High-efficiency decomposition of eggshell membrane by a keratinase from Meiothermus taiwanensis.

Eggshell membrane (ESM), a plentiful biological waste, consists of collagen-like proteins and glycosaminoglycans (GAGs) such as hyaluronic acid (HA). Here we used a keratinase (oeMtaker)-mediated system to decompose ESM. The best reaction condition was established by incubating the solution containing oeMtaker, sodium sulfite, and ESM with a weight ratio of 1:120:600. ESM enzymatic hydrolysate (ESM-EH) showed a high proportion of essential amino acids and type X collagen peptides with 963-2259 Da molecular weights. The amounts of GAGs and sulfated GAGs in ESM-EH were quantified as 6.4% and 0.7%, respectively. The precipitated polysaccharides with an average molecular weight of 1300-1700 kDa showed an immunomodulatory activity by stimulating pro-inflammatory cytokines (IL-6 and TNF-α) production. In addition, a microorganism-based system was established to hydrolyze ESM by Meiothermus taiwanensis WR-220. The amounts of GAGs and sulfated GAGs in the system were quantified as 0.9% and 0.1%, respectively. Based on our pre-pilot tests, the system shows great promise in developing into a low-cost and high-performance process. These results indicate that the keratinase-mediated system could hydrolyze ESM more efficiently and produce more bioactive substances than ever for therapeutical applications and dietary supplements.

Capsular Polysaccharide Is Involved in NLRP3 Inflammasome Activation by Klebsiella pneumoniae Serotype K1.

Klebsiella pneumoniae (strain 43816, K2 serotype) induces interleukin-1ß (IL-1ß) secretion, but neither the bacterial factor triggering the activation of these inflammasome-dependent responses nor whether they are mediated by NLRP3 or NLRC4 is known. In this study, we identified a capsular polysaccharide (K1-CPS) in K. pneumoniae (NTUH-K2044, K1 serotype), isolated from a primary pyogenic liver abscess (PLA K. pneumoniae), as the Klebsiella factor that induces IL-1ß secretion in an NLRP3-, ASC-, and caspase-1-dependent manner in macrophages. K1-CPS induced NLRP3 inflammasome activation through reactive oxygen species (ROS) generation, mitogen-activated protein kinase phosphorylation, and NF-κB activation. Inhibition of both the mitochondrial membrane permeability transition and mitochondrial ROS generation inhibited K1-CPS-mediated NLRP3 inflammasome activation. Furthermore, IL-1ß secretion in macrophages infected with PLA K. pneumoniae was shown to depend on NLRP3 but also on NLRC4 and TLR4. In macrophages infected with a K1-CPS deficiency mutant, an lipopolysaccharide (LPS) deficiency mutant, or K1-CPS and LPS double mutants, IL-1ß secretion levels were lower than those in cells infected with wild-type PLA K. pneumoniae. Our findings indicate that K1-CPS is one of the Klebsiella factors of PLA K. pneumoniae that induce IL-1ß secretion through the NLRP3 inflammasome.

Bamboo vinegar decreases inflammatory mediator expression and NLRP3 inflammasome activation by inhibiting reactive oxygen species generation and protein kinase C-α/δ activation.

Bamboo vinegar (BV), a natural liquid derived from the condensation produced during bamboo charcoal production, has been used in agriculture and as a food additive, but its application to immune modulation has not been reported. Here, we demonstrated that BV has anti-inflammatory activities both in vitro and in vivo. BV reduced inducible nitric oxide synthase expression and nitric oxide levels in, and interleukin-6 secretion by, lipopolysaccharide-activated macrophages without affecting tumor necrosis factor-α secretion and cyclooxygenase-2 expression. The mechanism for the anti-inflammatory effect of BV involved decreased reactive oxygen species production and protein kinase C-α/δ activation. Furthermore, creosol (2-methoxy-4-methylphenol) was indentified as the major anti-inflammatory compound in BV. Impaired cytokine expression and NLR family, pyrin domain-containing 3 (NLRP3) inflammasome activation was seen in mice treated with creosol. These findings provide insights into how BV regulates inflammation and suggest that it may be a new source for the development of anti-inflammatory agents or a healthy supplement for preventing and ameliorating inflammation- and NLRP3 inflammasome-related diseases, including metabolic syndrome.

Lipopolysaccharide/adenosine triphosphate-mediated signal transduction in the regulation of NLRP3 protein expression and caspase-1-mediated interleukin-1ß secretion.

OBJECTIVE: Reactive oxygen species (ROS) plays a critical role in the regulation of NLRP3 inflammasome activation. However, the ROS-mediated signaling pathways controlling NLRP3 inflammasome activation are not well defined. METHODS: Using lipopolysaccharide (LPS) and adenosine triphosphate (ATP) activated murine macrophages as the testing model, cytokine release and protein expression were quantified by enzyme-linked immunosorbent assay and Western blot, respectively. ROS was scavenged by N-acetyl cysteine; NADPH oxidase, the major source of ROS, was inhibited by diphenyliodonium, apocynin or gp91-phox siRNA transfection; and protein kinase was inhibited by its specific inhibitor. RESULTS: LPS-induced NLRP3 protein expression was regulated through the NADPH oxidase/ROS/NF-κB-dependent, JAK2/PI3-kinase/AKT/NF-κB-dependent, and MAPK-dependent pathways, while ATP-induced caspase-1 activation was regulated through the NADPH oxidase/ROS-dependent pathway. CONCLUSIONS: These results demonstrate that ROS regulates not only the priming stage, but also the activation stage, of NLRP3 inflammasome activation in LPS + ATP-activated macrophages.

