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The bone inducing factor derived from BF osteosarcoma was purified in the following manner. Step 1. The sarcoma, grown in CBA mice, was excised and lyophilized. Step 2. The powder was washed with chilled acetone. Step 3. The acetone-treated powder was then homogenized with chilled distilled water. Step 4. Washing with 0.15M KCl. Step 5. The precipitate was incubated in in 0.2 N NH2OH, pH7.0, for 48 H at 25 degrees. After Step 5, the bone-forming activity showed a slight increase; however, the factor remained insoluble. The properties of the factor were as follows. The factor is relatively relatively heat stable; the osteogenic activity survived the treatment at 75 degrees for 15 min or at 55 degrees for 19 h. The activity was easily lost by mechanical shaking. Incubation with DNase, RNase, neuraminidase, chondroitinase ABC and beta-galactosidase left the osteogenic activity intact, but treatment with either pronase or collagnease destroyed this activity. The results suggest that the factor may be a protein. The activity was seen with the lyophilized BF osteosarcoma cells (without matrix), and it is probable that the factor was exclusively synthesized in the cells. The bone formation, observed across a millipore filter when living BF osteosarcoma enclosed in a millipore chamber was implanted in mice, suggests the synthesis and secretion of the factor from the cells.

Phlebia sp. MG-60 is a white-rot fungus that produces ethanol with high efficiency from lignocellulosic biomass without additional enzymes. Through engineering of this powerful metabolic pathway for fermentation in Phlebia sp. MG-60, chemical compounds other than ethanol could be produced. Here, we demonstrate sugar accumulation from unbleached hardwood kraft pulp and conversion of xylose to xylitol by pyruvate decarboxylase (pdc)-negative Phlebia sp. MG-60. We isolated Phlebia sp. strain MG-60-P2 from protoplasts to unify the protoplast phenotypes of the regenerated strains. Homologous recombination achieved a stable pdc-knockout line, designated KO77. The KO77 line produced traces of ethanol, but accumulated xylitol from xylose or glucose from unbleached hardwood kraft pulp. These metabolic changes in the pdc-knockout strain reflect the potential of metabolic engineering in Phlebia sp. MG-60 for direct production of chemical compounds from lignocellulosic biomass.

This work investigates the potential of metabolic stimulators, firstly to enhance the production of beta-carotene, and later use of inhibitors of lycopene cyclase so as to accumulate lycopene in the fermentation medium. Various non-ionic surfactants, natural oils, stimulators such as amino-acids, tricarboxylic acid cycle (TCA) intermediates, vitamin A and antibiotics were investigated for improved production of beta-carotene using the zygomycete fungus Blakeslea trispora. Span 20 at 0.2% increased the beta-carotene production from 139 mg/l to 318 mg/l. Examination of the mycelial morphology of the B. trispora with span 20 showed a shorter mycelial length, which allowed a well-dispersed growth of B. trispora. Supplementation of the medium with 1000 ppm vitamin A acetate gave highest concentration of beta-carotene (830+/-6 mg/l). Several chemical inhibitors such as imidazole, pyridine, triethylamine, piperidine, and nicotinic acid were then evaluated to block the biosynthesis at lycopene. Piperidine at 500 ppm gave a 7.76-fold improvement, and produced high titers of lycopene (775+/-5 mg/l) in a medium supplemented with vitamin A acetate.

The effect of the methanolic fraction of Pluchea indica Less. root extract was evaluated on various models of inflammation and ulcer in vivo to assess the role of P. indica on the 5-lipoxygenase pathway of prostaglandin synthesis. Studies presented here reveal significant antiinflammatory activity of the fraction on Arachidonic acid, Platelet activation factor and Compound 48/80-induced paw oedema. There was significant inhibition of spontaneous as well as compound 48/80-induced histamine release from mast cells. Ulcer studies revealed significant protective action of the fraction on indomethacin, alcohol and indomethacin-alcohol induced ulceration. There was significant decrease of gastric volume and acidity in pylorus ligated rats. Studies also showed, significant protective action of the same on gastric mucosa.

The chemical and biological properties of the water column at a Tyrrhenian site (Isola del Giglio) were studied during a 3-year period. The results highlighted the oligotrophic features of the site, characterised by quite low concentrations of organic carbon (on average DOC 102 micromol/L and POC 9 micromol/L). Relevant bacterial biomass (on average 42.1 microg C/L) and a notable activity (in terms of frequency of dividing cells, on average more than 5%) were observed. However, remarkable changes for these parameters were seasonally recorded. The cyclic occurrence, generally during the late spring-summer period, of benthic mucilage indicated that localised distrophic processes may occur. In particular, the benthic mucilage events of 2000 and 2001 were investigated, although some comparative information was available also for 1999 and 2002. The mucilage aggregates generally showed high bacterial colonisation, which have remarkable effects on the organic matter cycle both inside the aggregates and in the surrounding seawater. During the benthic mucilage development, an increase of DOC and POC concentrations was observed (up to 129 and 18 micromol/L, respectively, in June 2000 and up to 145 and 10 micromol/L, respectively, in May and June 2001) in the water column adjacent to the bottom. However, a general decrease of the trophic value of particulate matter (in terms of C/N ratio) was also observed, especially in 2000 after the disappearance of the mucilage. The available energy and organic matter during the mucilage events led to an increased presence of bacteria in the bottom waters of the Isola del Giglio, with maximum biomass values in 2001. Similarly, the replicative activity of bacteria was higher in 2001 (frequency of dividing cells about 5% vs. 3% of 2000). The lower activity of 2000, in addition to the lower trophic value of organic matter and different environmental conditions (namely lower temperature), might be involved in the persistence of mucilage in 2000 with respect to the rapid disappearance observed in 2001.

