Inhibition of HSP27 phosphorylation by a cell-permeant MAPKAP Kinase 2 inhibitor

Heat shock protein 27 (HSP27) has been implicated in many intracellular signaling processes. Since the phosphorylation of HSP27 can modulate its activity, the ability to inhibit phosphorylation of HSP27 might have clinical relevance especially with regard to the treatment of fibrosis. We have developed a cell-permeant peptide inhibitor of MAPKAP Kinase 2 (MK2), an enzyme that phosphorylates HSP27, by combining a previously described peptide substrate of MK2 with a cell penetrating peptide. This novel MK2 inhibitor (MK2i) reduced HSP27 phosphorylation by MK2 in vitro. At 10 μM, MK2i inhibited TGF-β1-induced HSP27 phosphorylation in serum-starved human keloid fibroblasts. In addition, 10 μM MK2i decreased TGF-β1-induced expression of connective tissue growth factor and collagen type I within serum-starved keloid fibroblasts. Thus, MK2i represents a potential therapeutic for the treatment of fibrotic disorders.     

DNA Polymerases β and λ Mediate Overlapping and Independent Roles in Base Excision Repair in Mouse Embryonic Fibroblasts

Base excision repair (BER) is a DNA repair pathway designed to correct small base lesions in genomic DNA. While DNA polymerase beta (pol β) is known to be the main polymerase in the BER pathway, various studies have implicated other DNA polymerases in back-up roles. One such polymerase, DNA polymerase lambda (pol λ), was shown to be important in BER of oxidative DNA damage. To further explore roles of the X-family DNA polymerases λ and β in BER, we prepared a mouse embryonic fibroblast cell line with deletions in the genes for both pol β and pol λ. Neutral red viability assays demonstrated that pol λ and pol β double null cells were hypersensitive to alkylating and oxidizing DNA damaging agents. In vitro BER assays revealed a modest contribution of pol λ to single-nucleotide BER of base lesions. Additionally, using co-immunoprecipitation experiments with purified enzymes and whole cell extracts, we found that both pol λ and pol β interact with the upstream DNA glycosylases for repair of alkylated and oxidized DNA bases. Such interactions could be important in coordinating roles of these polymerases during BER.     

Some observations on moulting, growth and maturation of krill (Thysanoessa inermis) from the Barents Sea

The moulting interval of the euphausiid Thysanoessa inermisfrom the Barents Sea was found to be temperature dependent withinthe range of 1.5–8.0°C. Each moult accounted for a2.1% loss of dry body wt, equivalent to a 1.2–1.3% lossof body carbon or nitrogen. During 5 months of observation bothbody length and secondary sexual characters (thelycum and petasma)of specimens varied greatly, including positive and negativegrowth periods and progression and regression of maturity stages.Characteristics of eggs spawned by one female are described.     

Studies on a continuous recycle reactor for a lipase-catalysed processing of ricebran oil

The authors have developed a continuous recycle reactor which efficiently performs emulsion type enzymatic reactions. The reactor column is filled with immobilised lipase and the reactions are effected by pumping the pre-prepared oil-water emulsion through the bottom of the reactor. A part of the product was recycled back and this type of recycling greatly improves the productivity of fatty acid compared to continuous once-through reactor without recycling. The recycle reactor could be continuously run for 35 days without decrease in conversions. The performance of the reactor was interpreted by a model and the theoretical conversion was compared with the experimental data.List of Symbols F _AO mol/min feed rate - K _M g/l Michaelis constant - R recycle ratio - r ₅ mol/(ml · min) reaction rate - S ₀ g/l initial substrate concentration - V _max mol/(ml · min) maximum reaction velocity - V _R l void volume of the reactor - x _s fractional conversion - Standard deviation      

The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation

Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.     