Application of power plant flue gas in a photobioreactor to grow Spirulina algae, and a bioactivity analysis of the algal water-soluble polysaccharides.

A novel photobioreactor was developed with a total volume of 30 m(3) which required merely 100 m(3) of land footprint. The bioreactor was capable of utilizing CO(2) in the flue gas of a power plant as the carbon source for the growth of a freshwater alga, Spirulina platensis, mitigating the greenhouse effect caused by the same amount of CO(2) discharge. Results of the study indicated that the photobioreactor was capable of fixing 2,234 kg of CO(2) per annum. Upon deducting the energy consumption of operating the bioreactor unit, the estimated amount of CO(2) to be fixed by a scaled-up reactor would be 74 tons ha(-1)year(-1). In addition, the study prove that protein-free polysaccharides of S. platensis could induce the production of pro-IL-1 and IL-1 proteins through the mediation of ERK, JNK, and p38 MAPKs pathways. As a consequence, immunogenic activities of the macrophage cells were enhanced.

High glucose increases nitric oxide generation in lipopolysaccharide-activated macrophages by enhancing activity of protein kinase C-α/δ and NF-κB.

OBJECTIVE: Although several mechanisms by which hyperglycemia modulate inflammation have been proposed, it remains unclear how hyperglycemia regulates inflammation induced by lipopolysaccharide (LPS). METHODS: We hypothesized that hyperglycemia might interplay with LPS to modulate the generation of an inflammatory mediator. RAW 264.7 macrophages cultured in medium containing either normal glucose (5.5-mM) or high glucose (HG) (15- and 25-mM) were treated with LPS. The nitric oxide (NO) generation, inducible NO synthase (iNOS) expression and cytokine release were then quantified by Griess reaction, western blot, and enzyme-linked immunosorbent assay (ELISA) respectively. The effect of HG on the activation of kinase and Nuclear Factor-Kappa B (NF-κB) were measured by western blot and NF-κB reporter assay respectively. RESULTS: Without LPS stimulation, HG alone did not induce NO generation and cytokine secretion; but LPS-induced NO generation, iNOS expression, and interleukin-1beta (IL-1ß) secretion were higher in HG-cultured cells than in normal glucose-cultured cells. In contrast, LPS-induced interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) secretion were lower in HG-cultured cells than in normal glucose-cultured cells. Furthermore, HG increased iNOS expression and NO generation by enhancing phosphorylation levels of protein kinase C-alpha (PKC-α), protein kinase C-delta (PKC-δ), and p38 phosphorylation and NF-κB transcriptional activity. CONCLUSIONS: This study revealed a possible role of PKC-α and PKC-δ potentially involved in diabetes-promoted inflammation.

Composition, antioxidant and antimicrobial activities of leaf and twig essential oils of Litsea akoensis from Taiwan.

This study analyzed the hydrodistilled essential oils in the leaves and twigs of Litsea akoensis to determine composition and yield. Seventy-one and 40 compounds were identified in the leaf and twig oils, respectively. The main components of leaf oil were limonene (18.5%), thymol (10.1%), p-cymene (9.6%), beta-caryophyllene (8.9%), and carvacrol (8.2%). The main components of twig oil were beta-phellandrene (43.7%) and trans-beta-ocimene (10.4%). The results demonstrated that leaf oil had excellent antioxidant and antimicrobial activities, superior to those of twig oil.

Composition and anti-wood-decay fungal activities of the leaf essential oil of Machilus philippinensis from Taiwan.

The hydrodistilled leaf essential oil of Machilus philippinensis was analyzed to determine its composition and yield. Seventy compounds were identified, the main ones being beta-caryophyllene (13.6%), alpha-pinene (12.0%), alpha-cadinol (7.4%), cis-ocimene (7.0%), spathulenol (5.6%), (E)-nerolidol (5.3%), tau-cadinol (4.8%) and beta-pinene (4.5%). Monoterpene hydrocarbons (36.1%) and oxygenated sesquiterpenes (33.0%) were the predominant groups of compounds. The leaf oil exhibited excellent anti-wood-decay fungal activities.

n-Butylidenephthalide induced apoptosis in the A549 human lung adenocarcinoma cell line by coupled down-regulation of AP-2alpha and telomerase activity.

AIM: To investigate the role of hTERT gene expression and AP-2alpha in n-butylidenephthalide (n-BP)-induced apoptosis in A549 lung cancer cells. METHODS: Viability of A549 cells was measured by MTT assay. Protein expression was determined by Western blot. Telomerase activity was measured using the modified telomere repeat amplification protocol (TRAP) assay. Xenograft mice were used as a model system to study the cytotoxic effect of n-BP in vivo. The morphology of tumor was examined by immunohistochemical staining. RESULTS: The growth of A549 lung cancer cells treated with n-BP was significantly inhibited. Telomerase activity and hTERT mRNA expression were determined by telomeric repeat amplification protocol and reverse transcription-polymerase chain reaction, respectively. n-BP inhibited telomerase activity and hTERT mRNA expression in A549 cells while overexpression of hTERT could abolish BP-induced growth inhibition in the A549 cells. We also showed that hTERT promoter activity in the presence of n-BP was mediated via AP-2alpha. We saw an inhibition of tumor growth when nude mice carrying A549 subcutaneous xenograft tumors were treated with n-BP. Immunohistochemistry of this tumor tissue also showed a decrease in the expression of hTERT. CONCLUSION: The antiproliferative effects of n-BP on A549 cells in vitro and in vivo suggest a novel clinical application of this compound in the treatment of lung cancers.