Protein kinase R mediates intestinal epithelial gene remodeling in response to double-stranded RNA and live rotavirus

As sentinels of host defense, intestinal epithelial cells respond to the viral pathogen rotavirus by activating a gene expression that promotes immune cell recruitment and activation. We hypothesized that epithelial sensing of rotavirus might target dsRNA, which can be detected by TLR3 or protein kinase R (PKR). Accordingly, we observed that synthetic dsRNA, polyinosinic acid:cytidylic acid (poly(I:C)), potently induced gene remodeling in model intestinal epithelia with the specific pattern of expressed genes, including both classic proinflammatory genes (e.g., IL-8), as well as genes that are classically activated in virus-infected cells (e.g., IFN-responsive genes). Poly(I:C)-induced IL-8 was concentration dependent (2-100 mug/ml) and displayed slower kinetics compared with IL-8 induced by bacterial flagellin (ET(50) approximately 24 vs 8 h poly(I:C) vs flagellin, respectively). Although model epithelia expressed detectable TLR3 mRNA, neither TLR3-neutralizing Abs nor chloroquine, which blocks activation of intracellular TLR3, attenuated epithelial responses to poly(I:C). Conversely, poly(I:C)-induced phosphorylation of PKR and inhibitors of PKR, 2-aminopurine and adenine, ablated poly(I:C)-induced gene expression but had no effect on gene expression induced by flagellin, thus suggesting that intestinal epithelial cell detection of dsRNA relies on PKR. Consistent with poly(I:C) detection by an intracellular molecule such as PKR, we observed that both uptake of and responses to poly(I:C) were polarized to the basolateral side. Lastly, we observed that the pattern of pharmacologic inhibition of responses to poly(I:C) was identical to that seen in response to infection by live rotavirus, indicating a potentially important role for PKR in activating intestinal epithelial gene expression in rotavirus infection.     

Transient Inability to Manage Proteobacteria Promotes Chronic Gut Inflammation in TLR5-Deficient Mice

Colitis results from breakdown of homeostasis between intestinal microbiota and the mucosal immune system, with both environmental and genetic influencing factors. Flagellin receptor TLR5-deficient mice (T5KO) display elevated intestinal proinflammatory gene expression and colitis with incomplete penetrance, providing a genetically sensitized system to study the contribution of microbiota to driving colitis. Both colitic and noncolitic T5KO exhibited transiently unstable microbiotas, with lasting differences in colitic T5KO, while their noncolitic siblings stabilized their microbiotas to resemble wild-type mice. Transient high levels of proteobacteria, especially enterobacteria species including E.?coli, observed in close proximity to the gut epithelium were a striking feature of colitic microbiota. A Crohn's disease-associated E.?coli strain induced chronic colitis in T5KO, which persisted well after the exogenously introduced bacterial species had been eliminated. Thus, an innate immune deficiency can result in unstable gut microbiota associated with low-grade inflammation, and harboring proteobacteria can drive and/or instigate chronic colitis.     

Conformation of a cyclic decapeptide analog of a repeat pentapeptide sequence of elastin: cyclo-bis(valyl-prolyl-alanyl-valyl-glycyl)

The conformation of a cyclic decapeptide analog of a repeat sequence of elastin has been determined in the crystalline state using X-ray crystallographic techniques. Tetragonal crystals were grown from a solution of the decapeptide in water; space group P4(2)2(1)2, a = 19.439(2) & c = 13.602(1) A, with four formula units (C40H66N10O10.4H2O) per unit cell. The cyclic decapeptide in the crystal exhibits exact twofold symmetry. The asymmetric unit contains one pentapeptide and two water molecules for a total of 32 nonhydrogen atoms. The structure has been determined by the application of direct methods and refined by full-matrix least squares to an R index of 0.053 for 2272 reflections with intensities greater than 2 sigma(I). The backbone conformation of the asymmetric pentapeptide can be described as consisting of a double beta bend of Type III-I. The Type III turn has Pro (phi = -59.3 degrees, psi = -26.8 degrees) and Ala (phi = -65.9 degrees, psi = -23.1 degrees) at the corners while Type I turn has Ala (phi = -65.9 degrees, psi = -23.1 degrees) and Val (phi = -98.9 degrees, psi = 8.3 degrees) as the corner residues. The cyclic decapeptide has two such double bends linked together by Gly-Val bridges.     

Functionalization of cellulose acetate fibers with engineered cutinases

In the present work, we describe for the first time the specific role of cutinase on surface modification of cellulose acetate fibers. Cutinase exhibits acetyl esterase activity on diacetate and triacetate of 0.010 U and 0.007 U, respectively. An increase on the hydroxyl groups at the fiber surface of 25% for diacetate and 317% for triacetate, after a 24 h treatment, is estimated by an indirect assay. Aiming at further improvement of cutinase affinity toward cellulose acetate, chimeric cutinases are genetically engineered by fusing the 3′‐end coding sequence with a bacterial or a fungal carbohydrate‐binding module and varying the linker DNA sequence. A comparative analysis of these genetic constructions is presented showing that, the superficial regeneration of cellulose hydrophilicity and reactivity on highly substituted cellulose acetates is achieved by chimeric cutinases. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Comparison of the three-dimensional structures of human, yeast, and oat ubiquitin

The crystal structure of human ubiquitin has been solved by x-ray diffraction methods and refined by standard procedures to a conventional crystallographic R factor of 0.176 at 1.8-A resolution (Vijay-Kumar, S., Bugg, C.E., and Cook, W.J. (1987) J. Mol. Biol. 194, 525-538). Crystals of yeast and oat ubiquitin have been grown using human ubiquitin crystals as seeds. Diffraction data for yeast and oat ubiquitin have been collected to a resolution of 1.9 and 1.8 A, respectively. Difference Fourier electron-density maps reveal that the structures of yeast and oat ubiquitin are quite similar to human ubiquitin. All the amino acid changes are clustered in two small patches on one surface of the molecule. This surface is probably not involved in conjugation with proteins destined for ATP-dependent proteolysis.     

