Antioxidant compounds produced by endolichenic fungus Penicillium sp.-strain 1322P isolated from Pyxine subcinerea.

Endolichenic fungi are expecting for new bioresources of pharmacological compounds. However, the number of investigations targeting antioxidant compounds produced by endolichenic fungi remains limited. To discover new antioxidant compounds, we analyzed the antioxidant activity of the methanol extracts derived from isolated lichen mycobionts or endolichenic fungi induced from Pyxine subcinerea. We performed this analysis using the oxygen radical absorbance capacity (ORAC) method. As a result, we isolated from an endolichenic fungus identified as Penicillium sp.-stain 1322P in Pyxine subcinerea. This fungus produced a red pigment, and its chemical structure was determined to be sclerotioramine based on the analytical data obtained from NMR, LC-MS/MS, and HPLC-PDA. Sclerotioramine exhibited high antioxidant activity, and the ORAC values (mean ± SD) of sclerotioramine and sclerotiorin were 11.4 ± 0.36 and 4.86 ± 0.70 mmol TE per gram of the respective pure compound. Thus, the antioxidant activity of sclerotioramine was greater than twice that of sclerotiorin. This work represents the first report that the antioxidant activity of sclerotioramine is higher than that of the sclerotiorin.

Uphill energy transfer mechanism for photosynthesis in an Antarctic alga.

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Šresolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC's unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.

Time Course of Axon and Myelin Degeneration in Peripheral Nerves in Experimental Autoimmune Neuritis Rats.

Experimental autoimmune neuritis (EAN) is an animal model for Guillain-Barré syndrome (GBS), which results in neurological symptoms and histopathological changes in peripheral nerves. In this model, the correlation between the progression of the disease and the histopathological changes is not clear. To further examine histopathological changes in peripheral nerves in EAN rats, sciatic nerves were sampled at onset (day 10), peak (day 16), and recovery (days 22 and 25) of neurological symptoms in P2(57-81)-peptide-administered rats. Axon and myelin degeneration was observed by light microscopy at onset, degeneration became severe at peak, and persisted at recovery. Densities of myelinated nerve fibers and myelin areas decreased from day 10 to a minimum on day 22. Slight axon and myelin degeneration, such as accumulation of vesicles in axons and focal myelin splitting and folding, was observed by transmission electron microscopy at onset; severe degeneration, such as axonal loss, myelin ovoid, and demyelination, increased at peak; and regenerative changes, such as remyelination and enlargement of Schwann cell cytoplasm, occurred at recovery. These results suggest that EAN rats have histopathological similarities to some types of GBS patients and that EAN rats are a useful model to understand the pathogenesis of GBS.

The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota.

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast' polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast' polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes.

Norlichexanthone produced by cultured endolichenic fungus induced from Pertusaria laeviganda and its antioxidant activity.

Endolichenic fungi, nonobligate microfungi that live in lichen, are promising as new bioresources of pharmacological compounds. We found that norlichexanthone isolated from the endolichenic fungus in Pertusaria laeviganda exhibited high antioxidant activity. Norlichexanthone produced by endolichenic fungus had the antioxidant activity with same level of ascorbic acid. This is the first report of high antioxidant activity of norlichexanthone. Abbreviations: AAPH: 2,2'-azobis (2-methylpropionamidine) dihydrochloride; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FL: fluorescein sodium salt; HPLC-PDA: high-performance liquid chromatography with photodiode array; LC-ESI-MS: liquid chromatography with electrospray ionization mass spectrometry; ORAC: oxygen radical absorbance capacity; PB: phosphate buffer; ROS: reactive oxygen species; TLC: thin-layer chromatography.

A Chemical Genetics Strategy that Identifies Small Molecules which Induce the Triple Response in Arabidopsis.

To explore small molecules with ethylene-like biological activity, we conducted a triple response-based assay system for chemical library screening. Among 9600 compounds, we found N-[(1,3,5-trimethyl-1H-pyrazol-4-yl)methyl]-N-methyl-2-naphthalenesulfonamide (EH-1) displayed promising biological activity on inducing a triple response in Arabidopsis seedlings. Chemical synthesis and structure-activity relationship (SAR) analysis of EH-1 analogues with different substitution patterns on the phenyl ring structure of the sulfonamide group indicated that 3,4-dichloro-N-methyl-N-(1,3,5-trimethyl-1H-pyrazol-4-yl-methyl) benzenesulfonamide (8) exhibits the most potent biological activity. To determine the mechanism of action, we conducted RNA sequencing (RNA-Seq) analysis of the effect of EH-1 and 1-aminocyclopropane-1-carboxylate (ACC), the precursor of ethylene biosynthesis, following the quantitative real-time polymerase chain reaction (RT-PCR) confirmation. Data obtained from RNA-Seq analysis indicated that EH-1 and ACC significantly induced the expression of 39 and 48 genes, respectively (above 20 fold of control), among which five genes are up-regulated by EH-1 as well as by ACC. We also found 67 and 32 genes that are significantly down-regulated, respectively, among which seven genes are in common. For quantitative RT-PCR analysis. 12 up-regulated genes were selected from the data obtained from RNA-Seq analysis. We found a good correlation of quantitative RT-PCR analysis and RNA-Seq analysis. Based on these results, we conclude that the action mechanism of EH-1 on inducing triple response in Arabidopsis is different from that of ACC.

Memory formation and retrieval of neuronal silencing in the auditory cortex.

