Pancreatic lipase-related protein-2 (PLRP2) can contribute to dietary fat digestion in human newborns

In newborn mice, PLRP2 is essential for fat digestion. In human infants, the role of PLRP2 in fat digestion is unclear, as it has poor activity against long-chain triglycerides in vitro. Also, many infants carry a genetic polymorphism resulting in a truncated protein, PLRP2 W340X, which may impact function significantly. We re-examined the properties of recombinant human PLRP2 and studied the impact of W340X mutation on its function. In the presence of bile salt micelles and colipase, human PLRP2 hydrolyzed long-chain tri-, di-, and monoglycerides. It hydrolyzed triolein at a level much lower than that of pancreatic triglyceride lipase, but close to that of carboxyl ester lipase, after a long lag phase, which could be eliminated by the addition of oleic acids. Human PLRP2 W340X was poorly secreted and largely retained inside the cell. The retention of the mutant protein triggered endoplasmic reticulum stress and unfolded protein responses. Our results show that earlier studies underestimated human PLRP2 activity against triolein by employing suboptimal assay conditions. In vivo, dietary fat emulsions contain fatty acids as a result of the action of gastric lipase. Consequently, PLRP2 can contribute to fat digestion during early infancy. Furthermore, infants with homozygous W340X alleles will not secrete functional PLRP2 and may have inefficient dietary fat digestion, particularly when breastfeeding is unavailable. Additionally, the aberrant folding of W340X mutant may cause chronic cellular stress and increase susceptibility of pancreatic exocrine cells to other metabolic stressors.     

Purification and characterization of repressor of temperate S. aureus phage phi11

To gain insight into the structure and function of repressor proteins of bacteriophages of gram-positive bacteria, repressor of temperate Staphylococcus aureus phage phi11 was undertaken as a model system here and purified as an N-terminal histidine-tagged variant (His-CI) by affinity chromatography. A approximately 19 kDa protein copurified with intact His-CI (approximately 30 kDa) at low level was resulted most possibly due to partial cleavage at its Ala-Gly site. At approximately 10 nM and higher concentrations, His-CI forms significant amount of dimers in solution. There are two repressor binding sites in phi11 cI-cro intergenic region and binding to two sites occurs possibly by a cooperative manner. Two sites dissected by HincII digestion were designated operators O(L) and O(R), respectively. Equilibrium binding studies indicate that His-CI binds to O(R) with a little more strongly than O(L) and binding species is probably dimeric in nature. Interestingly His-CI binding affinity reduces drastically at elevated temperatures (32-42 degrees C). Both O(L) and O(R) harbor a nearly identical inverted repeat and studies show that phi11 repressor binds to each repeat efficiently. Additional analyses indicate that phi11 repressor, like lambda repressor, harbors an N-terminal domain and a C-terminal domain which are separated by a hinge region. Secondary structure of phi11 CI even nearly resembles to that of lambda, phage repressor though they differ at sequence level. The putative N-terminal HTH (helix-turn-helix) motif of phi11 repressor belongs to the HTH -XRE-family of proteins and shows significant identity to the HTH motifs of some proteins of evolutionary distant organisms but not to HTH motifs of most S. aureus phage repressors.     

Application of Absorption Modeling to Predict Bioequivalence Outcome of Two Batches of Etoricoxib Tablets

