A newly characterized exopolysaccharide from Sanghuangporus sanghuang.

Sanghuangporus sanghuang is a well-known pharmacodynamic and economically important edible fungus associated with mulberry (Morus spp.). A distinctly new exopolysaccharide (EPS), designated SHP-2 was obtained from S. sanghuang P0988 broth, and its structure and anti-aging prosperity were characterized. SHP-2 was found to be composed of a back-bone of →4)-ß-Manp-(1→4)-α-Araf-(1→3,4)-α-Glcp(1→3,4)-α-Glcp-(1→3,4)-α-Glcp-(1→3,4)-α-Glcp-(1→3,4)-α-Glcp-(1→6)-α-Galp-(1→4)-ß-Manp-(1→ and five branches, including four α-D-Glcp-(1→ and one α-D-Manp-(1→SHP-2 was shown to increase antioxidant enzyme activities including catalase (CAT) and superoxide dismutase (SOD) activities, as well as trolox equivalent antioxidant (TEAC) capacity in serum of mice pre-treated with D-Gal, while reducing lipofuscin levels. SHP-2 exerted a favorable influence on immune organ coefficients and ameliorated the histopathological hepatic lesions and apoptosis in hepatocytes of D-galactose-aged mice almost in a dose-dependent manner. Using the same analytical methods, on comparison with previously studied EPS compounds (i.e. SHP-1), SHP-2 was found to have more complex structure, larger molecule weight, and different anti-aging properties. The results presented here suggest that not only does EPS bioactivity vary with respect to molecular structures and molecule weight, but that multiple structures with different activity can be expressed by a single fungal strain. These results may help understanding the anti-aging prosperity of these polysaccharides for use in health foods or dietary supplements.

Structural characterization of two endopolysaccharides from Phellinus sp. and their immunologic effects by intragastric administration in a healthy mammalian model.

Two purified endopolysaccharides derived from cultured Phellinus sp., individually named SHIP-1 and 2, were structurally characterized, along with an evaluation of their in vivo influential immunomodulatory activity in a healthy mammalian model. The structure of SHIP-1 was mainly composed of →4)-α-d-Fucp-(1→, →3,6)-α-d-Araf-(1→ and →2,4)-ß-d-Galp-(→, with four residuals of α-d-Manp-(1→ and one α-d-Glcp-(1→ as sidegroups, while the planar structure and the heteronuclear multiple-bond correlation of SHIP-2 were not able to be analyzed. Biochemical analysis in the healthy mice model demonstrated that SHIP-1 increased the concentrations of the detected cytokines in a dosage-dependent manner but not in a time-dependent way. SHIP-2 exerted a positive effect in a dose-dependent manner over time for interferon gamma (IFN-γ) and interleukin (IL)-2 cytokine production at elevated dosages of 200, or 350 mg kg-1 d-1, while IFN-alpha(α) and IL-4 production was observed only in a dosage-dependent manner even at high dosages. Thus, SHIP-1 and 2 significantly improved the immune response through the intragastric administration of the tested high dosages by increasing the production of cytokines in the healthy mice, and these polysaccharides could possibly be used as an immunopotentiator in health foods or dietary supplements.

The influence of heavy metals on the bioremediation of polycyclic aromatic hydrocarbons in aquatic system by a bacterial-fungal consortium.