Sensory stimuli not only activate specific populations of cortical neurons but can also silence other populations. However, it remains unclear whether neuronal silencing per se leads to memory formation and behavioral expression. Here we show that mice can report optogenetic inactivation of auditory neuron ensembles by exhibiting fear responses or seeking a reward. Mice receiving pairings of footshock and silencing of a neuronal ensemble exhibited a fear response selectively to the subsequent silencing of the same ensemble. The valence of the neuronal silencing was preserved for at least 30 d and was susceptible to extinction training. When we silenced an ensemble in one side of auditory cortex for conditioning, silencing of an ensemble in another side induced no fear response. We also found that mice can find a reward based on the presence or absence of the silencing. Neuronal silencing was stored as working memory. Taken together, we propose that neuronal silencing without explicit activation in the cerebral cortex is enough to elicit a cognitive behavior.

Effects of Cu on the content of chlorophylls and secondary metabolites in the Cu-hyperaccumulator lichen Stereocaulon japonicum.

Understanding the relationship between Cu and Cu-hyperaccumulator lichens is important for their application in monitoring and assessing heavy metal pollution. We investigated the Cu-hyperaccumulator lichen Stereocaulon japonicum at several Cu-polluted and control sites in Japan, and found the lichen to be widely distributed. Its concentrations of Cu, chlorophylls, and secondary metabolites, chlorophyll-related indices, and absorption spectra were measured, and we observed negative effects of Cu on these concentrations and indices. For highly Cu-polluted samples (>100ppm dry weight), however, we found significant linear correlations between Cu and chlorophyll concentrations. This can be considered as the response of the photobiont in S. japonicum to Cu stress. In highly Cu-polluted samples the chlorophyll-related indices and concentration of total secondary metabolites were almost constant regardless of Cu concentration. This suggests that the increase in chlorophyll concentration with the increase in Cu concentration enhances photosynthetic productivity per unit biomass, which will allow the production of extra structure and energy for maintaining the chlorophyll-related indices under Cu stress. The relationship between the increase in chlorophyll concentration of S. japonicum and the decrease in secondary metabolite concentration of the lichen can be explained by considering the balance of carbohydrates in the lichen. We found that a spectral index A372-A394 can be a useful index of the concentrations of Cu and total secondary metabolites in S. japonicum. These findings show the adjustment of the content of chlorophylls and secondary metabolites in S. japonicum to Cu stress, and provide a better understanding of the relationship between Cu and the Cu-hyperaccumulator lichen.

Improvement of TiO2/dye/electrolyte interface conditions by positional change of alkyl chains in modified panchromatic Ru complex dyes.

A series of panchromatic ruthenium sensitizers (MJ sensitizers) with attached thiophene and phenyl units bearing alkyl chains was synthesized. A new synthetic route was used to examine all possible positions for the alkyl chains. The absorption spectra showed the sum of a ruthenium complex and peripheral organic chromophore units. The hypochromic effect and blueshift of the metal-to-ligand charge-transfer band observed in the modified ruthenium sensitizers were suppressed by changing the positions of the alkyl chains on the attached thiophene ring. Changing only one alkyl chain also influenced the performance of dye-sensitized solar cells. Ruthenium sensitizer MJ-10 with bulky substituent harvests visible and near-infrared light, and solar cells sensitized by MJ-10 exhibit an efficiency of 9.1% under 1 sun irradiation.

Thermostable direct hemolysin of Vibrio parahaemolyticus is a bacterial reversible amyloid toxin.

Thermostable direct hemolysin (TDH), a major virulence factor of Vibrio parahaemolyticus, is detoxified by heating at approximately 60-70 degrees C but is reactivated by additional heating above 80 degrees C. This paradoxical phenomenon, known as the Arrhenius effect, has remained unexplained for approximately 100 years. We now demonstrate that the effect is related to structural changes in the protein that produce fibrils. The native TDH (TDHn) is transformed into nontoxic fibrils rich in beta-strands by incubation at 60 degrees C (TDHi). The TDHi fibrils are dissociated into unfolded states by further heating above 80 degrees C (TDHu). Rapid cooling of TDHu results in refolding of the protein into toxic TDHn, whereas the protein is trapped in the TDHi structure by slow cooling of TDHu. Transmission electron microscopy indicates the fibrillar structures of TDHi. The fibrils show both the property of the nucleation-dependent elongation and the increase in its thioflavin T fluorescence. Formation of beta-rich structures of TDH was also observed in the presence of lipid vesicles containing ganglioside G(T1b), a putative TDH receptor. Congo red was found to inhibit the hemolytic activity of TDH in a dose-dependent manner. These data reveal that the mechanism of the Arrhenius effect which is tightly related to the fibrillogenicity of TDH.

Osmotic stress tolerance of transgenic tobacco expressing a gene encoding a membrane-located receptor-like protein from tobacco plants.

Tobacco (Nicotiana tabacum) genes regulated during the early stage of responses to wounding were screened by a modified fluorescence differential display method. Among 28 genes initially identified, a particular clone designated NtC7 was subjected to further analysis. Its transcripts were found to accumulate rapidly and transiently within 1 h upon treatments with not only wounding but also salt and osmotic stresses. However, jasmonic and abscisic acids and ethylene did not effectively induce NtC7 transcripts. Amino acid sequence analysis suggested NtC7 to be a new type of transmembrane protein that belongs to the receptor-like protein family, and a membrane location was confirmed in onion (Allium cepa) epidermis cells transiently expressing an NtC7-green fluorescent protein fusion protein. Seeds of transgenic tobacco overexpressing NtC7 normally germinated and grew in the presence of 500 mM mannitol, but not in the presence of 220 mM sodium chloride or 60 mM lithium chloride. Cuttings of mature transgenic leaf exhibited a marked tolerance upon treatment with 500 mM mannitol for 12 h, at which concentration wild-type counterparts were seriously damaged. These results suggested that NtC7 predominantly functions in maintenance of osmotic adjustment independently of ion homeostasis.