Calcium Concentration In Medium Research Articles

In vitro cluster buds regeneration and control of shoot tip necrosis in tissue cultures of Trichosanthes cucumerina L.

Trichosanthes cucumerina L. is a medicinal vine with great potential that can be used as a vegetable. In this study, an efficient in vitro regeneration system of T. cucumerina was established by inducing adventitious buds and controlling Shoot Tip Necrosis (STN) in the in vitro culture. The results of hormone single factor and orthogonal experiments showed that 6-BA had a significant effect on inducing adventitious buds. The best medium for bud proliferation was MS + 6-BA 3.0 mg·L− 1 + KIN 2.0 mg·L− 1 + NAA 0.5 mg·L− 1, and the proliferation capacity was 14.68. However, after three consecutive subcultures, a higher incidence of STN (78.86%) was observed. The results of the STN control experiment showed that reducing the concentration of inorganic salt, shortening the subculture interval, and increasing the Ca2+ in the medium could effectively control the occurrence of STN. However, reducing the concentration of inorganic salt and shortening the subculture interval excessively reduces the proliferation capacity significantly. Considering both the incidence of STN and proliferation capacity, the measures proposed in this study to alleviate STN were to keep the concentration of inorganic salt unchanged, adjust the subculture interval from 45 days to 40 days, and increase the Ca2+ concentration in the medium to 480 mg·L− 1. When the subculture interval was 40 d, the stem segments obtained the lowest STN incidence (8.3%) and higher shoot proliferation capacity (10.02) on the medium of MS + 3.0 mg·L− 1 6-BA + 2.0 mg·L− 1 KIN + 0.5 mg·L− 1 NAA + 360.36 mg·L− 1 Ca2+ (360 mg·L− 1 is the concentration of additional calcium to be added, and after this addition, the total calcium concentration in the MS medium was 480.48 mg·L− 1). The best rooting medium for stem segments was MS + 0.50 mg·L− 1 NAA; the rooting rate was 100%, and no STN occurred. After acclimation, the survival rate of the rooted plantlet reached 95% after 40 days in the soil matrix of coconut husk: humus soil: perlite = 2: 2: 1 (v/v). The results of this study not only reveal the mechanism of STN in T. cucumerina, but also provide technical support and a theoretical basis for its further breeding and cultivation.

High-frequency low-intensity semiconductor laser irradiation enhances osteogenic differentiation of human cementoblast lineage cells

PurposeLaser irradiation activates a range of cellular processes in the periodontal components and promotes tissue repair. However, its effect on osteogenic differentiation of human cementoblast lineage cells remains unclear. This study aimed to examine the effects of high-frequency semiconductor laser irradiation on the osteogenic differentiation of human cementoblast lineage (HCEM) cells.MethodsHCEM cells were cultured to reach 80% confluence and irradiated with a gallium-aluminum-arsenide (Ga-Al-As) semiconductor laser with a pulse width of 200 ns and wavelength of 910 at a dose of 0–2.0 J/cm2. The outcomes were assessed by analyzing the mRNA levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and type I collagen (COLL1) using real-time polymerase chain reaction (PCR) analysis 24 h after laser irradiation. Cell mineralization was evaluated using ALP activity, calcium deposition, and Alizarin Red staining.ResultsThe laser-irradiated HCEM cells showed significantly enhanced gene expression levels of ALP, RUNX2, and COLL1 as well as ALP activity and calcium concentration in the culture medium compared with the non-irradiated cells. In addition, enhanced calcification deposits were confirmed in the laser-irradiated group compared with the non-irradiated group at 21 and 28 days after the induction of osteogenic differentiation.ConclusionHigh-frequency semiconductor laser irradiation enhances the osteogenic differentiation potential of cultured HCEM cells, underscoring its potential utility for periodontal tissue regeneration.

Sustained Calcium(II)-Release to Impart Bioactivity in Hybrid Glass Scaffolds for Bone Tissue Engineering.

