Neurite Outgrowth-Promoting Activity of Compounds in PC12 Cells from Sunflower Seeds

In the current super-aging society, the establishment of methods for prevention and treatment of Alzheimer’s disease (AD) is an urgent task. One of the causes of AD is thought to be a decrease in the revel of nerve growth factor (NGF) in the brain. Compounds showing NGF-mimicking activity and NGF-enhancing activity have been examined as possible agents for improving symptoms. In the present study, sunflower seed extract was found to have neurite outgrowth-promoting activity, which is an NGF-enhancing activity, in PC12 cells. To investigate neurite outgrowth-promoting compounds from sunflower seed extract, bioassay-guided purification was carried out. The purified active fraction was obtained by liquid-liquid partition followed by some column chromatographies. Proton nuclear magnetic resonance and gas chromatography-mass spectrometry analyses of the purified active fraction indicated that the fraction was a mixture of β-sitosterol, stigmasterol and campesterol, with β-sitosterol being the main component. Neurite outgrowth-promoting activities of β-sitosterol, stigmasterol, campesterol and cholesterol were evaluated in PC12 cells. β-Sitosterol and stigmasterol showed the strongest activity of the four sterol compounds (β-sitosterol ≈ stigmasterol > campesterol > cholesterol), and cholesterol did not show any activity. The results indicated that β-sitosterol was the major component responsible for the neurite outgrowth-promoting activity of sunflower seeds. Results of immunostaining also showed that promotion by β-sitosterol of neurite formation induced by NGF was accompanied by neurofilament expression. β-Sitosterol, which showed NGF-enhancing activity, might be a candidate ingredient in food for prevention of AD

Simultaneous Imaging and Characterization of Polyunsaturated Fatty Acids, Carotenoids, and Microcrystalline Guanine in Single Aurantiochytrium limacinum Cells with Linear and Nonlinear Raman Microspectroscopy

Thraustochytrids are heterotrophic marine protists known for their high production capacity of various compounds with health benefits, such as polyunsaturated fatty acids and carotenoids. Although much effort has been focused on developing optimal cultivation methods for efficient microbial production, these high-value compounds and their interrelationships are not well understood at the single-cell level. Here we used spontaneous (linear) Raman and multiplex coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy to visualize and characterize lipids (e.g., docosahexaenoic acid) and carotenoids (e.g., astaxanthin) accumulated in single living Aurantiochytrium limacinum cells. Spontaneous Raman imaging with the help of multivariate curve resolution−alternating least-squares enabled us to make unambiguous assignments of the molecular components we detected and derive their intracellular distributions separately. Near-IR excited CARS imaging yielded the Raman images at least an order of magnitude faster than spontaneous Raman imaging, with suppressed contributions of carotenoids. As the culture time increased from 2 to 5 days, the lipid amount increased by a factor of ∼7, whereas the carotenoid amount did not change significantly. Furthermore, we observed a highly localized component in A. limacinum cells. This component was found to be mixed crystals of guanine and other purine derivatives. The present study demonstrates the potential of the linear−nonlinear Raman hybrid approach that allows for accurate molecular identification and fast imaging in a label-free manner to link information derived from single cells with strategies for mass culture of useful thraustochytrids

A calmodulin inhibitor, W-7 influences the effect of cyclic adenosine 3', 5'-monophosphate signaling on ligninolytic enzyme gene expression in Phanerochaete chrysosporium

The capacity of white-rot fungi to degrade wood lignin may be highly applicable to the development of novel bioreactor systems, but the mechanisms underlying this function are not yet fully understood. Lignin peroxidase (LiP) and manganese peroxidase (MnP), which are thought to be very important for the ligninolytic property, demonstrated increased activity in Phanerochaete chrysosporium RP-78 (FGSC #9002, ATCC MYA-4764™) cultures following exposure to 5 mM cyclic adenosine 3', 5'-monophosphate (cAMP) and 500 μM 3'-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that transcription of most LiP and MnP isozyme genes was statistically significantly upregulated in the presence of the cAMP and IBMX compared to the untreated condition. However, 100 μM calmodulin (CaM) inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which had insignificant effects on fungal growth and intracellular cAMP concentration, not only offset the increased activity and transcription induced by the drugs, but also decreased them to below basal levels. Like the isozyme genes, transcription of the CaM gene (cam) was also upregulated by cAMP and IBMX. These results suggest that cAMP signaling functions to increase the transcription of LiP and MnP through the induction of cam transcription

Mechanistic Insights into Indigo Reduction in Indigo Fermentation : A Voltammetric Study

Indigo is one of the oldest natural blue dyes. Microorganisms and their enzymatic activities are deeply involved in the traditional indigo staining procedure. To elucidate the mechanism of the microbial indigo reduction, we directly performed cyclic voltammetry on alkaline fermenting dye suspensions. A pair of characteristic redox peaks of leuco-indigo was observed in a supernatant fluid of the fermenting dye suspension. On the other hand, it was found that the indigo/leuco-indigo redox couple mediated two-way microbially catalyzed oxidation and reduction in a sediment-rich suspension of the fermenting suspension. Acetaldehyde was supposed to be the electron donor and acceptor of the catalytic reactions. In order to verify the bioelectrocatalytic reaction, we isolated indigo-reducing bacterium K2-3′ from the fermenting suspension, and the two-way bioelectrocatalysis was successfully restaged in a model system containing K2-3′ and methyl viologen (as a soluble mediator instead of indigo) as well as acetaldehyde at pH 10