As part of the overall product development and manufacturing strategy, pharmaceutical companies routinely change formulation and manufacturing site. Depending on the type and level of change and the BCS class of the molecule, dissolution data and/or bioequivalence (BE) may be needed to support the change for immediate release dosage forms. In this report, we demonstrate that for certain weakly basic low-solubility molecules which rapidly dissolve in the stomach, absorption modeling could be used to justify a BE study waiver even when there is failure to show dissolution similarity under some conditions. The development of an absorption model for etoricoxib is described here, which was then used to a priori predict the BE outcome of tablet batches manufactured at two sites. Dissolution studies in 0.01 N HCl media (pH 2.0) had demonstrated similarity of etoricoxib tablets manufactured at two different sites. However, dissolution testing at pH 4.5 and pH 6.8 media failed to show comparability of the tablets manufactured at the two sites. Single simulations and virtual trials conducted using the 0.01 N HCl dissolution showed similarity in AUC and C_max for all tablet strengths for batches manufactured at the two manufacturing sites. These predicted results were verified in a definitive bioequivalence study, which showed that both tablet batches were bioequivalent. Since the development of traditional in vitro–in vivo correlations (IVIVC) for immediate release (IR) products is challenging, in cases such as etoricoxib, absorption modeling could be used as an alternative to support waiver of a BE study.KEY WORDS: bioequivalence, dissolution, modeling, pharmacokinetics, SUPAC     

Heuristic RNA pseudoknot prediction including intramolecular kissing hairpins

Pseudoknots are an essential feature of RNA tertiary structures. Simple H-type pseudoknots have been studied extensively in terms of biological functions, computational prediction, and energy models. Intramolecular kissing hairpins are a more complex and biologically important type of pseudoknot in which two hairpin loops form base pairs. They are hard to predict using free energy minimization due to high computational requirements. Heuristic methods that allow arbitrary pseudoknots strongly depend on the quality of energy parameters, which are not yet available for complex pseudoknots. We present an extension of the heuristic pseudoknot prediction algorithm DotKnot, which covers H-type pseudoknots and intramolecular kissing hairpins. Our framework allows for easy integration of advanced H-type pseudoknot energy models. For a test set of RNA sequences containing kissing hairpins and other types of pseudoknot structures, DotKnot outperforms competing methods from the literature. DotKnot is available as a web server under http://dotknot.csse.uwa.edu.au.     

The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3C^pro cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3C^pro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3C^pro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3C^pro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition.     

Comparative RNAi screening reveals host factors involved in enterovirus infection of polarized endothelial monolayers

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Highlights? RNAi screens reveal 117 host molecules involved in enterovirus infection ? Components of immune signaling including MAPKs, Akts, and TLR8 restrict infection ? Adenylate cyclases are required for both PV and CVB infection ? Rab GTPases, Src tyrosine kinases, and tyrosine phosphatases control infection     

Multifunctional effect of epigallocatechin-3-gallate (EGCG) in downregulation of gelatinase-A (MMP-2) in human breast cancer cell line MCF-7

AimsThe tumor inhibiting property of green tea polyphenol epigallocatechin-3-gallate (EGCG) is well documented. Studies reveal that matrix-metalloproteinases (MMPs) play pivotal roles in tumor invasion through degradation of basement membranes and extracellular matrix (ECM). We studied the effect of EGCG on matrixmetalloproteinases-2 (MMP-2), the factors involved in activation, secretion and signaling molecules that might be involved in the regulation of MMP-2 in human breast cancer cell line, MCF-7.Main methodsMCF-7 was treated with EGCG (20 μM, 24 h), the effect of EGCG on MMP-2 expression, activity and its regulatory molecules were studied by gelatin zymography, Western blot, quantitative and semi-quantitative real time RT-PCR, immunoflourescence and cell adhesion assay.Key findingsEGCG treatment reduced the activity, protein expression and mRNA expression level of MMP-2. EGCG treatment reduced the expression of focal adhesion kinase (FAK), membrane type-1-matrix metalloproteinase (MT1-MMP), nuclear factor-kappa B (NF-kB), vascular endothelial growth factor (VEGF) and reduced the adhesion of MCF-7 cells to ECM, fibronectin and vitronectin. Real time RT-PCR revealed a reduced expression of integrin receptors α5, β1, αv and β3 due to EGCG treatment.SignificanceDown regulation of expression of MT1-MMP, NF-kB, VEGF and disruption of functional status of integrin receptors may indicate decreased MMP-2 activation; low levels of FAK expression might indicate disruption in FAK-induced MMP-2 secretion and decrease in activation of phosphatidyl-inositol-3-kinase (PI-3K), extracellular regulated kinase (ERK) indicates probable hindrance in MMP-2 regulation and induction. We propose EGCG as potential inhibitor of expression and activity of pro-MMP-2 by a process involving multiple regulatory molecules in MCF-7.     

