Amenyunnaosides A–C,Three New Neolignans Isolated from Amentotaxus yunnanensis and Their Anti-inflammatory Activities

Phytochemical study on the leaves of Amentotaxus yunnanensis led to the isolation of seventeen phenolic compounds including sixteen neolignans and lignans, and one flavone glycoside. Three among the isolates were previously unreported neolignans and named as amenyunnaosides A–C, respectively. Their structures were elucidated by extensive analyses of HR-ESI-MS, 1D and 2D NMR, and ECD spectra. The isolated neolignans potentially inhibited NO production in LPS-activated RAW264.7 cells with their IC₅₀ values ranging from 11.05 to 44.07 μM, compared to that of the positive control compound, dexamethasone, IC₅₀ value of 16.93 μM. Additionally, amenyunnaoside A dose-dependently reduced production of IL-6 and COX-2 but did not effect to that of TNF-α at concentrations of 0.8, 4, and 20 μM.     

EMMPRIN/CD147, an MMP modulator in cancer, development and tissue repair

Matrix metalloproteinases (MMPs) play a central role in normal tissue remodeling and disease, they regulate tumor microenvironment and their expression is increased in most human cancers. Targeting their activity remains a major challenge. Their production and activation is tightly regulated by complex mechanisms that include cytokines and growth factors, cell-matrix and cell-cell interactions. The observations of increased MMP level at the epithelio-stromal interface led to the identification of EMMPRIN/CD147, a membrane spanning molecule highly expressed in tumor cells, that stimulates MMPs production in neighboring fibroblasts. Later studies have shown that EMMPRIN can also induce MMP in the same population of cells. Elevated EMMPRIN level was detected in numerous malignant tumors and has been correlated with tumor progression in experimental and clinical conditions. The presence and modulation of EMMPRIN in normal tissues associated with increased MMP expression suggests that this EMMPRIN-mediated MMP induction could be a common mechanism in non-tumoral physiological and/or pathological situations. Targeting EMMPRIN in cancer and other pathological conditions such arthritis and ulceration appears a promising future therapeutic strategy, but requires a better understanding of its mode of action and regulation. Potential regulators that influence EMMPRIN level and its MMP inducing activity include growth factors, hormones, glycosylation and membrane shedding. This review will discuss the recent findings concerning these diverse regulatory mechanisms in various physiological and pathological situations.     

Zoonotic viruses associated with illegally imported wildlife products

The global trade in wildlife has historically contributed to the emergence and spread of infectious diseases. The United States is the world's largest importer of wildlife and wildlife products, yet minimal pathogen surveillance has precluded assessment of the health risks posed by this practice. This report details the findings of a pilot project to establish surveillance methodology for zoonotic agents in confiscated wildlife products. Initial findings from samples collected at several international airports identified parts originating from nonhuman primate (NHP) and rodent species, including baboon, chimpanzee, mangabey, guenon, green monkey, cane rat and rat. Pathogen screening identified retroviruses (simian foamy virus) and/or herpesviruses (cytomegalovirus and lymphocryptovirus) in the NHP samples. These results are the first demonstration that illegal bushmeat importation into the United States could act as a conduit for pathogen spread, and suggest that implementation of disease surveillance of the wildlife trade will help facilitate prevention of disease emergence.     

Reorganization of actin cytoskeleton of nuclear erythrocytes and leukocytes in fish,frogs, and birds during migration

Using the method of confocal laser scanning microscopy, changes in the spatial organization of actin filaments of nuclear erythrocytes and leukocytes in fish, frogs, and birds during migration were studied. It has been shown that, during movement, in erythrocytes, like in leukocytes, reorganization of cytoskeleton microfilaments occurs. In the course of migration, in amphibians and birds, red blood cells form pseudopodia filled with bundles of actin filaments arranged in parallel, whereas no pseudopodia are formed in fish erythrocytes. The change in the structure of the actin cytoskeleton of nuclear erythrocytes, like in leukocytes, is responsible for the capability of red blood cells to undergo reactions of migration and phagocytosis.     

Efficiency of ligation-mediated PCR and TAIL-PCR methods for isolation of RbcS promoter sequences from green microalgae Ankistrodesmus convolutus

Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus.     

