Fresh organically grown ginger (Zingiber officinale): composition and effects on LPS-induced PGE2 production

Gas chromatography in conjunction with mass spectrometry, a technique previously employed to analyze non-volatile pungent components of ginger extracts modified to trimethylsilyl derivatives, was applied successfully for the first time to analyze unmodified partially purified fractions from the dichloromethane extracts of organically grown samples of fresh Chinese white and Japanese yellow varieties of ginger, Zingiber officinale Roscoe (Zingiberaceae). This analysis resulted in the detection of 20 hitherto unknown natural products and 31 compounds previously reported as ginger constituents. These include paradols, dihydroparadols, gingerols, acetyl derivatives of gingerols, shogaols, 3-dihydroshogaols, gingerdiols, mono- and diacetyl derivatives of gingerdiols, 1-dehydrogingerdiones, diarylheptanoids, and methyl ether derivatives of some of these compounds. The thermal degradation of gingerols to gingerone, shogaols, and related compounds was demonstrated. The major constituent in the two varieties was [6]-gingerol, a chemical marker for Z. officinale. Mass spectral fragmentation patterns for all the compounds are described and interpreted. Anti-inflammatory activities of silica gel chromatography fractions were tested using an in vitro PGE2 assay. Most of the fractions containing gingerols and/or gingerol derivatives showed excellent inhibition of LPS-induced PGE2 production.     

rIL 2-induced proliferation of human circulating NK cells and T lymphocytes: synergistic effects of IL 1 and IL 2

Cells participating in the rIL 2-induced proliferation of resting PBMC were identified by using different methods of cell purification. NK cells recovered in the light density fraction of Percoll gradients responded, as already known, directly to rIL 2 by strong proliferation. In contrast, large T lymphocytes co-purifying with NK cells, and small T cells sedimenting in the high density area of the Percoll gradients, were virtually unresponsive when cultivated in the sole presence of rIL 2. However, the addition of either irradiated autologous monocytes or highly purified IL 1 allowed both kinds of T cells to undergo cell division. Stringent elimination of possibly contaminating NK cells (NKH-1+) and/or activated T cells (TNKTAR, Tac+, HLA-DR+) from the high density T cells by complement lysis did not impair rIL 2-induced cell proliferation, indicating entire responsiveness of these cells to the synergistic action of IL 1 plus IL 2. Both high density CD4+ and CD8+ participated in this phenomenon, with an apparent advantage for CD4+ cells. All Tac+ cells emerging in a 6-day culture of these cells expressed the WT31 antigen, which indicates that T cells involved in rIL 2-induced proliferation are conventional mature T cells. The relative precursor frequencies of NK cells, large T lymphocytes, and small T lymphocytes that proliferated in response to rIL 2 were analyzed by limiting dilution analysis. The frequencies of clonal growth of NK cells and low density T lymphocytes were approximately the same (1/103 vs 1/185), whereas that of high density T cells was four times lower (1/458). Thus, we clearly demonstrate that resting T cells, defined as such by morphological, density, and phenotypic criteria, are able to proliferate in response to IL 2 in the presence of IL 1 without antigenic or mitogenic triggering.     

Effects of disulfide bond and cholesterol derivatives on human calcitonin amyloid formation

Human calcitonin (hCT) is a 32-residue peptide that aggregates to form amyloid fibrils under appropriate conditions. In this study, we investigated the effect of the intramolecular disulfide bond formed at the N-terminal region of the peptide in the aggregation kinetics of hCT. Our results indicate that the presence of the disulfide bond in hCT plays a crucial role in forming the critical nucleus needed for fibril formation, facilitating the rate of hCT amyloidogenesis. Furthermore, we reported for the first time the effects of cholesterol, cholesterol sulfate, and 3β-[N-(dimethylaminoethane)carbamoyl]-cholesterol (DC-cholesterol) on the amyloid formation of oxidized hCT. Our results show that while cholesterol does not affect amyloidogenesis of oxidized hCT, high concentrations of cholesterol sulfate exhibits a moderate inhibiting activity on hCT amyloid formation. In particular, our results show that DC-cholesterol strongly inhibits amyloidogenesis of oxidized hCT in a dose-dependent manner. Further studies at different pH conditions imply the crucial impact of electrostatic and hydrogen bonding interactions in mediating the interplay of hCT and the surface of DC-cholesterol vesicles and the inhibiting function of DC-cholesterol on hCT fibrillization.     

