Bovine lactoferricin is anti‐inflammatory and anti‐catabolic in human articular cartilage and synovium

Bovine lactoferricin (LfcinB) is a multi‐functional peptide derived from proteolytic cleavage of bovine lactoferrin. LfcinB was found to antagonize the biological effects mediated by angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF‐2) in endothelial cells. However, the effect of LfcinB on human articular cartilage remained unknown. Here, our findings demonstrate that LfcinB restored the proteoglycan loss promoted by catabolic factors (interleukin‐1β) IL‐1β and FGF‐2 in vitro and ex vivo. Mechanistically, LfcinB attenuated the effects of IL‐1β and FGF‐2 on the expression of cartilage‐degrading enzymes (MMP‐1, MMP‐3, and MMP‐13), destructive cytokines (IL‐1β and IL‐6), and inflammatory mediators (iNOS and TLR2). LfcinB induced protective cytokine expression (IL‐4 and IL‐10), and downregulated aggrecanase basal expression. LfcinB specifically activated ERK MAPK and Akt signaling pathways, which may account for its anti‐inflammatory activity. We also revealed that LfcinB exerted similar protective effects on human synovial fibroblasts challenged by IL‐1β, with minimal cytotoxicity. Collectively, our results suggest that LfcinB possesses potent anti‐catabolic and anti‐inflammatory bioactivities in human articular tissues, and may be utilized for the prevention and/or treatment of OA in the future. J. Cell. Physiol. 228: 447–456, 2013. © 2012 Wiley Periodicals, Inc.     

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO₂). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO₂, a major greenhouse gas, making this a promising alternative for chemical CO₂ mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO₂ sequestration by mineral carbonation, a process with the potential to store large quantities of CO₂. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO₂ compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO₃) formation and exerted a striking impact on the maximal amount of CaCO₃ produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO₂ sequestration.     

Decline of dark coniferous stands in Baikal Region

The reasons for the decline in Siberian pine and fir in the Baikal Region (Khamar-Daban) were analyzed using remote sensing techniques, dendrochronology and GIS-technology methods, and in situ observations. It is found that a decrease in the value of the growth index (R ² = 0.69) and an decrease in the SPEI drought index (Standardized Precipitation–Evapotranspiration Index) (R ² = 0.72) has been observed since the 1980s. In the mid-2000s, the increase in aridity led to the division of Siberian pine trees into two cohorts: “survivors” and “decliners.” The spatial distribution of these cohorts is different: dead and declining stands are localized mainly on relief elements with increased risk of water stress (steep and convex slopes of southwestern exposure). The growth index of the trees is closely related to the dryness index in June (r ² = 0.55). Along with water stress, declining trees were also exposed to stem pests and plant pathogens. The primary cause of Siberian pine decline is water stress due to the increasing climate aridity. The weakened waterstressed trees were sensitized to pathogens. The synergism of climatic and biotic effects led to the decline of Siberian pine stands. On the whole, heavily damaged and declining stands (over 50% of dead and declining trees) within the Khamar-Daban ridge are 8–10% of the total area of dark coniferous forests.     

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation‐sensitive amplified polymorphism

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal‐contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation‐sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal‐contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal‐contaminated site compared to uncontaminated populations. Other genotypes from a different metal‐contaminated site within the same region appear to be recalcitrant to metal‐induced DNA alterations even ≥30 years of tree life exposure to nickel and copper . MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal‐contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.     

Association of CD38 with nonmuscle myosin heavy chain IIA and Lck is essential for the internalization and activation of CD38

Activation of CD38 in lymphokine-activated killer (LAK) cells involves interleukin-8 (IL8)-mediated protein kinase G (PKG) activation and results in an increase in the sustained intracellular Ca(2+) concentration ([Ca(2+)](i)), cADP-ribose, and LAK cell migration. However, direct phosphorylation or activation of CD38 by PKG has not been observed in vitro. In this study, we examined the molecular mechanism of PKG-mediated activation of CD38. Nonmuscle myosin heavy chain IIA (MHCIIA) was identified as a CD38-associated protein upon IL8 stimulation. The IL8-induced association of MHCIIA with CD38 was dependent on PKG-mediated phosphorylation of MHCIIA. Supporting these observations, IL8- or cell-permeable cGMP analog-induced formation of cADP-ribose, increase in [Ca(2+)](i), and migration of LAK cells were inhibited by treatment with the MHCIIA inhibitor blebbistatin. Binding studies using purified proteins revealed that the association of MHCIIA with CD38 occurred through Lck, a tyrosine kinase. Moreover, these three molecules co-immunoprecipitated upon IL8 stimulation of LAK cells. IL8 treatment of LAK cells resulted in internalization of CD38, which co-localized with MHCIIA and Lck, and blebbistatin blocked internalization of CD38. These findings demonstrate that the association of phospho-MHCIIA with Lck and CD38 is a critical step in the internalization and activation of CD38.     