Metabolic engineering of oleaginous fungus Mortierella alpina for high production of oleic and linoleic acids

The aim of this work was to study the molecular breeding of oleaginous filamentous Mortierella alpina for high production of linoleic (LA) or oleic acid (OA). Heterologous expression of the Δ12-desaturase (DS) gene derived from Coprinopsis cinerea in the Δ6DS activity-defective mutant of M. alpina increased the LA production rate as to total fatty acid to 5 times that in the wild strain. By suppressing the endogenous Δ6I gene expression by RNAi in the Δ12DS activity-defective mutant of M. alpina, the OA accumulation rate as to total fatty acid reached 68.0%. The production of LA and OA in these transformants reached 1.44 and 2.76 g/L, respectively, on the 5th day. The Δ6I transcriptional levels of the RNAi-treated strains were suppressed to 1/10th that in the parent strain. The amount of Δ6II RNA in the Δ6I RNAi-treated strain increased to 8 times that in the wild strain

Ethanol Enhances Astaxanthin Production by Aurantiochytrium sp.

Two-percent ethanol increased the astaxanthin productivity of heterotrophic microalgae Aurantiochytrium sp. O5-1-1 to 2.231 mg/L, 45-fold higher than under ethanol-free condition. Ethanol in the medium decreased at the same rate as spontaneous volatilization, suggesting that it was not a transient signaling factor but a continuous stress on the cells. The triply mutated strain OM3-3 produced 5.075 mg/L astaxanthin under 2% ethanol conditions. Furthermore, the astaxanthin accumulation of the mutant OM3-9 was 0.895 mg/g, which was 150-fold higher than that of strain O5-1-1 in ethanol-free condition. These results are beneficial for the commercial exploitation of carotenoids producing Aurantiochytrium spp

Neurite Outgrowth-Promoting Activity of Compounds in PC12 Cells from Sunflower Seeds

In the current super-aging society, the establishment of methods for prevention and treatment of Alzheimer’s disease (AD) is an urgent task. One of the causes of AD is thought to be a decrease in the revel of nerve growth factor (NGF) in the brain. Compounds showing NGF-mimicking activity and NGF-enhancing activity have been examined as possible agents for improving symptoms. In the present study, sunflower seed extract was found to have neurite outgrowth-promoting activity, which is an NGF-enhancing activity, in PC12 cells. To investigate neurite outgrowth-promoting compounds from sunflower seed extract, bioassay-guided purification was carried out. The purified active fraction was obtained by liquid-liquid partition followed by some column chromatographies. Proton nuclear magnetic resonance and gas chromatography-mass spectrometry analyses of the purified active fraction indicated that the fraction was a mixture of β-sitosterol, stigmasterol and campesterol, with β-sitosterol being the main component. Neurite outgrowth-promoting activities of β-sitosterol, stigmasterol, campesterol and cholesterol were evaluated in PC12 cells. β-Sitosterol and stigmasterol showed the strongest activity of the four sterol compounds (β-sitosterol ≈ stigmasterol > campesterol > cholesterol), and cholesterol did not show any activity. The results indicated that β-sitosterol was the major component responsible for the neurite outgrowth-promoting activity of sunflower seeds. Results of immunostaining also showed that promotion by β-sitosterol of neurite formation induced by NGF was accompanied by neurofilament expression. β-Sitosterol, which showed NGF-enhancing activity, might be a candidate ingredient in food for prevention of AD

Efficient production of biolipids by crude glycerol-assimilating fungi

The aim of this study was to isolate microorganisms utilizing crude glycerol as a carbon source efficiently and to evaluate their lipid productivity. Fusarium oxysporum W1 grew well on medium containing 20% crude glycerol as well as 50% pure glycerol. The dry cell weights and total fatty acids of F. oxysporum W1 reached 24.5 g/L and 12.4 g/L. Penicillium sp. N1 and P. citrinum N3 were found to accumulate free fatty acids to as much as 56.2 % and 48.5 % of total fatty acids, respectively, on cultivation in the crude glycerol-containing medium. These strains grew well on medium containing crude glycerol only heat-treated at 80-105°C without autoclave sterilization

Gene targeting in the oil-producing fungus Mortierella alpina 1S-4 and construction of a strain producing a valuable polyunsaturated fatty acid.

First online: 18 March 2015To develop an efficient gene-targeting system in Mortierella alpina 1S-4, we identified the ku80 gene encoding the Ku80 protein, which is involved in the nonhomologous end-joining pathway in genomic double-strand break (DSB) repair, and constructed ku80 gene-disrupted strains via single-crossover homologous recombination. The Δku80 strain from M. alpina 1S-4 showed no negative effects on vegetative growth, formation of spores, and fatty acid productivity, and exhibited high sensitivity to methyl methanesulfonate, which causes DSBs. Dihomo-γ-linolenic acid (DGLA)-producing strains were constructed by disruption of the Δ5-desaturase gene, encoding a key enzyme of bioconversion of DGLA to ARA, using the Δku80 strain as a host strain. The significant improvement of gene-targeting efficiency was not observed by disruption of the ku80 gene, but the construction of DGLA-producing strain by disruption of the Δ5-desaturase gene was succeeded using the Δku80 strain as a host strain. This report describes the first study on the identification and disruption of the ku80 gene in zygomycetes and construction of a DGLA-producing transformant using a gene-targeting system in M. alpina 1S-4