Interleukin-6 induces keratin expression in intestinal epithelial cells: potential role of keratin-8 in interleukin-6-induced barrier function alterations

Keratin 8 (K8) and keratin-18 (K18) are the major intermediate filament proteins in the intestinal epithelia. The regulation and function of keratin in the intestinal epithelia is largely unknown. In this study we addressed the role and regulation of K8 and K18 expression by interleukin 6 (IL-6). Caco2-BBE cell line and IL-6 null mice were used to study the effect of IL-6 on keratin expression. Keratin expression was studied by Northern blot, Western blot, and confocal microscopy. Paracellular permeability was assessed by apical-to-basal transport of a fluorescein isothiocyanate dextran probe (FD-4). K8 was silenced using the small interfering RNA approach. IL-6 significantly up-regulated mRNA and protein levels of K8 and K18. Confocal microscopy showed a reticular pattern of intracellular keratin localized to the subapical region after IL-6 treatment. IL-6 also induced serine phosphorylation of K8. IL-6 decreased paracellular flux of FD-4 compared with vehicle-treated monolayers. K8 silencing abolished the decrease in paracellular permeability induced by IL-6. Administration of dextran sodium sulfate (DSS) significantly increased intestinal permeability in IL-6-/- mice compared with wild type mice given DSS. Collectively, our data demonstrate that IL-6 regulates the colonic expression of K8 and K18, and K8/K18 mediates barrier protection by IL-6 under conditions where intestinal barrier is compromised. Thus, our data uncover a novel function of these abundant cytoskeletal proteins, which may have implications in intestinal disorders such as inflammatory bowel disease wherein barrier dysfunction underlies the inflammatory response.     

Methods of using click chemistry in the discovery of enzyme inhibitors

This protocol describes the step-by-step procedures for the efficient assembly of bidentate inhibitor libraries of a target enzyme, using the so-called 'click chemistry' between an alkyne-bearing core group and an azide-modified peripheral group, followed by direct biological screening for the identification of potential 'hits'. The reaction is highlighted by its modularity, high efficiency (approximately 100% yield in most cases) and tolerance toward many functional groups present in the fragments, as well as biocompatibility (typically carried out in aqueous conditions with small amounts of biocompatible catalysts). The approach consists of three steps: (i) chemical synthesis of alkyne-bearing protein tyrosine phosphatase or matrix metalloprotease core groups and diverse azide-modified peripheral groups; (ii) click chemistry to assemble the bidentate inhibitor libraries; and (iii) direct screening of the libraries with target enzymes using 384-well microplate assays. Following the chemical synthesis of the core and peripheral groups and optimization of the click chemistry conditions (approximately 1 week), steps (ii) and (iii) take 3 d to complete (approximately 1-2 d for library assembly and 1 d for inhibitor screening).     

A spatio-temporal mining approach towards summarizing and analyzing protein folding trajectories

This paper presents a software library, nicknamed BATS, for some basic sequence analysis tasks. Namely, local alignments, via approximate string matching, and global alignments, via longest common subsequence and alignments with affine and concave gap cost functions. Moreover, it also supports filtering operations to select strings from a set and establish their statistical significance, via z-score computation. None of the algorithms is new, but although they are generally regarded as fundamental for sequence analysis, they have not been implemented in a single and consistent software package, as we do here. Therefore, our main contribution is to fill this gap between algorithmic theory and practice by providing an extensible and easy to use software library that includes algorithms for the mentioned string matching and alignment problems. The library consists of C/C++ library functions as well as Perl library functions. It can be interfaced with Bioperl and can also be used as a stand-alone system with a GUI. The software is available at http://www.math.unipa.it/~raffaele/BATS/ under the GNU GPL.     

Sestrin2 regulates monocyte activation through AMPK-mTOR nexus under high-glucose and dyslipidemic conditions

The vicious cycle between hyperinsulinemia and insulin resistance results in the progression of atherosclerosis in the vessel wall. The complex interaction between hyperglycemia and lipoprotein abnormalities promotes the development of atherogenesis. In the early phase of atherosclerosis, macrophage-derived foam cells play an important role in vascular remodeling. Mechanistic target of rapamycin (mTOR) signaling pathway has been identified to play an essential role in the initiation, progression, and complication of atherosclerosis. Recently sestrin2, an antioxidant, was shown to modulate TOR activity and thereby regulating glucose and lipid metabolism. But the role of sestrin2 in monocyte activation is still not clearly understood. Hence, this study is focussed on investigating the role of sestrin2 in monocyte activation under hyperglycemic and dyslipidemic conditions. High-glucose and oxidized low-density lipoprotein (LDL) treatments mediated proinflammatory cytokine production (M1) with a concomitant decrease in the anti-inflammatory cytokine (M2) levels in human monocytic THP1 cells. Both glucose and oxidized LDL (OxLDL) in a dose and time-dependent manner increased the mTOR activation with a marked reduction in the levels of pAMPK and sestrin2 expression. Both high-glucose and OxLDL treatment increased foam cell formation and adhesion of THP1 cells to endothelial cells. Experiments employing activator or inhibitor of adenosine monophosphate kinase (AMPK) as well as overexpression or silencing of sestrin2 indicated that high-glucose mediated monocyte polarization and adhesion of monocytes to the endothelial cells were appeared to be programmed via sestrin2-AMPK-mTOR nexus. Our results evidently suggest that sestrin2 plays a major role in regulating monocyte activation via the AMPK–mTOR-pathway under diabetic and dyslipidemic conditions and also AMPK regulates sestrin2 in a feedback mechanism.     