Improved insulin sensitivity and adiponectin level after exercise training in obese Korean youth

Objective: The objective of this study was to investigate the association among adiposity, insulin resistance, and inflammatory markers [high‐sensitivity C‐reactive protein (hs‐CRP), interleukin (IL)‐6, and tumor necrosis factor (TNF)‐α] and adiponectin and to study the effects of exercise training on adiposity, insulin resistance, and inflammatory markers among obese male Korean adolescents. Research Methods and Procedures: Twenty‐six obese and 14 lean age‐matched male adolescents were studied. We divided the obese subjects into two groups: obese exercise group (N = 14) and obese control group (N = 12). The obese exercise group underwent 6 weeks of jump rope exercise training (40 min/d, 5 d/wk). Adiposity, insulin resistance, lipid profile, hs‐CRP, IL‐6, TNF‐α, and adiponectin were measured before and after the completion of exercise training. Results: The current study demonstrated higher insulin resistance, total cholesterol, LDL‐C levels, triglyceride, and inflammatory markers and lower adiponectin and HDL‐C in obese Korean male adolescents. Six weeks of increased physical activity improved body composition, insulin sensitivity, and adiponectin levels in obese Korean male adolescents without changes in TNF‐α, IL‐6, and hs‐CRP. Discussion: Obese Korean male adolescents showed reduced adiponectin levels and increased inflammatory cytokines. Six weeks of jump rope exercise improved triglyceride and insulin sensitivity and increased adiponectin levels.     

Characterization of Cold-Shock Protein A of Antarctic <Emphasis Type="Italic">Streptomyces</Emphasis> sp. AA8321

Abstact Polar organisms should have mechanisms to survive the extremely cold environment. Four genes encoding cold-shock proteins, which are small, cold-induced bacterial proteins, have been cloned from the Antarctic bacterium Streptomyces sp. AA8321. Since the specific functions of any polar bacterial or Streptomyces cold-shock proteins have not yet been determined, we examined the role of cold-shock protein A from Streptomyces sp. AA8321 (CspA_St). Gel filtration chromatography showed that purified CspA_St exists as a homodimer under physiological conditions, and gel shift assays showed that it binds to single-stranded, but not double-stranded, DNA. Overexpression of CspA_St in Escherichia coli severely impaired the ability of the host cells to form colonies, and the cells developed an elongated morphology. Incorporation of a deoxynucleoside analogue, 5-bromo-2′-deoxyuridine, into newly synthesized DNA was also drastically diminished in CspA_St-overexpressing cells. These results suggest that CspA_St play a role in inhibition of DNA replication during cold-adaptation.     

The effect of intestinal alkaline phosphatase on intestinal epithelial cells,macrophages and chronic colitis in mice

Aims

Intestinal alkaline phosphatase (IAP) is an intestinal brush border enzyme that is shown to function as a gut mucosal defense factor, but its defensive mechanism remains unclear. The aims of this study were to evaluate the effect of IAP on intestinal epithelial cells and macrophages, and on chronic colitis in interleukin-10-deficient (IL-10^−/−) mice.

Main methods

Human intestinal epithelial cells COLO 205 and peritoneal macrophages from IL-10^−/− mice were pretreated with IAP and then stimulated with lipopolysaccharide (LPS). IL-8 secretion from COLO205 cells and TNF-α, IL-6, IL-12 from peritoneal macrophages were measured by ELISA. Electrophoretic mobility shift assay was used to assess the DNA binding activity of NF-κB and IκBα phosphorylation/degradation was evaluated by immunoblot assay in COLO 205. For the in vivo study, colitis was induced in IL-10^−/− mice with piroxicam, the mice were then treated with 100 or 300 units of IAP by oral gavage for 2 weeks. Colitis was quantified by histopathologic scoring, and the phosphorylation of IκBα in the colonic mucosa was assessed using immunohistochemistry.

Key findings

IAP significantly inhibited LPS-induced inflammatory cytokine production in both IECs and peritoneal macrophages. IAP also attenuated LPS-induced NF-κB binding activity and IκBα phosphorylation/degradation in IECs. Oral administration of IAP significantly reduced the severity of colitis and down-regulated colitis-induced IκBα phosphorylation in IL-10^−/− mice.

Significance

IAP may inhibit the activation of intestinal epithelial cells and peritoneal macrophages, and may attenuate chronic murine colitis. This finding suggests that IAP supplementation is a potential therapeutic option for inflammatory bowel disease.     

Axin expression reduces staurosporine-induced mitochondria-mediated cell death in HeLa cells

Cytoplasmic axin expression frequently produces punctuate structures in cells, but the nature of axin puncta has not been fully elucidated. In an effort to analyze cytoplasmic axin puncta, we established HeLa cells expressing axin in a doxycycline-inducible manner (HeLa-Axin). We observed that axin accumulated in an aggregate-like pattern in perinuclear areas and appeared to be associated with mitochondria, Golgi apparatus, and endoplasmic reticulum (ER), but not lysosomes. Further biochemical analysis suggested that some part of the cytoplasmic axin pool was associated with mitochondria. In addition, mitochondrial proteins [i.e., cytochrome oxidase IV (CoxIV) and cytochrome c] were slightly higher in HeLa-Axin cells than in HeLa-EV cells, suggesting altered mitochondrial degradation. HeLa-Axin cells were then treated with staurosporine (STS) to determine if the mitochondria-induced apoptosis pathway was altered. Compared to STS-treated control cells (HeLa-EV), HeLa-Axin cells had less STS-induced cytotoxicity and reduced caspase-3 activation and PARP cleavage. Given that mitochondria outer membrane potential was unchanged, HeLa-Axin cells might be relatively resistant to STS-mediated mitochondrial damage. Mitochondria associated with axin aggregates were resistant to detergent-mediated permeabilization. These results suggest that axin forms aggregate-like structures in association with mitochondria, which render mitochondria resistant to STS-induced membrane damage and cytotoxicity.