente com esse antígeno. Demonstramos que CTSP-1 interage com a proteína CTCF em ensaios de duplo-híbrido em leveduras, pulldown e de co-localização e, em seguida, analisamos o impacto da superexpressão de CTSP-1 no controle da expressão de genes CT mediada por CTCF e na progressão do ciclo celular. Utilizando o CT NY-ESO-1 como modelo, demonstramos que a superexpressão de CTSP-1 não altera os níveis endógenos de NY-ESO-1 na linhagem celular tumoral H1299. Por outro lado, observamos que a superexpressão de CTSP-1 48h após as transfecções em H1299 induz um bloqueio do ciclo em G0/G1, reduzindo a capacidade clonogênica dessas células por um mecanismo dependente dos níveis de expressão de CTSP-1. Resultados semelhantes não foram observados em ensaios com clones superexpressando CTSP-1 estavelmente, o que sugere que eles tenham se originado de células que conseguiram escapar do bloqueio em G0/G1. Resultados preliminares sugerem que a redução da capacidade clonogênica das células H1299 que superexpressam CTSP-1 48h após as tansfecções não está associada à ocorrência de morte por apoptose.

Cancer-testis (CT) antigens are immunogenic proteins expressed in gametogenic tissues and in different histological types of tumors, being considered promising candidates for cancer immunotherapy. However, little is known about their role in tumorigenesis. In 2006, we identified CTSP-1 as a novel CT antigen, frequently expressed in different types of tumors. In this work, we investigated the functional role of CTSP-1 through the identification of proteins expressed in prostate tumors and that physically interact with this tumor antigen. We demonstrate that CTSP-1 interacts with the CTCF protein using the yeast two-hybrid system, pulldown and co-localization assays and have further analyzed the impact of CTSP-1 overexpression on the expression of CT genes mediated by CTCF and on the cell cycle progression. Using the CT antigen NY-ESO-1 as a model, we showed that the CTSP-1 overexpression does not alter the endogenous levels of NY-ESO-1 in the tumor cell line H1299. On the other hand, we observed that the overexpression of CTSP-1 in H1299 cells 48h after the transfections induces a cell cycle arrest in G0/G1 and reduces the clonogenic capacity of these cells by a mechanism dependent on the CTSP-1 expression levels. Similar results were not observed for cell clones stably overexpressing CTSP-1, suggesting that these clones have arisen from cells that managed to escape cell cycle arrest in G0/G1. Preliminary results suggest that the reduced clonogenic capacity of H1299 cells expressing CTSP-1 and analyzed 48h after the transfections is not associated with cell death by apoptosis.

Caracterização funcional da interação entre as proteínas CTSP-1 e CTCF / Functional characterization of the interaction between the proteins CTSP-1 and CTCF

Os antígenos cancer-testis (CT) são proteínas imunogênicas expressas em tecido gametogênico e em diferentes tipos de tumor, sendo considerados candidatos promissores para a imunoterapia do câncer. Entretanto, pouco se sabe sobre a função desses antígenos na tumorigênese. Em 2006, identificamos CTSP-1 como um novo antígeno CT, frequentemente expresso em vários tumores. Nesse trabalho, investigamos a função de CTSP-1 por meio da identificação de proteínas expressas em tumores de próstata e que são capazes de interagir fisicamente com esse antígeno. Demonstramos que CTSP-1 interage com a proteína CTCF em ensaios de duplo-híbrido em leveduras, pulldown e de co-localização e, em seguida, analisamos o impacto da superexpressão de CTSP-1 no controle da expressão de genes CT mediada por CTCF e na progressão do ciclo celular. Utilizando o CT NY-ESO-1 como modelo, demonstramos que a superexpressão de CTSP-1 não altera os níveis endógenos de NY-ESO-1 na linhagem celular tumoral H1299. Por outro lado, observamos que a superexpressão de CTSP-1 48h após as transfecções em H1299 induz um bloqueio do ciclo em G0/G1, reduzindo a capacidade clonogênica dessas células por um mecanismo dependente dos níveis de expressão de CTSP-1. Resultados semelhantes não foram observados em ensaios com clones superexpressando CTSP-1 estavelmente, o que sugere que eles tenham se originado de células que conseguiram escapar do bloqueio em G0/G1. Resultados preliminares sugerem que a redução da capacidade clonogênica das células H1299 que superexpressam CTSP-1 48h após as tansfecções não está associada à ocorrência de morte por apoptose

Cancer-testis (CT) antigens are immunogenic proteins expressed in gametogenic tissues and in different histological types of tumors, being considered promising candidates for cancer immunotherapy. However, little is known about their role in tumorigenesis. In 2006, we identified CTSP-1 as a novel CT antigen, frequently expressed in different types of tumors. In this work, we investigated the functional role of CTSP-1 through the identification of proteins expressed in prostate tumors and that physically interact with this tumor antigen. We demonstrate that CTSP-1 interacts with the CTCF protein using the yeast two-hybrid system, pulldown and co-localization assays and have further analyzed the impact of CTSP-1 overexpression on the expression of CT genes mediated by CTCF and on the cell cycle progression. Using the CT antigen NY-ESO-1 as a model, we showed that the CTSP-1 overexpression does not alter the endogenous levels of NY-ESO-1 in the tumor cell line H1299. On the other hand, we observed that the overexpression of CTSP-1 in H1299 cells 48h after the transfections induces a cell cycle arrest in G0/G1 and reduces the clonogenic capacity of these cells by a mechanism dependent on the CTSP-1 expression levels. Similar results were not observed for cell clones stably overexpressing CTSP-1, suggesting that these clones have arisen from cells that managed to escape cell cycle arrest in G0/G1. Preliminary results suggest that the reduced clonogenic capacity of H1299 cells expressing CTSP-1 and analyzed 48h after the transfections is not associated with cell death by apoptosis

Purification and functional characterization of two fibrinogenolytic enzymes from Bothrops alternatus venom

Two fibrinogenolytic enzymes, Bothrops alternatus metalloprotease isoform (BaltMP)-I and II, were purified from Bothrops alternatus venom using Diethylaminoethyl (DEAE) Sephacel, Sephadex G-75 and Heparin-Agarose column chromatography. Purified BaltMP-I and II ran as single protein bands on analytical polyacrylamide gel electrophoresis and showed molecular weights of 29000 and 36000, respectively, under reducing conditions in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). BaltMP-II, but not BaltMP-I, displayed blood-clotting activity in bovine plasma, which was about 10-fold higher than that of the crude venom. Both enzymes were proteolytically active against bovine fibrinogen as substrate. When fibrinogen and each enzyme were incubated at 37°C, at a ratio of 1:100 (w/w), BaltMP-II cleaved preferentially the Aalpha -chain and more slowly the Bbeta -chain. The action of BaltMP-I was similar, but lower. None of the proteases degraded the gamma-chain of fibrinogen. The fibrinogenolytic activity of the enzymes was inhibited by 1,10-phenanthroline, suggesting they are metalloproteases. Since both enzymes were found to cause defibrinogenation when intraperitoneally (i.p.) administered to mice, they can be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.(AU)