Use of liposome encapsulated hemoglobin as an oxygen carrier for fetal and adult rat liver cell culture

Engineering liver tissue constructs with sufficient cell mass for transplantation implies culturing large numbers of hepatocytes in a reduced volume; however, providing sufficient oxygen to dense cell cultures is still not feasible using only conventional culture medium. Liposome-encapsulated hemoglobin (LEH), an oxygen-carrying blood substitute originally designed for short-term perfusion, may be a good candidate as an oxygen carrier to cultured liver cells. In this study, we investigated the feasibility of maintaining long term hepatocyte cultures using LEH. Primary fetal and adult rat liver cells were directly exposed to LEH for 6 to 14 days in static culture or in a perfused flat plate bioreactor. The functions and viability of adult rat hepatocytes exposed to LEH were not adversely affected in static monolayer culture and were even improved in the bioreactor. However, some cytotoxicity of LEH was observed with fetal rat liver cells after 4 days of culture. LEH, though a suitable oxygen carrier for long-term culture of mature hepatocytes, is not suitable in its present form for perfusing fetal hepatocyte cultures in direct contact with the liposomes; either the LEH will have to be made less toxic or a more sophisticated bioreactor that prevents the direct contact between hepatocytes and perfusates will have to be designed if fetal cells are to be used for liver tissue engineering.     

Lipid peroxidation-derived cytotoxic aldehyde, 4-hydroxy-2-nonenal in smoked pork

The contents of 4-Hydroxy-2-nonenal (HNE), cytotoxic aldehyde, in smoked meat products (ham, bacon and sausage) were analyzed. All the samples analyzed contained HNE, although large differences in the contents between the different samples were observed. In one lot of ham (H1) and wiener sausage (WS1) a high level in HNE was observed. The changes of HNE contents of pork meats containing 0, 0.1, 0.5 and 1% Sugi wood vinegar (SWV) stored at 0?°C were also analyzed for 7 days. As an index of lipid peroxidation level, malonaldehyde (MA) contents were also analyzed in these samples. After 7 days of storage, HNE was detected only in pork meats containing 1% SWV and the level was similar to those of H1 and WS1. Judging from MA contents, SWV may act as a pro-oxidant in pork meats and HNE may accumulate in pork in which lipid peroxidation is in progress.     

Open dumping site in Asian developing countries: a potential source of polychlorinated dibenz-p-dioxins and polychlorinated dibenzofurans

Open landfill dumping areas for municipal wastes in Asian developing countries have recently received particular attention with regard to environmental pollution problems. Because of the uncontrolled burning of solid wastes, elevated contamination by various toxic chemicals including dioxins and related compounds in these dumping sites has been anticipated. In this study, concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and coplanar polychlorinated biphenyls (PCBs) were determined in soils from dumping sites in the Philippines, Cambodia, India, and Vietnam. Residue concentrations of PCDD/Fs and coplanar PCBs in dumping site soils were apparently greater than those in soils collected in agricultural or urban areas far from dumping sites, suggesting that dumping sites are potential sources of PCDD/Fs and related compounds. Observed PCDD/F concentrations in soils from dumping sites in the Philippines and Cambodia were comparable or higher than those reported for dioxin-contaminated locations in the world (e.g., near the municipal waste incinerators and open landfill dumping sites). Homologue profiles of PCDD/Fs in dumping site soils from the Philippines and, to a lesser extent, from Cambodia and India reflected patterns of samples representing typical emissions, while profiles of agricultural or urban soils were similar to those of typical environmental sinks. This result suggests recent formation of PCDD/Fs in dumping site areas and that open dumping sites are a potential source of dioxins in Asian developing countries. Uncontrolled combustions of solid wastes by waste pickers, generation of methane gas, and low-temperature burning can be major factors for the formation of dioxins in dumping sites. Elevated fluxes of PCDD/Fs to soils in dumping sites were encountered in the Philippines, Cambodia, India, and Vietnam-Hanoi, and these levels were higher than those reported for other countries. Considerable loading rates of PCDD/Fs in the dumping sites of these countries were observed, ranging from 20 to 3900 mg/yr (0.12-35 mg TEQ/yr). PCDD/F concentrations in some soil samples from the Philippines, Cambodia, India, and Vietnam-Hanoi exceeded environmental guideline values, suggesting potential health effects on humans and wildlife living near these dumping sites. The estimated intakes of dioxins via soil ingestion and dermal exposure for children were higher than those for adults, suggesting greater risk of dioxin exposure for children in dumping sites. To our knowledge, this is the first comprehensive study on PCDD/Fs contamination in open dumping sites of Asian developing countries. On the basis of the result of this study, we have addressed a new environmental issue that open dumping sites are potential sources of PCDD/Fs and related compounds, and dioxin contamination in dumping sites may become a key environmental problem in developing countries.     

