Osmotic Stabilizer Research Articles

An efficient protoplast isolation and regeneration system in Coprinus comatus

An efficient protocol for protoplast isolation and regeneration was first established inCoprinus Comatus. The highest yield of protoplasts was up to 8.9 × 106 cells/ml in digestion solution containing 2.0% lywallzyme and 0.6 M KCl after incubation of four-day-old mycelia at 30°C for 3 h; among which, about 1.4% protoplasts could be regenerated into mycelia after 4 - 6 days of incubation at 25°C in CYM medium with0.6 M mannitol as osmotic stabilizer. The results are beneficial for breeding new cultivars by the methods such as protoplast fusion, mutagenesis as well as transformation. Moreover, the stepwise procedure for protoplast liberation and regeneration could be referred in other species. Key words: Coprinus comatus, mycelia, lywallzyme, protoplast, liberation and regeneration.

Protoplast formation and regeneration in Lactobacillus delbrueckii.

Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii.

Isolation and fusion of protoplasts from the phytopathogenic fungus Sclerotium rolfsii (Sacc.)

Sclerotium rolfsii (Sacc.) is a serious plant pathogenic fungus and lacks perfect (basidial) stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x10(5)/g mycelia and 2.8x10(5)/g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30% (w/v) with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato.

Production and regeneration of protoplasts from orchid Mycorrhizal Fungi Epulorhiza repens and Ceratorhiza sp.

The aim of this work was to study the standardization of conditions to obtain and regenerate Epulorhiza repens and Ceratorhiza sp. protoplasts. For E. repens, the largest number of protoplasts (8.0 × 10(6) protoplasts/mL) was obtained in 0.6 M KCl, using 15 mg/mL of Lysing Enzymes, and 2-day-old fungal mycelium. When 0.5 M sucrose was used as osmotic stabilizer, the highest frequency of regeneration was achieved (8.5 %); 80.0 % of protoplasts were nucleated, and 20.0 % anucleated. For Ceratorhiza sp., the largest number of protoplasts (4.0 × 10(7) protoplasts/mL) was achieved in 0.6 M NaCl, when 15 mg/mL of Lysing Enzymes and 15mg/mL of Glucanex, with 2-day-old fungal mycelium were used. The highest frequency of regeneration was 6.7 % using 0.5 M sucrose as osmotic stabilizer; 88.8 % of protoplasts were nucleated, and 11.2 % anucleated.

Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum

The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h) in 0.6 mol l-1 NaCl as the osmotic stabilizer, with 30 mg ml-1 Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6) protoplatos ml-1 in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l-1 and 1 mol l-1 were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35% and 14.54%, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.

BacMam Virus Transduced Cardiomyoblasts Can Be Used for Myocardial Transplantation Using AP-PEG-A Microcapsules: Molecular Cloning, Preparation, andIn VitroAnalysis

The potential of genetically modified cardiomyoblasts in treating damaged myocardium is well known. However, efficient delivery of these cells is of major concern during treatment. The limiting factors are the massive cell death that occurs soon after their intramyocardial transplantation into the beating heart. To address these problems, we generated recombinant baculoviruses (BacMam viruses) which efficiently transduced cardiomyoblast cells under optimized conditions. These genetically modified cells were then protected in a new polymeric microcapsule using poly-ethylene-glycol (PEG), alginate, and poly-L-lysine (PLL) polymers for efficient delivery. Results showed that microcapsules maintain cell viability and support cell proliferation for at least 30 days. The capsules exhibit strong immunoprotective potential and have high mechanical and osmotic stability with more than 70% intact capsules. The encased transduced cells showed a rapid transgene expression inside the capsule for at least 15 days. However, preclinical studies are needed to further explore its long-term functional benefits.

Isolation and fusion of protoplasts from the phytopathogenic fungus sclerotium rolfsii(sacc.)

