Isolation of diallyl trisulfide inducible differentially expressed genes in human gastric cancer cells by modified cDNA representational difference analysis

Extensive epidemiologic studies indicated protective effects of consumption of garlic on reducing human gastric cancer (HGC) incidence. Diallyl trisulfide (DATS), a critical organic allyl sulfur component of garlic, was reported to have chemopreventive effects in inhibiting tumor process. We used DATS to treat HGC cell line BGC823 cells, and showed that DATS induces G1/S arrest and apoptosis in BGC823 cells demonstrated by a flow cytometric analysis. To further isolate DATS inducible differentially expressed genes in BGC823 cells, we combined a highly specific subtractive hybridization of cDNA representational difference analysis (cDNA RDA) with a sensitive bidirectional radioactive detection of mRNA differential display (mRNA DD) to develop a subtractive hybridization differential display (SHDD) method. This modified method adopted a first round of bidirectional subtractive hybridization between two sample cDNAs and a second round of bidirectional subtractive hybridization between the two resultant first-round difference products. Bidirectional subtractive hybridizations magnified the differences between the two sample cDNAs and favored isolating mRNA species with very small expression differences. We employed the SHDD method to detect DATS inducible differentially expressed genes in BGC823 cells. A total of 14 cDNA fragments (11 upregulated and 3 downregulated by DATS treatment) were isolated and confirmed by reverse Northern blot analysis. Our data show that SHDD is a powerful technique for identifying differentially expressed mRNA species between two sample cDNAs and provide useful cellular and molecular information for understanding the effects of garlic against human gastric cancer.     

Detection of gene mutations related with drug resistance in Mycobacterium leprae from leprosy patients using Touch-Down (TD) PCR

The lack of methods to identify Mycobacterium leprae with the resistance against multi-drugs quickly and specifically has hindered effective chemotherapy against M. leprae infection. To screen M. leprae with resistance against multi-drugs, the Touch-Down (TD)-PCR has been used in this study. Sequences of the folP, rpoA, B, and gyrA, B genes were analyzed for isolates of M. leprae from leprosy patients in Korea. We amplified designated region of several genes in M. leprae involved in drug resistance and could obtain the PCR products of each gene. The mutations in the particular region of folP, rpoB, and gyrB gene were certified by TD-PCR single-stranded conformational polymorphism and DNA sequencing, respectively.     

Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.     

The Arabidopsis RING E3 ubiquitin ligase AtAIRP2 plays combinatory roles with AtAIRP1 in abscisic acid-mediated drought stress responses

The ubiquitin (Ub)-26S proteasome pathway is implicated in various cellular processes in higher plants. AtAIRP1, a C3H2C3-type RING (for Really Interesting New Gene) E3 Ub ligase, is a positive regulator in the Arabidopsis (Arabidopsis thaliana) abscisic acid (ABA)-dependent drought response. Here, the AtAIRP2 (for Arabidopsis ABA-insensitive RING protein 2) gene was identified and characterized. AtAIRP2 encodes a cytosolic C3HC4-type RING E3 Ub ligase whose expression was markedly induced by ABA and dehydration stress. Thus, AtAIRP2 belongs to a different RING subclass than AtAIRP1 with a limited sequence identity. AtAIRP2-overexpressing transgenic (35S:AtAIRP2-sGFP) and atairp2 loss-of-function mutant plants exhibited hypersensitive and hyposensitive phenotypes, respectively, to ABA in terms of seed germination, root growth, and stomatal movement. 35S:AtAIRP2-sGFP plants were highly tolerant to severe drought stress, and atairp2 alleles were more susceptible to water stress than were wild-type plants. Higher levels of drought-induced hydrogen peroxide production were detected in 35S:AtAIRP2-sGFP as compared with atairp2 plants. ABA-inducible drought-related genes were up-regulated in 35S:AtAIRP2-sGFP and down-regulated in atairp2 progeny. The positive effects of AtAIRP2 on ABA-induced stress genes were dependent on SNF1-related protein kinases, key components of the ABA signaling pathway. Therefore, AtAIRP2 is involved in positive regulation of ABA-dependent drought stress responses. To address the functional relationship between AtAIRP1 and AtAIRP2, FLAG-AtAIRP1 and AtAIRP2-sGFP genes were ectopically expressed in atairp2-2 and atairp1 plants, respectively. Constitutive expression of FLAG-AtAIRP1 and AtAIRP2-sGFP in atairp2-2 and atairp1 plants, respectively, reciprocally rescued the loss-of-function ABA-insensitive phenotypes during germination. Additionally, atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation lines were more tolerant to dehydration stress relative to atairp1 and atairp2-2 single knockout plants. Overall, these results suggest that AtAIRP2 plays combinatory roles with AtAIRP1 in Arabidopsis ABA-mediated drought stress responses.     

