Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis

The optimization of submerged culture conditions for mycelial growth and exopolysaccharide (EPS) production in an edible mushroom Tremella fuciformis was studied in shake flasks and bioreactors. The temperature of 28 degrees C and pH 8 in the beginning of fermentation in agitated flasks was the most efficient condition to obtain maximum mycelial biomass and EPS. The optimal medium constituents were as follows (gL(-1)): glucose 20, tryptone 2, KH(2)PO(4) 0.46, K(2)HPO(4) 1 and MgSO(4).7H(2)O 0.5. The fungus was cultivated under various agitation and aeration conditions in a 5L stirred-tank bioreactor. The maximum cell mass and EPS production were obtained at a relatively high agitation speed of 200 rpm and at an aeration rate of 2 vvm. The flow behavior of the fermentation broth was Newtonian and the maximum apparent viscosity (35 cP) was observed at a highly aerated condition (2 vvm). The EPS productivity in an airlift reactor was higher than that in the stirred-tank reactor. The morphological study revealed that the fungus grows in mainly three different yeast-like forms: ovoid, elongated, and double yeast forms. The high population of the elongated yeast has a very close relationship to high EPS production. The EPS were protein-bound polysaccharides consisted of mainly mannose, xylose, and fucose. The molecular weights of EPS were determined to be (1.3-1.5)x10(6).     

Potent inhibitors of lipoprotein-associated phospholipase A(2): benzaldehyde O-heterocycle-4-carbonyloxime

A series of multi-substituted oximes were prepared and their potencies for inhibiting lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) activity were evaluated in vitro. Among them, compounds 3a, 3b, and 3m were identified to display a micromolar potency for inhibiting Lp-PLA(2) in whole human plasma and isolated human LDL. Based on these results, structure-activity relationship was studied on modification of three parts of R(1), R(2), and R(3) to identify a potent pharmacophore for Lp-PLA(2). In an attempt to introduce various functional groups at R(2) and R(3), we discovered that replacement of less lipophilic groups led to an increase of inhibitory activity. Among the tested oxime derivatives, cyano- and morpholino-substituted analogue 4f at R(2) and R(3) had the highest potency with an IC(50) value of 0.05 microM in whole human plasma.     

Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.     

Pyrogallol inhibits the growth of human pulmonary adenocarcinoma A549 cells by arresting cell cycle and triggering apoptosis

Pyrogallol (PG) is a polyphenol compound and has been known to be an O generator. We evaluated the effects of PG on the growth of human pulmonary A549 cells in relation to the cell cycle and apoptosis. Treatment with 50 or 100 μM PG significantly inhibited the cell growth of A549 for 72 h. DNA flow cytometric analysis indicated that PG slightly induced a G1 phase arrest of the cell cycle at 24 or 48 h, but did not induce the specific cell cycle arrest at 72 h. Intracellular GSH depletion was observed in PG‐treated cells. PG induced apoptosis in A549 cells, as evidenced by sub‐G1 cells, annexin V staining cells, and the loss of mitochondrial membrane potential (Δ Ψ_m). The intracellular ROS (reactive oxygen species) level including O increased in PG‐treated A549 cells at 24 and 48 h, and persisted at 72 h. The changes in GSH as well as ROS levels by PG affected the cell viability in A549 cells. In conclusion, PG inhibited the growth of human pulmonary A549 cells by inducing cell cycle arrest as well as triggering apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:36–42, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20263     

Reduction of Escherichia coli O157:H7 shedding in cattle by addition of chitosan microparticles to feed

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a significant human pathogen that resides in healthy cattle. It is thought that a reduction in the prevalence and numbers of EHEC in cattle will reduce the load of EHEC entering the food chain. To this end, an intervention strategy involving the addition of chitosan microparticles (CM) to feed in order to reduce the carriage of this pathogen in cattle was evaluated. Experiments with individual Holstein calves and a crossover study found that the addition of CM to feed decreased E. coli O157:H7 shedding. In the crossover study, CM resulted in statistically significant reductions in the numbers recovered from rectal swab samples (P < 0.05) and the duration of shedding (P < 0.05). The effects of feeding CM to calves differed, indicating that the optimal levels of CM may differ between animals or that other factors are involved in the interaction between CM and E. coli O157:H7. In vitro studies demonstrated that E. coli O157:H7 binds to CM, suggesting that the reduction in shedding may result at least in part from the binding of positively charged CM to negatively charged E. coli cells. Additional studies are needed to determine the impact of CM feeding on animal production, but the results from this study indicate that supplementing feed with CM reduces the shedding of E. coli O157:H7 in cattle.     

Insecticide susceptibility of Ephemera orientalis (Ephemeroptera: Ephemeridae) and two mosquito species,Anopheles sinensis and Culex pipiens in the Republic of Korea

Field-collected populations of mayflies, Ephemera orientalis were tested for susceptibility to 10 different insecticides using a direct-contact mortality bioassay. Ephemera orientalis subimagoes were susceptible to the insecticides chlorpyrifos, fenitrothion and chlorfenapyr with LD₅₀ values of 69.7, 78.8 and 81.9 μg/♀, and adults had LD₅₀ values of 71.9, 78.8 and 85.4 μg/♀, respectively. Susceptibility ratios (SRs) of subimagoes and adults of E. orientalis to the 10 insecticides were 1.0 to1.2 folds. The mayflies showed higher susceptibility to organophosphates than to pyrethroids. The SRs of Anopheles sinensis to E. orientalis were 514 to 1438 folds higher for organophosphates (LD₅₀ values of 0.05 to 0.23 μg/♀) and 62 to 1155 folds higher for pyrethroids (LD₅₀ values of 0.13 to 2.41 μg/♀). The SRs of Culex pipiens to E. orientalis were 606 to 3595 folds higher for organophosphates with LD₅₀ values of 0.02–0.17 μg/♀ and 81 to 1365 folds higher for pyrethroids with LD₅₀ values of 0.11–1.83 μg/♀. These results indicate that the use of ineffective insecticides will result in unsatisfactory control against field populations of the subimagoes and adults of E. orientalis.     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.     

Infection Status of Hospitalized Diarrheal Patients with Gastrointestinal Protozoa, Bacteria, and Viruses in the Republic of Korea

To understand protozoan, viral, and bacterial infections in diarrheal patients, we analyzed positivity and mixed-infection status with 3 protozoans, 4 viruses, and 10 bacteria in hospitalized diarrheal patients during 2004-2006 in the Republic of Korea. A total of 76,652 stool samples were collected from 96 hospitals across the nation. The positivity for protozoa, viruses, and bacteria was 129, 1,759, and 1,797 per 10,000 persons, respectively. Especially, Cryptosporidium parvum was highly mixed-infected with rotavirus among pediatric diarrheal patients (29.5 per 100 C. parvum positive cases), and Entamoeba histolytica was mixed-infected with Clostridium perfringens (10.3 per 100 E. histolytica positive cases) in protozoan-diarrheal patients. Those infected with rotavirus and C. perfringens constituted relatively high proportions among mixed infection cases from January to April. The positivity for rotavirus among viral infection for those aged ≤ 5 years was significantly higher, while C. perfringens among bacterial infection was higher for ≥ 50 years. The information for association of viral and bacterial infections with enteropathogenic protozoa in diarrheal patients may contribute to improvement of care for diarrhea as well as development of control strategies for diarrheal diseases in Korea.