Complete Genome Sequence of Pseudomonas sp. Strain KUIN-1, a Model Strain for Studies on the Production of Cell-Free Ice Nucleation Proteins.

Pseudomonas sp. (formerly Pseudomonas fluorescens) strain KUIN-1 is an ice-nucleating bacterium that was isolated from the leaves of field beans (Phaseolus vulgaris L.). This microorganism can release cell-free ice nucleation proteins and shows cold shock-induced freezing tolerance. Here, we report the 6,028,589-bp complete genome sequence of Pseudomonas sp. KUIN-1.

Monitoring of glutamate-induced excitotoxicity by mitochondrial oxygen consumption.

Dysfunction of mitochondrial activity is often associated with the onset and progress of neurodegenerative diseases. Membrane depolarization induced by Na+ influx increases intracellular Ca2+ levels in neurons, which upregulates mitochondrial activity. However, overlimit of Na+ influx and its prolonged retention ultimately cause excitotoxicity leading to neuronal cell death. To return the membrane potential to the normal level, Na+ /K+ -ATPase exchanges intracellular Na+ with extracellular K+ by consuming a large amount of ATP. This is a reason why mitochondria are important for maintaining neurons. In addition, astrocytes are thought to be important for supporting neighboring neurons by acting as energy providers and eliminators of excessive neurotransmitters. In this study, we examined the meaning of changes in the mitochondrial oxygen consumption rate (OCR) in primary mouse neuronal populations. By varying the medium constituents and using channel modulators, we found that pyruvate rather than lactate supported OCR levels and conferred on neurons resistance to glutamate-mediated excitotoxicity. Under a pyruvate-restricted condition, our OCR monitoring could detect excitotoxicity induced by glutamate at only 10 µM. The OCR monitoring also revealed the contribution of the N-methyl-D-aspartate receptor and Na+ /K+ -ATPase to the toxicity, which allowed evaluating spontaneous excitation. In addition, the OCR monitoring showed that astrocytes preferentially used glutamate, not glutamine, for a substrate of the tricarboxylic acid cycle. This mechanism may be coupled with astrocyte-dependent protection of neurons from glutamate-mediated excitotoxicity. These results suggest that OCR monitoring would provide a new powerful tool to analyze the mechanisms underlying neurotoxicity and protection against it.

Design and synthesis of galactose-conjugated fluorinated and non-fluorinated proline oligomers: towards antifreeze molecules.

Galactose-conjugated fluorinated and non-fluorinated proline oligomers that exhibit an α-helical structure with hydrophilic and lipophilic parts were designed as potential antifreeze molecules. These galactose-proline oligomers were synthesized and their physical properties were evaluated. Interestingly, the non-fluorinated galactose-proline oligomers showed in contrast to the fluorinated analogues weak antifreeze activity. The difference in antifreeze activity should be attributed to the fluorine gauche effect, which should induce a conformation in fluorinated prolines that is different from that of natural proline. The results obtained in this study thus suggest that the 3D conformation of the galactose-conjugated fluorinated and non-fluorinated proline oligomers is very important for their anti-freezing properties.

Anti-Ice Nucleation Activities of Tyrosine Peptide.

　We investigated whether trimers of serine, threonine, tyrosine, and phenylalanine which may interact with water molecules and ice, show anti-ice nucleation activity. Only tyrosine trimer had high levels of anti-ice nucleation activity (10.10±0.74℃) at a final concentration of 0.2 mM. This was constant at an activity of 2.0℃ between the 0.01-0.1 mM concentrations, and rapidly increased at 0.1 mM or more. At the final concentration of 0.2 mM or more, the activity of the tyrosine trimer was almost constant (from 9.2℃ to 10.2℃). Although it is lower than the activity against silver iodide, the tyrosine trimer showed an effect on the activity of the ice nucleating bacteria. This is the first report that revealed that trimer of amino acid, especially tyrosine has the supercooling-facilitating activity.

