Phosphatidylinositol‐4 kinase III beta and oxysterol‐binding protein accumulate unesterified cholesterol on poliovirus‐induced membrane structure

Studies on anti‐picornavirus compounds have revealed an essential role of a novel cellular pathway via host phosphatidylinositol‐4 kinase III beta (PI4KB) and oxysterol‐binding protein (OSBP) family I in poliovirus (PV) replication. However, the molecular role for this pathway in PV replication has yet to be determined. Here, viral and host proteins modulating production of phosphatidylinositol 4‐phosphate (PI4P) and accumulation of unesterified cholesterol (UC) in cells were analyzed and the role of the PI4KB/OSBP pathway in PV replication characterized. Virus protein 2BC was identified as a novel interactant of PI4KB. PI4KB and VCP/p97 bind to a partially overlapped region of 2BC with different sensitivity to a 2C inhibitor. Production of PI4P and accumulation of UC were enhanced by virus protein 2BC, but suppressed by virus proteins 3A and 3AB. In PV‐infected cells, a PI4KB inhibitor suppressed production of PI4P, and both a PI4KB inhibitor and an OSBP ligand suppressed accumulation of UC on virus‐induced membrane structure. Inhibition of PI4KB activity caused dissociation of OSBP from virus‐induced membrane structure in PV‐infected cells. Synthesis of viral nascent RNA in PV‐infected cells was not affected in the presence of PI4KB inhibitor and OSBP ligand; however, transient pre‐treatment of PV‐infected cells with these inhibitors suppressed viral RNA synthesis. These results suggest that virus proteins modulate PI4KB activity and provide PI4P for recruitment of OSBP to accumulate UC on virus‐induced membrane structure for formation of a virus replication complex.     

Phosphatidylinositol 4-kinase III beta is a target of enviroxime-like compounds for antipoliovirus activity

Enviroxime is an antienterovirus compound that targets viral protein 3A and/or 3AB and suppresses a step in enterovirus replication by unknown mechanism. To date, four antienterovirus compounds, i.e., GW5074, Flt3 inhibitor II, TTP-8307, and AN-12-H5, are known to have similar mutations in the 3A protein-encoding region causing resistance to enviroxime (a G5318A [3A-Ala70Thr] mutation in poliovirus [PV]) and are considered enviroxime-like compounds. Recently, antienterovirus activity of a phosphatidylinositol 4-kinase III beta (PI4KB) inhibitor, PIK93, was reported, suggesting that PI4KB is an important host factor targetable by antienterovirus compounds (N. Y. Hsu et al., Cell 141:799-811, 2010). In this study, we analyzed the inhibitory effects of previously identified enviroxime-like compounds (GW5074 and AN-12-H5) and a newly identified antienterovirus compound, T-00127-HEV1, on phosphoinositide (PI) kinases. We found that T-00127-HEV1 inhibited PI4KB activity with a higher specificity for than other PI kinases, in contrast to GW5074, which had a broad specificity for PI kinases. In contrast, AN-12-H5 showed no inhibitory effect on PI4KB activity and only moderate inhibitory effects on PI 3-kinase activity. Small interfering RNA (siRNA) screening targeting PI kinases identified PI4KB is a target of GW5074 and T-00127-HEV1, but not of AN-12-H5, for anti-PV activity. Interestingly, T-00127-HEV1 and GW5074 did not inhibit hepatitis C virus (HCV) replication, in contrast to a strong inhibitory effect of AN-12-H5. These results suggested that PI4KB is an enterovirus-specific host factor required for the replication process and targeted by some enviroxime-like compounds (T-00127-HEV1 and GW5074) and that enviroxime-like compounds may have targets other than PI kinases for their antiviral effect.     

