In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells

Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.     

X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway

The X protein from a chronic strain of hepatitis B virus (HBx) was determined to inhibit Fas-mediated apoptosis and promote cell survival. Fas-mediated apoptosis is the major cause of hepatocyte damage during liver disease. Experiments demonstrated that cell death caused by anti-Fas antibodies was blocked by the expression of HBx in human primary hepatocytes and mouse embryo fibroblasts. This effect was also observed in mouse erythroleukemia cells that lacked p53, indicating that protection against Fas-mediated apoptosis was independent of p53. Components of the signal transduction pathways involved in this protection were studied. The SAPK/JNK pathway has previously been suggested to be a survival pathway for some cells undergoing Fas-mediated apoptosis, and kinase assays showed that SAPK activity was highly up-regulated in cells expressing the HBx protein. Normal mouse fibroblasts expressing HBx were protected from death, whereas identical fibroblasts lacking the SEK1 component from the SAPK pathway succumbed to Fas-mediated apoptosis, whether HBx was present or not. Assays showed that caspase 3 and 8 activities and the release of cytochrome c from mitochondria were inhibited, in the presence of HBx, following stimulation with anti-Fas antibodies. Coprecipitation and confocal immunofluorescence microscopy experiments demonstrated that HBx localizes with a cytoplasmic complex containing MEKK1, SEK1, SAPK, and 14-3-3 proteins. Finally, mutational analysis of HBx demonstrated that a potential binding region for 14-3-3 proteins was essential for induction of SAPK/JNK activity and protection from Fas-mediated apoptosis.     

Low Incidence of Renal Dysfunction among HIV-Infected Patients on a Tenofovir-Based First Line Antiretroviral Treatment Regimen in Myanmar

Background

Since 2004, Médecins Sans Frontières-Switzerland has provided treatment and care for people living with HIV in Dawei, Myanmar. Renal function is routinely monitored in patients on tenofovir (TDF)-based antiretroviral treatment (ART), and this provides an opportunity to measure incidence and risk factors for renal dysfunction.

Methods

We used routinely collected program data on all patients aged ≥15 years starting first-line TDF-based ART between January 2012 and December 2013. Creatinine clearance (CrCl) was assessed at base line and six-monthly, with renal dysfunction defined as CrCl < 50ml/min/1.73m². We calculated incidence of renal dysfunction and used Cox regression analysis to identify associated risk factors.

Results

There were 1391 patients, of whom 1372 had normal renal function at baseline. Of these, 86 (6.3%) developed renal dysfunction during a median time of follow-up 1.14 years with an incidence rate of 5.4 per 100 person-years: 78 had CrCl between 30–50ml/min/1.73m² and were maintained on TDF–based ART, but 5 were changed to another regimen: 4 because of CrCl <30ml/min/1.73m². Risk factors for renal dysfunction included age ≥45 years, diagnosed diabetes, underlying renal disease, underweight and CD4 count <200cells/mm³. There were 19 patients with baseline renal dysfunction and all continued on TDF-based ART: CrCl stayed between 30–49 ml/min/1.73m² in five patients while the remainder regained normal renal function.

Conclusions

In a resource-poor country like Myanmar, the low incidence of renal toxicity in our patient cohort suggests that routine assessment of CrCl may not be needed and could be targeted to high risk groups if resources permit.     

Phytoconstituents from the root of Streptocaulon tomentosum and their chemotaxonomical relevance for separation from S. juventas

A new cardenolide, 17β-H-periplogenin-3-O-β-d-digitoxoside (1), and a new pregnane glycoside, Δ⁵-pregnene-3β,16α-diol-d-O-[2,4-O-diacetyl-β-digitalopyranosyl-(1 → 4)-β-d-cymaropyranoside]-16-O-[β-d-glucopyranoside] (2) were isolated from the roots of Streptocaulon tomentosum (Asclepiadaceae) together with a series of known compounds. Their chemotaxonomic significance for the separation of S. tomentosum from Streptocaulon juventas is discussed, suggesting a rather clear distinction of these species.     

