Sclerotial Formation of Polyporus umbellatus by Low Temperature Treatment under Artificial Conditions

Background

Polyporus umbellatus sclerotia have been used as a diuretic agent in China for over two thousand years. A shortage of the natural P. umbellatus has prompted researchers to induce sclerotial formation in the laboratory.

Methodology/Principal Finding

P. umbellatus cultivation in a sawdust-based substrate was investigated to evaluate the effect of low temperature conditions on sclerotial formation. A phenol-sulfuric acid method was employed to determine the polysaccharide content of wild P. umbellatus sclerotia and mycelia and sclerotia grown in low-temperature treatments. In addition, reactive oxygen species (ROS) content, expressed as the fluorescence intensity of mycelia during sclerotial differentiation was determined. Analysis of ROS generation and sclerotial formation in mycelia after treatment with the antioxidants such as diphenyleneiodonium chloride (DPI), apocynin (Apo), or vitamin C were studied. Furthermore, macroscopic and microscopic characteristics of sclerotial differentiation were observed. Sclerotia were not induced by continuous cultivation at 25°C. The polysaccharide content of the artificial sclerotia is 78% of that of wild sclerotia. In the low-temperature treatment group, the fluorescent intensity of ROS was higher than that of the room temperature (25°C) group which did not induce sclerotial formation all through the cultivation. The antioxidants DPI and Apo reduced ROS levels and did not induce sclerotial formation. Although the concentration-dependent effects of vitamin C (5–15 mg mL⁻¹) also reduced ROS generation and inhibited sclerotial formation, using a low concentration of vitamin C (1 mg mL⁻¹) successfully induced sclerotial differentiation and increased ROS production.

Conclusions/Significance

Exposure to low temperatures induced P. umbellatus sclerotial morphogenesis during cultivation. Low temperature treatment enhanced ROS in mycelia, which may be important in triggering sclerotial differentiation in P. umbellatus. Moreover, the application of antioxidants impaired ROS generation and inhibited sclerotial formation. Our findings may help to provide new insights into the biological mechanisms underlying sclerotial morphogenesis in P. umbellatus.     

Determination of optimal carbon source and pH value for sclerotial formation of Polyporus umbellatus under artificial conditions

Polyporus umbellatus is one of the precious medicinal fungi, with sclerotia used as a diuretic agent and antidote in China for many years. This has led to the present interest in producing sclerotia of P. umbellatus in the laboratory due to a decreased abundance in natural sources. Here, we investigated the determining factors for sclerotial formation in P. umbellatus. Five carbon sources, namely, maltose, fructose, glucose, sucrose and soluble starch with different initial pH values were evaluated for their effects on mycelial growth and sclerotial development of P. umbellatus. Maltose, fructose and glucose could induce sclerotial formation of P. umbellatus. Sucrose and soluble starch could stimulate growth of the fungus but had no effect on sclerotial formation. The most efficient sclerotial production occurred with maltose followed by fructose and a pH of 5. In addition, different macroscopically evident characteristics of sclerotial development of P. umbellatus induced by different carbon sources were also observed. Our findings could provide new insights into further research on sclerotial production in P. umbellatus under artificial cultivation.     

A new method to induce sclerotial differentiation in Polyporus umbellatus by split-plate culture

Polyporus umbellatus is one of the most valuable medicinal fungi, and its sclerotium has been used as a diuretic agent and an antidote in traditional Chinese medicine. In nature, Polyporus umbellatus has almost been depleted because of over-exploitation and lack of natural habitats. Thus, artificial sclerotia production has increased. This study aimed at finding an effective method to induce sclerotia, and selected the split-plate culture method. One side contained fructose agar medium (FAM), while the other side contained nutrient-limited medium. It was observed that sclerotia were only formed on the nutrient-limited medium side but scarcely emerged on the FAM side, even when the fructose concentration on both sides were the same. The sclerotial differentiation rate was 100% and the sclerotial yield was 106% higher than in the conventional way. In conclusion, the split-plate culture method is an effective way to induce P. umbellatus sclerotia in the laboratory.     