ACBD3-mediated recruitment of PI4KB to picornavirus RNA replication sites

Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for genome RNA replication of enteroviruses, small non-enveloped viruses belonging to the family Picornaviridae. Here, we demonstrated that PI4KB is also essential for genome replication of another picornavirus, Aichi virus (AiV), but is recruited to the genome replication sites by a different strategy from that utilized by enteroviruses. AiV non-structural proteins, 2B, 2BC, 2C, 3A, and 3AB, interacted with a Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3). Furthermore, we identified previously unknown interaction between ACBD3 and PI4KB, which provides a novel manner of Golgi recruitment of PI4KB. Knockdown of ACBD3 or PI4KB suppressed AiV RNA replication. The viral proteins, ACBD3, PI4KB, and phophatidylinositol-4-phosphate (PI4P) localized to the viral RNA replication sites. AiV replication and recruitment of PI4KB to the RNA replication sites were not affected by brefeldin A, in contrast to those in enterovirus infection. These results indicate that a viral protein/ACBD3/PI4KB complex is formed to synthesize PI4P at the AiV RNA replication sites and plays an essential role in viral RNA replication.     

Valosin-containing protein (VCP/p97) is required for poliovirus replication and is involved in cellular protein secretion pathway in poliovirus infection

Poliovirus (PV) modifies membrane-trafficking machinery in host cells for its viral RNA replication. To date, ARF1, ACBD3, BIG1/BIG2, GBF1, RTN3, and PI4KB have been identified as host factors of enterovirus (EV), including PV, involved in membrane traffic. In this study, we performed small interfering RNA (siRNA) screening targeting membrane-trafficking genes for host factors required for PV replication. We identified valosin-containing protein (VCP/p97) as a host factor of PV replication required after viral protein synthesis, and its ATPase activity was essential for PV replication. VCP colocalized with viral proteins 2BC/2C and 3AB/3B in PV-infected cells and showed an interaction with 2BC and 3AB but not with 2C and 3A. Knockdown of VCP did not suppress the replication of coxsackievirus B3 or Aichi virus. A VCP-knockdown-resistant PV mutant had an A4881G (a mutation of E253G in 2C) mutation, which is known as a determinant of a secretion inhibition-negative phenotype. However, knockdown of VCP did not affect the inhibition of cellular protein secretion caused by overexpression of each individual viral protein. These results suggested that VCP is a host factor required for viral RNA replication of PV among membrane-trafficking proteins and provides a novel link between cellular protein secretion and viral RNA replication.     

Oxysterol‐binding protein recruitment and activity at the endoplasmic reticulum‐Golgi interface are independent of Sac1

Oxysterol‐binding protein (OSBP) localizes to endoplasmic reticulum (ER)‐Golgi contact sites where it transports cholesterol and phosphatidylinositol 4‐phosphate (PI‐4P), and activates lipid transport and biosynthetic activities. The PI‐4P phosphatase Sac1 cycles between the ER and Golgi apparatus where it potentially regulates OSBP activity. Here we examined whether the ER‐Golgi distribution of endogenous or ectopically expressed Sac1 influences OSBP activity. OSBP and Sac1 co‐localized at apparent ER‐Golgi contact sites in response to 25‐hydroxycholesterol (25OH), cholesterol depletion and p38 MAPK inhibitors. A Sac1 mutant that is unable to exit the ER did not localize with OSBP, suggesting that sterol perturbations cause Sac1 transport to the Golgi apparatus. Ectopic expression of Sac1 in the ER or Golgi apparatus, or Sac1 silencing, did not affect OSBP localization to ER‐Golgi contact sites, OSBP‐dependent activation of sphingomyelin synthesis, or cholesterol esterification in the ER. p38 MAPK inhibition and retention of Sac1 in the Golgi apparatus also caused OSBP phosphorylation and OSBP‐dependent activation of sphingomyelin synthesis at ER‐Golgi contacts. These results demonstrate that Sac1 expression in either the ER or Golgi apparatus has a minimal impact on the PI‐4P that regulates OSBP activity or recruitment to contact sites.      

Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus

Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid-modifying pathway underlying formation of viral replication sites.     

Suppression of epidermal growth factor receptor‐mediated β‐catenin nuclear accumulation enhances the anti‐tumor activity of phosphoinositide 3‐kinase inhibitor in breast cancer