Sclerotium rolfsii (Sacc.) is a serious plant pathogenic fungus and lacks perfect (basidial) stage in production. Protoplast fusion technology was employed to reconstruct fusants from this fungus. Two strains designated as A and R were used. Maximum protoplast yields of 3.8x105/g mycelia and 2.8x105/g mycelia were formed in strains A and R respectively. Osmotic stabilizer sucrose 1M gave maximum yield. Lysing enzyme at the rate of 15mg/ml was found best for yield. Fusion of protoplasts from strains A and R was carried out in fusion media containing PEG 4000 30% (w/v) with 0.2mM CaCl2. Four fusants F1, F2, F3 and F4 were recovered. Morphological, physiological and pathogenic characters of fusants were compared with parent strains on carrots, beans and tomato.

Effect of solute hydrogen bonding capacity on osmotic stability of lysosomes

The effect of solute hydrogen bonding capacity on the osmotic stability of lysosomes was examined through measurement of free enzyme activity of lysosomes after their incubation in sucrose and poly(ethylene glycol) (PEG) (1500-6000 Da molecular mass) media. Free enzyme activity of the lysosomes was less in the PEG medium than that in the sucrose medium under the same hypotonic condition. The lysosomal enzyme latency loss decreased with increasing hydrogen bonding capacity of the solute. In addition, the lysosomes lost less latency at lower incubation temperature. The results indicate that solute hydrogen bonding capacity plays an important role in the osmotic protection of an incubation medium to lysosomes.

A novel carbon source-dependent genetic transformation system for the versatile cell factoryHypocrea jecorina (anamorphTrichoderma reesei)

Genetic transformation is an indispensable tool for basic fungal research, as well as a useful technique for directed improvement of industrial strains. Here we describe a simple and reproducible transformation system for the filamentous fungus Hypocrea jecorina. The system is based on hxk1 (encoding hexokinase) as selectable marker, a hexokinase-negative strain and D-mannitol, which is used as selective carbon source and osmotic stabilizer. Following transformation with the hxk1 gene, the obtained transformants were able to grow on D-mannitol as sole carbon source. Transformation efficiency achieved using D-mannitol as carbon source and osmotic stabilizer was roughly five times higher than that using D-sorbitol. The utility of this system was further demonstrated by transformation of H. jecorina with the egfp (encoding the enhanced green fluorescent protein) gene. Fluorescence microscopy revealed EGFP fluorescence in positive transformants. Our results demonstrated the feasibility of exploiting a carbon source metabolic pathway for the development of promising fungal transformation systems, which provides a new molecular toolbox for genetic modifications of the cell factory H. jecorina.

Yeast karyopherins Kap123 and Kap95 are related to the function of the cell integrity pathway

The characterization of mutant strains in the gene encoding karyopherin Kap123 has revealed several morphogenetic defects. Inactivation of KAP123 caused alterations in the actin cytoskeleton, resulting in hyperpolarization and resistance to the actin polymerization inhibitor latrunculin B. In fact, the level of actin filaments is increased in kap123 mutant cells. In addition to the defect in actin cytoskeleton, the kap123 mutant cells showed a weakened cell wall, cell lysis and a growth defect in either the presence of sodium dodecyl sulfate or at high temperatures, which is alleviated by osmotic stabilizers. These defects in cell integrity and the actin cytoskeleton suggested a relationship with the protein kinase C (PKC) cell integrity pathway. Slt2, the mitogen-activated protein kinase of the PKC cell integrity pathway, is constitutively activated in the absence of Kap123, which is consistent with the existence of cell integrity defects. Analysis of the subcellular localization of nuclear proteins involved in cell wall gene expression indicated that the localization of the Slt2 kinase and the transcription factors Rlm1, Swi6 and Paf1 was not affected by Kap123. Finally, we identified karyopherin Kap95 as the transport factor responsible for the nuclear import of Slt2 and Rlm1.