The relationship between copy numbers of internal transcribed spacer 1 (ITS1) and biomass or zoospore count of anaerobic fungi was studied to develop a quantitative real-time PCR-based monitoring method for fungal biomass or population in the rumen. Nine fungal strains were used to determine the relationship between ITS1 copy number and fungal biomass. Rumen fluid from three sheep and a cow were used to determine the relationship between ITS1 copy number and fungal population. ITS1 copy number was determined by real-time PCR with a specific primer set for anaerobic fungi. Freeze-dried fungal cells were weighed for fungal biomass. Zoospore counts were determined by the roll-tube method. A positive correlation was observed between both ITS1 copy number and dry weight and ITS1 copy number and zoospore counts, suggesting that the use of ITS1 copy numbers is effective for estimating fungal biomass and population density. On the basis of ITS1 copy numbers, fluctuations in the fungal population in sheep rumen showed that although the values varied among individual animals, the fungal population tended to decrease after feeding. In the present study, a culture-independent method was established that will provide a powerful tool for understanding the ecology of anaerobic fungi in the rumen.

Reproduction in fishes is sensitive to temperature. Elevated temperatures and anomalous ‘heat waves’ associated with climate change have the potential to impact fish reproductive performance and, in some cases, even induce sex reversals. Here we examine how thermal sensitivity in the hormone pathways regulating reproduction provides a framework for understanding impacts of warmer conditions on fish reproduction. Such effects will differ depending on evolved variation in temperature sensitivity of endocrine pathways regulating reproductive processes of sex determination/differentiation, gametogenesis and spawning, as well as how developmental timing of those processes varies with reproductive ecology. For fish populations unable to shift geographical range, persistence under future climates may require changes in temperature responsiveness of the hormone pathways regulating reproductive processes. How thermal sensitivity in those hormone pathways varies among populations and species, how those pathways generate temperature maxima for reproduction, and how rapidly reproductive thermal tolerances can change via adaptation or transgenerational plasticity will shape which fishes are most at risk for impaired reproduction under rising temperatures. This article is part of the theme issue ‘Endocrine responses to environmental variation: conceptual approaches and recent developments’.

In this study, we investigated the antiinflammatory effects of ethanol extracts of Potentilla. supina Linne (EPS) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and septic mice. EPS suppressed LPS-induced nitric oxide, prostaglandin E2 , TNF-α, interleukin-6 and interleukin-1β at production and mRNA levels in LPS-induced RAW 264.7 macrophages. Consistent with these observations, EPS attenuated the expressions of inducible nitric oxide synthase and cyclooxygenase-2 by downregulation of their promoter activities. Molecularly, EPS reduced the LPS-induced transcriptional activity and DNA-binding activity of nuclear factor-κB (NF-κB), and this was associated with a decrease of translocation and phosphorylation of p65 NF-κB by inhibiting the inhibitory κB-α degradation and IKK-α/β phosphorylation. Furthermore, EPS inhibited the LPS-induced activation of activator protein-1 by reducing the expression of c-Fos and c-Jun in nuclear. EPS also suppressed the phosphorylation of mitogen-activated protein kinase, such as p38 mitogen-activated protein kinase and c-Jun N-terminal kinase. In an LPS-induced endotoxemia mouse model, pretreatment with EPS reduced the mRNA levels of inducible nitric oxide synthase, cyclooxygenase-2 and proinflammatory cytokines and increased the survival rate of mice. Collectively, these results suggest that the antiinflammatory effects of EPS were associated with the suppression of NF-κB and activator protein-1 activation and support its possible therapeutic role for the treatment of endotoxemia. Copyright © 2017 John Wiley & Sons, Ltd.

The ability of cells to adapt low-dose or low-dose rate radiation is well known. High-LET radiation has unique characteristics, and the data concerning low doses effects and high-LET radiation remain fragmented. In this study, we assessed in vitro the ability of low doses of X-rays to induce an adaptive response (AR) to a subsequent challenging dose of heavy-ion radiation. Lymphoblastoid cells (TK6, AHH-1, NH32) were exposed to priming 0.02-0.1Gy X-rays, followed 6h later by challenging 1Gy heavy-ion radiation (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm). Pre-exposure of p53-competent cells resulted in decreased mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and different H2AX phosphorylation kinetics, as compared to cells exposed to challenging radiation alone. This phenomenon did not seem to be linked with cell cycle effects or radiation-induced apoptosis. Taken together, our results suggested the existence of an AR to mutagenic effects of heavy-ion radiation in lymphoblastoid cells and the involvement of double-strand break repair mechanisms.