The inhibitory effect of Ca2+ on the flavonoid production of Tetrastigma hemsleyanum suspension cells induced by metal elicitors

Tetrastigma hemsleyanum suspension cells were treated with four metal salts to screen suitable elicitors for the promotion of plant cell biomass and flavonoid production. The effects of calcium ions (Ca²⁺) on induction were also studied. It was found that the most effective elicitors were 50 μM of the heavy metal ion copper (Cu²⁺) and 100 μM of the rare earth element cerium (Ce³⁺). The maximal biomass levels under respective treatments over a 16-d culture period increased by 1.3- and 1.6-fold, and the total flavonoid content was 1.8- and 1.6-fold greater than the control, respectively. Reducing the exogenous Ca²⁺ concentration or adding Ca²⁺ antagonists (1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′,N-tetraacetc acid (EGTA) or 1 mM verapamil) strengthened inductive effects of metal elicitors and enhanced flavonoid production. However, 0.5 μM of the calcium ionophore A23187 showed contrary results. The increase in exogenous Ca²⁺ concentration in the presence of A23187 suppressed H₂O₂ bursts and peroxidase activity caused by metal elicitors. The results suggest that Ca²⁺ plays an inhibitory role in the plant cell response to metal elicitors. This suppression could have been caused by Ca²⁺ preventing the cells from absorbing metal ions and then easing the induction, or because the decrease of Ca²⁺ concentration worked as an induction signal. Therefore, reducing the Ca²⁺ concentration in culture medium, or adding Ca²⁺ antagonists could be used to improve flavonoid production and cell growth in combination with induction by metal elicitors during in vitro culture of T. hemsleyanum suspension cells.     

Analysis of phenomena involved in the apical growth of<Emphasis Type="Italic"> Phycomyces blakesleeanus</Emphasis>

The effects of the Ca²⁺/H⁺ exchanger A23187 and the K⁺/H⁺ exchanger nigericin, the electrogenic membrane-potential depleters valinomycin and CCCP, and the calcium channel blockers ruthenium red, nifedipine, and nitrendipine on the apical growth of Phycomyces blakesleeanus were analyzed. While all of the compounds inhibited the growth of germlings in liquid medium, the Ca²⁺ channel blockers were the least effective. Chitin synthesis in vivo was also sensitive to the inhibitors; here again, the calcium channel blockers were less efficient, and their effect occurred after a lag phase, in contrast to the electroneutral ionophores whose effects were immediate. The ionophores rapidly inhibited protein secretion, and reduced the number of secretory vesicles and chitosomes in the hyphal apex of P. blakesleeanus. The results suggest that not only tip-to-base calcium gradients but also transmembrane ionic gradients and membrane potential have a role in the apical growth of P. blakesleeanus. They are probably involved in the formation, migration, and/or fusion with the plasmalemma of secretory vesicles and chitosomes.     

Calcium Rapidly Down-Regulates Human Renal Epithelial Sodium Channels Via a W-7-Sensitive Mechanism

Increases in intracellular calcium (Ca²⁺) inhibit renal sodium (Na⁺) absorption in cortical collecting ducts, but the precise mechanism is unclear. We, therefore, studied the effects of raising intracellular Ca²⁺ (using 10 µmol/L A23187, a Ca²⁺ ionophore) on wild-type and Liddle-mutated human epithelial Na⁺ channels (hENaC) expressed in Xenopus oocytes, using the dual-electrode voltage clamp technique. A23187 decreased amiloride-sensitive Na⁺ current by 55 % in oocytes expressing wild-type hENaC, an effect prevented by co-exposure to 50 μmol/L W-7 (to inhibit the Ca²⁺/calmodulin complex). By contrast, co-exposure to 50 μmol/L calphostin (to inhibit protein kinase C) or 5 μmol/L KN-62 (to inhibit Ca²⁺/calmodulin-dependent protein kinase II) had no effect on the decrease in amiloride-sensitive Na⁺ current elicited by A23187 alone. Whereas A23187 reduced amiloride-sensitive Na⁺ current in oocytes expressing wild-type hENaC, it had no similar effect in those expressing Liddle-mutated hENaCs, suggesting that the activity of individual Na⁺ channels in situ was unchanged by the rise in intracellular Ca²⁺. These data suggest that the A23187-induced rise in intracellular Ca²⁺ inhibited wild-type hENaC through a W-7-sensitive mechanism, which likely reflected enhanced removal of Na⁺ channels from the cell membrane by endocytosis. We, therefore, propose that Na⁺ absorption in cortical collecting duct cells is inhibited by Ca²⁺, possibly when complexed with calmodulin.     