Synergistic effects of detergents and organic solvents on protein refolding: Control of aggregation and folding rates

This paper presents the synergistic enhancement of the refolding yield of denatured and reduced lysozyme by using detergents as aggregation inhibitors and water-miscible organic cosolvents as modulators for the detergents. Adding only cetyltrimethylammonium bromide (CTAB) led to a slight increase in the refolding yield (up to 13%). Further addition of dimethylsulfoxide (DMSO) with CTAB drastically increased the refolding yield up to 35%, a value which was higher than the simple sum of the refolding yields in the presence of only CTAB or DMSO. The synergistic enhancement was also observed in the coexistence of other detergents, such as polyethylene glycol monooleyl ether (n = 50) and N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and cosolvents, such as N,N-dimethylformamide and N,N-dimethylacetamide. Experimental data and a kinetic analysis revealed the guideline for selecting a couple of additives; detergents which can adequately inhibit the aggregation of proteins by binding to hydrophobic surfaces of refolding intermediates should be employed as an aggregation inhibitor, and cosolvents which can properly prevent both protein–protein and protein–detergent interactions act as effective modulators for the aggregation inhibitor, resulting in a desirable balance between folding and aggregation rates.     

Large-scale genome reorganization in Saccharomyces cerevisiae through combinatorial loss of mini-chromosomes

A highly efficient technique, termed PCR-mediated chromosome splitting (PCS), was used to create cells containing a variety of genomic constitutions in a haploid strain of Saccharomyces cerevisiae. Using PCS, we constructed two haploid strains, ZN92 and SH6484, that carry multiple mini-chromosomes. In strain ZN92, chromosomes IV and XI were split into 16 derivative chromosomes, seven of which had no known essential genes. Strain SH6484 was constructed to have 14 mini-chromosomes carrying only non-essential genes by splitting chromosomes I, II, III, VIII, XI, XIII, XIV, XV, and XVI. Both strains were cultured under defined nutrient conditions and analyzed for combinatorial loss of mini-chromosomes. During culture, cells with various combinations of mini-chromosomes arose, indicating that genomic reorganization could be achieved by splitting chromosomes to generate mini-chromosomes followed by their combinatorial loss. We found that although non-essential mini-chromosomes were lost in various combinations in ZN92, one mini-chromosome (18kb) that harbored 12 genes was not lost. This finding suggests that the loss of some combination of these 12 non-essential genes might result in synthetic lethality. We also found examples of genome-wide amplifications induced by mini-chromosome loss. In SH6484, the mitochondrial genome, as well as the copy number of genomic regions not contained in the mini-chromosomes, was specifically amplified. We conclude that PCS allows for genomic reorganization, in terms of both combinations of mini-chromosomes and gene dosage, and we suggest that PCS could be useful for the efficient production of desired compounds by generating yeast strains with optimized genomic constitutions.     

Temperature dependence for anammox bacteria enriched from freshwater sediments

The anoxic ammonium oxidation (anammox) process has been regarded as an attractive alternative process to treat wastewater containing high ammonium concentrations. By the implementation of anammox process at moderately low temperatures (<25°C), the anammox process will be applied to more various industrial wastewater treatments. In this study, we established enrichment cultures of anammox bacteria from freshwater sediments by using an up-flow column reactor equipped with porous polyester nonwoven fabric at moderately low temperatures. Their nitrogen conversion rates reached 0.07-0.26kg-N/m(3)/d. Phylogenetic analysis based on 16S rRNA gene from enrichment cultures revealed the presence of various anammox bacteria affiliated with unknown anammox bacteria as well as known anammox candidates, i.e., Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia fulgida, Candidatus Scalindua wagneri. Anammox bacterial populations were influenced by enrichment conditions, i.e., seed sediments and temperature.     