Response of green alder (Alnus viridis subsp. fruticosa) patch dynamics and plant community composition to fire and regional temperature in north‐western Canada

Aim Feedbacks between climate warming and fire have the potential to alter Arctic and sub‐Arctic vegetation. In this paper we assess the effects and interactions of temperature and wildfire on plant communities across the transition between the Arctic and sub‐Arctic. Location Mackenzie Delta region, Northwest Territories, Canada. Methods We sampled air temperatures, green alder (Alnus viridis ssp. fruticosa) cover, growth, reproduction and age distributions, and overall plant community composition on burned and unburned sites across a latitudinal gradient. Results Mean summer temperature across the study area decreased by 3 °C per degree of increasing latitude (6 °C across the study area). In the northern part of the study area, where seed viability was low, alder was less dominant than at southern sites where seed viability was high. The age structure of alder populations across the temperature gradient was highly variable, except in the northern part of the forest–tundra transition, where populations were dominated by young individuals. Alder growth and reproduction were significantly greater on burned sites (38–51 years following fire) than on unburned sites. North to south across the temperature gradient, vegetation changed from a community dominated by dwarf shrubs and fruticose lichens to one characterized by black spruce (Picea mariana), alder and willows (Salix spp.). Regardless of the position along the temperature gradient, burned sites were dominated by tall shrubs. Main conclusions Temperature limitation of alder abundance and repro‐duction, combined with evidence of recent recruitment on unburned sites, indicates that alder is likely to respond to increased temperature. Elevated alder growth and reproduction on burned sites shows that wildfire also has an important influence on alder population dynamics. The magnitude of alder’s response to fire, combined with observations that burns at the southern margin of the low Arctic are shrub dominated, suggest that increases in the frequency of wildfire have the potential to alter northern vegetation on decadal scales. By creating new seedbeds, fire provides opportunities for colonization that may facilitate the northward movement of tall shrubs. Feedbacks between the global climate system and low Arctic vegetation make understanding the long‐term impact of increasing fire frequency critical to predicting the response of northern ecosystems to global change.     

Use of aqueous two-phase systems in sample preparation for polymerase chain reaction-based detection of microorganisms

An aqueous two-phase system, consisting of poly(ethylene glycol) (PEG) and dextran, was employed to separate polymerase chain reaction (PCR)-inhibitory substances from bacterial cells. The PCR inhibition of four soft cheeses was examined and three of them were found to be strongly PCR-inhibitory. Extraction of the PCR-inhibitory soft cheeses inoculated with Listeria monocytogenes in an aqueous two-phase system containing 8% (w/w) PEG 400 and 8% (w/w) dextran 500, was found to lower the PCR detection level of L. monocytogenes by more than four orders of magnitude in two of the cheeses compared to the case where no such sample pretreatment was performed. Depending on the type of cheese used, the PCR-inhibitory factors were found to be enriched in either the top or botton phase in the aqueous two-phase system. These results show that different soft cheeses contain different types and amounts of PCR-inhibitory substances.     

Compartment differences of inflammatory activity in chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is associated with local and systemic inflammation. The knowledge of interaction and co-variation of the inflammatory responses in different compartments is meagre.

Method

Healthy controls (n = 23), smokers with (n = 28) and without (n = 29) COPD performed spirometry and dental examinations. Saliva, induced sputum, bronchoalveolar lavage (BAL) fluid and serum were collected. Inflammatory markers were assessed in all compartments using ELISA, flow cytometry and RT-PCR.

Results

Negative correlations between lung function and saliva IL-8 and matrix metalloproteinase-9 (MMP-9) were found in smokers with COPD. IL-8 and MMP-9 in saliva correlated positively with periodontal disease as assessed by gingival bleeding in non-smokers.Tumor necrosis factor-α (TNF-α) in saliva, serum and TNF-α mRNA expression on macrophages in BAL-fluid were lower in smokers than in non-smokers. There were positive correlations between soluble TNF-α receptor 1 (sTNFR1) and soluble TNF-α receptor 2 (sTNFR2) in sputum, BAL-fluid and serum in all groups. Sputum interleukin-8 (IL-8) or interleukin-6 (IL-6) was positively correlated with sTNFR1 or sTNFR2 in non-smokers and with sTNFR2 in COPD.

