Micropropagation of ‘Durondeau’ pear in modified-gelled medium

Summary The influence of partial substitution of agar by galactomannans (GMs) in culture media was studied in pear (Pyrus communis L. cv. ‘Durondeau’) micropropagation. GMs. extracted from seeds of Cassia fastuosa (cassia) or Cyamopsis tetragonolobus (guar gum, a commercial GM), were mixed in equal proportions with agar to a final concentration of 0.3% (w/v) for each type of gelling agent. The production of multiple shoots and the formation of roots from shoots were compared with the control solidified with agar alone at a concentration of 0.6% (w/v). In the media solidified with the mixtures of agar/guar and agar/cassia GMs, an, increase of 32 and 17%, respectively, was obtained in the number of regenerated shoots. The modified media promoted a higher number of roots and increased the rooting percentage. A maximum of 91% rooting was obtained in the medium solidified with the agar/cassia GM and containing 9.80 μM indole-3-butyric acid. Less callus formation at the base of the shoot was also observed on this medium. The improved in vitro performance of shoot formation and rooting, combined with a significantly lower cost, suggests a potential use of agar/GM gels in plant tissue culture.     

с-Met receptor can be activated by extracellular alkaline medium

Receptor tyrosine kinase (RTK) Met or c-Met is a target of hepatocyte growth factor (HGF) and it plays an important role under normal and pathological conditions. Activation of Met signaling pathway is associated with several cellular processes, such as proliferation, survival, motility, angiogenesis, invasion, and metastasis. In this article, we describe the ability of Met to activate upon a mild alkali treatment. To identify potential alkali-regulated proteins, CAKI-1 cells were treated with alkaline media and further tested for protein phosphorylation changes. By anti-phosphotyrosine antibody precipitation and lectin chromatography, we identified Met as a major cytoplasmic membrane protein that responded to pH changes by its phosphorylation. The activation of Met by alkali occurred at pH >8.0 and was dose-dependent. Specificity of the Met response to alkali was confirmed by the treatment with Met kinase inhibitor SU11274 and also by Met receptor knockout using CRISPR/CAS9 genome editing system. Both approaches completely blocked the Met phosphorylation response in CAKI-1 cells. Similar pH-dependent Met activation was observed in the HeLa cell line. Our data suggest existence of ligand-independent mechanism of Met receptor activation.     

Can cell culture medium costs be reduced? Strategies and possibilities

The complex media needed for the culture of animal cells is expensive and is one factor that puts cell culture at a great economic disadvantage compared with other microbial systems. Viewing these costs in a proper perspective shows that this disadvantage is often exaggerated, but even so there are several ways in which significant savings can be made.     

The cell culture medium — a functional extracellular compartment of suspension-cultured cells

The spent medium of suspension-cultured cells of Lupinus polyphyllus was analyzed by capillary GC and GC-MS and shown to contain ethanol (up to 160 mmol l^-1), organic acids (lactate, benzoate, succinate, fumarate, malate), amino acids (main components: alanine, glycine, serine, aspartate, ornithine, glutamate), and quinolizidine alkaloids (lupanine and an uncharacterized malonylderivative). In addition, cells obviously secrete polysaccharides and enzymes (acid phosphatase, phosphodiesterase, DNAse, esterase, -mannosidase, -galactosidase, -glucosidase, lipase, protease and peroxidase) into the medium. Typically these enzymes are localized in the vacuole of intact cells. Cytosolic enzymes, such as glutamate dehydrogenase and malate dehydrogenase were retained by the cells. Peroxidase is overexpressed in suspension-cultured lupin cells but only one basic isoenzyme is secreted, whereas the others are retained in the vacuole. In lupin leaves this isoenzyme is sequestered in the vacuole, implying that secretion is selective and needs a change in the sorting signals of the peroxidase protein. The cell culture medium shares many features of the vacuole. We assume therefore that the medium functions as a lytic compartment. In addition it provides a sink-source system for nutrients and metabolites.Abbreviations ADH alcohol dehydrogenase - FW fresh weight - GC gas chromatography - GC-MS gas chromatography — mass spectrometry - POD peroxidase - QA quinolizidine alkaloids     

Do light/dark cycles of medium frequency enhance phytoplankton productivity?