In this study, we integrated different calcium sources into sol-gel hybrid glass scaffolds with the aim of producing implants with long-lasting calcium release while maintaining mechanical strength of the implant. Calcium(II)-release was used to introduce bioactivity to the material and eventually support implant integration into a bone tissue defect. Tetraethyl orthosilicate (TEOS) derived silica sols were cross-linked with an ethoxysilylated 4-armed macromer, pentaerythritol ethoxylate and processed into macroporous scaffolds with defined pore structure by indirect rapid prototyping. Triethyl phosphate (TEP) was shown to function as silica sol solvent. In a first approach, we investigated the integration of 1 to 10% CaCl2 in order to test the hypothesis that small CaCl2 amounts can be physically entrapped and slowly released from hybrid glass scaffolds. With 5 and 10% CaCl2 we observed an extensive burst release, whereas slightly improved release profiles were found for lower Calcium(II) contents. In contrast, introduction of melt-derived bioactive 45S5 glass microparticles (BG-MP) into the hybrid glass scaffolds as another Calcium(II) source led to an approximately linear release of Calcium(II) in Tris(hydroxymethyl)aminomethane (TRIS) buffer over 12 weeks. pH increase caused by BG-MP could be controlled by their amount integrated into the scaffolds. Compression strength remained unchanged compared to scaffolds without BG-MP. In cell culture medium as well as in simulated body fluid, we observed a rapid formation of a carbonated hydroxyapatite layer on BG-MP containing scaffolds. However, this mineral layer consumed the released Calcium(II) ions and prevented an additional increase in Calcium(II) concentration in the cell culture medium. Cell culture studies on the different scaffolds with osteoblast-like SaOS-2 cells as well as bone marrow derived mesenchymal stem cells (hMSC) did not show any advantages concerning osteogenic differentiation due to the integration of BG-MP into the scaffolds. Nonetheless, via the formation of a hydroxyapatite layer and the ability to control the pH increase, we speculate that implant integration in vivo and bone regeneration may benefit from this concept.

Hemocyanin Modification of Chitosan Scaffolds with Calcium Phosphate Phases Increase the Osteoblast/Osteoclast Activity Ratio-A Co-Culture Study.

The ongoing research on biomaterials that support bone regeneration led to the quest for materials or material modifications that can actively influence the activity or balance of bone tissue cells. The bone biocompatibility of porous chitosan scaffolds was modified in the present study by the addition of calcium phosphates or hemocyanin. The first strategy comprised the incorporation of calcium phosphates into chitosan to create a biomimetic chitosan—mineral phase composite. The second strategy comprised dip-coating of chitosan scaffolds with hemocyanin extracted from crayfish hemolymph. The cytocompatibility was assessed in a mono-culture of human bone marrow stromal cells (hBMSCs) and their differentiation to osteoblasts; in a mono-culture of human monocytes (hMs) and their maturation to osteoclasts; and in a co-culture of hBMSC/osteoblasts—hM/osteoclasts. Mineral incorporation caused an increase in scaffold bioactivity, as shown by reduced calcium concentration in the cell culture medium, delayed differentiation of hBMSCs, and reduced osteoclastic maturation of hMs in mono-culture. Dip-coating with hemocyanin led to increased proliferation of hBMSCs and equivalent osteoclast maturation in mono-culture, while in co-culture, both an inhibitory effect of mineral incorporation on osteoblastogenesis and stimulatory effects of hemocyanin were observed. It was concluded that highly bioactive scaffolds (containing mineral phases) restrain osteoblast and osteoclast development, while hemocyanin coating significantly supports osteoblastogenesis. These influences on the osteoblasts/osteoclasts activity ratio may support scaffold-driven bone healing in the future.

Calcium Binding by Arabinogalactan Polysaccharides Is Important for Normal Plant Development.

Arabinogalactan proteins (AGPs) are a family of plant extracellular proteoglycans involved in many physiological events. AGPs are often anchored to the extracellular side of the plasma membrane and are highly glycosylated with arabinogalactan (AG) polysaccharides, but the molecular function of this glycosylation remains largely unknown. The β-linked glucuronic acid (GlcA) residues in AG polysaccharides have been shown in vitro to bind to calcium in a pH-dependent manner. Here, we used Arabidopsis (Arabidopsis thaliana) mutants in four AG β-glucuronyltransferases (GlcAT14A, -B, -D, and -E) to understand the role of glucuronidation of AG. AG isolated from glcat14 triple mutants had a strong reduction in glucuronidation. AG from a glcat14a/b/d triple mutant had lower calcium binding capacity in vitro than AG from wild-type plants. Some mutants had multiple developmental defects such as reduced trichome branching. glcat14a/b/e triple mutant plants had severely limited seedling growth and were sterile, and the propagation of calcium waves was perturbed in roots. Several of the developmental phenotypes were suppressed by increasing the calcium concentration in the growth medium. Our results show that AG glucuronidation is crucial for multiple developmental processes in plants and suggest that a function of AGPs might be to bind and release cell-surface apoplastic calcium.