Tiliroside, a glycosidic flavonoid, inhibits carbohydrate digestion and glucose absorption in the gastrointestinal tract

Scope Recent studies have reported that tiliroside, a glycosidic flavonoid, possesses anti‐diabetic activities. In the present study, we investigated the effects of tiliroside on carbohydrate digestion and absorption in the gastrointestinal tract. Methods and results This study showed that tiliroside inhibits pancreatic α‐amylase (IC₅₀ = 0.28 mM) in vitro. Tiliroside was found as a noncompetitive inhibitor of α‐amylase with K_i values of 84.2 μM. In male ICR mice, the increase in postprandial plasma glucose levels was significantly suppressed in the tiliroside‐administered group. Tiliroside treatment also suppressed hyperinsulinemia after starch administration. Tiliroside administration inhibited the increase of plasma glucose levels in an oral glucose tolerance test, but not in an intraperitoneal glucose tolerance test. In human intestinal Caco‐2 cells, the addition of tiliroside caused a significant dose‐dependent inhibition of glucose uptake. The inhibitory effects of both sodium‐dependent glucose transporter 1 (SGLT1) and glucose transporter 2 (GLUT2) inhibitors (phlorizin and phloretin, respectively) on glucose uptake were significantly inhibited in the presence of tiliroside, suggesting that tiliroside inhibited glucose uptake mediated by both SGLT1 and GLUT2. Conclusion These findings indicate that the anti‐diabetic effects of tiliroside are at least partially mediated through inhibitory effects on carbohydrate digestion and glucose uptake in the gastrointestinal tract.     

A method for the determination of acrylamide in a broad variety of processed foods by GC-MS using xanthydrol derivatization

A novel GC-MS method was developed for the determination of acrylamide, which is applicable to a variety of processed foods, including potato snacks, corn snacks, biscuits, instant noodles, coffee, soy sauces and miso (fermented soy bean paste). The method involves the derivatization of acrylamide with xanthydrol instead of a bromine compound. Isotopically labelled acrylamide (d?-acrylamide) was used as the internal standard. The aqueous extract from samples was purified using Sep-Pak? C?? and Sep-Pak? AC-2 columns. For amino acid-rich samples, such as miso or soy sauce, an Extrelut? column was used for purification or extraction. After reaction with xanthydrol, the resultant N-xanthyl acrylamide was determined by GC-MS. The method was validated for various food matrices and showed good linearity, precision and trueness. The limit of detection and limit of quantification ranged 0.5-5 and 5-20?μg?kg?1), respectively. The developed method was applied as an exploratory survey of acrylamide in Japanese foods and the method was shown to be applicable for all samples tested.     

Immunomodulatory effect of mushrooms on cytotoxic activity and cytokine production of intestinal lamina propria leukocytes does not necessarily depend on β-glucan contents

We evaluated the effects of seven mushroom extracts (Grifola frondosa, Pholiota nameko, Panellus serotinus, Hypsizygus marmoreus, Pleurotus cornucopiae, Armillaria mellea, and Flammulina velutipes) on cytotoxic activity and cytokine production of lamina propria leukocytes (LPLs) isolated from rat small (S) and large (L) intestinal mucosa. Boiling water extracts from seven species of mushrooms showed no direct cytotoxicity against the YAC-1 target cells. However, prominent increases of cytotoxicity were observed in S- and L-LPLs co-cultured with P. serotinus extract. Cytokine production (TNFα, IFNγ, IL-12 p70, and IL-4) of S- and L-LPLs was stimulated in response to P. cornucopiae extract. Mushroom extracts contributed to target cell adhesion and/or cytokine production in the effector cells. The promotion of cytotoxic activity in S- and L-LPLs was not necessarily related to β-glucan content of the mushroom.