Effect of cryopreservation and microencapsulation of lactic acid bacterium Enterococcus faecium MC13 for long-term storage

The aim of this work was to investigate the effect of cryoprotectants on the survival of probiotic bacterium Enterococcus faecium MC13 during freeze drying and storage. The maximum relative cell viabilities were observed when cells were freeze dried and stored at −20 °C, which is optimum temperature for the preservation of E. faecium. At all storage temperatures, trehalose was found to be retaining the highest relative cell viability than other cryoprotectants. In addition, alginate–chitosan capsules were produced to encapsulate E. faecium with the aim of enhancing survival of probiotic cells and keeping the probiotic during exposure to the harsh gastro-intestinal conditions. Encapsulation of probiotic into alginate–chitosan capsules found to be retaining higher survival of probiotic cells (4.342 ± 0.26 Log CFU mL⁻¹) at −20 °C for six months. Microencapsulated cells were resistant to simulated gastric (pH 2.0) and intestinal fluids (pH 7.5), resulting in significantly enhanced survival when compared with free cells. During in vivo treatment, capsules were broken and probiotic cells were directly released into the intestinal tract of rat. This result showed that microencapsulation of E. faecium MC13 with alginate and a chitosan coating offers an effective means of delivery of viable cells to the colon and maintains their survival during the adverse gastro-intestinal conditions.     

Chitosan and a fungal elicitor inhibit tracheary element differentiation and promote accumulation of stress lignin-like substance in Zinnia elegans xylogenic culture

We investigated the effect of elicitors on xylem differentiation and lignification using a Zinnia elegans xylogenic culture system. Water-soluble chitosan and a fungal elicitor derived from Botrytis cinerea were used as elicitors. Elicitor addition at the start of culturing inhibited tracheary element (TE) differentiation in a concentration-dependent manner, and 30 μg mL^?1 of chitosan or 16.7 μg mL^?1 of the fungal elicitor strikingly inhibited TE differentiation and lignification. Addition of chitosan (at 50 μg mL^?1) or the fungal elicitor (at 16.7 μg mL^?1) during the culturing period also inhibited TE differentiation without inhibiting cell division, except for immature TEs undergoing secondary wall thickening. Elicitor addition after immature TE appearance also caused the accumulation of an extracellular lignin-like substance. It appears that elicitor addition at the start of culturing inhibits the process by which dedifferentiated cells differentiate into xylem cell precursors. Elicitor addition during culturing also appears to inhibit the transition from xylem cell precursors to immature TEs, and induces xylem cell precursors or xylem parenchyma cells to produce an extracellular stress lignin-like substance.     

Enterococcus faecalis immunoregulates osteoclastogenesis of macrophages

Persistent apical periodontitis (PAP) is characterized by refractory inflammation and progressive bone destruction. Enterococcus faecalis infection is considered an important etiological factor for the development of PAP, although the exact mechanisms remain unknown. This study aimed at investigating the role of E. faecalis in cell proliferation, inflammatory reactions and osteoclast differentiation of macrophages using an in vitro infection model of osteoclast precursor RAW264.7 cells. A cell viability assay of cultured RAW264.7 cells exposed to live E. faecalis at a multiplicity of infection of 100 for 2 h, indicated that the infection exhibited no cytotoxic effect. Transmission electron microscopy images revealed no apoptotic changes but a rise of metabolic activity and phagocytic features in the infected RAW264.7 cells. Confocal laser scanning microscopic and flow cytometric analysis indicated that the phagocytosis of RAW264.7 cells was activated by E. faecalis infection. Furthermore, quantitative real-time PCR assays demonstrated that the expression of inflammatory cytokines was remarkably elevated in infected RAW264.7 cells. Differentiation of infected RAW264.7 cells into osteoclasts was remarkably attenuated, and expression of osteoclast marker genes as well as fusogenic genes significantly dropped. In summary, E. faecalis appears to attenuate osteoclastic differentiation of RAW264.7 precursor cells, rather stimulates them to function as macrophages.     

