Polygonatum cyrtonema belongs to the plant family Liliaceae, and its dried rhizome is one of the sources of Chinese traditional medicine of Polygonati Rhizoma. It possesses the dual function as both medicine and food. Its main chemical components are polysaccharides and saponins. In order to understand the biosynthesis pathway of polysaccharides and diosgenin in P. cyrtonema, the corresponding transcriptomic data were obtained by extracting and sequencing the RNA of four parts of P. cyrtonema, namely, leaves, stems, rhizomes and roots. By adopting BGISEQ-500 sequencing platform, 42.03 Gb data were retrieved. Subsequently, the de novo assembly was carried out by Trinity software to obtain 137 233 transcripts, of which 68.13% of unigenes were annotated in seven databases including KEGG, GO, NR, NT, SwissProt, Pfam and KOG. Transcripts that may be involved in the biosynthesis of polysaccharides and diosgenin were analyzed by data mining. With help of qPCR, we validated expression data of four genes that were possibly involved in the biosynthesis of target metabolites. This experiment provides data for the study of biosynthetic pathways of P. cyrtonema secondary metabolites and the clarification of related structural gene functions.
Biosynthetic Pathways ; Diosgenin/metabolism* ; Gene Expression Profiling ; Phytochemicals/biosynthesis* ; Polygonatum/metabolism* ; Polysaccharides/biosynthesis* ; Transcriptome

OBJECTIVE: To investigate the effects of Ganoderma lucidum polysaccharides (GL-PS) on human fibroblasts and skin wound healing in Kunming male mice and to explore the putative molecular mechanism. METHODS: Primary human skin fibroblasts were cultured. The viability of fibroblasts treated with 0, 10, 20, 40, 80, and 160 μg/mL of GL-PS, respectively were detected by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-Htetrazolium bromide (MTT). The migration ability of fibroblasts treated with 0, 10, 20, and 40 μg/mL of GL-PS were measured by transwell assay. The secretion of the C-terminal peptide of procollagen type I (CICP) and transforming growth factor-β1 (TGF-β1) in the cell supernatant was tested by enzyme-linked immunosorbent assay. The expression of β-catenin was detected by Western blot. Furthermore, the Kunming mouse model with full-layer skin resection trauma was established, and was treated with 10, 20, and 40 mg/mL of GL-PS, respectively as external use. The size of the wound was measured daily, complete healing time in each group was recorded and the percentage of wound contraction was calculated. RESULTS: Compared with the control group, 10, 20, and 40 μg/mL of GL-PS significantly increased the viability of fibroblasts, promoted the migration ability of fibroblasts, and up-regulated the expressions of CICP and TGF-β1 in fibroblasts (Plt;0.05 or Plt;0.01). The expression of β-catenin in fibroblasts treated with 20 and 40 μg/mL of GL-PS was significantly higher than that of the control group (Plt;0.01). Furthermore, after external use of 10, 20, and 40 mg/mL of GL-PS, the rates of wound healing in mice were significantly higher and the wound healing time was significantly less than the control group (Plt;0.05 or Plt;0.01). CONCLUSION A certain concentration of GL-PS may promote wound healing via activation of the Wnt/β-catenin signaling pathway and up-regulation of TGF-β1, which might serve as a promising source of skin wound healing.
Animals ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Collagen Type I ; biosynthesis ; Fibroblasts ; drug effects ; Humans ; Male ; Mice ; Polysaccharides ; pharmacology ; Reishi ; chemistry ; Skin ; drug effects ; injuries ; Transforming Growth Factor beta1 ; physiology ; Wound Healing ; drug effects ; beta Catenin ; physiology

In order to understand the function of GDP-mannose pyrophosphorylase(GMPP) function and its regulation in polysaccharide biosynthesis mechanism in Dendrobium. D. huoshanense was used to clone GMPP gene. GMPP gene expression in D. huoshanense,D. officinale and D. moniliforme was also determined by qPCR. The results showed that the length of D. huoshanense GMPP gene c DNA sequence is 1 867 bp,containing 1 245 bp open reading frame(ORF),encoding 415 amino acids. Phylogenetic tree analysis showed that D. huoshanense,D. officinale and D. moniliforme are closely related with GMPP taken into consideration. Bioinformatics analysis demonstrated that GMPP sequence similarity among the three species reached as high as 99%. qPCR results indicated that GMPP genes was highly expressed in stem of D. huoshanense compared with its leaf,flower and root. According to GMPP gene expression profile in D. huoshanense,D. officinale and D. moniliforme grown in Huoshan area,it was clear that GMPP in D. huoshanense showed the highest expression level. Furthermore,our findings of GMPP gene expression profile will facilitate future researches into its polysaccharide biosynthetic mechanism.
Base Sequence ; Cloning, Molecular ; Dendrobium ; enzymology ; genetics ; Nucleotidyltransferases ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Polysaccharides ; biosynthesis

