Eicosapentaenoic acid inhibits TNF-alpha-induced matrix metalloproteinase-9 expression in human keratinocytes, HaCaT cells

Eicosapentaenoic acid (EPA) is an omega-3 (ω-3) polyunsaturated fatty acid (PUFA), which has anti-inflammatory and anti-cancer properties. Some reports have demonstrated that EPA inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α or lipopolysaccharide (LPS) in various cells. However, its detailed mode of action is unclear. In this report, we investigated whether EPA inhibits the expression of TNF-α-induced matrix metalloproteinases (MMP)-9 in human immortalized keratinocytes (HaCaT). TNF-α induced MMP-9 expression by NF-κB-dependent pathway. Pretreatment of EPA inhibited TNF-α-induced MMP-9 expression and p65 phosphorylation. However, EPA could not affect IκB-α phosphorylation, nuclear translocation of p65, and DNA binding activity of NF-κB. EPA inhibited TNF-α-induced p65 phosphorylation through p38 and Akt inhibition and this inhibition was IKKα-dependent event. Taken together, we demonstrate that EPA inhibits TNF-α-induced MMP-9 expression through inhibition of p38 and Akt activation.     

Cathepsin L expression is directed to secretory vesicles for enkephalin neuropeptide biosynthesis and secretion

Proteases within secretory vesicles are required for conversion of neuropeptide precursors into active peptide neurotransmitters and hormones. This study demonstrates the novel cellular role of the cysteine protease cathepsin L for producing the (Met)enkephalin peptide neurotransmitter from proenkephalin (PE) in the regulated secretory pathway of neuroendocrine PC12 cells. These findings were achieved by coexpression of PE and cathepsin L cDNAs in PC12 cells with analyses of PE-derived peptide products. Expression of cathepsin L resulted in highly increased cellular levels of (Met)enkephalin, resulting from the conversion of PE to enkephalin-containing intermediates of 23, 18-19, 8-9, and 4.5 kDa that were similar to those present in vivo. Furthermore, expression of cathepsin L with PE resulted in increased amounts of nicotine-induced secretion of (Met)enkephalin. These results indicate increased levels of (Met)enkephalin within secretory vesicles of the regulated secretory pathway. Importantly, cathespin L expression was directed to secretory vesicles, demonstrated by colocalization of cathepsin L-DsRed fusion protein with enkephalin and chromogranin A neuropeptides that are present in secretory vesicles. In vivo studies also showed that cathepsin L in vivo was colocalized with enkephalin. The newly defined secretory vesicle function of cathepsin L for biosynthesis of active enkephalin opioid peptide contrasts with its function in lysosomes for protein degradation. These findings demonstrate cathepsin L as a distinct cysteine protease pathway for producing the enkephalin member of neuropeptides.     

The promoter of the pepper pathogen-induced membrane protein gene <Emphasis Type="Italic">CaPIMP1</Emphasis> mediates environmental stress responses in plants

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 was analyzed by an Agrobacterium-mediated transient expression assay in tobacco leaves. Several stress-related cis-acting elements (GT-1, W-box and ABRE) are located within the CaPIMP1 promoter. In tobacco leaf tissues transiently transformed with a CaPIMP1 promoter-β-glucuronidase (GUS) gene fusion, serially 5′-deleted CaPIMP1 promoters were differentially activated by Pseudomonas syringae pv. tabaci, ethylene, methyl jasmonate, abscisic acid, and nitric oxide. The −1,193 bp region of the CaPIMP1 gene promoter sequence exhibited full promoter activity. The −417- and −593 bp promoter regions were sufficient for GUS gene activation by ethylene and methyl jasmonate treatments, respectively. However, CaPIMP1 promoter sequences longer than −793 bp were required for promoter activation by abscisic acid and sodium nitroprusside treatments. CaPIMP1 expression was activated in pepper leaves by treatment with ethylene, methyl jasmonate, abscisic acid, β-amino-n-butyric acid, NaCl, mechanical wounding, and low temperature, but not with salicylic acid. Overexpression of CaPIMP1 in Arabidopsis conferred hypersensitivity to mannitol, NaCl, and ABA during seed germination but not during seedling development. In contrast, transgenic plants overexpressing CaPIMP1 exhibited enhanced tolerance to oxidative stress induced by methyl viologen during germination and early seedling stages. These results suggest that CaPIMP1 expression may alter responsiveness to environmental stress, as well as to pathogen infection. The nucleotide sequence data reported here has been deposited in the GenBank database under the accession number DQ356279.     