Inhibition of mechanical strain-induced fetal rat lung cell proliferation by gadolinium,a stretch-activated channel blocker

Normal growth of the fetal lung is dependent upon fetal breathing movements. We have previously demonsrated that mechanical strain, simulating fetal breathing movements, stimulated DNA synthesis and cell division by reaggregated alveolar-like structures of fetal rat lung cells. Herein, we report that both intracellular and extracellular calcium modulate strain-induced proliferative activity. Strain-induced cell proliferation was inhibited by BAPTA/AM, an intracellular calcium chelator. The intracellular calcium modulators, cyclopiazonic acid and 2,5-di-(tert-butyl)-1, 4-benzohydroquinone, increased DNA synthesis of unstrained cultures and partially reduced strain-induced cell growth activity. A similar effect was noted with the calcium ionophore A23187. Extracellular Ca²⁺ increased DNA synthesis in unstrained cultures in a concentration-dependent fashion. The stimulatory effect of strain on DNA synthesis was also dependent on the calcium concentration in the medium. Furthermore, strain-enhanced DNA synthesis was inhibited by the presence of a divalent ion chelator, EGTA, in the medium. Mechanical strain increased ⁴⁵Ca²⁺ influx within 1 min after the onset strain. This rapid entry of calcium was not affected by calcium channel blockers, such as verapamil or Ni²⁺. Calcium channel blockers verapamil, nifedipine, Ni²⁺, Co²⁺, or La³⁺ also did not inhibit strain-induced cell growth activity. In contrast, gadolinium, a stretch-activated channel blocker, inhibited strain-induced ⁴⁵Ca²⁺ influx and suppressed strain-enhanced DNA synthesis. We conclude that the entry of calcium into cells through stretch-activated ion channels plays a critical role in strain-induced fetal lung cell proliferation. © 1994 Wiley-Liss, Inc.     

The Role of Calcium in Differentiation of Human Adipose-Derived Stem Cells to Adipocytes

Differentiation process of mesenchymal stem cells (MSCs) into adipocyte is involved in obesity. Multiple factors such as Ca²⁺ play important roles in different stages of this process. Because of the complicated roles of Ca²⁺ in adipogenesis, the aim of present investigation was to study the influx and efflux of Ca²⁺ into and out of the cells during adipogenesis. Adipose-derived MSCs were used to differentiate into adipocytes. MSCs were exposed to 2.5 mM Ca²⁺ or 1.8 mM Ca²⁺ plus calcium ionophore, A23187, for 3 days. Lipid staining, triglycerides (TG) content, and glyceraldehyde phosphate dehydrogenase (GAPDH) activity were evaluated to confirm the efficiency of the differentiation. Gene expression of GLUT4, PPARγ2, RAR-α, and calreticulin, as well as the protein levels of GLUT4 and PPARγ2 were determined. Ca²⁺ and in particular Ca²⁺ plus A23187 significantly lowered the efficiency of differentiation accompanied by decrease in intracellular TG deposits, GAPDH activity and alleviation of gene, and protein levels of GLUT4 and PPARγ2. While calreticulin and RAR-α were remarkably upregulated in A23187 group. This study showed the inhibitory effects of calcium in adipogenesis. Additionally, it indicated the greater inhibitory effect of calreticulin and RAR-α in controlling adipogenesis by higher levels of calcium.     

Red Blood Cell and Serum Magnesium Levels Among Children and Adolescents With Sickle Cell Anemia

This study investigated neurotoxicity of chronic fluorosis in the rat hippocampus. Newly weaning, male, Sprague-Dawley (SD) rats were administered 15, 30, and 60 mg/L sodium fluoride (NaF) solution (fluorine ion concentration 8.25, 16.50, and 33.00 mg/L, respectively), and tap water, for 18 months. The neurotoxicological mechanism was examined with a focus on intracellular calcium overload. Results showed that as the fluoride concentration increased, calcium ion concentration [Ca²⁺], the expression of calcium/calmodulin-dependent protein kinase II α (CaMKIIα), and the expression of catus proto-oncogene protein c-fos (c-fos) all tend to increase. Compared to the control group, Ca²⁺, CaMKIIα, and c-fos significantly increased (P < 0.05) in the moderate-fluoride and the high-fluoride groups. These results indicate that Ca²⁺/CaMKIIα/c-fos channel signal may be the molecular mechanism of central nervous system damage caused by chronic fluoride intoxication. Moreover, elevated Ca²⁺ concentration in the hippocampus may be the initiating factor of neuronal apoptosis induced by fluoride.     