Impact of myo‐inositol trispyrophosphate (ITPP) on tumour oxygenation and response to irradiation in rodent tumour models

Tumour hypoxia is a well‐established factor of resistance in radiation therapy (RT). Myo‐inositol trispyrophosphate (ITPP) is an allosteric effector that reduces the oxygen‐binding affinity of haemoglobin and facilitates the release of oxygen by red blood cells. We investigated herein the oxygenation effect of ITPP in six tumour models and its radiosensitizing effect in two of these models. The evolution of tumour pO₂ upon ITPP administration was monitored on six models using 1.2 GHz Electron Paramagnetic Resonance (EPR) oximetry. The effect of ITPP on tumour perfusion was assessed by Hoechst staining and the oxygen consumption rate (OCR) in vitro was measured using 9.5 GHz EPR. The therapeutic effect of ITPP with and without RT was evaluated on rhabdomyosarcoma and 9L‐glioma rat models. ITPP enhanced tumour oxygenation in six models. The administration of 2 g/kg ITPP once daily for 2 days led to a tumour reoxygenation for at least 4 days. ITPP reduced the OCR in six cell lines but had no effect on tumour perfusion when tested on 9L‐gliomas. ITPP plus RT did not improve the outcome in rhabdomyosarcomas. In 9L‐gliomas, some of tumours receiving the combined treatment were cured while other tumours did not benefit from the treatment. ITPP increased oxygenation in six tumour models. A decrease in OCR could contribute to the decrease in tumour hypoxia. The association of RT with ITPP was beneficial for a few 9L‐gliomas but was absent in the rhabdomyosarcomas.     

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: implication of their membrane fluidizing action

Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called "green notes". The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of HPOD on yeast membrane. In addition, we determined by infrared spectroscopy measurement that this compound rapidly diffused into model phospholipids [1, 2-Dimyristoyl-D54-sn-Glycero-3-Phosphocholine (DMPC-D54)] bilayer, modifying their general physical state and their phase transition. In the presence of various concentrations of HPOD, the phase transition of DMPC-D54 occurred with an increase of both the corresponding wave number shift and the temperature range but the phase transition temperature was not modified. These results show that the toxic effects of HPOD on the yeast Yarrowia lipolytica may be initially linked to a strong interaction of this compound with the cell membrane phospholipids and components.     

Optimization of the isolation procedure and culturing conditions for hepatic stellate cells obtained from mouse

Liver fibrosis (LF) mortality rate is approximately 2 million per year. Irrespective of the etiology of LF, a key element in its development is the transition of hepatic stellate cells (HSCs) from a quiescent phenotype to a myofibroblast-like cell with the production of fibrotic proteins. It is necessary to define optimal isolation and culturing conditions for good HSCs yield and proper phenotype preservation for studying the activation of HSCs in vitro. In the present study, the optimal conditions of HSC isolation and culture were examined to maintain the HSC’s undifferentiated phenotype. HSCs were isolated from Balb/c mice liver using Nycodenz, 8, 9.6, and 11%. The efficiency of the isolation procedure was evaluated by cell counting and purity determination by flow cytometry. Quiescent HSCs were cultured in test media supplemented with different combinations of fetal bovine serum (FBS), glutamine (GLN), vitamin A (vitA), insulin, and glucose. The cells were assessed at days 3 and 7 of culture by evaluating the morphology, proliferation using cell counting kit-8, lipid storage using Oil Red O (ORO) staining, expression of a-smooth muscle actin, collagen I, and lecithin-retinol acyltransferase by qRT-PCR and immunocytochemistry (ICC). The results showed that Nycodenz, at 9.6%, yielded the best purity and quantity of HSCs. Maintenance of HSC undifferentiated phenotype was achieved optimizing culturing conditions (serum-free Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with glucose (100 mg/dl), GLN (0.5 mM), vitA (100 μM), and insulin (50 ng/ml)) with a certain degree of proliferation allowing their perpetuation in culture. In conclusion, we have defined optimal conditions for HSCs isolation and culture.     

Anti-Inflammatory Properties of Longifuran A,a New Benzofuran from the Stems of Amomum longiligulare

In this study, the following compounds were isolated from the dichloromethane fraction of the stems of Amomum longiligulare and then characterized: a new benzofuran, namely, longifuran A ( 1 ); five other phenolic compounds, namely, 4-methoxycinnamic acid ( 2 ), 2,5-dimethoxyphenol ( 3 ), eudesmic acid ( 4 ), 1,7-bis(4-hydroxyphenyl)-1,4,6-heptatrien-3-one ( 5 ), and 4,4’-dihydroxychalcone ( 6 ); and two triterpenoids, namely, 24-methylcycloartan-3β-ol ( 7 ) and 24-methylencycloartan-3β-ol ( 8 ). They were evaluated in terms of their inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophages. Results indicated that 1 and 5 exhibited promising inhibitory activities against NO generation with IC₅₀ of 10.47±1.02 μM and 8.51±1.14 μM, respectively. Enzymatic assays demonstrated that they remarkably suppressed the secretion of two pro-inflammatory cytokines (i. e., IL-6 and TNF-α). They also dose-dependently inhibited the expression of inducible nitric oxide synthase and cyclooxygenase-2, two important enzymes modulating inflammation. Therefore, 1 and 5 could be targets for the development of new anti-inflammatory therapeutics