Urban structure degradation caused by growth of plants and microbial activity

The purpose of this study was to isolate microorganisms associated to surface-affected concrete structures and to measure the in vitro dissolution of concrete based on the release of elements such as calcium and silicon. Although many microorganisms were detected only a fungus was capable of significantly decreasing the culture medium pH and releasing both elements. The molecular characterization allowed to identify the microorganism as Aspergillus carbonaurius, a citric-acid producing fungus that dissolved concrete in the in vitro test. After seven days of incubation, the soluble calcium concentration in the uninoculated culture medium containing concrete was 172.3 mg/L, while in the inoculated medium it was 525.0 mg/L. The soluble silicon concentration in the uninoculated medium was 10.3 mg/L, while in the inoculated medium it was 50.1 mg/L. These findings showed that plants and microorganisms rendered a synergistic effect accelerating the biodeterioration of concrete.

Calcium Concentration in Culture Medium as a Nondestructive and Rapid Marker of Osteogenesis

Artificial bones made of β-tricalcium phosphate (β-TCP) combined with bone marrow-derived mesenchymal stromal cells (BM-MSCs) are used for effective reconstruction of bone defects caused by genetic defects, traumatic injury, or surgical resection of bone tumors. However, the selection of constructs with high osteogenic potential before implantation is challenging. The purpose of this study was to determine whether the calcium concentration in BM-MSC culture medium can be used as a nondestructive and simple osteogenic marker for selecting tissue-engineered grafts constructed using β-TCP and BM-MSCs. We prepared three cell passages of BM-MSCs derived from three 7-week-old, male Fischer 344 rats; the cells were cultured in osteoinductive medium in the presence of β-TCP for 15 days. The medium was replaced with fresh medium on day 1 in culture and subsequently changed every 48 h; it was collected for measurement of osteocalcin secretion and calcium concentration by enzyme-linked immunosorbent assay and X-ray fluorescence spectrometry, respectively. After cultivation, the constructs were implanted subcutaneously into the backs of recipient rats. Four weeks after implantation, the alkaline phosphatase (ALP) activity and osteocalcin content of the constructs were measured. A strong inverse correlation was observed between the calcium concentration in the medium and the ALP activity and osteocalcin content of the constructs, with Pearson's correlation coefficients of 0.92 and 0.90, respectively. These results indicate that tissue-engineered bone with high osteogenic ability can be selected before implantation based on low calcium content of the culture medium, resulting in successful bone formation after implantation. This nondestructive, simple method shows great promise for assessing the osteogenic ability of tissue-engineered bone.

Evaluation of Calcium Release from Pulp-capping Material Using International Organization for Standardization Guidelines

Aims and Objectives: Cytotoxicity assays are one of the methods used to assess the biocompatibility of dental materials. The objective of this study was to determine if surface area/volume ratios designated by the International Organization for Standardization (ISO) for biological evaluation of medical devices affects cytotoxicity and calcium release in an endodontic pulp-capping materials. Material and Methods: Human periodontal ligament fibroblasts (PDLFb) were treated with extractables derived from the calcium silicate pulp-capping material TheraCal LC at pellet surface area/culture medium volume ratios of 0.2, 0.5, and 1.0 cm2/mL. A colorimetric cytotoxicity assay (MTT) and phase-contrast microscopy was used to assess biocompatibility against the fibroblasts. The concentration of calcium leached into culture medium from TheraCal LC at pellet surface area/culture medium volume ratios of 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, and 6.0 cm2/mL after 24 hours was determined by colorimetric assay. Results: Both MTT assays and microscopy demonstrate that at a pellet surface area/culture medium volume ratio of 1.0 cm2/mL TheraCal LC extractable challenge to fibroblasts results in a significant decrease in cell viability ( P < 0.001) compared to control. TheraCal LC calcium release into culture medium varied with the surface area/volume ratio, with concentrations ranging from 2 to 8 times the calcium concentration in culture medium. Conclusion: Pellet surface area/culture medium volume ratios impacted parameters of TheraCal LC used in in vitro studies to determine biocompatibility with oral tissues. The ISO guidelines should be followed for all in vitro cytotoxicity studies of dental materials.