Protein O -Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans

Aspergillus nidulans possesses three pmt genes encoding protein O-d-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology 150:1973-1982, 2004). In the present paper, we describe the characterization of the pmtA paralogues pmtB and pmtC. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A pmtB disruptant showed wild-type (wt) colony formation at 30 degrees C but slightly repressed growth at 42 degrees C. Conidiation of the pmtB disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. A pmtA and pmtB double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single pmt mutants, the pmtC disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42 degrees C further enabled the pmtC disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three pmt disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth.

Loss of Cardiolipin Leads to Longevity Defects That Are Alleviated by Alterations in Stress Response Signaling

Perturbation of cardiolipin (CL) synthesis in yeast cells leads to defective respiratory chain function and mitochondrial DNA loss, both of which have been implicated in aging in mammals. The availability of yeast CL mutants enabled us to directly investigate the role of CL synthesis in aging. In this report, we show that the replicative life span of pgs1Delta, which lacks both CL and the precursor phosphatidylglycerol (PG), was significantly decreased at 30 degrees C. The life span of crd1Delta, which has PG but not CL, was unaffected at 30 degrees C but reduced at 37 degrees C. Life span extension induced by calorie restriction was not affected by the loss of CL. However, mild heat and osmotic stress, which extend life span in wild type cells, did not increase longevity in CL mutants, suggesting that the stress response is perturbed in these mutants. Consistent with this, longevity defects in pgs1Delta were alleviated by down-regulation of the high osmolarity glycerol stress response pathway, as well as by promoting cell integrity with the osmotic stabilizer sorbitol or via genetic suppression with the kre5(W1166X) mutant. These findings show for the first time that perturbation of CL synthesis leads to decreased longevity in yeast, which is restored by altering signaling through stress response pathways.

A cell-based screening system for detection of inhibitors toward mycobacterial cell wall core

Mycobacterium tuberculosis and nonpathogenic bacteria, Corynebacterium glutamicum, possess a common and unusual cell wall architecture. A cell-based screening system was designed to identify novel compounds interacting with the synthesis, assembly or regulation of the M. tuberculosis cell wall. C. glutamicum was tested in a paired medium assay in 96-well plates with natural product extracts and pure chemical compounds in the presence and absence of the osmotic stabilizer, sorbitol and some ions. Growth was visually examined over a 12-h period and detected with a microplate reader for absorbance at 544 nm. Screening hits from the osmotic stabilizer rescue were then examined by mycolic acid analysis to confirm the effect on cell wall integrity.

Optimization of different factors for efficient protoplast release from entomopathogenic fungusMetarhizium anisopliae

Optimization of different factors for efficient protoplast release fromMetarhizium anisopliae was investigated. Factors like culture media, age of the mycelium, incubation time, different enzymatic combinations and osmotic stabilizer were studied. Mycelium harvested at 40th h in Sabouraud Dextrose broth showed the best protoplast yield over the other media and age of mycelium tested. An incubation time of 3 h and Lysing enzyme at a concentration of 10 mg/ml was the best among the different enzymatic concentrations and combinations tested and yielded a protoplast release of 7.3×108 protoplasts/ml. The most suitable osmotic stabilizer for efficient protoplast release was 0.7M KCl.

Two Distinct Mechanisms of Transport Through the Plasmodial Surface Anion Channel

The plasmodial surface anion channel (PSAC) is a voltage-dependent ion channel on erythrocytes infected with malaria parasites. To fulfill its presumed function in parasite nutrient acquisition, PSAC is permeant to a broad range of charged and uncharged solutes; it nevertheless excludes Na(+) as required to maintain erythrocyte osmotic stability in plasma. Another surprising property of PSAC is its small single-channel conductance (<3 pS in isotonic Cl(-)) in spite of broad permeability to bulky solutes. While exploring the mechanisms underlying these properties, we recently identified interactions between permeating solutes and PSAC inhibitors that suggest the channel has more than one route for passage of solutes. Here, we explored this possibility with 22 structurally diverse solutes and found that each could be classified into one of two categories based on effects on inhibitor affinity, the temperature dependence of these effects and a clear pattern of behavior in permeant solute mixtures. The clear separation of these solutes into two discrete categories suggests two distinct mechanisms of transport through this channel. In contrast to most other broad-permeability channels, selectivity in PSAC appears to be complex and cannot be adequately explained by simple models that invoke sieving through rigid, noninteracting pores.