Growth of AM fungi on in vitro root organ culture of Sorghum vulgare and Saccharum officinarum

Spores of Gl mosseae and Gig gigantea germinated on minimal medium produced extraradical mycelium. Gl. mosseae infected roots of S. officinarum in in vitro condition were inoculated in M medium with in vitro cultured roots of Sorghum vulgare (test roots). From the infected root of S. officinarum, the mycelium developed and it infected the test roots. The roots developed new mycelia and further the mycelia produced a few hyaline spores. In MS medium combined with soil extract, root exudate, thiamine HCl and inositol combination, spore germination and germ tube growth were higher when compared with other media.     

Landing strategies in honeybees, and possible applications to autonomous airborne vehicles

Insects, being perhaps more reliant on image motion cues than mammals or higher vertebrates, are proving to be an excellent organism in which to investigate how information on optic flow is exploited to guide locomotion and navigation. This paper describes one example, illustrating how bees perform grazing landings on a flat surface. A smooth landing is achieved by a surprisingly simple and elegant strategy: image velocity is held constant as the surface is approached, thus automatically ensuring that flight speed is close to zero at touchdown. No explicit knowledge of flight speed or height above the ground is necessary. The feasibility of this landing strategy is tested by implementation in a robotic gantry, and its applicability to autonomous airborne vehicles is discussed.     

Functional site of bukatoxin, an alpha-type sodium channel neurotoxin from the Chinese scorpion (Buthus martensi Karsch) venom: probable role of the (52)PDKVP(56) loop

Alpha-toxins from scorpion venoms prolong the action potential of excitable cells by blocking sodium channel inactivation. We have purified bukatoxin, an alpha-toxin from scorpion (Buthus martensi Karsch) venom, to homogeneity. Bukatoxin produced marked relaxant responses in the carbachol-precontracted rat anococcygeus muscle (ACM), which were mediated through the L-arginine-nitric oxide synthase-nitric oxide pathway, consequent to a neuronal release of nitric oxide. Based on the presence of proline residues in the flanking segments of protein-protein interaction sites, we predicted the site between (52)PP(56) to be the potential interaction site of bukatoxin. A homology model of bukatoxin indicated the presence of this site on the surface. Buka11, a synthetic peptide designed based on this predicted site, produced a concentration-dependent nitric oxide-mediated relaxant response in ACM. Using alanine-substituted peptides, we have shown the importance (53)DKV(55) flanked by proline residues in the functional site of bukatoxin.     

Non-iron metalloporphyrins: potent antibacterial compounds that exploit haem/Hb uptake systems of pathogenic bacteria

A new group of potent antibacterial compounds, non-iron metalloporphyrins (MPs), is described. MPs possess a strong antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and mycobacteria. Anaerobically grown bacteria and microorganisms that do not respire and/or express haem uptake systems were resistant to MPs. Antibacterial activity of MPs was not affected by known antibiotic resistance mechanisms operating in bacteria. The most potent MP against Y. enterocolitica, methicillin-resistant S. aureus and M. smegmatis was gallium protoporphyrin IX (Ga-PPIX). When tested alone, Ga ions and metal-free porphyrins had approximately 100-fold higher minimum inhibitory concentration (MIC) values for these organisms. Ga-PPIX was not degraded by MP-sensitive bacteria, indicating that the whole molecule is responsible for antibacterial activity. MPs are antibacterial 'Trojan horses', as they exploit haem transport systems of Gram-negative bacteria as portals of entry into the cell. Bacterial mutants in superoxide dismutases, catalases and stationary-phase sigma factors were hypersensitive to Ga-PPIX. The extreme sensitivity of sod mutants to MPs and the requirement for active respiration for MP activity suggests that these compounds stimulate the production of reactive oxygen radicals in bacteria. Ga-PPIX was not toxic to primary human fibroblasts, several established cell lines and experimental animals at concentrations > 100-fold higher than the MIC for sensitive bacteria.