Retinoic Acid-induced Gene-1 (RIG-I) Associates with the Actin Cytoskeleton via Caspase Activation and Recruitment Domain-dependent Interactions

The actin cytoskeleton serves as a barrier that protects mammalian cells from environmental pathogens such as bacteria, fungi, and viruses. Several components of antimicrobial signaling pathways have been shown to associate directly with the actin cytoskeleton, indicating that the cytoskeleton may also serve as a platform for immune-associated molecules. Here we report that retinoic acid-induced gene-I (RIG-I), an important viral RNA recognition molecule, is associated with the actin cytoskeleton and localizes predominantly to actin-enriched membrane ruffles in non-polarized epithelial cells. Subcellular localization and fractionation experiments revealed that the association between RIG-I and the actin cytoskeleton was mediated by its N-terminal caspase activation and recruitment domains (CARDs), which were necessary and sufficient to induce cytoskeletal association. We also show that RIG-I plays a role in cellular migration, as ectopic expression of RIG-I enhanced cellular migration in a wound healing assay and depletion of endogenous RIG-I significantly reduced wound healing. We further show that in both cultured intestinal epithelial cells (IEC) and human colon and small intestine biopsies, RIG-I is enriched at apico-lateral cell junctions and colocalizes with markers of the tight junction. Depolymerization of the actin cytoskeleton in polarized IEC led to the rapid relocalization of RIG-I and to the induction of type I interferon signaling. These data provide evidence that RIG-I is associated with the actin cytoskeleton in non-polarized epithelial cells and with the junctional complex in polarized IECs and human intestine and colon biopsies and may point to a physiological role for RIG-I in the regulation of cellular migration.The actin cytoskeleton is not only a fundamental component of cellular homeostasis, but in many ways also serves as the first line of host defense against an invading pathogen. Cortical actin filaments directly below the cell membrane form a complex network that provides a barrier to the penetration of viral and bacterial pathogens. This network must therefore be modulated for a pathogen to gain entry into the cell cytoplasm, most commonly via endocytic vesicles. It is therefore not surprising that many pathogens have evolved highly varied strategies to dissolve or modulate the cortical actin meshwork to facilitate cell entry and/or trafficking (1, 2). Thus, the actin cytoskeleton would be ideally suited to act as a platform for immune-associated molecules to sense an invading pathogen and mount an immediate response to promote an antimicrobial state.Consistent with this, previous studies have shown that several components of the inflammatory pathway interact either directly or indirectly with the actin cytoskeleton. The Gram-positive and -negative bacterial recognition molecule nucleotide oligimerization domain 2 (NOD2)² associates with the actin cytoskeleton (3) and localizes to cell-cell junctions in intestinal epithelial cells (IECs) (4). Moreover, the proinflammatory caspase caspase-11 directly interacts with the actin interacting protein (Aip1) to promote actin depolymerization mediated by cofilin (5) and its activity is regulated by interaction with Flightless-1, a component of actin remodeling (6). The p65 subunit of nuclear factor (NF)-κB has also been shown to interact with actin filaments, suggesting its activity may also be regulated via this association (7). These findings suggest that components of the inflammatory response tightly associate with the actin cytoskeleton and that this association may regulate inflammatory signal activation. Disruption of the actin cytoskeleton by the use of actin depolymerizing agents (such as cytochalasin D (cytoD)) activates NFκB signaling in monocytes (8), human embryonic kidney (3), and polarized intestinal epithelial cells (9). Activation of NFκB often correlates with the induction of an inflammatory state, indicating that inflammatory signals are generated in response to dramatic alterations in actin cytoskeletal architecture. Taken together, these findings support a model of inflammatory signaling in response to perturbations of the actin cytoskeleton, possibly due to the activation of actin-associated inflammatory components.The innate immune system responds to essential functional components of microorganisms, which are thus broadly expressed within classes of pathogens (10). Two functionally related intracellular viral recognition molecules recognize cytoplasmic double-stranded RNA that is produced as a replication intermediate in the life cycle of many RNA viruses. Retinoic acid-induced gene-I (RIG-I) and melanoma differentiation associated gene 5 (MDA5) binding to double-stranded RNA initiates signaling events resulting in translocation of interferon (IFN) regulatory factors (IRF)-3 and -7 into the nucleus and subsequent induction of type I IFNs, a key component of antiviral host defense (11, 12). RIG-I and MDA5 each contain two N-terminal caspase activation and recruitment domains (CARDs) and a C-terminal DEXD/H-box RNA helicase domain. The C-terminal domain serves as a regulatory repressor domain that masks the exposure of the CARDs to prevent downstream signaling in the absence of stimulus. Upon RNA binding, structural changes release this domain leading to exposure of the CARD domains and in the induction of downstream antiviral signals.It remains unclear if either RIG-I or MDA5 serve functions beyond that of the antimicrobial response. Deletion of RIG-I in mice leads to the development of a colitis-like phenotype characterized by severe inflammation of the colon mucosa (13) (in another study, RIG-I-deficient mice die in utero (14)). RIG-I has also been implicated as a negative regulator of granulocytic differentiation in mice (15). These studies would suggest that RIG-I may serve an essential role beyond that of viral RNA recognition and may actively participate in some aspect of development and/or mucosal signaling. However, the precise nature of this role remains undefined.In this study, we show that RIG-I is concentrated at sites of actin-rich membrane ruffles in non-polarized and polarized epithelial cells and plays a role in cell migration. We further show that RIG-I is enriched at apico-lateral cell junctions of both cultured IECs and human colon biopsies where it colocalizes with markers of the apical tight junction (TJ) complex. The localization of RIG-I to lamellipodia and its association with the actin cytoskeleton is mediated by CARD-dependent interactions, as these domains are both necessary and sufficient to promote membrane ruffle association. Our results show that in addition to serving an important role in innate immune signaling, RIG-I is closely associated with the actin cytoskeleton and may participate in the regulation of cellular motility and migration.     