Co-contamination of polycyclic aromatic hydrocarbons (PAHs) with heavy metals (HMs) in aquatic environments is a global threat; however, little is understood about PAH biodegradation in these sites. In this study, PAHs' biodegradation in the presence of HMs in water by a metal-tolerant consortium composed of Bacillus subtilis and fungus Acremonium sp. was investigated. The consortium demonstrated higher tolerance to the tested HMs (Fe2+, Al3+, Ni2+, Cu2+, Mn2+ and Zn2+) than the individual consortium components, and the tolerance to individual metals decreased with increasing metal concentrations. In the absence of HMs in aquatic systems, the consortium efficiently degraded naphthalene, fluorine, phenanthrene, anthracene and fluoranthene individually (50 mmol/L) over 10 days. However, while Ni2+ supplementation (5 mmol/L) suppressed phenanthrene and anthracene removal (p ≤ 0.01), enhanced fluoranthene degradation relative to the control was observed. Cu2+, Zn2+, Fe2+ and Al3+ supplementation demonstrated significant inhibition against individual phenanthrene, anthracene and fluoranthene removal, and Cu2+ showed a more significant effect on the degradation of these PAH compounds compared to other metals. Conversely, Mn2+ significantly enhanced the removal of fluorene, phenanthrene and fluoranthene, but inhibited anthracene degradation. HM contamination in aquatic systems did not show any effect on naphthalene bioremediation, possible due to its rapid degradation over a short time. Thus, metals affect PAH aquatic biodegradation by consortia, depending on metal species and PAH compound, underlining the complex nature of co-contaminated systems containing HMs and PAHs. To our knowledge, this is the first study to examine the influence of HMs on PAHs' bioremediation by such PAH-degrading consortia in water.

Structural characterization and anti-aging activity of a novel extracellular polysaccharide from fungus Phellinus sp. in a mammalian system.

Little is known about the chemical structure of purified extracellular polysaccharides from Phellinus sp., a fungal species with known medicinal properties. A combination of IR spectroscopy, methylation analysis and NMR were performed for the structural analysis of a purified extracellular polysaccharide derived from Phellinus sp. culture, denoted as SHP-1, along with an evaluation of the anti-aging effect in vivo of the polysaccharide supplementation. The structure of SHP-1 was established, with a backbone composed of →2,4)-α-d-glucopyranose-(1→ and →2)-ß-d-mannopyranose-(1→ and two terminal glucopyranose branches. Biochemical analysis from mammalian animal experiments demonstrated that SHP-1 possesses the ability to enhance antioxidant enzyme activities, such as catalase (CAT) and superoxide dismutase (SOD) activities, Trolox equivalent antioxidant capacity (TEAC) in serum of d-galactose-aged mice, while reducing lipofuscin levels, another indicator of cell aging, indicating a potential association with anti-aging activities in a dose dependent manner. This compound had a favourable influence on immune organ indices, and a marked amelioration ability of histopathological hepatic lesions such as necrosis, karyolysis and reduced inflammation and apoptosis in mouse hepatocytes. These results suggest that SHP-1 has strong antioxidant activities and a significant protective effect against oxidative stress or hepatotoxicity induced by d-galactose in mice and it could be developed as a food ingredient or a pharmaceutical to prevent many age-associated diseases such as major depressive disorder and hepatotoxicity. To our knowledge, this is the first report on the antioxidant effects of a novel purified exopolysaccharide derived from Phellinus sp.

Heavy metals species affect fungal-bacterial synergism during the bioremediation of fluoranthene.

The co-occurrence of polycyclic aromatic hydrocarbons (PAHs) with heavy metals (HMs) is very common in contaminated soils, but the influence of HMs on fungal-bacterial synergism during PAH bioremediation has not been investigated. The bioremediation of fluoranthene-contaminated sand using co-cultures of Acremonium sp. P0997 and Bacillus subtilis showed increases of 109.4 and 9.8 % in degradation compared to pure bacterial and fungal cultures, respectively, removing 64.1 ± 1.4 % fluoanthene in total. The presence of Cu(2+) reduced fluoranthene removal to 53.7 ± 1.7 %, while inhibiting bacterial growth, and reducing translocation of bacteria on fungal hyphae by 49.5 %, in terms of the bacterial translocation ratio. Cu(2+) reduced bacterial diffusion by 46.8 and 31.9 %, as reflected by D (a bulk random motility diffusional coefficient) and D eff (the effective one-dimensional diffusion coefficient) compared to the control without HM supplementation, respectively. However, Mn(2+) resulted in a 78.2 ± 1.9 % fluoranthene degradation, representing an increase of 21.9 %, while enhancing bacterial growth and bacterial translocation on fungal hyphae, showing a 12.0 % increase in translocation ratio, with no observable impact on D and D eff. Hence, the presence of HMs has been shown to affect fungal-bacterial synergism in PAH degradation, and this effect differs with HM species.