Evidence on antimicrobial properties and mode of action of a chitosan obtained from crustacean exoskeletons on Pseudomonas syringae pv. tomato DC3000

Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) causes bacterial speck of tomato, a widely spread disease that causes significant economical losses worldwide. It is representative of many bacterial plant diseases for which effective controls are still needed. Despite the antimicrobial properties of chitosan has been previously described in phytopathogenic fungi, its action on bacteria is still poorly explored. In this work, we report that the chitosan isolated from shrimp exoskeletons (70 kDa and 78 % deacetylation degree) exerts cell damage on Pto DC3000. Chitosan inhibited Pto DC3000 bacterial growth depending on its concentration, medium-pH, and presence of metal ion (Mg⁺²). Biochemical and cellular changes resulting in cell aggregation and impaired bacterial growth were also viewed. In vivo studies using fluorescent probes showed cell aggregation, increase in membrane permeability, and cell death, suggesting the chitosan antibacterial activity is due to its interaction as a polycation with Pto DC3000 membranes. Transmission electron microscopic analysis revealed that chitosan also caused morphological changes and damage in bacterial surfaces. Also, the disease incidence in tomato inoculated with Pto DC3000 was significantly reduced in chitosan pretreated seedlings, revealing a promising action of chitosan as nontoxic biopesticide in tomato plants. Indeed, a wider comprehensive knowledge of the mechanism of action of chitosan in phytopathogenic bacterial cells will increase the chances of its successful application to the control of spread disease in plants.     

Interleukin-17A induces cathepsin K and MMP-9 expression in osteoclasts via celecoxib-blocked prostaglandin E2 in osteoblasts

Interleukin-17 (IL-17) is a cytokine secreted primarily by T_H-17 cells that can stimulate the development of osteoclasts (osteoclastogenesis) in the presence of osteoblasts. IL-17, through osteoblasts, has indirect effects on the expression of bone resorption-related enzymes in osteoclasts, which have not been well clarified. Here, using MC3T3-E1 cells and RAW264.7 cells as osteoblasts and osteoclast precursors, we aimed to clarify these effects of IL-17A. MC3T3-E1 cells were cultured in the presence or absence of IL-17A for 72 h and the conditioned media collected (in the presence of soluble receptor activator of NF-кB ligand) and used to culture RAW264.7 cells. To assess osteoclast differentiation, adherent cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). Our analyses demonstrated that the number of TRAP-positive multinucleated cells increases after 3 days of culture in conditioned medium from IL-17A-treated cells compared to untreated controls. In addition, we observed that the levels of cathepsin K and MMP-9 increase in the conditioned medium from IL-17A-treated cells, whereas CA II expression levels remain unaffected. PGE₂ production from MC3T3-E1 cells increased in the presence of IL-17A. Celecoxib, a specific inhibitor of cyclooxygenase-2 (COX-2), blocked both the IL-17A-stimulated increase in TRAP-positive multinucleated cells and the expression of cathepsin K and MMP-9. Furthermore, when MC3T3-E1 cells were transformed with small interfering RNA to silence COX-2 expression before IL-17A treatment, the resulting conditioned medium was less effective at inducing cathepsin K and MMP-9 expression in RAW264.7 cells. These results suggest that IL-17A induces the differentiation and function of osteoclasts via celecoxib-blocked prostaglandin, mainly PGE₂, in osteoblasts.     

Loss of differences in mesangial cell phenotype between diabetic and normal rats: Role of culture passages

In mesangial cells (MC) isolated from streptozotocin (STZ)-induced diabetic rat kidneys, sensitivity to bradykinin (BK) for the induction of cell division and collagen synthesis, was found to be lower than in normal MC. Nevertheless, decreased activities could be reverted in vitro by insulin, at non-proliferative concentration (Girolami et al (1995), Can J Physiol Pharmacol 73, 848–853). The aim of the present study was to determine whether differences in the properties of diabetic MC could be ascribed to the diabetic state per se, and/or to experimental conditions, ie culture replating. Through successive cell replating, normal and diabetic types of MC were compared in terms of proliferation, contraction, free calcium concentration in response to KCl depolarization, and in relation to the expression of two cytoskeleton proteins specific to muscle cells, myosin and dystrophin. Studies of proliferation, contraction and free calcium concentration consistently showed that passage 5 was a limit beyond which differences between the two MC types were very small and sometimes non-significant. We found that the mean maximum contraction (MMC) and especially the proportion of contractile cells (PCC) among diabetic cells was lower than in normal MC. In addition, loss in proliferation activity and in [Ca²⁺]_i concentrations were also found to occur during these five early passages. Dystrophin, a new marker of contractile phenotype recently described in smooth muscle cell (Leis et al (1994) Cell Biol Toxicol 10, 305), was first localized in MC and was compared with myosin also expressed in MC. However, during the course of cell replating and/or with the diabetic state, no visible quantitative changes were detected in the expression of the two contractile proteins. We conclude that cultured mesangial cells undergo phenotype modulations, as observed in other cells, in particular smooth muscle cells and consequently, comparative studies between normal and diabetic MC should not be carried out after the 5th passage.     