Characterization of Anti-Ice Nucleation Activity of the Extract from Coffee Refuse.

　The supercooling-facilitating (SCF) activities, that is, the anti-ice nucleation activity of the hot water extracts from five types of processed food refuse was examined. The extract with the highest activity among five hot water extracts was coffee refuse, showing 1.50℃ of SCF activity at a final concentration of 0.1 mg/ml. From the hot water extract of coffee refuse, the coffee refuse extract containing various polyphenols was prepared by the ultrafiltration (less than MWCO 10,000), a solvent fractionation of ethyl acetate. The yield of coffee refuse extract was 0.9% (w/w) from dried coffee refuse. The SCF activity of the coffee refuse extract at a final concentration of 1.0 mg/ml was 4.2℃. HPLC analysis of the coffee refuse extract showed that caffeine and chlorogenic acid, which are major components of coffee, could be found at 173 and 62.3 µg/ml, respectively. However, the SCF activities of both compounds (0.70 and 1.06℃) at a final concentration of 0.1 mg/ml were lower than those of ferulic acid and coumaric acid, respectively at 3.40 and 2.35℃. This is the first report to our knowledge on the SCF activity of caffeine. The SCF activity of caffeine at a final concentration of 1.0 mg/ml was 2.3℃. The specificity of caffeine against various ice nuclei containing calcium oxalate, 9-fluorenon, and ice nucleating bacteria was examined. Caffeine at a final concentration of 1.0 mg/ml could inhibit the ice nucleation activity of calcium oxalate, and Pseudomonas fluorescens KUIN-1 at the same level that of as silver iodide. From these results, it was suggested that the extract could be able to be applied to the field to control the frost damage of the vegetables and that the harvested vegetables might be stored unfrozen even at 0℃ or less.

Anti-Ice Nucleation Activities of Adenine and Poly-A Nucleotides.

　Most of the ice nucleation activity inhibitor reported so far are compounds processing the hydroxyl group such as the polyphenolic derivative. After examining the anti-ice nucleation activity of the purine base, the highest compound is theophylline, and the activity showed 3.80±0.32℃ at a final concentration of 0.1 mg/ml. We found that the activity of the adenine which was essential to genome information DNA was higher than that of guanine. After examining effect of adenine concentration, high activity showed 9.1±1.2℃ and became approximately constant above 0.1 mg/ml. This active rise is a result of effect of concentration under alkaline condition. Therefore after examining effect of pH on the activity of adenine, this activity rose under an alkaline condition. The active rise predicts that an electric charge of adenine is a factor. Among four kinds of nucleotide of 6 bases, poly-A nucleotide was higher and showed 1.33±0.42℃ at a final concentration of 0.1 mg/ml. This activity of poly-A were proportional to the number of the base. From these results, it was suggested that the poly-A and adenine could be able to be applied to the field to preserve the blood and tissue which differentiated in the generative medicine.

The Importance of 11α-OH, 15-oxo, and 16-en Moieties of 11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Its Inhibitory Activity on Melanogenesis.

Cosmetic industries have an interest in exploring and developing materials that have the potential to regulate melanin synthesis in human skin. Although melanin protects the skin from ultraviolet irradiation, excess melanin can be undesirable, particularly on the face where spots or freckles are associated with an appearance of aging. In this study, we found that ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic acid (11α-OH KA) in Pteris dispar Kunze strongly inhibited melanin synthesis by suppressing tyrosinase gene expression. The melanogenic transcription factor microphthalmia-associated transcription factor (MITF) is required for this suppression. However, 11α-OH KA did not modulate the expression level or activity of MITF. Structure-activity relationship analyses suggested that the 11α-OH, 15-oxo, and 16-en moieties of 11α-OH KA are essential for the suppression of melanin synthesis. On the other hand, the 19-COOH moiety is important for preventing cellular toxicity associated with 11α-OH KA and its related compounds. These results suggest that 11α-OH KA is an attractive target for potential use in the production of cosmetic items.