Characterization of a novel protease fromBacillus cereusand evaluation of an eco-friendly hydrolysis of a brewery byproduct

Proteases and proteolytic enzymes constitute one of the most important groups of enzymes and are attracting worldwide attention in attempts to exploit their physiological and biotechnological applications. In this study, partial purifications and biochemical and antimicrobial characterizations of a protease from Bacillus cereus spp., originally isolated from fermented cabbage, were carried out. The crude extract obtained after purification, involving ammonium sulphate precipitation and dialysis, was designated as a partially purified protease (PPP). The obtained PPP had a specific activity of 0.395–2.539 U/g at 32 °C, with maximum activities for the fractions precipitated at 60 and 80% ammonium sulphate. The PPP activity ranged between 20 and 55 °C, with an optimum temperature at 40 °C. At 60 °C, the PPP retained more than 30% of its activity. The optimum pH for the PPP was achieved at pH 9, indicating the alkaline source of the enzyme. Protease production was specifically dependent on the calcium concentration in the culture medium. Also the robustness of the protease on brewer's spent grain hydrolysis was demonstrated. This suggests a potential eco-friendly application of the enzyme. Finally, it was found that the PPP inhibited the growth of Escherichia coli O157:H7. This novel property of the PPP liberated by the B. cereus spp. could provide important future benefits to industry. Copyright © 2015 The Institute of Brewing & Distilling

Presence of a thapsigargin-sensitive calcium pump in Trypanosoma evansi: Immunological, physiological, molecular and structural evidences

In higher eukaryotes, the sarco-endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) is characterized for its high sensitivity to low concentrations of thapsigargin (TG), a very specific inhibitor. In contrast, SERCA-like enzymes with different sensitivities to TG have been reported in trypanosomatids. Here, we characterized a SERCA-like enzyme from Trypanosoma evansi and evaluated its interaction with TG. Confocal fluorescence microscopy using BODIPY FL TG and specific anti-SERCA antibodies localized the T. evansi SERCA-like enzyme in the ER and confirmed its direct interaction with TG. Moreover, the use of either 1 μM TG or 25 μM 2′,5′-di (tert-butyl)-1,4-benzohydroquinone prevented the reuptake of Ca2+ and consequently produced a small increase in the parasite cytosolic calcium concentration in a calcium-free medium, which was released from the ER pool. A 3035 bp-sequence coding for a protein with an estimated molecular mass of 110.2 kDa was cloned from T. evansi. The corresponding gene product contained all the invariant residues and conserved motifs found in other P-type ATPases but lacked the calmodulin binding site. Modeling of the three-dimensional structure of the parasite enzyme revealed that the amino acid changes found in the TG-SERCA binding pocket do not compromise the interaction between the enzyme and the inhibitor. Therefore, we concluded that T. evansi possesses a SERCA-like protein that is inhibited by TG.

Effect of Calcium Concentration in Growth Medium on Oxalate Content and Evaluation of the Role of Guttation in the Regulation of Oxalate in Eddo

The effects of calcium concentrations in the growth medium on oxalate content of leaf blades, petioles and corms and the involvement of guttation in the regulation of oxalate homeostasis were investigated in eddo (Colocasia esculenta (L.) Schott var. antiquorum Hubbard & Rehder). The plants were grown hydroponically in solutions containing 0 mM calcium, 1 mM calcium nitrate (control), 15 mM calcium nitrate or 15 mM calcium chloride. Total oxalate content (soluble plus insoluble) of leaf blades, petioles and corms did not differ with the calcium concentration in solutions containing 1 mM or 15 mM calcium nitrate or 0 mM calcium. The soluble oxalate content of these parts decreased as the calcium concentration of the solution was increased. Solutions containing 15 mM calcium nitrate or 15 mM calcium chloride gave a significantly lower proportion of soluble oxalate content to total oxalate content in each part, especially in leaf blades than 0 mM calcium or 1 mM calcium nitrate. In contrast, a positive correlation was found between insoluble oxalate content and calcium concentration in the solution. These results demonstrate that high calcium concentrations in the growth medium reduce soluble oxalate content of the plant. Soluble oxalate was detected in eddo guttation fluid. Soluble oxalate content in this fluid (mg mL−1) and the amount of soluble oxalate exuded by guttation (mg leaf−1 night−1) were significantly lower in the solutions containing 15 mM calcium than in those containing 0 mM and 1 mM calcium. These results indicate that guttation may affect the concentration of soluble oxalate in the plant bodies although not strongly contributing to a decrease in soluble oxalate content in eddo grown under high calcium conditions.