Carboxymethyl dextran-trimethyl chitosan coated superparamagnetic iron oxide nanoparticles: An effective siRNA delivery system for HIV-1 Nef

Gene therapy, including small interfering RNA (siRNA) technology, is one of the leading strategies that help to improve the outcomes of the current therapeutic systems against HIV-1 infection. The successful therapeutic application of siRNAs requires their safe and efficient delivery to specific cells. Here, we introduce a superparamagnetic iron oxide nanoparticle (SPION) for delivering siRNA against HIV-1 nef (anti-nef siRNA) into two cell lines, HEK293 and macrophage RAW 264.7. SPIONs were coated with trimethyl chitosan (TMC), and thereafter, different concentrations of SPION–TMC were coated with different ratios of a carboxymethyl dextran (CMD) to modify the physicochemical properties and improve the biological properties of the nanocarriers. The nanoparticles exhibited a spherical shape with an average size of 112 nm. The obtained results showed that the designed delivery route enhanced the uptake of siRNA into both HEK293 and RAW 264.7 cells compared with control groups. Moreover, CMD–TMC–SPIONs containing anti-nef siRNA significantly reduced the expression of HIV-1 nef in HEK293 stable cells. The modified siRNA-loaded SPIONs also displayed no toxicity or apoptosis-inducing effects on the cells. The CMD–TMC–SPIONs are suggested as potential nanocarriers for siRNA delivery in gene therapy of HIV-1 infection.     

A comparison of Thai silk fibroin-based and chitosan-based materials on in vitro biocompatibility for bone substitutes

The novel hybrid scaffolds fabricated from silk fibroin, gelatin, low deacetylation degree chitosan and hydroxyapatite were investigated for their in vitro biocompatibility and osteoconductivity to mouse pre-osteoblast cell line (MC3T3-E1) and rat bone marrow-derived stem cells (MSC). We found that gelatin-conjugated silk fibroin films and scaffolds dominantly promoted cell adhesion and proliferation. Film and scaffold prepared from gelatin-conjugated silk fibroin with hydroxyapatite grown crystals effectively enhanced osteogenic differentiation of both cell types, as evaluated by alkaline phosphatase activity and calcium content. However the blend of hydroxyapatite/low deacetylation degree chitosan hybrid materials did not support cell growth. Furthermore, the blended hydroxyapatite in the bulk scaffold was found to be less effective for osteogenic differentiation than the scaffold with hydroxyapatite grown crystals. The comparative study between MC3T3-E1 and MSC showed that both cell types had similar trend of proliferation and osteogenic differentiation on the same material. Also, higher proliferative rate of MC3T3-E1 than MSC was observed.     

Chitosan gallate as potential antioxidant biomaterial

Chitosan gallate were synthesized using a free radical-induced grafting reaction. Chitosan gallate showed enhanced water-solubility compared to plain chitosan, and exhibited good thermal stability. The IC₅₀ value of chitosan gallate against 2,2-diphenyl-1-picrylhydrazyl (DPPH) was 17.86 μg/mL. In addition, chitosan gallate effectively inhibited the generation of intracellular reactive oxygen species (ROS), and also suppressed lipid peroxidation in RAW264.7 macrophage cells. Chitosan gallate also exhibited the protection effect on genomic DNA damage by induced hydroxyl radical, and up-regulated the protein expression of antioxidant enzymes including superoxide dismutase-1 and glutathione reductase under H₂O₂-mediated oxidative stress in RAW264.7 macrophage cells. These results indicate that chitosan gallate might be potential antioxidant biomaterials.     

Ferric iron reduction by Thiobacillus ferrooxidans at extremely low pH-values

When ferrous iron and sulfur were supplied, cells of T. ferrooxidans in a well-aerated medium started growth by oxidizing ferrous iron. After ferrous iron depletion a lagphase followed before sulfur oxidation started. During sulfur oxidation at pH-values below 1.3 (±0,2) the ferrous iron concentration increased again, although the oxygen saturation of the medium amounted to more than 95%. The number of viable cells did not increase. Thus resting cells of T. ferrooxidans, which are oxidizing sulfur to maintain their proton balance, reduce ferric to ferrous iron. The ferrous iron-oxidizing system seemed to be inhibited at pH-values below 1.3. At a pH-value of 1.8 the ferrous iron was reoxidized at once. A scheme for the linkage of iron- and sulfur metabolism is discussed.     