Cation-permeable vacuolar ion channels in the moss Physcomitrella patens: a patch-clamp study

Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca²⁺ on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K⁺ (E _K), indicating K⁺ selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at ?80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na⁺ and K⁺ selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na⁺ influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca²⁺ revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca²⁺ activated SV channels, vacuolar Ca²⁺ inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca²⁺ was also determined. These channels were active even without Ca²⁺ (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca²⁺ on the cytoplasmic side. Apart from monovalent cations (K⁺ and Na⁺), SV channels were permeable to divalent cations (Ca²⁺ and Mg²⁺). Both monovalent and divalent cations passed through the channels in the same direction—from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.     

Calcium in the Regulation of Gravitropism by Light

The red light requirement for positive gravitropism in roots of corn (Zea mays cv “Merit”) provides an entry for examining the participation of calcium in gravitropism. Applications of calcium chelators inhibit the light response. Calcium channel blockers (verapamil, lanthanum) can also inhibit the light response, and a calcium ionophore, A23187, can substitute for light. One can substitute for red light by treatments which have elsewhere been shown to trigger Ca²⁺ influx into the cytosol, e.g. heat or cold shock. Agents which are known to be agonists of the phosphatidylinositol second messenger system (serotonin, 2,4-dichlorophenoxyacetic acid, deoxycholate) can each partially substitute for the red light, and Li⁺ can inhibit the light effect. These experiments suggest that the induction of positive gravitropism by red light involves a rise in cytoplasmic Ca²⁺ concentration, and that a contribution to this end may be made by the phosphatidylinositol second messenger system.     

Calcium antagonists and calmodulin inhibitors block cytokinin-induced bud formation in Funaria

The plant hormone cytokinin stimulates nuclear migration followed by an asymmetric cell division in target cells of the protonema of the moss Funaria hygrometrica, leading to bud formation. The role of calcium in this developmental event was investigated by examining the effects of various calcium antagonists on the cytokinin-induced division. Calcium-free medium (buffered with EGTA), the extracellular Ca²⁺ antagonist La³⁺ (lanthanum), and the Ca²⁺ channel inhibitors D 600 and verapamil all block bud formation. These inhibitions are partially reversed by washing the cells or by raising the extracellular [Ca²⁺]. The Ca²⁺ ionophore A23187 partially reversed the effects of D 600 and verapamil. Bud formation is also inhibited by the intracellular Ca²⁺ antagonist TMB-8 (8-diethylamino)ocytl 3,4,5-trimethoxybenzoate HCl), and this inhibition is partially reversed by washing or raising the extracellular [Ca²⁺]. The cross walls of both the filaments and bud initial cells formed during TMB-8 exposure exhibit a distorted morphology. High concentrations of TMB-8 block nuclear migration. The calmodulin inhibitor trifluoperazine stops cytokinin-induced budding more effectively than the related compound chlorpromazine. Low concentrations of these two compounds do not affect nuclear migration; however, the target cell does not enter mitosis. These results support the hypothesis that a rise in intracellular calcium mediates cytokinin-induced bud formation in Funaria. It is concluded that the proposed cytokinin-induced rise in intracellular calcium may be effected in part by the activation of calmodulin. The essential source of Ca²⁺ appears to be extracellular, because blocking Ca²⁺ uptake with Ca²⁺ transport inhibitors can block both nuclear migration and subsequent division.     