It has been suggested that turbulence with the resultant light/dark cycle and light gradient through which phytoplankton move, enhances their productivity. The stationary bottle incubation technique for estimating rates of primary productivity has mainly been criticized because of bottle effects, the elimination of natural turbulence and the presence of photo-inhibition. In a series of experiments where productivity was measured over static profiles and compared to the productivity in a mixed system, no definite conclusion could be reached regarding the effect of varying light/dark cycles of medium frequency (seconds to minutes). It appeared as though the ratio of the euphotic depth to mixing depth (Z _eu/Z _m) influenced productivity more than the duration of the light/dark cycle. The static bottle incubation method gave higher integral productivities than the mixed samples at low ratio's ofZ _eu/Z _m. It is suggested that mixing has two separate, but synergistic effects i.e. it not only moves the phytoplankton cells through a light/dark cycle, but also decreases the boundary layer, which increases the rate of exchange through the cell wall of nutrients and metabolites. In doing so more nutrients are available and light could be utilized more efficiently and therefore, productivity is increased.     

Recovery of 6-α-methylprednisolone from biotransformation medium by tangential flow filtration

Tangential flow filtration of a biotransformation medium of 6--methylhydrocortisone to 6--methylprednisolone (medrol) was carried out to study the recovery of the product, from heattreated cell suspensions of Arthrobacter simplex. A higher product recovery rate was achieved when concentration of the medium was performed prior to the diafiltration. The concentration of the product in the permeate was also higher when concentration is the first mode of operation. Combined anionic and cationic microsized polymeric particles were used as filtration aids. These resins flocculated cells of A. simplex and allowed a 5-fold enhancement in the recovery rate of the product, through a 0.22 m Durapore membrane, by increasing the diafiltration flux from 20 dm³/hm² to 125 dm³/hm². The medrol rejection coefficient remained constant when the polymeric particles were used, being highly dependent on the cell concentration.     

Development and characterization of butanol — Resistant strain ofClostridium acetobutylicum in molasses medium

The production of acetone—butanol—ethanol solvents from cane molasses by locally isolated culture ofClostridium acetobutylicum was limited by butanol toxicity (1.6 mol/L). The butanol tolerance of the isolated culture was increased up to 4.8 mol/L by a serial enrichment method. The butanol-resistant strain had greater efficiency for the conversion of saccharides to mixed solvents and produced 52% more butanol at the expense of acetone and ethanol than the original strain. Moreover, the fermentation profile of parent and butanol-resistant strains in anerobic fermentation of cane molasses demonstrated the superiority of the latter in terms of growth rate, time of onset of butanol production, sugar utilization, final butanol concentration and other parameters.     

Galanthamine production in “shoot-clump” cultures of Narcissus confusus in liquid-shake medium

Galanthamine (GAL) is increasingly used in the treatment of Alzheimer's disease. We have attempted to develop a method of producing this alkaloid using in vitro cultures of Narcissus confusus plants. The “shoot-clump” culture in liquid medium was shown to be an appropriate method for the micropropagation of this bulbous plant. The complete process included three steps:

1. culture of “twin-scales” starting from the bulbs;
2. culture of the newly formed shoots in a medium for bud proliferation (Murashige Skoog+1 mg l^-1 of 2,4-dichlorophenoxyacetic acid+5 mg l^-1 of benzyladenine), and
3. culture of “shoot-clumps” in a liquid-shake medium. Here we describe the effect of the addition of trans-cinnamic acid, a precursor in the biosynthesis of the Amaryllidaceae alkaloids, on the production of galanthamine and related alkaloids, and also on the growth of the “shoot-clump” culture. The production of galanthamine was found to be inhibited by the addition of the precursor, which promoted the production of the other alkaloid in the same biosynthetic pathway, N-formyl-norgalanthamine. The total production of galanthamine in the control cultures in day-long photoperiod was 2.50 mg per culture, of which 1.97 mg per culture were released into the medium.

     

Behaviour of dehydrated baker’s yeast during reduction reactions in a biphasic medium

The behaviour of dry bakers yeast (Saccharomyces cerevisiae type II, Sigma) used as biocatalyst without preliminary growth for the synthesis of 2-heptanol from 2-heptanone in a biphasic system is presented. Cells undergo intracellular trehalose consumption with a stoichiometric ethanol production during the first 15 h of the process. This metabolism is then replaced by acetate accumulation. These reactions are disconnected from the biocatalytic reaction and do not provide reduced cofactors. 2-Heptanone is metabolised by two pathways. The first leads to 2-heptanol (molar yield close to 55%, enantioselectivity higher than 99%, with a slight decrease at the end of the process) and the second corresponds to material incorporation into the biomass. This latter phenomenon is assumed to provide the biocatalyst with the reduced cofactors needed for the reduction process. Overall, the process yielded ca. 1.4 g/l 2-heptanol in 50 h reaction, which is close to that observed with fresh cells previously grown for 15 h.     