Protoplast from β-carotene-producing fungus Blakeslea trispora: Preparation, regeneration and validation

The effects of key parameters on the preparation and regeneration of protoplast from the β-carotene-producing fungus Blakeslea trispora were discussed in this paper, including the combination of various enzymes, mycelial age, digesting time and temperature, pH value, osmotic stabilizers, pretreatment, culture medium and culture method. Under the condition of mixed enzymes in osmotic stabilizer (0.6 M NaCl) combined with 2% lysozyme, 3% cellulase and 3% snailase, the highest protoplast yield, as high as 7.48×106 protoplasts/mL, was obtained when mycelial age was 60 h at pH 5.0–6.0 with digesting for 14–16 h at 28 °C. After purification of the obtained protoplasts, they were regenerated in PDA regenerative medium using bilayer plate culture method. To validate the usability of the protoplasts, a novel plasmid with green fluorescent protein (GFP) was used in transformation for easy visual observation. The results showed that the protoplasts prepared by the optimized method were active and applicable in further gene manipulation experiments.

Characteristics of protoplast inter, intra-fusant and regeneration of antagonistic fungi Trichoderma harzianum and Trichoderma viride

A standard method for isolation, fusion and regeneration of protoplasts from Trichoderma harzianum and Trichoderma viride was developed. The protoplasts from T. harzianum and T. viride were isolated using Novozym 234 as lytic enzyme and potassium chloride as osmotic stabilizer. The maximum number of protoplasts (9.37 × 105/ml) was obtained from 16 h old mycelium of T. harzianum and 11.1 × 105/ml for T. viride from 14 h old mycelium at pH 5.5, 28oC for 3 h. The interspecific and intraspecific fusion frequency was determined using 40% polyethylene glycol (PEG) as fusogen. The intrafusants were selected based on their growth, sporulation, pigmentation on chitin and cellulose amended media, where as the interfusants were selected on fungicide resistance as a marker. The protoplast fusion frequency was found to be 1.92% for interspecific fusion. In the case of intraspecific fusion it was about 6.2 and 7.2%, respectively, for T. harzianum and T. viride. The protoplast regeneration frequency of intrafusant was 17% for T. harzianum on chitin medium and 19.2% for T. viride on cellulose medium after two days. The regeneration frequency of 11% for interfusants on fungicide amended medium was observed after three days. The regenerated fusants morphology, growth, sporulation and pigmentation were compared with parental strains.

Depletion of the MobB and CotA complex in Aspergillus nidulans causes defects in polarity maintenance that can be suppressed by the environment stress

Polarized growth is a central feature in eukaryotes. Establishment and maintenance of cell polarity are coordinated by signaling pathways. In this study, we have identified MobB is required for the regulation of cell polarity in Aspergillus nidulans. Depletion of MobB by alcA (p) promoter repression or deletion of MobB abolished conidiation completely, and induced severe growth defects. mobB mutants showed abnormal nuclear segregation with increased number of nuclei in spores, but the formation of septa occurred among dividing cells. The phenotype of mobB in A. nidulans is similar to that of cotA. Furthermore, we verified that MobB interacted with CotA to function as a complex. Interestingly, both mobB and cotA deletion mutants clearly exhibited filament elongation by using environmental osmotic stress in the media. However, calcium channel blocker or chelator inhibited phenotype suppression of mobB or cotA mutants. These results suggest that Ca 2+ is potentially involved in the response to the suppression coupled with osmotic stabilizer. This is the first report of the function of MobB in A. nidulans. We propose that the MobB/CotA complex, a component in the conserved RAM-signaling pathway, serves an important role in cell morphogenesis.