GAPDH(glyceraldehyde-3-phosphate dehydrogenase) gene is a key enzyme gene in carbohydrate metabolism and always used as reference gene. To clarify and complete the biosynthetic pathway of polysaccharide, the GAPDH gene in Codonopsis pilosula, named CpGAPDH, was cloned according to the transcriptome of pilosula, using the GAPDH gene in potato as query. The CpGAPDH contained a 1 014 bp open reading frame(ORF) and encoded a protein with 337 amino acids. Bioinformatic analysis clearly suggested that CpGAPDH shared high similarity with GAPDH among other plants, and had the closest relatives to potato and danshen. The predicted protein did not have signal peptide, which indicated that it might be located in the cytoplasm. According to the existing of several phosphorylation sites and the conserved domains analysis, we predicted that it belonged to Gp_dh_N superfamily. Prokaryotic expression showed that the recombinant expressed a 44.3 kDa protein, which was corresponding to the theoretical relative molecular mass. However, the relative transcript level of the CpGAPDH did not have significant differences in different tissues and roots at different developmental stages of pilosula. Moreover, the stability of the CpGAPDH was analyzed by BestKeeper, geNorm, and NormFinder and RefFinder software, which showed that the CpGAPDH was more stable and could be used as a new reference gene. All these lay a foundation for the expression analysis of the gene relative to the polysaccharide synthesis.
Codonopsis ; enzymology ; genetics ; Glyceraldehyde-3-Phosphate Dehydrogenases ; genetics ; Plant Proteins ; genetics ; Polysaccharides ; biosynthesis ; Transcriptome

Agitator is one of the essential factors to realize high efficient fermentation for high aerobic and viscous microorganisms, and the influence of different impeller combination on the fermentation process is very important. Welan gum is a microbial exopolysaccharide produced by Alcaligenes sp. under high aerobic and high viscos conditions. Computational fluid dynamics (CFD) numerical simulation was used for analyzing the distribution of velocity, shear rate and gas holdup in the welan fermentation reactor under six different impeller combinations. The best three combinations of impellers were applied to the fermentation of welan. By analyzing the fermentation performance, the MB-4-6 combination had better effect on dissolved oxygen and velocity. The content of welan was increased by 13%. Furthermore, the viscosity of production were also increased.
Alcaligenes ; metabolism ; Fermentation ; Hydrodynamics ; Industrial Microbiology ; methods ; Oxygen ; Polysaccharides, Bacterial ; biosynthesis ; Viscosity

A novel Pleurotus nebrodensis polysaccharide (PN-S) was purified and characterized, and its immune-stimulating activity was evaluated in RAW264.7 macrophages. PN-S induced the proliferation of RAW264.7 cells in a dose-dependent manner, as determined by the MTT assay. After exposure to PN-S, the phagocytosis of the macrophages was significantly improved, with remarkable changes in morphology being observed. Flow cytometric analysis demonstrated that PN-S promoted RAW264.7 cells to progress through S and G2/M phases. PN-S treatment enhanced the productions of interleukin-6 (IL-6), nitric oxide (NO), interferon gamma (INF-γ), and tumor necrosis factor-α (TNF-α) in the macrophages, with up-regulation of mRNA expressions of interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), interferon gamma(INF-γ) and tumor necrosis factor-α (TNF-α) being observed in a dose-dependent manner, as measured by qRT-PCR. In conclusion, these results suggest that the purified PN-S can improve immunity by activating macrophages.
Animals ; Cell Cycle ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Fungal Polysaccharides ; pharmacology ; Immunity ; drug effects ; Interferon-gamma ; biosynthesis ; metabolism ; Interleukin-6 ; biosynthesis ; metabolism ; Macrophages ; immunology ; metabolism ; Mice ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; metabolism ; Pleurotus ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; biosynthesis ; metabolism ; Up-Regulation

The polysaccharides from pumpkin fruit (PP) were obtained and purified by hot-water extraction, anion-exchange chromatography, and gel column chromatography. The physicochemical properties of PP were determined by gel filtration chromatography, gas chromatography, fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy. Results indicated that the molecular weight of PP was about 23 kDa and PP was composed of D-Arabinose, D-Mannose, D-Glucose, and D-Galactose with a molar ratio of 1 : 7.79 : 70.32 : 7.05. FTIR and NMR spectra indicated that PP was the polysaccharide containing pyranose ring. Additionally, PP protected islets cells from streptozotocin (STZ) injury in vitro via increasing the levels of super-oxide dismutase (SOD) and malondialdehyde (MDA) and reducing the production of NO. The experiment of reverse transcriptase-polymerase chain reaction further proved that PP inhibited apoptosis via modulating the expression of Bax/Bcl-2 in STZ-damaged islet cells. In conclusion, PP could be explored as a novel agent for the treatment of diabetes mellitus.
Animals ; Apoptosis ; drug effects ; Chromatography, Gas ; Chromatography, Gel ; Cucurbita ; chemistry ; Diabetes Mellitus, Experimental ; drug therapy ; Islets of Langerhans ; drug effects ; injuries ; Magnetic Resonance Spectroscopy ; Malondialdehyde ; analysis ; Molecular Weight ; Monosaccharides ; analysis ; Nitric Oxide ; biosynthesis ; Polysaccharides ; chemistry ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Spectroscopy, Fourier Transform Infrared ; Superoxide Dismutase ; drug effects ; bcl-2-Associated X Protein ; drug effects