Establishment of Functional Acinar-like Cultures from Human Salivary Glands

Disorders of human salivary glands resulting from therapeutic radiation treatment for head and neck cancers or from the autoimmune disease Sjögren syndrome (SS) frequently result in the reduction or complete loss of saliva secretion. Such irreversible dysfunction of the salivary glands is due to the impairment of acinar cells, the major glandular cells of protein, salt secretion, and fluid movement. Availability of primary epithelial cells from human salivary gland tissue is critical for studying the underlying mechanisms of these irreversible disorders. We applied 2 culture system techniques on human minor salivary gland epithelial cells (phmSG) and optimized the growth conditions to achieve the maintenance of phmSG in an acinar-like phenotype. These phmSG cells exhibited progenitor cell markers (keratin 5 and nanog) as well as acinar-specific markers—namely, α-amylase, cystatin C, TMEM16A, and NKCC1. Importantly, with an increase of the calcium concentration in the growth medium, these phmSG cells were further promoted to acinar-like cells in vitro, as indicated by an increase in AQP5 expression. In addition, these phmSG cells also demonstrated functional calcium mobilization, formation of epithelial monolayer with high transepithelial electrical resistance (TER), and polarized secretion of α-amylase secretion after β-adrenergic receptor stimulation. Taken together, suitable growth conditions have been established to isolate and support culture of acinar-like cells from the human salivary gland. These primary epithelial cells can be useful for study of molecular mechanisms involved in regulating the function of acinar cells and in the loss of salivary gland function in patients.

Physiological and ultrastructural studies on calcium oxalate crystal formation in some plants

Results from light, scanning, and transmission electron microscopic examination of CaOx crystal formation in 2 plant species, namely Corchorus olitorius L. and Malva parviflora L., revealed the presence of crystalline deposits. These deposits were of prismatic type in the case of C. olitorius and of druse type in the case of M. parviflora. Ultrastructurally, some differences were observed between crystal idioblast and noncrystal cells. Moreover, the effect of heavy metals and different calcium concentrations on the growth of calcium oxalate crystals in leaves of the 2 plants was investigated. Different analytical techniques were used to determine the influence of exogenous cadmium (Cd), lead (Pb), copper (Cu), and zinc (Zn) on CaOx deposition and to detect the presence of these metals in CaOx crystals. We found a positive correlation between the calcium concentration in the nutrient medium and the production of calcium oxalate crystals in the leaves of hydroponically grown plants. On the other hand, addition of the heavy metals to the nutrient medium decreased the number of crystals. Our investigation suggests that CaOx crystals do not play a major role in heavy metal detoxification in these 2 plant species but do play an important role in bulk calcium regulation.

Shear stress-induced improvement of red blood cell deformability

Classically, it is known that red blood cell (RBC) deformability is determined by the geometric and material properties of these cells. Experimental evidence accumulated during the last decade has introduced the concept of active regulation of RBC deformability. This regulation is mainly related to altered associations between membrane skeletal proteins and integral proteins, with the latter serving to anchor the skeleton to the lipid matrix. It has been hypothesized that shear stress induces alterations of RBC deformability: the current study investigated the dynamics of the transient improvement in deformability induced by shear stress at physiologically-relevant levels. RBC were exposed to various levels of shear stress (SS) in a Couette type shearing system that is part of an ektacytometer, thus permitting the changes in RBC deformability during the application of SS to be monitored. Initial studies showed that there is an increase in deformability of the RBC subjected to SS in the range of 5-20 Pa, with kinetics characterized by time constants of a few seconds. Such improvement in deformability, expressed by an elongation index (EI), was faster with higher levels of SS and hence yielded shorter time constants: absolute values of EI increased by 3-8% of the starting level. Upon the removal of the shear stress, this response by RBC was reversible with a slower time course compared to the increase in EI during application of SS. Increased calcium concentration in the RBC suspending medium prevented the improvement of deformability. It is suggested that the improvement of RBC deformability by shear forces may have significant effects on blood flow dynamics, at least in tissues supplied by blood vessels with impaired vasomotor reserve, and may therefore serve as a compensating mechanism for the maintenance of adequate microcirculatory perfusion.