The influence of naphthaleneacetic acid (NAA) and coumarin on flavonoid production by fungus Phellinus sp.: modeling of production kinetic profiles.

For the purpose of improving the fungal production of flavonoids, the influence of naphthaleneacetic acid (NAA) and coumarin on flavonoid production by fungus Phellinus sp. P0988 was investigated by developing the corresponding kinetics of flavonoid production in a 7-L bioreactor. Phellinus sp. was confirmed to form flavonoids in pellets and broth when cultivated in basic medium, and the optimum concentration of NAA and coumarin in medium for flavonoid production were determined to be 0.03 and 0.02 g/L, respectively. The developed unstructured mathematical models were in good agreement with the experimental results with respect to flavonoid production kinetic profiles with NAA and coumarin supplementation at optimum levels and revealed significant accuracy in terms of statistical consistency and robustness. Analysis of these kinetic processes indicated that NAA and coumarin supplementations imposed a stronger positive influence on flavonoid production and substrate consumption compared to their effects on cell growth. The separate addition of NAA and coumarin resulted in enhancements in final product accumulation and productivity, achieving final flavonoid concentrations of 3.60 and 2.75 g/L, respectively, and glucose consumption showed a significant decrease compared to the non-supplemented control as well. Also, the separate presence of NAA and coumarin respectively decreased maintenance coefficients (M s) from 2.48 in the control to 1.39 and 0.22, representing decreases of 43.9 and 91.1 %, respectively. The current study is the first known application of mathematical kinetic models to explore the influence of medium components adding on flavonoid production by fungi.

Bioaugmentation of soil contaminated with high-level crude oil through inoculation with mixed cultures including Acremonium sp.

Heavy contamination of soil with crude oil has caused significant negative environmental impacts and presents substantial hazards to human health. To explore a highly efficient bioaugmentation strategy for these contaminations, experiments were conducted over 180 days in soil heavily contaminated with crude oil (50,000 mg kg(-1)), with four treatments comprised of Bacillus subtilis inoculation with no further inoculation (I), or reinoculation after 100 days with either B. subtilis (II), Acremonium sp.(III), or a mixture of both organisms (IV). The removal values of total petroleum hydrocarbons were 60.1 ± 2.0, 60.05 ± 3.0, 71.3 ± 5.2 and 74.2 ± 2.7 % for treatment (I-IV), respectively. Treatments (III-IV) significantly enhanced the soil bioremediation compared with treatments (I-II) (p < 0.05). Furthermore, significantly (p < 0.05) greater rates of degradation for petroleum hydrocarbon fractions were observed in treatments (III-IV) compared to treatments (I-II), and this was especially the case with the degradative rates for polycyclic aromatic hydrocarbons and crude oil heavy fractions. Dehydrogenase activity in treatment (III-IV) containing Acremonium sp. showed a constant increase until the end of experiments. Therefore reinoculation with pure fungus or fungal-bacterial consortium should be considered as an effective strategy in bioaugmentation for soil heavily contaminated with crude oil.

Heavy metal ions affecting the removal of polycyclic aromatic hydrocarbons by fungi with heavy-metal resistance.

The co-occurrence of polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HMs) is very common in contaminated environments. It is of paramount importance and great challenge to exploit a bioremediation to remove PAHs in these environments with combined pollution. We approached this question by probing the influence of HMs coexisting with PAHs on the removal of PAHs by Acremonium sp. P0997 possessing metal resistance. A removal capability for naphthalene, fluorene, phenanthrene, anthracene, and fluoranthenepresentalone (98.6, 99.3, 89.9, 60.4, and 70 %, respectively) and in a mixture (96.9, 71.8, 67.0, 85.0, and 87.9 %, respectively) was achieved in mineral culture inoculated with Acremonium sp. P0997, and this strain also displayed high resistance to the individual HMs (Mn(2+), Fe(2+), Zn(2+), Cu(2+), Al(3+), and Pb(2+)). The removal of individual PAHs existing in a mixture was differently affected by the separately tested HMs. Cu(2+)enhanced the partition process of anthracene to dead or alive mycelia and the contribution of the biosorption by this strain but imposed a little negative influence on the contribution of biodegradation to the total removal of anthracene individually in a culture. However, Mn(2+) had an inhibitory effect on the partition process of anthracene to dead or alive mycelia and decreased the contributions of both biosorption and biodegradation to the total anthracene removal. This work showcased the value of fungi in bioremediation for the environments with combined pollution, and the findings have major implications for the bioremediation of organic pollutants in metal-organic mixed contaminated sites.