Enhanced secretion of recombinant α-cyclodextrin glucosyltransferase from E. coli by medium additives

The purpose of this study was to investigate the effect of medium additives on the secretion of recombinant α-cyclodextrin glucosyltransferase (α-CGTase) into the culture media of Escherichia coli. It is found that supplementation of the E. coli culture with SDS, glycine, Ca²⁺ or Na⁺, individually, facilitated the secretion of α-CGTase. Orthogonal experiment showed that the optimal condition to achieve maximal secretion of α-CGTase was the supplementation with 0.03% SDS, 400 mM Na⁺, 0.3% glycine and 10 mM Ca²⁺ together. Under this condition, extracellular enzyme activity reached 12.89 U/ml, which is 15 times higher than that of the culture without any additives. Further analysis showed that the permeability, fluidity and phosphatidylglycerol content of the E. coli cell membrane under the optimized condition were significantly increased in comparison to those under the control condition. These might be the potential mechanisms for the increased secretion of α-CGTase from the periplasmic compartment into the culture medium.     

Penicillin acylase immobilization depending on macromolecular crowding and catalysis in aqueous–organic medium

To enhance penicillin acylase (PA) performance, it was immobilized in mesocellular silica foams (MCFs) depended on macromolecular crowding and applied to catalysis in non-aqueous medium. Ficoll 70, dextran 10,000, dextran 40,000 and bovine serum albumin were co-assembled with PA. It was observed that specific activity of PA assembled in MCFs with dextran 10,000 in 80% cyclohexane (v/v) was 233.2 U/mg, 200% as that of PA assembled in MCFs in 80% cyclohexane and 323.5% as that of free PA in full aqueous medium. As content of alkane increased, activity of PA in MCFs with macromolecules varied slightly. In addition, PA co-immobilized with dextran 10 in MCFs retained 58.2% of its initial activity after heating at 50°C for 4 h, 1.2 times higher than that of PA immobilized alone in MCFs. The results showed that macromolecular crowding was favorable for immobilization of PA and its catalysis in suitable aqueous–organic medium.     

Measurement of one-bond 15N-13C′ dipolar couplings in medium sized proteins

A simple and accurate method is described for measurement of ¹ J _CN splittings in isotopically enriched proteins. The method is of the quantitative J correlation type, and the ¹ J _CN splitting is derived from the relative intensity in two 3D TROSY-HNCO spectra with ¹ J _CN dephasing intervals of 1/(2¹ J _CN) (reference intensity) and 1/¹ J _CN (residual intensity). If the two spectra are recorded under identical conditions and with the same number of scans, the random error in the ¹ J _CN value extracted in this manner is inversely related to the signal-to-noise (S/N) in the reference spectrum. A S/N of 30:1 in the reference spectrum yields random errors of less than 0.2 Hz in the extracted ¹ J _CN value. Dipolar couplings obtained from the difference in ¹ J _CN splitting in the isotropic and liquid crystalline phase for the C-terminal domain of calmodulin are in excellent agreement with its 1.68-Å crystal structure, but agree considerably less with the 2.2-Å structure.     

Statistical optimization of production medium for β-1,3-glucanase synthesis by Trichoderma harzianum

Response surface methods were used to determine the optimum concentration of medium nutrients for extracellular -1,3-glucanase production by Trichoderma harzianum in shakeflask culture. A Plackett-Burman design was used to screen the important variables which were then grouped and studied by a central composite design. The optimum levels of the medium constituents were determined by the complex algorithm of Box.     

Does the zona pellucida select spermatozoa from the medium with higher fertilizing potential?