Plants sensitivity on nickel under different conditions of iron or calcium concentration in the nutrient medium

The sensitivity of six vegetable plants on nickel at early stages of their growth was investigated by index of tolerance. Besides the possibility of nickel fitostabilization by additional application of iron or calcium was tested. The experiment was conducted on Petri dishes. Different concentrations of nickel (0; 0,03; 0,06mM Ni as nickel sulphate), iron (0,05; O,OlmM Fe as Fe<sup>2+</sup> citrate) and calcium (0,50; 0,75; lmM Ca as calcium carbonate) were added. Taking into consideration the sensitivity, investigated vegetables can be ordered in the following way: <i>Cucurbita pepo conv. giromontiina </i>L.><i>Lactuca sativa</i> L.><i>Sinapis alba</i> L.><i>Spinacia oleracea</i> L.=<i>Zea mays</i> var. <i>saccharata</i> Kcke.><i>Phaseolus vulgaris</i> L. Positive, statistically significant effect ofnickel fitostabilization (0,03 or 0,06mM Ni) on elongative growth by the iron application (0,10mM Fe) was shown for <i>Zea mays</i> var. <i>saccharata</i> Kcke independently of Ni concentration in the nutrient medium as well as for <i>Sinapis alba</i> L. and <i>Phaseolus vulgaris</i> L. in 0,06mM Ni. Addition as much as 0,75mM Ca in the presence 0,03mM Ni had positive result on Sinapis alba L and <i>Phaseolus vulgaris</i> L. seedlings as well as on Zea mays var. saccharata Kcke and <i>Lactuca sativa</i> L. roots and <i>Cucurbita pepo</i> convar. <i>giromontiina</i> L. shoots. Addition of 0,75mM Ca in the presence 0,06mM Ni promoted elongative growth of <i>Zea mays</i> var. <i>saccharata</i> Kcke seedlings. Application lmM Ca resulted in the promotion of elongative growth of <i>Zea mays var. saccharata</i> Kcke. roots (0,03mM Ni) as well as <i>Spinacia oleracea</i> L. roots (0,06mM Ni).

The calcium-dependent regulation of spheroid formation and cardiomyogenic differentiation for MSCs on chitosan membranes

Mesenchymal stem cells (MSCs) were recently found to form three-dimensional (3D) multicellular spheroids on chitosan membranes. The exact mechanism of spheroid formation, however, remains unclear. In this study, the regulation of spheroid formation for adipose derived adult stem cells (ADAS) grown on chitosan membranes was examined. By varying the membrane thickness, calcium concentration in culture medium, and acetylation extent of chitosan, the physico-chemical characteristics of chitosan that modulated spheroid formation was elucidated. The capacity of cardiomyogenic differentiation was further evaluated. Results suggested that the calcium binding capacity of chitosan may affect the cell–substrate and cell–cell interactions and critically influence the dynamics of spheroid formation. The intracellular calcium level was elevated for ADAS spheroids on chitosan. Chitosan-bound calcium was observed to enter the cells. The expression of N-cadherin was upregulated for ADAS spheroids on chitosan, evidenced by quantitative RT-PCR and Western blot. After the induction by 5-aza, the expression levels of cardiac marker genes (Gata4, Nkx2.5, Tnnt2, and Myh6) were remarkably enhanced (about four-fold) for ADAS on chitosan vs. tissue culture polystyrene or polyvinyl alcohol. Immunofluorescence staining confirmed the expression of cardiac-associated tight junction protein ZO-1 for ADAS grown on chitosan membranes. The gene expression of Wnt11 was significantly upregulated for ADAS spheroids on chitosan at 3 days and 12 days. We suggested that Wnt11 may be involved in the spheroid formation and cardiomyogenic differentiation of MSCs on chitosan membranes. Spheroids formed on the acetylated chitosan or polyvinyl alcohol membranes failed to show such behavior. The properties of MSC spheroids were therefore determined by the culture substrate.