Antifreeze Activity of Xylomannan from the Mycelium and Fruit Body of Flammulina velutipes.

An identified class of antifreeze, a xylomannan-based thermal hysteresis (TH)-producing glycolipid, has been discovered from diverse taxa, including plants, insects, and amphibians. We isolated xylomannan from the mycelium and fruit body of the basidiomycete Flammulina velutipes using successive hot extraction with water, 2% and 25% aqueous KOH, and gel filtration chromatography. The xylomannan from the fruit body had a recrystallization inhibiting (RI) activity (RI=0.44) at 0.5 mg/mL. The dried weight yield of the fruit body (7.7×10(-2)%, w/w) was higher than that of the mycelium. Although the purified xylomannan from both soures were composed of mannose and xylose in a 2 : 1 molar ratio, the molecular weight of the xylomannan from the mycelium and fruit body was 320,000 and 240,000, respectively. The RI activity of mycelial xylomannan was higher than that from the fruit body (RI=0.57) at 45 µg/mL. Although this RI activity was able to remain constant after exposure to various conditions, we confirmed that the decrease of RI activity was stimulated by the decrease of molecular weight that was caused by heating during the alkaline condition. The survival rate of the CHO cells at -20℃ for two days increased to 97% due to the addition of 20 µg/mL of purified xylomannan. This was the first report to indicate that xylomannan from the mycelium of Flammulina velutipes had a high level of ice recrystallization inhibiting activity like antifreeze proteins from plants and had rhe potential to become a new material for cell storage.

Simple chronic colitis model using hypopigmented mice with a Hermansky-Pudlak syndrome 5 gene mutation.

Pigmentation in mammals is important for protection of skin and eyes from ultraviolet radiation. Dysregulation of pigmentation is often associated with other conditions that are not directly linked to pigmentation. Here, we isolated spontaneously occurring hypopigmented mice that occasionally experienced severe diarrhea during lactation. Treatment of these mice with dextran sulfate sodium salt, a conventional method to induce acute colitis, caused chronic diarrhea with granulomatous colitis. Gene mapping and sequencing revealed that the mice had a nonsense mutation in the Hermansky-Pudlak syndrome (Hps)5 gene. As some HPS patients can develop granulomatous colitis, the simple induction of chronic colitis in spontaneously mutated Hps5-deficient mice may become an invaluable model for exploring treatment options in patients with HPS as well as other patients with inflammatory bowel disease.

Callicarpa longissima extract, carnosol-rich, potently inhibits melanogenesis in B16F10 melanoma cells.

Cosmetic industries focus on developing materials and resources that regulate skin pigmentation. Melanin, the major pigment in human skin, protects the skin against damage from ultraviolet light. An ethanolic extract of the leaves of Callicarpa longissima inhibits melanin production in B16F10 mouse melanoma cells by suppressing microphthalmia-associated transcription factor (MITF) gene expression. Following purification and analysis using liquid chromatography-mass spectrometry (LC-MS), NMR, and biochemical assays, carnosol was determined to be responsible for the major inhibitory effect of the C. longissima extract on melanin production. Carnosol is an oxidative product of carnosic acid, whose presence in the extract was also confirmed by an authentic reference. The carnosol and carnosic acid content in the extract was approximately 16% (w/w). These results suggest that C. longissima is a novel, useful, and attractive source of skin-whitening agents.

Altered Actions of Memantine and NMDA-Induced Currents in a New Grid2-Deleted Mouse Line.