Enhancing exopolysaccharide antioxidant formation and yield from Phellinus species through medium optimization studies.

The study was conducted to enhance exopolysaccharide (EPS) antioxidant formation (as measured by Trolox-Equivalent Antioxidant Capacity, TEAC) by Phellinus sp. P0988 through medium studies, while jointly increasing EPS yield. Out of seven medium components, four significant variables were identified based on linear models with statistical significance (p<0.05), namely rutin, FeSO4, and aspartate, which influenced TEAC, and malt extract, which influenced yield. Based on regression analysis of the experimental results from central composite design in response surface models, two second-order polynomial models adequately representing the relationship between these variables and EPS TEAC and yield were obtained, and the optimum concentration of these variables were determined and validated. Variable combinations for high EPS TEAC and yield were optimized through a desirability function test. The maximum EPS TEAC and yield reached 8.49 ± 0.32 mM and 10.92 ± 0.68 g/L, respectively, in a 7 L bioreactor under the optimum variable combinations.

Effect of bioconversion conditions on vanillin production by Amycolatopsis sp. ATCC 39116 through an analysis of competing by-product formation.

This study investigated the effects of transformation conditions such as initial pH, the initial concentration of glucose and yeast extract in the medium, and the separate addition of ferulic acid and vanillic acid, on the production of vanillin through an analysis of competing by-product formation by Amycolatopsis sp. ATCC 39116. The extent and nature of by-product formation and vanillin yield were affected by initial pH and different initial concentrations of glucose and yeast extract in the medium, with a high yield of vanillin and high cell density obtained at pH 8.0, 10 g/l glucose, and 8 g/l yeast extract. High concentrations of ferulic acid were found to negatively affect cell density. Additional supplementation of 100 mg/l vanillic acid, a metabolically linked by-product, was found to result in a high concentration of vanillin and guaiacol, an intermediate of vanillin. Via an analysis of the effect of these transformation conditions on competing by-product formation, high concentrations of ferulic acid were transformed with a molar yield to vanillin of 96.1 and 95.2 %, by Amycolatopsis sp. ATCC 39116 and Streptomyces V1, respectively, together with a minor accumulation of by-products. These are among the highest performance values reported in the literature to date for Streptomyces in batch cultures.

Transformation of ferulic acid to vanillin using a fed-batch solid-liquid two-phase partitioning bioreactor.

Amycolatopsis sp. ATCC 39116 (formerly Streptomyces setonii) has shown promising results in converting ferulic acid (trans-4-hydroxy-3-methoxycinnamic acid; substrate), which can be derived from natural plant wastes, to vanillin (4-hydroxy-3-methoxybenzaldehyde). After exploring the influence of adding vanillin at different times during the growth cycle on cell growth and transformation performance of this strain and demonstrating the inhibitory effect of vanillin, a solid-liquid two-phase partitioning bioreactor (TPPB) system was used as an in situ product removal technique to enhance transformation productivity by this strain. The thermoplastic polymer Hytrel(®) G4078W was found to have superior partitioning capacity for vanillin with a partition coefficient of 12 and a low affinity for the substrate. A 3-L working volume solid-liquid fed-batch TPPB mode, using 300 g Hytrel G4078W as the sequestering phase, produced a final vanillin concentration of 19.5 g/L. The overall productivity of this reactor system was 450 mg/L. h, among the highest reported in literature. Vanillin was easily and quantitatively recovered from the polymers mostly by single stage extraction into methanol or other organic solvents used in food industry, simultaneously regenerating polymer beads for reuse. A polymer-liquid two phase bioreactor was again confirmed to easily outperform single phase systems that feature inhibitory or easily further degraded substrates/products. This enhancement strategy might reasonably be expected in the production of other flavor and fragrance compounds obtained by biotransformations.