Pretreatment efficiency and structural characterization of rice straw by an integrated process of dilute-acid and steam explosion for bioethanol production

The combined pretreatment of rice straw using dilute-acid and steam explosion followed by enzymatic hydrolysis was investigated and compared with acid-catalyzed steam explosion pretreatment. In addition to measuring the chemical composition, including glucan, xylan and lignin content, changes in rice straw features after pretreatment were investigated in terms of the straw's physical properties. These properties included crystallinity, surface area, mean particle size and scanning electron microscopy imagery. The effect of acid concentration on the acid-catalyzed steam explosion was studied in a range between 1% and 15% acid at 180°C for 2 min. We also investigated the influence of the residence time of the steam explosion in the combined pretreatment and the optimum conditions for the dilute-acid hydrolysis step in order to develop an integrated process for the dilute-acid and steam explosion. The optimum operational conditions for the first dilute-acid hydrolysis step were determined to be 165°C for 2 min with 2% H(2)SO(4) and for the second steam explosion step was to be carried out at 180°C for 20 min; this gave the most favorable combination in terms of an integrated process. We found that rice straw pretreated by the dilute-acid/steam explosions had a higher xylose yield, a lower level of inhibitor in the hydrolysate and a greater degree of enzymatic hydrolysis; this resulted in a 1.5-fold increase in the overall sugar yield when compared to the acid-catalyzed steam explosion.     

Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL

PINK1 is a mitochondrial kinase mutated in some familial cases of Parkinson's disease. It has been found to work in the same pathway as the E3 ligase Parkin in the maintenance of flight muscles and dopaminergic neurons in Drosophila melanogaster and to recruit cytosolic Parkin to mitochondria to mediate mitophagy in mammalian cells. Although PINK1 has a predicted mitochondrial import sequence, its cellular and submitochondrial localization remains unclear in part because it is rapidly degraded. In this study, we report that the mitochondrial inner membrane rhomboid protease presenilin-associated rhomboid-like protein (PARL) mediates cleavage of PINK1 dependent on mitochondrial membrane potential. In the absence of PARL, the constitutive degradation of PINK1 is inhibited, stabilizing a 60-kD form inside mitochondria. When mitochondrial membrane potential is dissipated, PINK1 accumulates as a 63-kD full-length form on the outer mitochondrial membrane, where it can recruit Parkin to impaired mitochondria. Thus, differential localization to the inner and outer mitochondrial membranes appears to regulate PINK1 stability and function.     

Suppression of thymus- and activation-regulated chemokine (TARC/CCL17) production by 1,2,3,4,6-penta-O-galloyl-β-d-glucose via blockade of NF-κB and STAT1 activation in the HaCaT cells

Keratinocytes, one of major cell types in the skin, can be induced by TNF-α and IFN-γ to express thymus- and activation-regulated chemokine (TARC/CCL17), which is considered to be a pivotal mediator in the inflammatory responses during the development of inflammatory skin diseases, such as atopic dermatitis (AD). In this study, we examined the effect of 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), isolated from the barks of Juglans mandshurica, on TNF-α/IFN-γ induced CCL17 expression in the human keratinocyte cell line HaCaT. Pretreatment of HaCaT cells with PGG suppressed TNF-α/IFN-γ-induced protein and mRNA expression of CCL17. PGG significantly inhibited TNF-α/IFN-γ-induced NF-κB activation as well as STAT1 activation. Furthermore, pretreatment with PGG resulted in significant reduction in expression of CXCL9, 10, and 11 in the HaCaT cells treated with IFN-γ. These results suggest that PGG may exert anti-inflammatory responses by suppressing TNF-α and/or IFN-γ-induced activation of NF-κB and STAT1 in the keratinocytes and might be a useful tool in therapy of skin inflammatory diseases.     

Metformin sensitizes insulin signaling through AMPK-mediated PTEN down-regulation in preadipocyte 3T3-L1 cells

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.     

Feasibility of hydrogen production from ripened fruits by a combined two-stage (dark/dark) fermentation system

Anaerobic fermentation for hydrogen (H₂) production was studied in a two-stage fermentation system fed with different ripened fruit feedstocks (apple, pear, and grape). Among the feedstocks, ripened apple was the most efficient substrate for cumulative H₂ production (4463.7 mL-H₂ L⁻¹-culture) with a maximum H₂ yield (2.2 mol H₂ mol⁻¹ glucose) in the first stage at a hydraulic retention time (HRT) of 18 h. The additional cumulative biohydrogen (3337.4 mL-H₂ L⁻¹-culture) was produced in the second stage with the reused residual substrate from the first stage. The major byproducts in this study were butyrate, acetate, and ethanol, and butyrate was dominant among them in all test runs. During the two-stage system, the energy efficiency (H₂ conversion) obtained from mixed ripened fruits (RF) increased from 4.6% (in the first stage) to 15.5% (in the second stage), which indicated the energy efficiency can be improved by combined hydrogen production process. The RF could be used as substrates for biohydrogen fermentation in a two-stage (dark/dark) fermentation system.