Memantine is a non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, and is an approved drug for the treatment of moderate-to-severe Alzheimer's disease. We identified a mouse strain with a naturally occurring mutation and an ataxic phenotype that presents with severe leg cramps. To investigate the phenotypes of these mutant mice, we screened several phenotype-modulating drugs and found that memantine (10 mg/kg) disrupted the sense of balance in the mutants. Moreover, the mutant mice showed an attenuated optokinetic response (OKR) and impaired OKR learning, which was also observed in wild-type mice treated with memantine. Microsatellite analyses indicated that the Grid2 gene-deletion is responsible for these phenotypes. Patch-clamp analysis showed a relatively small change in NMDA-dependent current in cultured granule cells from Grid2 gene-deleted mice, suggesting that GRID2 is important for correct NMDA receptor function. In general, NMDA receptors are activated after the activation of non-NMDA receptors, such as AMPA receptors, and AMPA receptor dysregulation also occurs in Grid2 mutant mice. Indeed, the AMPA treatment enhanced memantine susceptibility in wild-type mice, which was indicated by balance sense and OKR impairments. The present study explores a new role for GRID2 and highlights the adverse effects of memantine in different genetic backgrounds.

Stabilization of astaxanthin by a novel biosurfactant produced by Rhodotorula mucilaginosa KUGPP-1.

We found that a novel biosurfactant from the cultured broth of red yeast, Rhodotorula mucilaginosa KUGPP-1, originating in the Antarctic, has dispersive power against astaxanthin. The novel biosurfactant was purified from extracts to the ultrafiltration state by acetone precipitation and chromatography on a DEAE-Toyopearl 650 M, and gel filtration on a Sephacryl S-400 HR. The molecular mass of the novel biosurfactant was estimated to be about 730,000 by gel filtration chromatography. The novel biosurfactant was comprised of sugar and protein in an approximate molar ratio of 9 : 1. The sugars were comprised of mannose, galactose and glucose. The particle size of the astaxanthin (0.13 µ g/ml) micelle was about 410 nm. Astaxanthin was stable to oxidation in the novel biosurfactant micelles. To our knowledge, this is the first report on a glycoprotein type of biosurfactant with astaxanthin-stabilizing ability.

Induction of melanogenesis by 4'-O-methylated flavonoids in B16F10 melanoma cells.

Agents to control melanogenesis are in demand for the development of cosmetics to improve pigmentation disorders of skin and hair. In this study, we examined and evaluated the effects of flavonoids on melanogenesis in the melanogenic cells model, murine B16F10 melanoma cells. In the course of this study, we found that incubation of the cells in a medium containing 10 µM of the 4'-O-methylated flavonoids, diosmetin (4'-O-methylluteolin), acacetin (4'-O-methylapigenin) or kaempferide (4'-O-methylkaempferol), increased the melanin contents of the cells 3- to 7-fold higher than the control cells. The concentration-dependence test revealed that 20 µM acacetin showed the highest effect, up to 33-fold higher than the vehicle. On the other hand, the corresponding 4'-OH-type flavonoids, luteolin, apigenin and kaempferol, had a significantly smaller effect. Furthermore, by evaluating the melanogenic proteins, we found that the cells treated with 4'-O-methylated flavonoids showed higher tyrosinase activity, as well as upregulation of tyrosinase expression, preceded by activation of cAMP response element binding protein (CREB) and extracellular signal-regulated kinases types 1 and 2 (ERK1/2). These results indicate that the 4'-O-methyl group of flavonoids plays an important role in the induction of melanogenesis by activating its major signal transduction pathway through the upregulation of phospho-CREB in murine B16F10 melanoma cells.

Characterization of Afp1, an antifreeze protein from the psychrophilic yeast Glaciozyma antarctica PI12.

The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at -12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.

Involvement of SIK3 in glucose and lipid homeostasis in mice.

Salt-inducible kinase 3 (SIK3), an AMP-activated protein kinase-related kinase, is induced in the murine liver after the consumption of a diet rich in fat, sucrose, and cholesterol. To examine whether SIK3 can modulate glucose and lipid metabolism in the liver, we analyzed phenotypes of SIK3-deficent mice. Sik3(-/-) mice have a malnourished the phenotype (i.e., lipodystrophy, hypolipidemia, hypoglycemia, and hyper-insulin sensitivity) accompanied by cholestasis and cholelithiasis. The hypoglycemic and hyper-insulin-sensitive phenotypes may be due to reduced energy storage, which is represented by the low expression levels of mRNA for components of the fatty acid synthesis pathways in the liver. The biliary disorders in Sik3(-/-) mice are associated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoic acid plays a role in cholesterol and bile acid homeostasis, wheras ALDH1a which produces retinoic acid, is expressed at low levels in Sik3(-/-) mice. Lipid metabolism disorders in Sik3(-/-) mice are ameliorated by the treatment with 9-cis-retinoic acid. In conclusion, SIK3 is a novel energy regulator that modulates cholesterol and bile acid metabolism by coupling with retinoid metabolism, and may alter the size of energy storage in mice.