Influence of rutin, FeSO4, Tween 80, aspartate and complex vitamins on synthesis of fungal exopolysaccharide.

The influence of several components on exopolysaccharide (EPS) production and antioxidative activity (TEAC, Trolox-Equivalent Antioxidant Capacity) as well as their effects on the morphological development and cell viability of Phellinus sp. P0988 was determined. Rutin, FeSO(4), Tween 80 and complex vitamins were found to impose a stronger influence on EPS production and TEAC compared to their effects on the mycelia growth of Phellinus sp. P0988. The relative effects of these components on EPS activity were found to be different from that on EPS yield. Rutin and aspartate significantly affected EPS TEAC (P<0.05), while FeSO(4) and Tween 80 significantly influenced EPS production (P<0.05). These results yielded the optimum culture medium composition, with an EPS yield and TEAC of 6.2±0.2 g/L and 5.5±0.1 mM, respectively.

HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner.

BACKGROUND: Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory. RESULTS: An anoikis-resistant clone (HEK293ar), derived from anoikis-sensitive parental Human Embryonic Kidney 293 cells, survived anoikis by the formation of cell-cell contacts. The expression of HAb18G/CD147 (a member of the CD147 family) was upregulated and the protein was located at cell-cell junctions. Upregulation of HAb18G/CD147 in suspended HEK293ar cells suppressed anoikis by mediating the formation of cell-cell adhesions. Anoikis resistance in HEK293ar cells also required E-cadherin-mediated cell-cell contacts. Knock-down of HAb18G/CD147 and E-cadherin inhibited cell-cell contacts formation and increased anoikis sensitivity respectively. When HAb18G/CD147 was downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of E-cadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no significant effect on HAb18G/CD147 expression. Finally, pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K/AKT) inhibitor, disrupted cell-cell contacts and decreased cell number, but this was not the case in cells treated with the extracellular signal-regulated kinase (ERK) inhibitor PD98059. CONCLUSIONS: Our results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis resistance in an E-cadherin-dependent manner; and cell-cell contact mediated resistance to anoikis implicates PI3K pathway in a highly relevant cell model (HEK293ar). Understanding of the role of HAb18G/CD147 cell-cell contacts in anoikis resistance may help in understanding the survival of cells in anchorage-independent growth, such as cells in tumor metastasis and suspension culture produced for biomedical engineering. Our results also contribute to a better understanding of the biology of HEK293 cell spheroids, a major workhorse for producing human therapeutic agents and viral vaccines.

Annexin II promotes invasion and migration of human hepatocellular carcinoma cells in vitro via its interaction with HAb18G/CD147.

HAb18G/CD147, a member of the immunoglobulin family enriched on the surface of tumor cells, is reported to be correlated with invasion, metastasis, growth, and survival of malignant cells. Here, we found that annexin II, a 36-kDa Ca(2+)- and phospholipid-binding protein and in vivo substrate for tyrosine kinase and PKC, is a new interaction protein of HAb18G/CD147 in human hepatocellular carcinoma (HCC) cells. In the present study, we explored the unclear role of annxin II in HCC invasion and migration and the interaction effects between HAb18G/CD147 and annexin II. Our data show that downregulation of annexin II in HCC cells significantly decreased the secretion of MMP, migration ability, and invasive potential, and affected the cytoskeleton rearrangement of tumor cells. The MMP-2 level and invasive potential of HCC cells were regulated by both annexin II and HAb18G/CD147. Also, interaction effects exist between the two molecules in tumor progression, including MMP-2 production, migration, and invasion. These results suggest that annexin II promotes the invasion and migration of HCC cells in vitro, and annexin II and HAb18G/CD147 interact with each other in the same signal transduction pathway working as a functional complex in tumor progression.

Contribution of cyclophilin A to the regulation of inflammatory processes in rheumatoid arthritis.