Mycobacterium kansasii-induced death of murine macrophages involves endoplasmic reticulum stress responses mediated by reactive oxygen species generation or calpain activation

Although pathogenic mechanisms of tuberculosis have been extensively studied, little is known about the pathogenic mechanisms of Mycobacterium kansasii. In this work the influence of virulence and ER-stress mediated apoptosis of macrophages during two different strains of M. kansasii infection was investigated. We show that M. kansasii infection is associated with ER stress-mediated apoptosis in the murine macrophage cell line RAW 264.7. Infection of RAW 264.7 cells in vitro with apoptosis-inducing a clinical isolate of M. kansasii SM-1 (SM-1) resulted in strong induction of ER stress responses compared with M. kansasii type strain (ATCC 12478)-infected RAW 264.7 cells. Interestingly, inhibition of calpain prevented the induction of CHOP and Bip in ATCC 12478-infected RAW 264.7 cells but not in RAW 264.7 cells infected with SM-1. In contrast, reactive oxygen species (ROS) were significantly increased only in RAW 264.7 cells infected with SM-1. We propose that ROS generation is important for triggering ER stress-mediated apoptosis during SM-1 infection, whereas ATCC 12478-induced, ER stress-mediated apoptosis is associated with calpain activation. Our results demonstrate that the ER stress pathway plays important roles in the pathogenesis of M. kansasii infections, and that different strains of M. kansasii induce different patterns of ER stress-mediated apoptosis.     

Effects of Cystamine on antioxidant activities and regulatory T cells in lupus‐prone mice

Attenuated antioxidant activities, irregular cytokines expressions and reduced regulatory T cells, are strongly associated with the pathogenesis of systemic lupus erythematosus (SLE). Despite the well‐established beneficial effects of cystamine on lupus‐prone mice, the extent to which cystamine contributes to antioxidant activity and the reduction of regulatory T cells has seldom been investigated. Therefore, this study elucidates how cystamine affects anti‐oxidant activities in NZB/W F1 mice by performing assays of Glutathione (GSH), 1,1‐diphenyl‐2‐ picryl‐hydrazyl (DPPH) and malondialdehyde thiobarbituric acid (MDA). In addition, investigations of the effects of cystamine on CD4⁺/CD25⁺ regulatory T cells and interleukin‐6 (IL6)/STAT‐3 signalling were performed with flow cytometry and immunoblots. Experimental results reveal more significantly reduced MDA and increased GSH and DPPH in NZB/W F1 mice receiving cystamine than in those mice receiving PBS. Meanwhile, CD4⁺/CD25⁺ regulatory T cells more significantly increase in NZB/W F1 mice receiving cystamine than in those mice receiving PBS, accompanied by significantly reduced IL‐6/phosphorylated STAT‐3 expression. The above findings suggest the beneficial effects of cystamine in terms of increasing antioxidant activities and CD4⁺/CD25⁺ regulatory T cells in lupus‐prone mice by suppressing IL‐6/STAT3 signalling.     

Mass transfer in blood oxygenators using blood analogue fluids

Mass transfer correlations for hollow fiber blood oxygenators have been determined experimentally using Newtonian and non-Newtonian blood analogue fluids. The Newtonian fluids consisted of deionized water and glycerol/water mixtures. The non-Newtonian fluids were prepared by adding small amounts of xanthan gum to the Newtonian blood analogue fluids. The rheological behavior of the non-Newtonian blood analogue fluids was modeled using the power law. The diffusion of oxygen into and out of the Newtonian and non-Newtonian blood analogue fluids has been studied. The liquid stream flowed outside and across bundles of woven hollow fibers, while the gas stream flowed inside the fibers.     

A novel pathway responsible for lipopolysaccharide-induced translational regulation of TNF-α and IL-6 expression involves protein kinase C and fascin

Fascin, as a substrate of protein kinase C (PKC), is a well-known cytoskeletal regulatory protein required for cell migration, invasion, and adhesion in normal and cancer cells. In an effort to identify the role of fascin in PKC-mediated cellular signaling, its expression was suppressed by stable transfection of specific short hairpin RNAs (shRNAs) in mouse monocytic leukemia RAW264.7 cells. Suppression of fascin expression resulted in impaired cellular migration and invasion through extracellular matrix proteins. Unexpectedly, the specific shRNA transfectants exhibited a marked reduction in LPS-induced expression of TNF-α and IL-6 by blocking the translation of their mRNAs. Transient transfection assay using a luciferase expression construct containing the 3' untranslated region of TNF-α or IL-6 mRNA revealed a significant reduction in both LPS- and PMA- (the direct activator of PKC) induced reporter activity in cells transfected with fascin-specific shRNA, indicating that fascin-mediated translational regulation targeted 3' untranslated region. Furthermore, LPS-induced translational activation of reporter expression was blocked by a pharmacological inhibitor of PKC, and the dominant-negative form of PKCα attenuated LPS-induced translational activation. The same type of regulation was also observed in the human monocytic leukemia cell line THP-1 and in mouse peritoneal macrophages. These data demonstrate the involvement of fascin in the PKC-mediated translational regulation of TNF-α and IL-6 expression during the LPS response.     

Study of a lipophilic captopril analogue binding to angiotensin I converting enzyme

Human ACE is a central component of the renin–angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.