A potent inhibitor of SIK2, 3, 3', 7-trihydroxy-4'-methoxyflavon (4'-O-methylfisetin), promotes melanogenesis in B16F10 melanoma cells.

Flavonoids, which are plant polyphenols, are now widely used in supplements and cosmetics. Here, we report that 4'-methylflavonoids are potent inducers of melanogenesis in B16F10 melanoma cells and in mice. We recently identified salt inducible kinase 2 (SIK2) as an inhibitor of melanogenesis via the suppression of the cAMP-response element binding protein (CREB)-specific coactivator 1 (TORC1). Using an in vitro kinase assay targeting SIK2, we identified fisetin as a candidate inhibitor, possibly being capable of promoting melanogenesis. However, fisetin neither inhibited the CREB-inhibitory activity of SIK2 nor promoted melanogenesis in B16F10 melanoma cells. Conversely, mono-methyl-flavonoids, such as diosmetin (4'-O-metlylluteolin), efficiently inhibited SIK2 and promoted melanogenesis in this cell line. The cAMP-CREB system is impaired in A(y)/a mice and these mice have yellow hair as a result of pheomelanogenesis, while Sik2(+/-); A(y)/a mice also have yellow hair, but activate eumelanogenesis when they are exposed to CREB stimulators. Feeding Sik2(+/-); A(y)/a mice with diets supplemented with fisetin resulted in their hair color changing to brown, and metabolite analysis suggested the presence of mono-methylfisetin in their feces. Thus, we decided to synthesize 4'-O-methylfisetin (4'MF) and found that 4'MF strongly induced melanogenesis in B16F10 melanoma cells, which was accompanied by the nuclear translocation of TORC1, and the 4'-O-methylfisetin-induced melanogenic programs were inhibited by the overexpression of dominant negative TORC1. In conclusion, compounds that modulate SIK2 cascades are helpful to regulate melanogenesis via TORC1 without affecting cAMP levels, and the combined analysis of Sik2(+/-) mice and metabolites from these mice is an effective strategy to identify beneficial compounds to regulate CREB activity in vivo.

Downregulation of SIK2 expression promotes the melanogenic program in mice.

cAMP response element-binding protein (CREB) promotes melanogenesis by inducing microphthalmia-associated transcription factor (Mitf ) gene expression. We report here that the CREB-specific coactivator TORC and its repressor, salt-inducible kinase 2 (SIK2), are fundamental determinants of the melanogenic program in mice. Exposure of B16 melanoma cells to ultraviolet (UV) light results in the immediate nuclear translocation of TORC1, which is inhibited by SIK2. Overexpression of dominant-negative TORC1 also inhibits UV-induced Mitf gene expression and melanogenesis. α-MSH signaling regulates hair pigmentation, and the decrease in α-MSH activity in hair follicle melanocytes switches the melanin synthesis from eumelanin (black) to pheomelanin (yellow). Mice with the lethal yellow allele of agouti (A(y)) have yellow hair because of impaired activation of the α-MSH receptor. To examine the involvement of SIK2 in the regulation of the melanogenesis switch in vivo, we prepared SIK2-knockout mice, and the Sik2(-/-) genotype was introduced into A(y)/a mice. The resultant Sik2(-/-); A(y)/a mice had brown hair, indicating that SIK2 represses eumelanogenesis in mice.

Cryoprotection and cryosterilization effects of type I antifreeze protein on E. coli cells.