Phosphoinositide 3‐kinase (PI3K) signaling is frequently deregulated in breast cancer and plays a critical role in tumor progression. However, resistance to PI3K inhibitors in breast cancer has emerged, which is due to the enhanced β‐catenin nuclear accumulation. Until now, the mechanisms underlying PI3K inhibition‐induced β‐catenin nuclear accumulation remains largely unknown. In the present study, we found inhibition of PI3K with LY294002 promoted β‐catenin nuclear accumulation in MCF‐7 and MDA‐MB‐231 breast cancer cells. Combining PI3K inhibitor LY294002 with XAV‐939, an inhibitor against β‐catenin nuclear accumulation, produced an additive anti‐proliferation effect against breast cancer cells. Subsequent experiments suggested β‐catenin nuclear accumulation induced by PI3K inhibition depended on the feedback activation of epidermal growth factor receptor (EGFR) signaling pathway in breast cancer cells. Inhibition of EGFR phosphorylation with Gefitinib enhanced anti‐proliferation effect of PI3K inhibitor LY294002 in MCF‐7 and MDA‐MB‐231 cells. Taken together, our findings may elucidate a possible mechanism explaining the poor outcome of PI3K inhibitors in breast cancer treatment.     

TLR3/TRIF signalling pathway regulates IL‐32 and IFN‐β secretion through activation of RIP‐1 and TRAF in the human cornea

Toll‐like receptor‐3 (TLR3) and RNA helicase retinoic‐acid‐inducible protein‐1 (RIG‐I) serve as cytoplasmic sensors for viral RNA components. In this study, we investigated how the TLR3 and RIG‐I signalling pathway was stimulated by viral infection to produce interleukin (IL)‐32‐mediated pro‐inflammatory cytokines and type I interferon in the corneal epithelium using Epstein–Barr virus (EBV)‐infected human cornea epithelial cells (HCECs/EBV) as a model of viral keratitis. Increased TLR3 and RIG‐I that are responded to EBV‐encoded RNA 1 and 2 (EBER1 and EBER2) induced the secretion of IL‐32‐mediated pro‐inflammatory cytokines and IFN‐β through up‐regulation of TRIF/TRAF family proteins or RIP‐1. TRIF silencing or TLR3 inhibitors more efficiently inhibited sequential phosphorylation of TAK1, TBK1, NF‐κB and IRFs to produce pro‐inflammatory cytokines and IFN‐β than RIG‐I‐siRNA transfection in HCECs/EBV. Blockade of RIP‐1, which connects the TLR3 and RIG‐I pathways, significantly blocked the TLR3/TRIF‐mediated and RIG‐I‐mediated pro‐inflammatory cytokines and IFN‐β production in HCECs/EBV. These findings demonstrate that TLR3/TRIF‐dependent signalling pathway against viral RNA might be a main target to control inflammation and anti‐viral responses in the ocular surface.     

Essential domains of phosphatidylinositol‐4 kinase III β required for enterovirus replication

Phosphatidylinositol‐4 kinase III β (PI4KB) is a host factor that is required for enterovirus (EV) replication. In this study, the importance of host proteins that interact with PI4KB in EV replication was analyzed by trans complementation with PI4KB mutants in a PI4KB‐knockout cell line. Ectopically expressed PI4KB mutants, which lack binding regions for ACBD3, RAB11, and 14‐3‐3 proteins, rescued replication of poliovirus and enterovirus 71. These findings suggest that interaction of PI4KB with these host proteins is not essential for EV replication once PI4KB has been expressed and that PI4KB is functionally independent from these host proteins regarding EV replication.     

Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

RNA viruses can rapidly mutate and acquire resistance to drugs that directly target viral enzymes, which poses serious problems in a clinical context. Therefore, there is a growing interest in the development of antiviral drugs that target host factors critical for viral replication, since they are unlikely to mutate in response to therapy. We recently demonstrated that phosphatidylinositol-4-kinase IIIβ (PI4KIIIβ) and its product phosphatidylinositol-4-phosphate (PI4P) are essential for replication of enteroviruses, a group of medically important RNA viruses including poliovirus (PV), coxsackievirus, rhinovirus, and enterovirus 71. Here, we show that enviroxime and GW5074 decreased PI4P levels at the Golgi complex by directly inhibiting PI4KIIIβ. Coxsackievirus mutants resistant to these inhibitors harbor single point mutations in the non-structural protein 3A. These 3A mutations did not confer compound-resistance by restoring the activity of PI4KIIIβ in the presence of the compounds. Instead, replication of the mutant viruses no longer depended on PI4KIIIβ, since their replication was insensitive to siRNA-mediated depletion of PI4KIIIβ. The mutant viruses also did not rely on other isoforms of PI4K. Consistently, no high level of PI4P could be detected at the replication sites induced by the mutant viruses in the presence of the compounds. Collectively, these findings indicate that through specific single point mutations in 3A, CVB3 can bypass an essential host factor and lipid for its propagation, which is a new example of RNA viruses acquiring resistance against antiviral compounds, even when they directly target host factors.     