INTRODUCTION: Previous studies show that cyclophilin A (CypA) acts as a strong chemotactic cytokine to neutrophils, eosinophils, and monocytes in rheumatoid arthritis (RA). METHODS: In this study, monocytes were stimulated by purified CypA and the production of matrix metalloproteinase (MMPs), the cell invasion and the release of inflammatory cytokines were detected respectively by gelatin zymography, invasion assay, and cytometric bead array FCM. RESULTS: The elevated level of inflammatory cytokine IL-8 was also detected. Results showed that CypA significantly promoted the invasion of THP-1 cells and increased the production of MMP-2 and MMP-9, which displayed a biphasic concentration dependency. In vivo experiments found that the cartilage erosion scores in CypA injection group were significantly higher than those in control group (P < 0.05). CONCLUSION: Our findings suggest that CypA significantly enhances the secretion of MMP-2 and MMP-9, the cell invasion, and the inflammatory cytokines production of monocytes. Our findings may shed some new light on the inflammatory process and the degradation of cartilage and bone in RA.

HOXB13 homeodomain protein is cytoplasmic throughout fetal skin development.

Substantial evidence suggests that HOX homeobox genes regulate aspects of body development, including hair formation. We initially isolated the HOXB13 gene from human fetal skin in experiments designed to identify candidate genes that regulate scarless fetal wound healing. Although the HOX homeodomain proteins have been proposed to function as transcription factors, we have demonstrated previously that substantial fractions of the HOXB6 and HOXB4 proteins are localized to the cytoplasm throughout epidermal development. The purpose of the current study was to identify HOXB13 protein expression patterns in developing skin to elucidate potential mechanisms by which this protein might regulate aspects of tissue development and healing. HOXB13 protein expression was detected throughout the developing epidermis, with weaker signal observed in the early developing dermis. Epidermal HOXB13 signal was detected over the entire body surface, but surprisingly, essentially all of the signal was cytoplasmic in developing skin. Low-level HOXB13 protein expression was detected in adult skin and within the telogen hair follicle, and a portion of the residual signal in adult epidermis was nuclear. Expression in hyperproliferative skin conditions remained cytoplasmic with the exception of epidermis associated with Kaposi's sarcoma, which showed strong HOXB13 expression that was partially localized to the nucleus.

HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation.

The HOX homeodomain proteins are fundamental regulators of organ and tissue development, where they are thought to function as transcription factors, and HOX gene expression has been associated with numerous types of cancers. Previous studies have demonstrated that enforced expression of the HOXB4 protein transforms cultured fibroblasts and leads to a selective expansion of the hematopoietic stem cell pool, suggesting that this protein might play a role in cellular proliferation. In support of this concept, we now show that enforced expression of HOXB4 in human neonatal keratinocytes results in increased cellular proliferation and colony formation as well as decreased expression of the alpha-2-integrin and CD44 cell surface adhesion molecules. We previously have reported HOXB4 gene expression in the basal and suprabasal layers of developing human skin and now show extensive HOXB4 mRNA in psoriatic skin and basal cell carcinoma. In fetal human skin HOXB4 protein expression was both nuclear and cytoplasmic within epidermal basal cells and in hair follicle inner and outer root sheath cells, whereas strong nuclear signals were observed in the bulge region. In adult skin, HOXB4 protein expression was both nuclear and cytoplasmic, but was predominantly localized to the intermediate and differentiated cell layers. In contrast to the striking gradient patterns of HOX gene and protein expression previously described in developing spinal cord and limb, HOXB4 protein was uniformly detected in all regions of the fetal and adult skin. Although little HOXB4 signal localized to proliferative cell layers, as marked by proliferating cell nuclear antigen (PCNA) staining, in normal adult epidermis, nuclear HOXB4 protein expression substantially overlapped with PCNA-positive cell in a series of samples of hyperproliferative skin. Taken together, these data suggest that nuclear HOXB4 protein may play a role in the regulation of cellular proliferation/adhesion in developing fetal human epidermis and in hyperproliferation conditions, including cancers, in adult epidermis. Published 2002 Wiley-Liss, Inc.