Conclusion

Saliva which is convenient to collect and analyse, may be suitable for biomarker assessment of disease activity in COPD. An attenuated TNF-α expression was demonstrated by both protein and mRNA analyses in different compartments suggesting that TNF-α response is altered in moderate and severe COPD. Shedding of TNFR1 or TNFR2 is similarly regulated irrespective of airflow limitation.     

Quantifying turbulent wall shear stress in a stenosed pipe using large eddy simulation

Large eddy simulation was applied for flow of Re=2000 in a stenosed pipe in order to undertake a thorough investigation of the wall shear stress (WSS) in turbulent flow. A decomposition of the WSS into time averaged and fluctuating components is proposed. It was concluded that a scale resolving technique is required to completely describe the WSS pattern in a subject specific vessel model, since the poststenotic region was dominated by large axial and circumferential fluctuations. Three poststenotic regions of different WSS characteristics were identified. The recirculation zone was subject to a time averaged WSS in the retrograde direction and large fluctuations. After reattachment there was an antegrade shear and smaller fluctuations than in the recirculation zone. At the reattachment the fluctuations were the largest, but no direction dominated over time. Due to symmetry the circumferential time average was always zero. Thus, in a blood vessel, the axial fluctuations would affect endothelial cells in a stretched state, whereas the circumferential fluctuations would act in a relaxed direction.     

MAIT Cells Detect and Efficiently Lyse Bacterially-Infected Epithelial Cells

Mucosal associated invariant T cells (MAIT) are innate T lymphocytes that detect a large variety of bacteria and yeasts. This recognition depends on the detection of microbial compounds presented by the evolutionarily conserved major-histocompatibility-complex (MHC) class I molecule, MR1. Here we show that MAIT cells display cytotoxic activity towards MR1 overexpressing non-hematopoietic cells cocultured with bacteria. The NK receptor, CD161, highly expressed by MAIT cells, modulated the cytokine but not the cytotoxic response triggered by bacteria infected cells. MAIT cells are also activated by and kill epithelial cells expressing endogenous levels of MRI after infection with the invasive bacteria Shigella flexneri. In contrast, MAIT cells were not activated by epithelial cells infected by Salmonella enterica Typhimurium. Finally, MAIT cells are activated in human volunteers receiving an attenuated strain of Shigella dysenteriae-1 tested as a potential vaccine. Thus, in humans, MAIT cells are the most abundant T cell subset able to detect and kill bacteria infected cells.     

MAIT Cell Recognition of MR1 on Bacterially Infected and Uninfected Cells

Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant αβ TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.     

Post-translational Modifications of Recombinant Human Lysyl Oxidase-like 2 (rhLOXL2) Secreted from Drosophila S2 Cells

Human lysyl oxidase-like 2 (hLOXL2) is highly up-regulated in metastatic breast cancer cells and tissues and induces epithelial-to-mesenchymal transition, the first step of metastasis/invasion. hloxl2 encodes four N-terminal scavenger receptor cysteine-rich domains and the highly conserved C-terminal lysyl oxidase (LOX) catalytic domain. Here, we assessed the extent of the post-translational modifications of hLOXL2 using truncated recombinant proteins produced in Drosophila S2 cells. The recombinant proteins are soluble, in contrast to LOX, which is consistently reported to require 2–6 m urea for solubilization. The recombinant proteins also show activity in tropoelastin oxidation. After phenylhydrazine derivatization and trypsin digestion, we used mass spectrometry to identify peptides containing the derivatized lysine tyrosylquinone cross-link at Lys-653 and Tyr-689, as well as N-linked glycans at Asn-455 and Asn-644. Disruption of N-glycosylation by site-directed mutagenesis or tunicamycin treatment completely inhibited secretion so that only small quantities of inclusion bodies were detected. The N-glycosylation site at Asn-644 in the LOX catalytic domain is not conserved in human LOX (hLOX), although the LOX catalytic domain of hLOX shares ∼50% identity and ∼70% homology with hLOXL2. The catalytic domain of hLOX was not secreted from S2 cells using the same expression system. These results suggest that the N-glycan at Asn-644 of hLOXL2 enhances the solubility and stability of the LOX catalytic domain.