Molecular insights on analogs of HIV PR inhibitors toward HTLV‐1 PR through QM/MM interactions and molecular dynamics studies: comparative structure analysis of wild and mutant HTLV‐1 PR

Retroviruses HTLV‐1 and HIV‐1 are the primary causative agents of fatal adult T‐cell leukemia and acquired immune deficiency syndrome (AIDS) disease. Both retroviruses are similar in characteristics mechanism, and it encodes for protease that mainly involved in the viral replication process. On the basis of the therapeutic success of HIV‐1 PR inhibitors, the protease of HTLV‐1 is mainly considered as a potential target for chemotherapy. At the same time, structural similarities in both enzymes that originate HIV PR inhibitors can also be an HTLV‐1 PR inhibitor. But the expectations failed because of rejection of HIV PR inhibitors from the HTLV‐1 PR binding pocket. In this present study, the reason for the HIV PR inhibitor rejection from the HTLV‐1 binding site was identified through sequence analysis and molecular dynamics simulation method. Functional analysis of M37A mutation in HTLV PR clearly shows that the MET37 specificity and screening of potential inhibitors targeting MET37 is performed by using approved 90% similar HIV PR inhibitor compounds. From this approach, we report few compounds with a tendency to accept/donate electron specifically to an important site residue MET37 in HTLV‐1 PR binding pocket. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural insights into Acyl‐coenzyme A binding domain containing 3 (ACBD3) protein hijacking by picornaviruses

Many picornaviruses hijack the Golgi resident Acyl‐coenzyme A binding domain containing 3 (ACBD3) protein in order to recruit the phosphatidylinositol 4‐kinase B (PI4KB) to viral replication organelles (ROs). PI4KB, once recruited and activated by ACBD3 protein, produces the lipid phosphatidylinositol 4‐phosphate (PI4P), which is a key step in the biogenesis of viral ROs. To do so, picornaviruses use their small nonstructural protein 3A that binds the Golgi dynamics domain of the ACBD3 protein. Here, we present the analysis of the highly flexible ACBD3 proteins and the viral 3A protein in solution using small‐angle X‐ray scattering and computer simulations. Our analysis revealed that both the ACBD3 protein and the 3A:ACBD3 protein complex have an extended and flexible conformation in solution.     

Sterol transfer,PI4P consumption,and control of membrane lipid order by endogenous OSBP

The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol‐binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans‐Golgi network (TGN). Using the inhibitor OSW‐1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4‐kinase PI4KIIIβ triggers large periodic traveling waves of PI4P across the TGN. These waves are cadenced by long‐range PI4P production by PI4KIIα and PI4P consumption by OSBP. Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4‐kinases to control the lipid composition of subcellular membranes.     

Phosphatidylinositol 3‐kinase‐mediated regulation of neuronal apoptosis and necrosis by insulin and IGF‐I

We examined effects of two insulin‐like growth factors, insulin and insulin‐like growth factor‐I (IGF‐I), against apoptosis, excitotoxicity, and free radical neurotoxicity in cortical cell cultures. Like IGF‐I, insulin attenuated serum deprivation‐induced neuronal apoptosis in a dose‐dependent manner at 10–100 ng/mL. The anti‐apoptosis effect of insulin against serum deprivation disappeared by addition of a broad protein kinase inhibitor, staurosporine, but not by calphostin C, a selective protein kinase C inhibitor. Addition of PD98059, a mitogen‐activated protein kinase kinase (MAPKK) inhibitor, blocked insulin‐induced activation of extracellular signal‐regulated protein kinases (ERK1/2) without altering the neuroprotective effect of insulin. Cortical neurons underwent activation of phosphatidylinositol (PI) 3‐kinase as early as 1 min after exposure to insulin. Inclusion of wortmannin or LY294002, selective inhibitors of PI 3‐K, reversed the insulin effect against apoptosis. In contrast to the anti‐apoptosis effect, neither insulin nor IGF‐I protected excitotoxic neuronal necrosis following continuous exposure to 15 μM N‐methyl‐d ‐aspartate or 40 μM kainate for 24 h. Surprisingly, concurrent inclusion of 50 ng/mL insulin or IGF‐I aggravated free radical‐induced neuronal necrosis over 24 h following continuous exposure to 10 μM Fe²⁺ or 100 μM buthionine sulfoximine. Wortmannin or LY294002 also reversed this potentiation effect of insulin. These results suggest that insulin‐ like growth factors act as anti‐apoptosis factor and pro‐oxidant depending uon the activation of PI 3‐kinase. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 536–546, 1999     

