Metabolites of Marine-derived Mangrove Endophytic Fungus #2492 from the South China Sea

Two metabolites （A and B） were isolated from the mycelium of mangrove endophytic fungus Stysanus like sp. （#2492） from the South China Sea. Their structures were identified by spectral data as N-（2-hydroxytetracosyl）-2-amino-1,3,4-trihydroxyoctadecane （A） and γ -stearolactone （B）. It is the first report that γ -stearolactone （B） is isolated from marine fungus as natural product.

A new naphthoquinoneimine derivative from the marine algal-derived endophytic fungus Aspergillus niger EN-13

Cultivation of an endophytic fungus Aspergillus niger EN-13 that was isolated from the inner tissue of the marine brown alga Colpomenia sinuosa resulted in the characterization of a new naphthoquinoneimine derivative, namely, 5,7-dihydroxy-2-[1-（4- methoxy-6-oxo-6H-pyran-2-yl）-2-phenylethylamino]-[ 1,4]naphthoquinone. The structure of the new compound was established on the basis of various NMR spectroscopic analyses including 2D NMR techniques, El-MS, and HR-ESI-MS. This compound displayed moderate antifungal activity.

Probiotic yeasts: Anti-inflammatory potential of various non-pathogenic strains in experimental colitis in mice

AIM: To evaluate the in vitro immunomodulation capacity of various non-pathogenic yeast strains and to investigate the ability of some of these food grade yeasts to prevent experimental colitis in mice.METHODS: In vitro immunomodulation was assessed by measuring cytokines [interleukin (IL)-12p70,IL-10,tumor necrosis factor and interferon γ] released by human peripheral blood mononuclear cells after 24 h stimulation with 6 live yeast strains (Saccharomyces ssp.) and with bacterial reference strains.A murine model of acute 2-4-6-trinitrobenzene sulfonic acid (TNBS)-colitis was next used to evaluate the distinct prophylactic protective capacities of three yeast strains compared with the performance of prednisolone treatment.RESULTS: The six yeast strains all showed similar non-discriminating anti-inflammatory potential when tested on immunocompetent cells in vitro .However,although they exhibited similar colonization patterns in vivo ,some yeast strains showed significant anti-inflammatory activities in the TNBS-induced colitis model,whereas others had weaker or no preventive effect at all,as evidenced by colitis markers (body-weight loss,macroscopic and histological scores,myeloperoxidase activities and blood inflammatory markers).CONCLUSION: A careful selection of strains is required among the biodiversity of yeasts for specific clinical studies,including applications in inflammatory bowel disease and other therapeutic uses.

Reaction Characteristics of Asymmetric Synthesis of (2S,5S)-2,5-Hexanediol Catalyzed with Baker’s Yeast Number 6

Baker’s yeast number 6 was selected by screening. It showed good catalytic activity and enantioselec-tivity for asymmetric reduction of 2,5-hexanedione to produce (2S,5S)-2,5-hexanediol. Gas chromatography-mass spectrometry (GC-MS) revealed that the intermediate was (S)-5-hydroxyhexane-2-one. Reduction of 2,5-hexanedione proceeded in a two-step reaction. The hydroxyketone was initially formed, and this intermediate was further re-duced to the diol. Factors influencing the product yield and the enantiomeric excess of the reduction of 2,5-hexandione catalyzed by baker’s yeast number 6 were investigated. Higher concentration (≤100 mmol·L-1) of 2,5-hexandione did not influence 5-hydroxyhexane-2-one production, but 2,5-hexanediol production was inhibited by excess accumulation (>30 mmol·L-1) of intermediate. The optimal conditions were glucose as the co-substrate at an initial glucose concentration of 20 g·L-1, 34°C, pH 7.0 and cell concentration 60 g·L-1 (cell dry mass). Under the optimal condition and an initial substrate concentration of 30 mmol·L-1, the yield of 2,5-hexandiol was 78.7% and the enantiomeric excess of (2S,5S)-2,5-hexandiol was 94.4% for 24-h reduction.

Interaction of 14-3-3σ with KCMF1 suppresses the proliferation and colony formation of human colon cancer stem cells

AIM: To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells. METHODS: Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybridscreen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells. RESULTS: The bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1 , quinone oxidore-ductase (NQO2 ), hydroxyisobutyrate dehydrogenase (HIBADH ) and 14-3-3σ . For the subsequent coimmu-noprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully constructed, and the sequences were further confirmed by DNA sequencing. The Fugene 6 reagent was used to transfect the plasmids, and fluorescence-activated cell sorting analysis showed the transfection efficiency was 97.8% after 48 h. The HEK 293FT cells showed the stable expression of the PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH plasmids. After anti-Myc antibody immunoprecipitation with Myc-KCMF1, Myc-NQO2 and Myc-HIBADH from cell lysates, the presence of Flag-14-3-3σ protein in the immuno-precipitated complex was determined by western blot analysis. The knock-down expression of the 14-3-3σ and KCMF1 proteins significantly inhibited cell proliferation and colony formation of SW1116csc. CONCLUSION: Genes of the proteins that interactedwith 14-3-3σ were successfully screened from a HeLa cDNA library. KCMF1 and 14-3-3σ protein may affect the proliferation and colony formation of human colon cancer stem cells.