Is it possible to track intracellular chitosan nanoparticles using magnetic nanoparticles as contrast agent?

Drug delivery systems prepared with nanostructures are able to overcome biological barriers. However, one of the main challenges in the use of these nanosystems is their internalization by macrophages. This study aims to prepare and characterize chitosan nanoparticles incorporating maghemite nanoparticles and investigate their intracellular tracking in RAW 264.7 macrophages in vitro. Then, maghemite nanoparticles were encapsulated within chitosan nanoparticles by ionotropic gelification method. The images from transmission electron microscopy were used to investigate the intracellular penetration of conjugated nanoparticles by macrophages using different times. Our data suggests that magnetic nanoparticles are suitable to act as a contrast agent to investigate the cellular internalization of chitosan nanoparticles.     

Cell labeling with magnetic nanoparticles: Opportunity for magnetic cell imaging and cell manipulation

This tutorial describes a method of controlled cell labeling with citrate-coated ultra small superparamagnetic iron oxide nanoparticles. This method may provide basically all kinds of cells with sufficient magnetization to allow cell detection by high-resolution magnetic resonance imaging (MRI) and to enable potential magnetic manipulation. In order to efficiently exploit labeled cells, quantify the magnetic load and deliver or follow-up magnetic cells, we herein describe the main requirements that should be applied during the labeling procedure. Moreover we present some recommendations for cell detection and quantification by MRI and detail magnetic guiding on some real-case studies in vitro and in vivo.     

In-vitro antioxidative,antiinflammatory properties of Aurea helianthus leaf extract a Korean traditional medicinal plant

The leaf of Aurea helianthus (A. helianthus Jinhuakui) is popularly used in China traditional medicine, however, scientific evidence on its antioxidant properties rarely studied. In this study, biological activities of A. helianthus leave’s 80% ethanol extract (AHL) were investigated. The measured total polyphenol and flavonoid content of AHL was 184.24 ± 5.01 mg GAE/g and 102.53 ± 0.98 mg NAR/g. AHL showed the highest α, α-diphenyl-β-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS) radical scavenging activities of 98.30 ± 0.18% at 1000 µg/mL. DPPH and ABTS radical scavenging activities significantly increased in a AHL concentration-dependent manner. AHL treatment significantly suppressed the generation of pro-inflammatory mediators, including nitric oxide (NO), in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. AHL demonstrated strong anti-inflammatory activity that reduced NO production in LPS-stimulated RAW 264.7 cells. To test the potential protective effect of AHL, the antioxidant capacity, on the cell growth, viability of a human hepatoma cell (HepG2) and Raw 264.7 cell were investigated. AHL also enhanced cytotoxicity on the proliferation of HepG2 cells and was capable of inhibiting 56% against LPS at 400 µg/mL. The results of this study the potential of AHL as an excellent antioxidant substance for inhibiting inflammatory mediators. Therefore, AHL may be used as a therapeutic approach to various inflammatory diseases.     

Evaluation of Electrostatic Interaction between Lysolecithin and Chitosan in Two-Layer Tuna Oil Emulsions by Nuclear Magnetic Resonance (NMR) Spectroscopy

The main objective of this work was to investigate the electrostatic interaction between lysolecithin and chitosan in two-layer tuna oil-in-water emulsions using nuclear magnetic resonance (NMR) spectroscopy. The influence of chitosan concentration on the stability and properties of these emulsions was also evaluated. The 5 wt% tuna oil one-layer emulsion (lysolecithin-stabilized oil droplets without chitosan) and two-layer emulsions (lysolecithin-chitosan stabilized oil droplets) containing 5 wt% tuna oil, 1 wt% lysolecithin and various chitosan concentrations (0.025–0.40 wt%) were prepared. The one-dimensional (1D) ³¹P and ¹H NMR spectra of emulsions were then recorded at 25 °C. The results showed that addition of chitosan affected the stability and properties of lysolecithin-stabilized one-layer emulsions. The ³¹P NMR peak of the choline head group on lysolecithin molecules disappeared when chitosan was added at concentrations above neutralization concentration (> 0.05 wt%). The ¹H NMR peak intensity monitoring free amino groups (?NH ₃ ⁺) of chitosan showed a strong positive linear relationship to the chitosan concentration with a high correlation coefficient (R² ≈ 0.99). This ¹H NMR peak in emulsions could not be detected for chitosan in emulsions lower than saturation concentration (< 0.15 wt%). These phenomena indicate an electrostatic interaction between lysolecithin and chitosan at droplet surface in emulsion and were consistent with the results from zeta-potential measurements. The T ₂* relaxation time of the choline head group (N-(CH ₃)₃) signal of lysolecithin also confirmed that lysolecithin-chitosan electrostatic interaction occurs at the surface of oil droplets in two-layer emulsions. The results suggest that NMR spectroscopy can be used as an alternative method for monitoring the electrostatic interaction between surfactant and oppositely charged electrolytes or biopolymers in two-layer emulsions.     