Hemoglobin induces the expression of indoleamine 2,3‐dioxygenase in dendritic cells through the activation of PI3K,PKC, and NF‐κB and the generation of reactive oxygen species

Indoleamine 2,3‐dioxygenase (IDO) is the rate‐limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme‐free globin (Apo Hb), induced IDO expression in bone marrow‐derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of IκBα. Hb‐induced IDO expression was inhibited by inhibitors of PI3‐kinase (PI3K), PKC and nuclear factor (NF)‐κB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb‐induced IDO expression was inhibited by anti‐oxidant N‐acetyl‐L ‐cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3‐amino‐1,2,4‐triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O, H₂O₂, and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF‐κB through the PI3K‐PKC‐ROS and PI3K‐Akt pathways is required for the Hb‐induced IDO expression in BMDCs. J. Cell. Biochem. 108: 716–725, 2009. © 2009 Wiley‐Liss, Inc.     

1‐Aminobenzocyclobutene‐1‐phosphonic Acid and Related Compounds as Inhibitors of Phenylalanine Ammonia‐Lyase

Five new geminal aminocycloalkanephosphonic acids ( 4 – 8 ) containing both an aromatic ring and a cycloalkane ring were synthesized and evaluated as potential inhibitors of buckwheat phenylalanine ammonia‐lyase (PAL). Within the set of compounds which are related to 2‐aminoindane‐2‐phosphonic acid (AIP, 3 ), a known powerful inhibitor of PAL, racemic 1‐aminobenzocyclobutene‐1‐phosphonic acid ( 4 ), was six times weaker than AIP as an in vitro inhibitor of buckwheat PAL, but six times stronger than AIP as an in vivo inhibitor of phenylalanine‐derived anthocyanin synthesis in buckwheat.     

Synthesis,Biological Evaluation,and Molecular Docking Studies of Novel 4‐[4‐Arylpyridin‐1(4H)‐yl]benzoic Acid Derivatives as Anti‐HIV‐1 Agents

The structural similarities between N1 substituted 1,4‐dihydropyridines and the known gp41 inhibitors, NB ‐2 and NB ‐64 , were considered in the current research for the design of some novel anti‐HIV‐1 agents. A series of novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid derivatives were synthesized and after a comprehensive structural elucidation were screened for in vitro anti‐HIV‐1 activity. Most of the tested compounds displayed moderate to good inhibitory activity against HIV‐1 growth and were evaluated for in vitro cytotoxic activity using XTT assay at the concentration of 100 μm . Among the tested compounds, 1c , 1d and 1e showed potent anti‐HIV‐1 activity against P24 expression at 100 μm with inhibition percentage of 84.00%, 76.42% and 80.50%, respectively. All the studied compounds possessed no significant cytotoxicity on MT‐2 cell line. The binding modes of these compounds to gp41 binding site were determined through molecular docking study. Docking studies proved 1a as the most potent compound and binding maps exhibited that the activities might be attributed to the electrostatic and hydrophobic interactions and additional H‐bonds with the gp41 binding site. The Lipinski's ‘rule of five’ and drug‐likeness criteria were also calculated for the studied compounds. All derivatives obeyed the Lipinski's ‘rule of five’ and had drug‐like features. The findings of this study suggest that novel 4‐[4‐arylpyridin‐1(4H)‐yl]benzoic acid might be a promising scaffold for the discovery and development of novel anti‐HIV‐1 agents.     