MassCode liquid arrays as a tool for multiplexed high-throughput genetic profiling

Multiplexed detection assays that analyze a modest number of nucleic acid targets over large sample sets are emerging as the preferred testing approach in such applications as routine pathogen typing, outbreak monitoring, and diagnostics. However, very few DNA testing platforms have proven to offer a solution for mid-plexed analysis that is high-throughput, sensitive, and with a low cost per test. In this work, an enhanced genotyping method based on MassCode technology was devised and integrated as part of a high-throughput mid-plexing analytical system that facilitates robust qualitative differential detection of DNA targets. Samples are first analyzed using MassCode PCR (MC-PCR) performed with an array of primer sets encoded with unique mass tags. Lambda exonuclease and an array of MassCode probes are then contacted with MC-PCR products for further interrogation and target sequences are specifically identified. Primer and probe hybridizations occur in homogeneous solution, a clear advantage over micro- or nanoparticle suspension arrays. The two cognate tags coupled to resultant MassCode hybrids are detected in an automated process using a benchtop single quadrupole mass spectrometer. The prospective value of using MassCode probe arrays for multiplexed bioanalysis was demonstrated after developing a 14plex proof of concept assay designed to subtype a select panel of Salmonella enterica serogroups and serovars. This MassCode system is very flexible and test panels can be customized to include more, less, or different markers.     

Polymerase II promoter strength determines efficacy of microRNA adapted shRNAs

Since the discovery of RNAi and microRNAs more than 10 years ago, much research has focused on the development of systems that usurp microRNA pathways to downregulate gene expression in mammalian cells. One of these systems makes use of endogenous microRNA pri-cursors that are expressed from polymerase II promoters where the mature microRNA sequence is replaced by gene specific duplexes that guide RNAi (shRNA-miRs). Although shRNA-miRs are effective in directing target mRNA knockdown and hence reducing protein expression in many cell types, variability of RNAi efficacy in cell lines has been an issue. Here we show that the choice of the polymerase II promoter used to drive shRNA expression is of critical importance to allow effective mRNA target knockdown. We tested the abundance of shRNA-miRs expressed from five different polymerase II promoters in 6 human cell lines and measured their ability to drive target knockdown. We observed a clear positive correlation between promoter strength, siRNA expression levels, and protein target knockdown. Differences in RNAi from the shRNA-miRs expressed from the various promoters were particularly pronounced in immune cells. Our findings have direct implications for the design of shRNA-directed RNAi experiments and the preferred RNAi system to use for each cell type.     

5-Aminomethylbenzimidazoles as potent ITK antagonists

Benzamide 1 demonstrated good potency as a selective ITK inhibitor, however the amide moiety was found to be hydrolytically labile in vivo, resulting in low oral exposure and the generation of mutagenic aromatic amine metabolites. Replacing the benzamide with a benzylamine linker not only addressed the toxicity issue, but also improved the cellular and functional potency as well as the drug-like properties. SAR studies around the benzylamines and the identification of 10n and 10o as excellent tools for proof-of-concept studies are described.     

Discovery of potent inhibitors of interleukin-2 inducible T-cell kinase (ITK) through structure-based drug design

Interleukin-2 inducible T-cell kinase (ITK) is a member of the Tec kinase family and is involved with T-cell activation and proliferation. Due to its critical role in acting as a modulator of T-cells, ITK inhibitors could provide a novel route to anti-inflammatory therapy. This work describes the discovery of ITK inhibitors through structure-based design where high-resolution crystal structural information was used to optimize interactions within the kinase specificity pocket of the enzyme to improve both potency and selectivity.     