Neural cell adhesion molecule (NCAM) is a glycoprotein expressing on the surface of neurons, glial cells, bone cells and natural killer cells. NCAM plays an important role in the process of cell - cell adhesion and cell migration, and is also a model protein to study polysialic acid. In this paper, NCAM gene from mouse mammary gland cells (NMuMG) was cloned into eukaryotic expression vectors pcDNA3.1(+) and transfected into mutant Chinese hamster ovary cells ldlD-14. The stable transfection over-expressing NCAM was obtained through the G418 selection and confirmed by Western blotting. Due to unique characters of ldlD-14 cells, carbohydrate chain of NCAM molecule can be easily manipulated with or without adding galactose in the serum free medium, and this modification can provide the basis for further studies on the effect of glycosylation on NCAM molecular function.
Animals ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Female ; Galactose ; Glycosylation ; Mammary Glands, Animal ; cytology ; Mice ; Neural Cell Adhesion Molecules ; biosynthesis ; Polysaccharides ; chemistry ; Sialic Acids ; chemistry ; Transfection

The objective of the study was to analyse Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media: Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy (CLSM)-based analyses of the microbial vitality, respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride, CTC) and production of extracellular polysaccharides (EPS) were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters: culture growth, vitality, CTC activity and EPS production. However, xylitol exposure caused a difference in gene expression compared to the control. GtfC was upregulated only in the presence of xylitol. Under xylitol exposure, gtfB was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three. Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the cariogenic potential of S. mutans biofilms.
Bacterial Load ; drug effects ; Bacteriological Techniques ; Biofilms ; drug effects ; Cariogenic Agents ; metabolism ; pharmacology ; Culture Media ; Dental Enamel ; microbiology ; Fluorescent Dyes ; Gene Expression Regulation, Bacterial ; drug effects ; Gene Expression Regulation, Enzymologic ; drug effects ; Genotype ; Glucose ; metabolism ; Glucosyltransferases ; metabolism ; Humans ; Microbial Viability ; drug effects ; Microscopy, Confocal ; Polysaccharides, Bacterial ; biosynthesis ; Streptococcus mutans ; drug effects ; enzymology ; metabolism ; Sucrose ; metabolism ; pharmacology ; Sweetening Agents ; metabolism ; pharmacology ; Tetrazolium Salts ; Time Factors ; Up-Regulation ; Xylitol ; metabolism ; pharmacology

OBJECTIVETo study the effect of laminarin polysaccharide (LP) on the activity of matrix metalloproteinase of photoaging skins.

METHODKunming SPF mice were prepared with back hair shaved, and randomly divided into the control group, the model group, the LP low does group (LP-L, 1 mg x kg(-1)), the LP high dose group (LP-H, 5 mg x kg(-1)) and the Vit E (100 mg x kg(-1)) group. They were abdominally injected with drugs twice on a daily basis. Except for the control group, all groups were exposed to ultraviolet rays for 1 hour every day, five times on a weekly basis, with accumulated exposure dose of UVB being 21.60 J x cm(-2) and accumulated exposure dose of UVA being 84.02 J x cm(-2). Eight weeks later, exposed back skins were collected to detect thickness of dermis by HE stain, content of hydroxyproline (Hyp) by chemical colorimetry, and serum MMP-1 and TIMP-1 content by ELISA. In addition, matrix metalloproteinase-1 (MMP-1) mRNA and relative content of tissue inhibitor of metalloproteinase-1 (TIMP1) mRNA was analyzed with Real-time PCR.

RESULTCompared with the model group, the LP-H group could significantly increase the thickness of dermis, skin Hyp content and serum TIMP-1 level, and decrease relative content of MMP-1 mRNA in skin and MMP-1 content in serum.

CONCLUSIONLP can regulate the metabolism of collagen photoaging skins by adjusting the activity of matrix metalloproteinase.

Animals ; Female ; Glucans ; Matrix Metalloproteinase 13 ; biosynthesis ; genetics ; metabolism ; Mice ; Plant Extracts ; chemistry ; pharmacology ; Plants, Medicinal ; chemistry ; Polysaccharides ; chemistry ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Skin Aging ; drug effects ; physiology ; radiation effects ; Tissue Inhibitor of Metalloproteinase-1 ; biosynthesis ; genetics ; metabolism ; Ultraviolet Rays