Gene expression profiling of the plant pathogenic basidiomycetous fungus Rhizoctonia solani AG 4 reveals putative virulence factors

Rhizoctonia solani is a ubiquitous basidiomycetous soilborne fungal pathogen causing damping-off of seedlings, aerial blights and postharvest diseases. To gain insight into the molecular mechanisms of pathogenesis a global approach based on analysis of expressed sequence tags (ESTs) was undertaken. To get broad gene-expression coverage, two normalized EST libraries were developed from mycelia grown under high nitrogen-induced virulent and low nitrogen/methylglucose-induced hypovirulent conditions. A pilot-scale assessment of gene diversity was made from the sequence analyses of the two libraries. A total of 2280 cDNA clones was sequenced that corresponded to 220 unique sequence sets or clusters (contigs) and 805 singlets, making up a total of 1025 unique genes identified from the two virulence-differentiated cDNA libraries. From the total sequences, 295 genes (38.7%) exhibited strong similarities with genes in public databases and were categorized into 11 functional groups. Approximately 61.3% of the R. solani ESTs have no apparent homologs in publicly available fungal genome databases and are considered unique genes. We have identified several cDNAs with potential roles in fungal pathogenicity, virulence, signal transduction, vegetative incompatibility and mating, drug resistance, lignin degradation, bioremediation and morphological differentiation. A codon-usage table has been formulated based on 14694 R. solani EST codons. Further analysis of ESTs might provide insights into virulence mechanisms of R. solani AG 4 as well as roles of these genes in development, saprophytic colonization and ecological adaptation of this important fungal plant pathogen.