Determination of <i>α</i>-1,3-Linked Mannose Residue in the Cell Wall Mannan of <i>Candida tropicalis</i>NBRC 1400 Strain

To investigate the chemical structure of cell wall mannan obtained from pathogenic yeast, Candida tropicalis NBRC 1400 (former antigenic standard strain, IFO 1400). As a result of two-dimensional NMR analysis, it was shown that the mannan of this strain is composed of α-1,6-, α-1,3-, α-1,2- and β-1,2-linked mannose residues. In this research, the mannan was subjected to three degradation procedures, acid-treatment, α-mannosidase, and acetolysis under two conditions in order to determine the chemical structure of the antigenic oligomannosyl side chains in this molecule. The 1H-nuclear magnetic resonance spectra of resultant oligosaccharides, pentaose and hexaose, demonstrated the existence of the oligomannosyl side chains corresponding to Manα1-3Manα1-2Manα1-2Manα1-2Man and Manα1-3Manα1-2Manα1-2Manα1-2Manα1-2Man, respectively, which have previously also been found in Candida albicans serotype A strain mannans. These findings indicate that C. tropicalis and C. albicans serotype A have no significant difference in the chemical structure of these cell wall mannans. Therefore, it can be interpreted that it is extremely difficult to distinguish both species by targeting the antigenic group in these mannans.

An overview of probiotic health booster-kombucha tea

Traditional herbal medicine(THM)is a significant division of traditional Chinese medicine(TCM)that plays an important role in maintaining health and disease prevention.WHO has consistently highlighted the significance of traditional,complementary,and alternative medicine in human healthcare.Most people in Eastern Asia will start their day with a cup of tea.The tea provides a nourishing effect,and it has become an inevitable part of life.There are several types of tea,like black tea,green tea,oolong tea,white tea,and herbal tea.Besides the refreshments,it is important to consume beverages that benefit health.One such alternative is a healthy probiotic drink called kombucha,a fermented tea.Kombucha tea is aerobically fermented by infusing sweetened tea with a cellulose mat/pellicle called SCOBY(symbiotic culture of bacteria and yeast).Kombucha is a source of bioactive compounds that include organic acids and amino acids,vitamins,probiotics,sugars,polyphenols,and antioxidants.Currently,studies on kombucha tea and SCOBY are gaining attention for their remarkable properties and applications in the food and health industries.The review gives an overview of the production,fermentation,microbial diversity,and metabolic products of kombucha.The possible implications for human health are also discussed.

Production of β-1,3-glucanase and chitinase of two biocon-trol agents and their possible modes of action

Pichia membranefaciens Hansen and Candida guilliermondii (Cast) Langeronet Guerra are two antagonists of R. stolonifer on harvested nectarine and peach fruits. In this study, β-1,3-glucanase and chitinase activities of the antagonists were induced in vitro and in vivo. The highest β-1, 3-glucanase activity was detected in Lilly-Barnett minimal salt medium supplemented with glucose in combination with CWP of R. stolonifer as a carbon source. The β-1,3-glucanase activity of P. membranefaciens reached the maximum level, being 114.0 SU (specific activity unit), and that of C. guilliermondii reached 103.2 SU. The lowest β-1,3-glucanase activity was observed in the medium containing glucose as sole car-bon source. P. membranefaciens was able to produce signifi-cantly higher levels of chitinase (exochitinase and endochiti-nase) in vitro than C. guilliermondii grown in Czapeck mini-mal medium. An increase in β-1,3-glucanase and chitinase activity was also triggered by wounding, adding of carbon sources

SGK and 14-3-3 protein are involved in the serine/threonine phosphorylation mechanism for TPO/MPL signal transduction

Thrombopioetin (TPO), the critical regulator of platelet production, acts by binding to its cell surface receptor, c-Mpl. Yeast two-hybrid screening was performed to isolate the proteins interacting with the cytoplasmic domain of c-Mpl. 48 positive clones were isolated from 5 xxxxxxxxxx106 independent transformants. The results of sequence analysis demonstrate that they represent 13 different protein encoding sequences. Among them there are a partial coding sequence of serine/threonine protein kinase SGK (serum and glucocorticoid-inducible kinase ) and 14-3-3 theta protein partial coding sequence. GST-pull-down assay and co-im-munoprecipitation in mammal cells have confirmed the interaction between these two proteins and c-Mpl. By constructing a series of deleted c-Mpl cytoplasmic domain, the interaction region in c-Mpl cytoplasmic tail was localized in amino acids 523-554. At the same time, the directed interaction between SGK and 14-3-3 proteins also has been verified by yeast two-hybrid assay. The

Identification of proteins that interact with murine cytomegalovirus early protein Ml12-113 in brain

Background Murine cytomegalovirus （MCMV） early protein Ml12-113 is involved in viral DNA replication and believed to play a crucial role in the viral pathogenesis. To investigate the biological function of Ml12-113 protein in the pathogenesis of the brain disorders caused by cytomegalovirus （CMV）, a screening for proteins interacting with Ml12-113 was performed by a yeast two-hybrid system. Methods Bait plasmid pGBKT7-M112-113 was constructed and transformed into AH109 yeast. After confirmation of the expression of MCMV Ml12-113 in yeast, the bait yeast was mated with a prey yeast containing mouse brain cDNA library plasmid to screen the proteins interacting with M 112-113. Interactions between Ml12-113 and the obtained proteins were verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion. Results Two proteins interacting with M112-113 were identified, including metastasis-associated 1 （MTA1） and zinc finger, CCHC domain containing 18 （ZCCHC18）. Ml12-113 protein could interact with MTAt or ZCCHC18 in yeast and mammalian cells. Conclusion The interactions of Ml12-113 with MTA1 or ZCCHC18 may be related to the pathogenesis of MCMV-associated disease in central nervous system.