Evaluation of efficacy on RANKL induced osteoclast from RAW264.7 cells

Established RAW264.7 cell lines for osteoclastic differentiation has been widely engaged in bone homeostasis research, however, the efficacy of RANKL independently stimulating has rarely been defined, because protocols were usually developed and modified by various laboratories. Otherwise, problematic issues are also lie in the cell's seeding density, RANKL stimulating time point, and distinguishing osteoclastogenesis ability of RANKL-treated RAW264.7 cells. Therefore, in the current study, we examined the efficacy of various concentrations of RANKL-treated RAW264.7 for its osteoclastic differentiation with or without pretreated other costimulators such as: LPS and/or M-CSF. The oteoclastogenesis ability of RANKL-treated RAW264.7 cells was demonstrated by bone resorption pit, F-actin, and osteoclastogenesis specific marker studies. Besides that, through tartrate-resistant acid phosphatase (TRAP) staining, we clarified to start the treatment with 30 ng/ml RANKL at 12 hr after seeded RAW264.7 with the density of 6.25 × 10 ³ cells/cm ² manifested an significantly increased number of multinucleated osteoclastic cells. Overall, our results establishing an optimal method for RANKL independently inducing RAW 264.7 cell osteoclastic differentiation, which could efficiently generate osteoclasts in vitro for significant advances in our understanding of bone biology.     

Identification and biological evaluation of flavonoids from the fruits of Prunus mume

This Letter describes the identification of potent antioxidant and anti-osteoporosis agents from the fruits of Prunus mume. From the methanol extract, a novel flavan dimer, characterized as 2β,3β-epoxy-5,7,4′-trihydroxyflavan-(4α → 8)-epicatechin (1), was isolated along with five known flavonoids (2–6). Their structures were determined based on extensive spectroscopic analysis, including IR, HRESIMS, 1D- and 2D-NMR, and CD spectra. The antioxidant activities of compounds 1–6 were evaluated in terms of their peroxyl radical-scavenging (Trolox equivalent) and reducing capacities. All isolates showed potent peroxyl radical-scavenging and reducing activities at concentrations of 1–10 μM. Among them, compounds 1 and 2 were the most active at 1 μM. Anti-osteoporosis activities were investigated using both murine osteoblastic MC3T3-E1 cells and osteoclastic RAW 264.7 cells. Compounds 2, 3, and 6 significantly stimulated the differentiation of osteoblastic MC3T3-E1 cells to increase collagen synthesis or mineralization functions of osteoblasts. Compounds 1, 3, 4, and 6 significantly suppressed tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastic RAW 264.7 macrophage cells.     

Fucoidan from Sargassum sp. and Fucus vesiculosus reduces cell viability of lung carcinoma and melanoma cells in vitro and activates natural killer cells in mice in vivo

Fucoidan is known to exhibit crucial biological activities, including anti-tumor activity. In this study, we examined the influence of crude fucoidan extracted from Sargassum sp. (MTA) and Fucus vesiculosus (SIG) on Lewis lung carcinoma cells (LCC) and melanoma B16 cells (MC). In vitro studies were performed using cell viability analysis and showed that SIG and MTA fucoidans significantly decreased the viable number of LCC and MC cells in a dose-response fashion. Histochemical staining showed morphological changes of melanoma B16 cells after exposure to fucoidan. The observed changes were indicative of crude fucoidan induced apoptosis. Male C57BL/6JJCL mice were subjected to daily i.p. injections over 4 days with either SIG or MTA fucoidan (50 mg/kg body wt.). The cytolytic activity of natural killer (NK) cells was enhanced by crude fucoidan in a dose-dependent manner as indicated by ⁵¹Cr labeled YAC-1 target cell release. This study provides substantial indications that crude fucoidan exerts bioactive effects on lung and skin cancer model cells in vitro and induces enhanced natural killer cell activity in mice in vivo.     