IL-6 and IL-10 induction from dendritic cells in response to Mycobacterium tuberculosis is predominantly dependent on TLR2-mediated recognition

The initial TLR-mediated interaction between Mycobacterium tuberculosis and dendritic cells is critical, since the cytokine production that ensues can greatly influence the class of adaptive immunity that is generated to the pathogen. In this study, we therefore determined the dependency on TLR2 and TLR4 for M. tuberculosis-induced cytokine production by murine dendritic cells. A key new finding of this study is that production of IL-6 and IL-10 from dendritic cells in response to M. tuberculosis is principally dependent on TLR2. The study also indicates that M. tuberculosis can induce IL-12 production in the absence of either TLR2 or TLR4, suggesting redundancy or possibly involvement of other receptors in IL-12 production. In addition, the data also reveal that lack of TLR2 or TLR4 does not impact on dendritic cell maturation or on their ability to influence the polarity of differentiating naive T cells. Collectively, data presented here provide a mechanistic insight for the contribution of TLR2 and TLR4 to tuberculosis disease progression and offer strategies for regulating IL-6 and IL-10 production in dendritic cell-based vaccine strategies.     

Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin

Obesity and type 2 diabetes are associated with nonalcoholic steatohepatitis (NASH), but an obese/diabetic animal model that mimics human NASH remains undefined. We examined the induction of steatohepatitis and liver fibrosis in obese and type 2 diabetic db/db mice in a nutritional model of NASH and determined the relationship of the expressions of osteopontin (OPN) and leptin receptors to the pathogenesis of NASH. db/db mice and the corresponding lean and nondiabetic db/m mice were fed a diet deficient in methionine and choline (MCD diet) or control diet for 4 wk. Leptin-deficient obese and diabetic ob/ob mice fed similar diets were used for comparison. MCD diet-fed db/db mice exhibited significantly greater histological inflammation and higher serum alanine aminotransferase levels than db/m and ob/ob mice. Trichrome staining showed marked pericellular fibrosis in MCD diet-fed db/db mice but no significant fibrosis in db/m or ob/ob mice. Collagen I mRNA expression was increased 10-fold in db/db mice, 4-fold in db/m mice, and was unchanged in ob/ob mice. mRNA expressions of OPN, TNF-alpha, TGF-beta, and short-form leptin receptors (Ob-Ra) were significantly increased in db/db mice compared with db/m or ob/ob mice. Parallel increases in OPN and Ob-Ra protein levels were observed in db/db mice. Cultured hepatocytes expressed only Ob-Ra, and leptin stimulated OPN mRNA and protein expression in these cells. In conclusion, our results demonstrate the development of an obese/diabetic experimental model for NASH in db/db mice and suggest an important role for Ob-Ra and OPN in the pathogenesis of NASH.     

Synthesis of β‐Ketoamide Curcumin Analogs for Anti‐Diabetic and AGEs Inhibitory Activities

Two different series of novel β‐ketoamide curcumin analogs enriched in biological activities have been synthesized. The synthesized compounds were screened for their in vitro anti‐diabetic and AGEs inhibitory activities and exhibited potent to good anti‐diabetic and AGEs inhibitory activities. The molecular docking study was also performed with the α‐amylase enzyme.     

Feeding ecology of the glacier lanternfish Benthosema glaciale (Actinopterygii, Myctophidae) in the Flemish Cap (North Atlantic Ocean)

The feeding dynamics of the glacier lanternfish Benthosema glaciale were studied during the summer of 2010 in the Flemish Cap. B. glaciale constituted a majority of the deep scattering layer (DSL), which was located between 300 and 650 m during daylight hours; at night, a part of the DSL migrated to the surface. B. glaciale is a zooplanktivore, feeding primarily on copepods (Calanus hyperboreus, Pareuchaeta norvegica, Metridia longa and Calanus finmarchicus), followed by amphipods (Themisto compressa) and krill (primarily Nematoscelis spp.). Although crustaceans dominated the diet, other invertebrates, such as chaetognaths, gastropods, polychaetes, ostracods and appendicularians, also contributed. The geographical differences in the composition of the diet resulted from the association of prey with distinct water masses. In the colder water at the north of the bank, Arctic species represented a higher proportion of the diet, but this situation was reversed in areas influenced by the warmer water from the south. B. glaciale fed more intensively towards the centre of the Flemish Cap. The diet of B. glaciale shifted ontogenetically, showing increasing prey size and prey diversity with increasing predator size.