Actin is Involved in Pollen Tube Tropism Through Redefining the Spatial Targeting of Secretory Vesicles

In order to accurately target the embryo sac and deliver the sperm cells, the pollen tube has to find an efficient path through the pistil and respond to precise directional cues produced by the female tissues. Although many chemical and proteic signals have been identified to guide pollen tube growth, the mechanism by which the tube changes direction in response to these signals is poorly understood. We designed an experimental setup using a microscope-mounted galvanotropic chamber that allowed us to induce the redirection of in vitro pollen tube growth through a precisely timed and calibrated external signal. Actin destabilization, reduced calcium concentration in the growth medium and inhibition of calcium channel activity decreased the responsiveness of the pollen tube to a tropic trigger. An increased calcium concentration in the medium enhanced this response and was able to rescue the effect of actin depolymerization. Time-lapse imaging revealed that the motion pattern of vesicles and the dynamics of the subapical actin array undergo spatial reorientation prior to the onset of a tropic response. Together these results suggest that the precise targeting of the delivery of new wall material represents a key component in the growth machinery that determines directional elongation in pollen tubes.

Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling

BackgroundSkin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes.MethodsWe have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods.ResultsCOCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional in vitro models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability.ConclusionsWe propose that COCA constitutes a good experimental system for in vitro and in vivo skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.

Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines

In this study, we determined the LD50 (50% lethal dose) for cell death, and the ED50 (50% of cell population staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat, U937, CHO-K1, and GH3) exposed to 10-ns electric pulses (EP). We found that the LD50 varied substantially across the cell lines studied, increasing from 51 J/g for Jurkat to 1861 J/g for HeLa. PS externalized at doses equal or lower than that required for death in all cell lines ranging from 51 J/g in Jurkat, to 199 J/g in CHO-K1. Pr uptake occurred at doses lower than required for death in three of the cell lines: 656 J/g for CHO-K1, 634 J/g for HeLa, and 142 J/g for GH3. Both Jurkat and U937 had a LD50 lower than the ED50 for Pr uptake at 780 J/g and 1274 J/g, respectively. The mechanism responsible for these differences was explored by evaluating cell size, calcium concentration in the exposure medium, and effect of trypsin treatment prior to exposure. None of the studied parameters correlated with the observed results suggesting that cellular susceptibility to injury and death by 10-ns EP was largely determined by cell physiology. In contrast to previous studies, our findings suggest that permeabilization of internal membranes may not necessarily be responsible for cell death by 10-ns EP. Additionally, a mixture of Jurkat and HeLa cells was exposed to 10-ns EP at a dose of 280 J/g. Death was observed only in Jurkat cells suggesting that 10-ns EP may selectively kill cells within a heterogeneous tissue.

Calcium concentration in vitrification medium affects the developmental competence of in vitro matured ovine oocytes

The present study was designed to determine whether different calcium concentrations in the vitrification solutions could improve the developmental competence of in vitro matured ovine oocytes after cryopreservation. In vitro matured oocytes were vitrified with 16.5% ethylene glycol (EG) + 16.5% dimethylsulfoxide (DMSO) vitrification media. The base media contain different calcium concentrations, so that five experimental groups were obtained: TCM/FCS (TCM 199 + 20% fetal calf serum (FCS), [Ca 2+] 9.9 mg/dl); PBS/FCS (Dulbecco Phosphate Buffered Saline (PBS) + 20% FCS, [Ca 2+] 4.4 mg/dl); PBS CaMg free/FCS (PBS without Ca 2+ and Mg 2+ + 20% FCS [Ca 2+] 2.2 mg/dl); PBS/BSA (PBS + 0.4% bovine serum albumin (BSA), [Ca 2+] 3.2 mg/dl) and PBS CaMg free/BSA (PBS without Ca 2+ and Mg 2+ +0.4% BSA, [Ca 2+] 0.4 mg/dl). After warming, the oocytes from the five experimental groups were assessed for survival, spontaneous parthenogenetic activation and developmental capacity via in vitro fertilization. Oocyte survival after vitrification procedures was better preserved in group PBS CaMg free/FCS compared to the others (P < 0.05). In addition, a positive correlation was found between calcium concentration in vitrification solutions and spontaneous parthenogenetic activation (correlation index 0,82; P < 0.001). Development of vitrified oocytes was significantly affected by vitrification media composition (P < 0.01). In particular, oocytes from group PBS CaMg free/FCS led to higher cleavage rates and blastocyst rate compared to the others. Our data showed that lowering calcium concentration in the vitrification medium improves the blastocyst rate of vitrified ovine oocytes, probably reducing the effect of EG + DMSO during vitrification. On the contrary, the replacement of FCS with BSA dramatically reduces the developmental potential of these oocytes.