A second calcium regulator of rod outer segment membrane guanylate cyclase, ROS-GC1: neurocalcin.

ROS-GC represents a membrane guanylate cyclase subfamily whose distinctive feature is that it transduces diverse intracellularly generated Ca(2+) signals into the production of the second messenger cyclic GMP. An intriguing feature of the first subfamily member, ROS-GC1, is that it is both stimulated and inhibited by these signals. The inhibitory signals are processed by the cyclase activating proteins, GCAPs. The only known stimulatory signal is by the Ca(2+)-dependent guanylate cyclase activating protein, CD-GCAP. There are two GCAPs, 1 and 2, which link the cyclase with phototransduction, and one CD-GCAP, which is predicted to link ROS-GC1 with its retinal synaptic activity. Individual switches for these GCAPs and CD-GCAP have been respectively defined as CRM1, CRM3, and CRM2. This report defines the identity of a new ROS-GC1 regulator: neurocalcin. A surprising feature of the regulator is that it structurally is a GCAP but functionally behaves as a CD-GCAP. Recombinant neurocalcin stimulates ROS-GC1 in a dose-dependent fashion; the stimulation is Ca(2+)-dependent with an EC(50) of 20 microM; and the modulated domain resides at the C-terminal segment, between amino acids 731 and 1054. Previously, the residence of CRM2 has also been defined in this segment of the cyclase. However, the present study shows that the neurocalcin-regulated domain is distinct from CRM2. This is now designated as CRM4. Thus, the signal transduction mechanisms of neurocalcin and CD-GCAP are different, occurring through different modules of ROS-GC1. Neurocalcin signaling of ROS-GC1 is highly specific. It does not influence the activity of its second subfamily member, ROS-GC2, and of the other retinal guanylate cyclase, atrial natriuretic factor-receptor guanylate cyclase. In conclusion, the findings extend the concept of ROS-GC1's sensing diverse Ca(2+) signals, reveal the identity of its unexpected new Ca(2+) regulator, and show that the regulator acts through its specific cyclase domain. This represents an additional transduction mechanism of Ca(2+) signaling via ROS-GC1.     

Selective ablation of matrix metalloproteinase-2 exacerbates experimental colitis: contrasting role of gelatinases in the pathogenesis of colitis

The matrix metalloproteinases (MMPs), MMP-2 and MMP-9, share structural and substrate similarities and are up-regulated during human as well as animal models of inflammatory bowel disease. We recently demonstrated that epithelial-derived MMP-9 is an important mediator of inflammation and tissue damage in colitis. In this study, we examined the role of MMP-2 in acute colitis. Colitis was induced using two models, administration of dextran sodium sulfate (DSS) and Salmonella enterica subsp. serovar Typhimurium (S.T.). Bone marrow chimeras were performed using bone marrow cells from wild-type (WT) and MMP-2(-/-) mice. Colitis was evaluated by clinical symptoms, myeloperoxidase assay, and histology. MMP-2 protein expression and activity were up-regulated in WT mice treated with DSS or S.T. MMP-2(-/-) mice were highly susceptible to the development of colitis induced by DSS (or S.T.) compared with WT. During inflammation, MMP-2 expression was increased in epithelial cells as well as in the infiltrating immune cells. Bone marrow chimera demonstrated that mucosa-derived MMP-2 was required for its protective effects toward colitis. Furthermore, we demonstrate that severe colitis in MMP-2(-/-) is not due to a compensatory increase in MMP-9. Finally, we show that MMP-2 regulates epithelial barrier function. In contrast to MMP-9, mucosa-derived MMP-2 may be a critical host factor that is involved in the prevention or cessation of the host response to luminal pathogens or toxins, an important aspect of healing and tissue resolution. Together, our data suggest that a critical balance between the two gelatinases determines the outcome of inflammatory response during acute colitis.     

EC3, a novel heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4 and alpha5 integrins in an RGD-independent manner

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing alpha4beta1 and alpha4beta7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6-30 nM, adhesion of K562 cells (alpha5beta1) to fibronectin with IC50 = 150 nM, and adhesion of alphaIIbbeta3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alphavbeta3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 microM), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mM), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5-10 mM) in contrast to RGDS peptide (IC50 = 3 mM), inhibiting completely at 10 mM.     

Is Predisposition to NAFLD and Obesity Communicable?

A forthcoming article in Nature reveals that inflammasome deficiency disturbs gut microbiota and predisposes to diet-induced obesity and liver disease. The aberrant microbiota and its consequences could be transferred by cohousing mutant and wild-type mice, suggesting that some aspects of metabolic syndrome can be communicable (Henao-Mejia et?al., 2012).