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Picornavirus infectivity is dependent on the RNA poly(A) tail, which binds the poly(A) binding protein (PABP). PABP was reported to stimulate viral translation and RNA synthesis. Here, we studied encephalomyocarditis virus (EMCV) and poliovirus (PV) genome expression in Krebs-2 and HeLa cell-free extracts that were drastically depleted of PABP (96%-99%). Although PABP depletion markedly diminished EMCV and PV internal ribosome entry site (IRES)-mediated translation of a polyadenylated luciferase mRNA, it displayed either no (EMCV) or slight (PV) deleterious effect on the translation of the full-length viral RNAs. Moreover, PABP-depleted extracts were fully competent in supporting EMCV and PV RNA replication and virus assembly. In contrast, removing the poly(A) tail from EMCV RNA dramatically reduced RNA synthesis and virus yields in cell-free reactions. The advantage conferred by the poly(A) tail to EMCV synthesis was more pronounced in untreated than in nuclease-treated extract, indicating that endogenous cellular mRNAs compete with the viral RNA for a component(s) of the RNA replication machinery. These results suggest that the poly(A) tail functions in picornavirus replication largely independent of PABP.     

A TLC bioautographic method for the detection of α‐ and β‐glucosidase inhibitors in plant extracts

Introduction – Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC assay has previously been established for the detection of β‐glucosidase inhibitors but not for α‐glucosidase. Nonetheless, α‐glucosidase inhibition is an important target for therapeutic agents against of type 2 diabetes and anti‐viral infections. Objective – To develop a TLC bioautographic method to detect α‐ and β‐glucosidase inhibitors in plant extracts. Methodology – The enzymes α‐ and β‐d ‐glucosidase were dissolved in sodium acetate buffer. After migration of the samples, the TLC plate was sprayed with enzyme solution and incubated at room temperature for 60 min in the case of α‐d ‐glucosidase, and 37°C for 20 min in the case of β‐d ‐glucosidase. For detection of the active enzyme, solutions of 2‐naphthyl‐α‐D‐glucopyranoside or 2‐naphthyl‐β‐D‐glucopyranoside and Fast Blue Salt were mixed at a ratio of 1 : 1 (for α‐d ‐glucosidase) or 1 : 4 (for β‐d ‐glucosidase) and sprayed onto the plate to give a purple background colouration after 2–5 min. Results – Enzyme inhibitors were visualised as white spots on the TLC plates. Conduritol B epoxide inhibited α‐d ‐glucosidase and β‐d ‐glucosidase down to 0.1 µg. Methanol extracts of Tussilago farfara and Urtica dioica after migration on TLC gave enzymatic inhibition when applied in amounts of 100 µg for α‐glucosidase and 50 µg for β‐glucosidase. Conclusion – The screening test was able to detect inhibition of α‐ and β‐glucosidases by pure reference substances and by compounds present in complex matrices, such as plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Tissue inhibitor of metalloproteinases‐1‐induced scattered liver metastasis is mediated by host‐derived urokinase‐type plasminogen activator

Paradoxically, not only proteinases but also their inhibitors can correlate with bad prognosis of cancer patients, underlining the evolving concept of the protease web as the complex interplay between proteinases, their inhibitors and effector molecules. Elevated levels of tissue inhibitor of metalloproteinases‐1 (TIMP‐1) render the liver more susceptible to metastasis by triggering urokinase plasminogen activator (uPA) expression as well as hepatocyte growth factor (HGF) signalling, thereby leading to the fatal scattered infiltration of metastasizing tumour cells throughout the parenchyma of the target organ. Here, we investigated whether host uPA is a crucial protagonist for the TIMP‐1‐induced modulation of a pro‐metastatic microenvironment in the liver. Indeed, in livers of uPA‐ablated mice elevated TIMP‐1 levels did not trigger HGF signalling and did not promote metastasis of a murine T‐lymphoma cell line. In contrast, lack of tumour cell‐derived uPA induced by gene silencing did not interfere with this pro‐metastatic pathway. Furthermore, host uPA was necessary for the recruitment of neutrophilic granulocytes and the associated increase of HGF in livers with elevated TIMP‐1 levels. This newly identified co‐operation between TIMP‐1 and host uPA suggests that therapies, simultaneously interfering with pro‐ and anti‐proteolytic pathways may be beneficial for patients with metastatic disease.