To establish the effects of type I antifreeze protein (AFP) on E. coli cells, we have focused on the survival rate of the E. coli cells using type I AFP at various concentrations under rapid cooling conditions using liquid N2 at atmospheric or low pressure. The survival rate of E. coli was enhanced by the addition of type I AFP at a concentration of 10 microg/ml, and its value shifted from 0.73% to 2.96%. When the concentration of type I AFP was 100 microg/ml, the cell survival rate markedly decreased to 0.090%. This low survival rate was further decreased (0.022%) by the application of the same freeze-thaw treatment for four times. Also, the effect of type I AFP as a bactericidal agent did not vary according to the varying initial cell densities from 10(4) to 10(8) cells / ml. Furthermore, the effects of using type I AFP at 1.0 MPa with N2 gas under conditions of low pressure and low oxygen tension using a simple device were examined. When the actions of type I AFP as a cryoprotectant were stimulated, the survival rate of the E. coil cells increased to 57.8%. In addition, the bactericidal effect of type I AFP at 100 micro g/ml of protein concentration could also be enhanced. The survival rate using 100 g/ml of type I AFP under low pressure was 0.35% of that using 10 microg/ml under the same conditions. This is the first report on the cryoprotectant and cryosterilization effects of type I AFP of E. coli cells under various conditions.

Antifreeze activity of cold acclimated Japanese radish and purification of antifreeze peptide.

Japanese radish tuber and leaf produced antifreeze proteins (AFPs) having thermal hysteresis activity (TH) and ice recrystallization inhibiting activity (RI). Upon cold acclimation, the apoplastic fluid of the Japanese radish exhibited hexagonal crystal growth, indicating the presence of an antifreeze protein. The induction patterns of protein and the TH activity of apoplastic fraction from both samples were different. The TH activities of apoplastic fraction from tuber and leaves were 0.20 +/- 0.03 and 0.18 +/- 0.02 degree C, respectively. Also, the TH and RI activities of apoplastic fraction of leaves were activated by autoclave treatment at pH 10.0. An antifreeze peptide (molecular weight 1320), was purified using chromatography. Furthermore, the chitinase and beta-1, 3-glucanase activities in the apoplastic fraction of its tuber were induced by the cold acclimation. Some proteins in this apoplastic fraction were reacted with the anti-glucanase-like protein (GLP) antiserum and anti-chitinase-like protein (CLP) antiserum produced against isolated winter rye AFPs. This is the first report on the presence and characterization of AFPs from Japanese radish tuber.

Cryoprotective activity of mannoprotein from the cell membrane of Pichia anomala.

Some organisms like bacteria and plants have a cryoprotective protein to cryopreserve the freeze-labile enzyme under stable conditions having high activity. By screening of the cryoprotective activities of various food-industrial yeasts, we found that the cell membrane component that was a glucanase extractable component in Pichia anomala NBRC 141 had a high level of cryoprotective activity against freeze-labile lactate dehydrogenase (LDH). The absorption of the active compound in the crude extract by ConA-Sepharose chromatography suggested that this active compound might be a glycoprotein (COGP). Strain NBRC had the COGP constantly without the treatment of cold acclimation. The active compound, that is, a COGP, could be homogeneously purified using DEAE-TOYOPEARL and Sephacryl S-400 chromatography. The purified COGP had a cryoprotective activity of 50.9% at a sugar concentration of 17.9 microg/ml. The molecular weight of purified COGP was 83,000, which was composed of one protein with a molecular weight of 22,000 and polysaccharide. Furthermore, the constituent sugar of COGP was only D-mannose based on HPLC analysis of its acid hydrolysate. Also, we confirmed that the cryoprotective activity of COGP was higher than those of the commercial cell membrane components. The CP50 of COGP was 0.28 microM, which was half to the CP50 of BSA. This is the first report, to our knowledge, that the cell membrane component of Pichia anomala had a high level of cryoprotective activity against a freeze-labile enzyme.