Effect of copper-induced oxidative stress on sclerotial differentiation and antioxidant properties of Penicillium thomii PT95 strain

Penicillium thomii PT95 strain was able to form abundant orange, sand-shaped sclerotia in which carotenoids were accumulated. The aim of this work was to determine the effects of copper-induced oxidative stress on the sclerotial differentiation and antioxidant properties of PT95 strain. The results showed that the time of exudates initiation, sclerotial initiation and sclerotial maturation of PT95 strain were advanced in 1–2 days under the copper-induced oxidative stress growth conditions. The analytical results of sclerotial biomass, carotenoids content in sclerotia showed that copper-induced oxidative stress favored the sclerotial differentiation and biosynthesis of carotenoids. Under the copper-induced oxidative stress growth conditions, the total phenolics content and DPPH free radical scavenging activity of sclerotia of this fungus were decreased as compared with the control. However, the oxidative stress induced by a lower amount of CuSO₄ in media could enhance significantly the reducing power of sclerotia.     

ROS and trehalose regulate sclerotial development in Rhizoctonia solani AG-1 IA

Rhizoctonia solani AG-1 IA is the causal agent of rice sheath blight (RSB) and causes severe economic losses in rice-growing regions around the world. The sclerotia play an important role in the disease cycle of RSB. In this study, we report the effects of reactive oxygen species (ROS) and trehalose on the sclerotial development of R. solani AG-1 IA. Correlation was found between the level of ROS in R. solani AG-1 IA and sclerotial development. Moreover, we have shown the change of ROS-related enzymatic activities and oxidative burst occurs at the sclerotial initial stage. Six genes related to the ROS scavenging system were quantified in different sclerotial development stages by using quantitative RT-PCR technique, thereby confirming differential gene expression. Fluorescence microscopy analysis of ROS content in mycelia revealed that ROS were predominantly produced at the hyphal branches during the sclerotial initial stage. Furthermore, exogenous trehalose had a significant inhibitory effect on the activities of ROS-related enzymes and oxidative burst and led to a reduction in sclerotial dry weight. Taken together, the findings suggest that ROS has a promoting effect on the development of sclerotia, whereas trehalose serves as an inhibiting factor to sclerotial development in R. solani AG-1 IA.     

TRPV-5 Mediates a Receptor Activator of NF-κB (RANK) Ligand-induced Increase in Cytosolic Ca2+ in Human Osteoclasts and Down-regulates Bone Resorption

Most of the signaling effectors located downstream of receptor activator of NF-κB (RANK) activation are calcium-sensitive. However, the early signaling events that lead to the mobilization of intracellular calcium in human osteoclasts are still poorly understood. The Ca²⁺-sensitive fluorescent probe Fura2 was used to detect changes in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) in a model of human osteoclasts. Stimulating these cells with receptor activator of NF-κB ligand (RANKL) induced a rapid and significant increase in [Ca²⁺]_i. Adding extracellular Ca²⁺ chelators, depleting intracellular stores, and the use of a phospholipase C inhibitor all indicated that the Ca²⁺ was of extracellular origin, suggesting the involvement of a Ca²⁺ channel. We showed that none of the classical Ca²⁺ channels (L-, T-, or R-type) were involved in the RANKL-induced Ca²⁺ spike. However, the effect of high doses of Gd³⁺ did suggest that TRP family channels were present in human osteoclasts. The TRPV-5 channel was expressed in osteoclasts and was mainly located in the cellular area in contact with the bone surface. Furthermore, the RNA inactivation of TRPV-5 channel completely inhibited the RANKL-induced increase in [Ca²⁺]_i, which was accompanied in the long term by marked activation of bone resorption. Overall, our results show that RANKL induced a significant increase in [Ca²⁺]_i of extracellular origin, probably as a result of the opening of TRPV-5 calcium channels on the surface of human osteoclasts. Our findings suggest that TRPV-5 contributes to maintaining the homeostasis of the human skeleton via a negative feedback loop in RANKL-induced bone resorption.     

Methyl jasmonate-induced lateral root formation in rice: The role of heme oxygenase and calcium

Lateral roots (LRs) play important roles in increasing the absorptive capacity of roots as well as to anchor the plant in the soil. Therefore, understanding the regulation of LR development is of agronomic importance. In this study, we examined the effect of methyl jasmonate (MJ) on LR formation in rice. Treatment with MJ induced LR formation and heme oxygenase (HO) activity. As well, MJ could induce OsHO1 mRNA expression. Zinc protoporphyrin IX (the specific inhibitor of HO) and hemoglobin [the carbon monoxide/nitric oxide (NO) scavenger] reduced LR formation, HO activity and OsHO1 expression. LR formation and HO activity induced by MJ was reduced by the specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-oxide. The effects of Ca²⁺ chelators, Ca²⁺-channel inhibitors, and calmodulin (CaM) antagonists on LR formation induced by MJ were also examined. All these inhibitors were effective in reducing the action of MJ. However, Ca²⁺ chelators and Ca²⁺ channel inhibitors induced HO activity when combining with MJ further. It is concluded that Ca²⁺ may regulate MJ action mainly through CaM-dependent mechanism.     