Investigation of MC3T3-E1 cell behavior on the surface of GRGDS-coupled chitosan

The GRGDS (Gly-Arg-Gly-Asp-Ser) peptide has intermediate affinity to alphaVbeta3 and alphaIIbbeta3, which are the integrins most reported to be involved in bone function. In this study, biomimetic chitosan films modified with GRGDS peptide were prepared and were used as a substrate for the in vitro culture of MC3T3-E1 cells in order to investigate the effect of GRGDS modification on MC3T3-E1 cell behavior. The results of electron spectroscopy for chemical analysis (ESCA), attenuated total reflection-Fourier transform infrared spectra (ATR-FTIR), and amino acid analysis (AAA) demonstrated that the chitosan films were successfully modified with GRGDS peptides and that the surface density of the immobilized GRGDS was on the order of 10(-9) mol/cm2. The immobilization of the GRGDS sequence on chitosan as well as the peptide concentration play a significant role in MC3T3-E1 cell behavior. MC3T3-E1 cell attachment, proliferation, migration, differentiation, and mineralization were remarkably greater on GRGDS-coupled chitosan than on unmodified chitosan. Besides, the degree of acceleration of these biological processes was found to be dependent on peptide density. Competitive inhibition of MC3T3-E1 cell attachment using soluble GRGDS peptides indicated that the interaction of MC3T3-E1 cells with the surface of the materials was ligand-specific. Cytoskeleton organization in the fully spread MC3T3-E1 cells was highly obvious on GRGDS-coupled chitosan when compared to the lack of actin fibers noted in the round MC3T3-E1 cells on unmodified chitosan. These results suggest that MC3T3-E1 cell function can be modulated, in a peptide density-dependent manner, by the immobilization of GRGDS peptide on chitosan used for scaffold-based bone tissue engineering.     

Formulation of Pseudomonas chlororaphis strains for improved shelf life

Formulations of Pseudomonas strains with long-term shelf life are needed for commercial use in biological disease control and growth promotion in crops. In the present work Pseudomonas chlororaphis (Pc) 63-28 formulated with coconut fiber [moisture content (MC) of 80%], talc (MC 8%) or peat (MC 40%), with or without the addition of carboxymethylcellulose or xanthan gum, and formulations of Pc 63-28 and P. chlororaphis TX-1 in coconut fiber with water contents (v:v) of 75%, 45%, and 25%, were evaluated in terms of shelf life and cell viability. The shelf life of Pc 63-28 was longer when formulated in coconut fibre with a MC was 80% than in the other formulations and longer at 3 ± 1 °C compared to 22 ± 1 °C. Densities of viable Pc 63-28 cells in coconut fiber stored at 3 ± 1 °C did not decline significantly during 224 days when the MC was 80% and within 120 days at 75% MC. Densities of Pc TX-1 in coconut fiber of 75% MC did not decline within 60 days at 3 ± 1 °C. P. chlororaphis 63-28 survived longer in deionized water and buffer than in canola oil. Cells of Pc 63-28 cells formulated in coconut fibre of 80% MC after storage for 140 days at 3 ± 1 °C in coconut fiber improved hydroponic growth of hydroponic lettuce and better than cells freshly recovered from culture. We conclude that coconut fiber is a carrier of superior performance in maintaining shelf life of Pseudomonas strains. The observed shelf life would be sufficient for practical use of Pseudomonas strains as tools for disease control and growth promotion in crops.     

Siderophore Production by Pathogenic Mucorales and Uptake of Deferoxamine B

Clinical reports have established that mucormycosis, mainly caused by Rhizopus spp., frequently occurs in patients treated with deferoxamine B (DFO, Desferal^®) which is misappropriated by these fungi. Siderophore production by twenty mucoralean isolates was therefore investigated using a commercial iron-depleted culture medium. Siderophore production was detected for most of the isolates. Our experiments confirmed that feroxamine B (iron chelate of DFO) promoted in vitro growth of Rhizopus arrhizus. Electrophoretic analysis of somatic extracts revealed iron-regulated proteins of 60 and 32 kDa which were lacking in iron-depleted culture conditions. Using a fluorescent derivative of deferoxamine B, we showed by fluorescence microscopy the entry of the siderophore within the fungal cells, thus suggesting a shuttle mechanism encompassing the uptake of the entire siderophore-ion complex into the cell. This useful tool renders possible a better understanding of iron metabolism in Mucorales which could lead to the development of new diagnostic method or new antifungal therapy using siderophores as imaging contrast agents or active drug vectors.