The present study was carried out to test whether the zona pellucida selects spermatozoa with higher fertilization potential. Fertilization rates of mouse oocytes after sperm microinjection under the zona pellucida (SMUZ) of zona-bound spermatozoa and of spermatozoa incubated in the absence of oocytes and treated (acid-treated group) or not (control group) with acid Tyrode's solution were compared. SMUZ was performed at 15, 30, 60, and 90 min after the insemination of fresh oocytes required for selecting spermatozoa bound to the zona pellucida. At these times, the percentages of acrosome-reacted spermatozoa (PARS) were evaluated using the chlortetracycline fluorescence method. Fertilization rate in the three groups analysed increased from 25.9% to 47.3% in the control group, from 29.3% to 44.0% in the acid-treated group, and from 19.5% to 40.0% in the zona-bound group from 15 to 90 min after insemination, respectively. The global fertilization potential was significantly lower in the zona-bound group compared to the other two groups. The PARS in the zona-bound group at 15 (11.48 ± 3.02); 30 (16.74 ± 3.71), and 90 (19.68 ± 3.68) min after insemination were significantly (P < 0.05) lower than those found in the acid-treated group (39.26 ± 6.69, 38.20 ± 6.24, and 42.83 ± 5.39, respectively). At 90 min after insemination, the PARS in the zona-bound group was also significantly (P 0.05) lower than the control group (36.72 ± 4.51). No significant correlation between either time and PARS or PARS and fertilization rate was observed. It appears that the zona pellucida does not select from the medium spermatozoa with higher fertilization potential. © 1993 Wiley-Liss, Inc.     

Evaluation of a chromogenic agar medium for the detection of extended-spectrum ß-lactamase-producing Enterobacteriaceae

Aim: To compare the performance of a new chromogenic agar medium CHROMagar ESBL (KC‐ESBL) to chromID ESBL (SB‐ESBL) for the detection and presumptive identification of extended‐spectrum β‐lactamase (ESBL)‐producing Enterobacteriaceae directly from clinical specimens. Methods and Results: A total of 256 specimens were screened for ESBL producers. Also, the genotypes of the ESBLs and plasmid‐mediated AmpC β‐lactamases (pAmpCBLs) were characterized by PCR and sequencing. Among the 256 specimens, 17 (6·6%) ESBL producers were isolated on both media. The sensitivity, specificity, positive predictive value and negative predictive value were higher for KC‐ESBL (100, 93·3, 51·5 and 100%, respectively) than for SB‐ESBL (88·2, 92·9, 46·9 and 99·1%, respectively) (P = 0·72). Enterobacteriaceae harbouring pAmpCBL genes as well as chromosomal cephalosporinase‐ and penicillinase‐hyperproducing Enterobacteriaceae and Pseudomonas aeruginosa accounted for the false‐positive results. Conclusion: KC‐ESBL can detect ESBL producers from clinical specimens with good selectivity and rapid presumptive identification by means of colony colour at 24 h. Significance and Impact of the Study: This is the first study that has evaluated the performance of KC‐ESBL that enables the detection and presumptive identification of ESBL producers from clinical specimens.     

Self-assembly of β-lactoglobulin and the soluble fraction of gum tragacanth in aqueous medium

Spectrophotometric and light scattering measurements, along with optical microscopy, were used to follow the complexation and coacervation process that occur when β-lactoglobulin (BLG)/tragacanthin (T) mixed dispersions (0.3 wt.% total concentration; BLG:T ratio of 2:1) were brought from pH 6 to pH 2. In addition, the coupling of slow in situ acidification of the mixture and rheometry was utilised to gain deeper insights into pH-induced structural transitions during the assembly process. The results obtained by this multi-methodological approach allowed the associative phase separation process to be parameterised in terms of a set of characteristic pH values (~5.3, ~4.8, ~4.5, ~4.15, ~4, ~3.8, ~2.5) at which critical structural changes took place. Investigation of the absorbance profiles of complexed/coacervated systems as a function of time revealed that several transitions could occur at different time scales. Morphological changes in the assemblies and the subsequent formation of some flocculant substances during the late stage of process were clearly visualised using microscopy.     

Production of α-amylase in a low cost medium by Bacillus licheniformis TCRDC-B13

A low cost synthetic medium producing large quantities of α-amylase has been developed. Bacillus licheniformis TCRDC-B13 isolated from soil was used for α-amylase production. The α-amylase enzyme of this strain showed excellent stability at high temperatures and over a wide pH range. The low cost medium produced 5 times more enzyme than the high cost synthetic medium (using yeast extract and peptone) in shake flasks. In a 2.6-l fermentor, the enzyme production further doubled.     

Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi

An efficient system for Agrobacterium-mediated transformation of Lilium × formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 μM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.