Capping and receptor-mediated endocytosis of cell-bound verotoxin (shiga-like toxin) 1: Chemical identification of an amino acid in the B subunit necessary for efficient receptor glycolipid binding and cellular internalization

The glycolipid globotriaosylceramide (Gb₃) is the plasma membrane receptor that mediates the internalization of verotoxin (VT1) into susceptible cells by capping and receptor-mediated endocytosis (RME). Internalization of fluorescein isothiocyanate-conjugated holotoxin into Daudi lymphoma cells was found to be slower than the pentameric receptor binding B subunit alone, suggesting that the A subunit may interact with the membrane to compromise the lateral mobility of the receptor bound B subunit. 3-D reconstruction of fluorescent images by confocal microscopy confirmed the complete internalization of holotoxin. VT1 internalization and cytotoxicity was inhibited by monodansyl cadavarine, which supports a role for clathrin coated pits in the RME of VT1. Biotinylation of the B subunit (in contrast to fluorescein labelling) was found to prevent toxin internalization. This effect correlated with reduced binding of Gb₃ and reduced cytotoxicity in vitro. By cleavage of the B subunit at the single tryptophan residue, the reduced Gb₃ binding and lack of cellular internalization was shown to be due to the biotinylation of lysine 53 in the VT1 B subunit. This residue was not labelled with fluorescein isothiocyanate in the native protein. This conclusion was confirmed by the finding that biotinylation of VT2c (which contains lys 53) prevented glycolipid receptor binding, whereas biotinylation of VT2e (in which lys 53 is substituted by ile) had no effect. © 1994 Wiley-Liss, Inc.     

Functional significance of globotriaosyl ceramide in interferon-alpha(2)/type 1 interferon receptor-mediated antiviral activity

The N-terminus of the type 1 interferon receptor subunit, IFNAR1, has high amino acid sequence similarity to the receptor binding B subunit of the Escherichia coli-derived verotoxin 1, VT1. The glycolipid, globotriaosyl ceramide (Gb(3): Gal alpha(1) --> 4 Gal beta 1 --> 4 Glu beta 1 --> 1 Cer) is the specific cell receptor for VT1. Gb(3)-deficient variant cells selected for VT resistance are cross-resistant to interferon-alpha (IFN-alpha)-mediated antiproliferative activity. The association of eIFNAR1 with Gal alpha 1 --> 4 Gal containing glycolipids has been previously shown to be important for the receptor-mediated IFN-alpha signal transduction for growth inhibition. The crucial role of Gb(3) for the signal transduction of IFN-alpha-mediated antiviral activity is now reported. IFN-alpha-mediated antiviral activity, nuclear translocation of activated Stat1, and increased expression of PKR were defective in Gb(3)-deficient vero mutant cells, although the surface expression of IFNAR1 was unaltered. The VT1B subunit was found to inhibit IFN-alpha-mediated antiviral activity, Stat1 nuclear translocation and PKR upregulation. Unlike VT1 cytotoxicity, IFN-alpha-induced Stat1 nuclear translocation was not inhibited when RME was prevented, suggesting that the accessory function of Gb(3) occurs at the plasma membrane. IFN-alpha antiviral activity was also studied in Gb(3)-positive MRC-5 cells, which are resistant to IFN-alpha growth inhibition, partially resistant to VT1 but still remain fully sensitive to IFN-alpha antiviral activity, and two astrocytoma cell lines expressing different Gb(3) fatty acid isoforms. In both systems, long chain fatty acid-containing Gb(3) isoforms, which are less effective to mediate VT1 cytotoxicity, were found to correlate with higher IFN-alpha-mediated antiviral activity. Inhibition of Gb(3) synthesis in toto prevented IFN-alpha antiviral activity in all cells. We propose that the long chain Gb(3) fatty isoforms preferentially remain in the plasma membrane, and by associating with IFNAR1, mediate IFN-alpha antiviral signaling, whereas short chain Gb(3) fatty acid isoforms are preferentially internalized to mediate VT1 cytotoxicity and IFNAR1-dependent IFN-alpha growth inhibition.