Fungal species residing in the sclerotia of <Emphasis Type="Italic">Polyporus umbellatus</Emphasis>

Sclerotia of Polyporus umbellatus is a traditional Chinese herb. The sclerotia can survive in soils for long time and their growth depends upon a symbiotic association with Armillariella mellea. But it is unclear whether other fungi reside or play a role in the sclerotia. In this study, wild sclerotial samples were collected from seven provinces, which span southwest to northeast China. A total of 148 fungal isolates were recovered from the sclerotia of P. umbellatus and classified into 19 morphological taxa. Seventeen belonged to five genera: Fusarium, Eurotium, Penicillium, Geomyces and Mucor. The fungi found within the sclerotia varied depending on the province from which they were collected. The possible role of these fungi is discussed.     

Overproduction of poly(β-malic acid) (PMA) from glucose by a novel Aureobasidium sp. P6 strain isolated from mangrove system

After over 100 strains of Aureobasidium spp isolated from mangrove system were screened for their ability to produce poly(β-malic acid) (PMA), it was found that Aureobasidium sp. P6 strain among them could produce high level of Ca²⁺-PMA. Fourteen percent glucose and 6.5 % CaCO₃ in the medium were the most suitable for Ca²⁺-PMA production. Then, 100.7 g/l of Ca²⁺-PMA was produced using Aureobasidium sp. P6 strain within 168 h at flask level. During 10-l batch fermentation, when the medium contained 12.0 % glucose, 98.7 g/l of Ca²⁺-PMA in the culture and 14.7 g/l of cell dry weight were obtained within 156 h, leaving 0.34 % reducing sugar in the fermented medium. When glucose concentration in the fermentation medium was 14.0 %, 118.3 g/l of Ca²⁺-PMA in the culture and 16.4 g/l of cell dry weight were obtained within 168 h, leaving 0.4 % reducing sugar in the fermented medium. After purification of Ca²⁺-PMA from the culture and acid hydrolysis of the pure Ca²⁺-PMA, analysis of HPLC showed that Aureobasidium sp. P6 strain only produced two main components of Ca²⁺-PMA and minor amount of calcium malate and that the hydrolysate of PMA was mainly composed of calcium malate. This is the first time to report that the novel yeast strain Aureobasidium sp. P6 strain isolated from the mangrove systems can produce such high amount of Ca²⁺-PMA.     

Effects of extracellular calcium on viability and osteogenic differentiation of bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) have been extensively used for tissue engineering. However, the effect of Ca²⁺ on the viability and osteogenic differentiation of BMSCs has yet to be evaluated. To determine the dose-dependent effect of Ca²⁺ on viability and osteogenesis of BMSCs in vitro, BMSCs were cultured in calcium-free DMEM medium supplemented with various concentrations of Ca²⁺ (0, 1, 2, 3, 4, and 5 mM) from calcium citrate. Cell viability was analyzed by MTT assay and osteogenic differentiation was evaluated by alkaline phosphatase (ALP) assay, Von Kossa staining, and real-time PCR. Ca²⁺ stimulated BMSCs viability in a dose-dependent manner. At slightly higher concentrations (4 and 5 mM) in the culture, Ca²⁺ significantly inhibited the activity of ALP on days 7 and 14 (P < 0.01 or P < 0.05), significantly suppressed collagen synthesis (P < 0.01 or P < 0.05), and significantly elevated calcium deposition (P < 0.01) and mRNA levels of osteocalcin (P < 0.01 or P < 0.05) and osteopontin (P < 0.01 or P < 0.05). Therefore, elevated concentrations of extracellular calcium may promote cell viability and late-stage osteogenic differentiation, but may suppress early-stage osteogenic differentiation in BMSCs.