Genotypic variation for drought tolerance in cotton

Increasing scarcity of irrigational water is a major threat to sustainable production of cotton (Gossypium hirsutum L.). It could be resolved by developing drought-tolerant cultivars. Osmotic adjustment and cellular membrane stability are well-documented traits that help to sustain yield under drought in cereals. However, their utility in cotton is not well established. Here, we studied genotypic variability and relationships among osmotic adjustment, cell membrane stability and productivity traits under field-induced water stress at the flowering stage. We evaluated a set of cotton germplasm comprising 32 cotton genotypes under contrasting water regimes for measurements of productivity including seedcotton yield, number of bolls per plant and boll weight, and physiological attributes such as osmotic adjustment and cell membrane stability in two field trials. The mean reduction in seedcotton yield due to water deficit was 20 and 43% in 2003 and 2004, respectively. Genotypes differed considerably for relative yield losses due to water stress ranging from 20 to 74%. Significant association between number of bolls and seedcotton yield under a water-limited regime suggests boll retention as the principal determinant of yield in a water-deficit-stress environment. Cell membrane stability varied significantly among the cotton genotypes; however, its association with productivity measurements was not significant in the water-limited regime. The significant positive correlation found between cell membrane stability and osmotic adjustment implicates the role of osmolytes in the protection of various cellular functions, including those associated with cellular membranes. Moderate but significant differences for osmotic adjustment were found among the genotypes in both years. Osmotic adjustment was positively associated with seedcotton yield under the water-limited regime and inversely correlated with the drought susceptibility index. These results demonstrated the contribution of osmotic adjustment in sustaining yield under water-deficit stress in cotton. Thus, like cereals, osmotic adjustment may be useful as a selection criterion in breeding programs with the objective of improving drought tolerance and yield in cotton under water-limited environments; however, the role of cell membrane stability as a drought-tolerant trait requires further investigation.

The Putative α-1,2-Mannosyltransferase AfMnt1 of the Opportunistic Fungal Pathogen Aspergillus fumigatus Is Required for Cell Wall Stability and Full Virulence

Proteins entering the eukaryotic secretory pathway commonly are glycosylated. Important steps in this posttranslational modification are carried out by mannosyltransferases. In this study, we investigated the putative alpha-1,2-mannosyltransferase AfMnt1 of the human pathogenic mold Aspergillus fumigatus. AfMnt1 belongs to a family of enzymes that comprises nine members in Saccharomyces cerevisiae but only three in A. fumigatus. A Deltaafmnt1 mutant is viable and grows normally at 37 degrees C, but its hyphal cell wall appears to be thinner than that of the parental strain. The lack of AfMnt1 leads to a higher sensitivity to calcofluor white and Congo red but not to sodium dodecyl sulfate. The growth of the mutant is abrogated at 48 degrees C but can be restored by osmotic stabilization. The resulting colonies remain white due to a defect in the formation of conidia. Electron and immunofluorescence microscopy further revealed that the observed growth defect of the mutant at 48 degrees C can be attributed to cell wall instability resulting in leakage at the hyphal tips. Using a red fluorescence fusion protein, we localized AfMnt1 in compact, brefeldin A-sensitive organelles that most likely represent fungal Golgi equivalents. The tumor necrosis factor alpha response of murine macrophages to hyphae was not affected by the lack of the afmnt1 gene, but the corresponding mutant was attenuated in a mouse model of infection. This and the increased sensitivity of the Deltaafmnt1 mutant to azoles, antifungal agents that currently are used to treat Aspergillus infections, suggest that alpha-1,2-mannosyltransferases are interesting targets for novel antifungal drugs.