Alfalfa embryo production in airlift vessels via direct somatic embryogenesis

A procedure for the development of alfalfa (Medicago falcata L.) somatic embryos to the torpedo stage in air-lift vessels is described. Embryos were initiated from chopped leaf explants and were formed by direct somatic embryogensis. The system produced a high number of torpedo stage embryos. The effect of various inoculation densities on embryo development was studied. A procedure for the development and maturation of embryos in aerated liquid media was established. The rate of conversion of the torpedo stage embryos formed in the vessels was 83%.Abbreviations ABA abscisic acid - B5 Gamborgs B5 medium (Gamborg et al. 1968) - COT cotyledon embryo state - 2,4-d 2,4-dichlorophenoxyacetic acid - FW fresh weight - ID internal diameter - MS Murashige and Skoog medium (Murashige & Skoog 1962) - PEG polyethylene glycol - POLY polyembryos - VVM volume of gas/volume of bioreactor     

Natural products with anti-aging potential: Affected targets and molecular mechanisms

In recent years, there has been a great deal of attention toward the molecular machinery relevant to age-related progression controlled through the external intervention of polyphenols- an epigenetic-modulating diet. Natural products modulate cellular longevity through histone post-translational modification and can also induce the upregulation of autophagy, thus reducing the level of acetyl coenzyme A (AcCoA). In addition, the effect of caloric restriction (CR) on cancer-related chronic inflammation is of great significance in aging. In line with this, SIRT1 protein levels are expanded in response to calorie restriction mimetics (CRM), in this way acting as autophagy inducers relevant to cancer prevention.     

Alkaloid variability in Leucojum aestivum from wild populations

Leucojum aestivum (summer snowflake) is a plant species used for the extraction of galanthamine, an acetylcholinesterase inhibitor for the treatment of Alzheimer's disease. Extracts from bulbs collected from 18 Bulgarian populations and from shoot-clumps obtained in vitro from 8 different populations showed variations in their alkaloid composition. Nineteen alkaloids were detected in the studied samples by GC-MS. Typically, the alkaloid fractions of L. aestivum bulbs were dominated by galanthamine type compounds, but lycorine, haemanthamine and homolycorine type alkaloids were also found as dominant compounds in some of the samples. Extracts from the shoot-clumps obtained in vitro were found to contain galanthamine or lycorine as main alkaloids. The galanthamine content ranged from 28 to 2104 microg/g dry weight in the bulbs, and from traces to 454 microg/g dry weight in the shoot-clumps.     

Batch and fed-batch production of betalains by red beet (Beta vulgaris) hairy roots in a bubble column reactor

Hairy root cultures from red beet (Beta vulgaris L.), which could be used for the commercial production of biologically active betalain pigments, were cultivated in a 3 L bubble column bioreactor in batch mode with various rates of air supply. Both the growth of the roots and betalain volumetric yields were highest (12.7 g accumulated dry biomass/L and 330.5 mg/ L, respectively) with a 10 L/h (0.083 vvm) air supply. The air flow rate also influenced the betacyanins/betaxanthins ratios in the cultures. Growth and betalains production were then examined in two fed-batch regimes (with a 10 L/h air supply), in which nutrient medium was fed just once or on five occasions, designated FBI and FBII, respectively. The root mass accumulation was increased in the FBI feeding regime (to 13.3 g accumulated dry biomass/ L), while in FBII the betalains content was ca. 11% higher (15.1 mg betacyanins/g dry weight and 14.0 mg betaxanthins/g dry weight) than in the most productive batch regime. Data on the time course of the utilization of major components in the medium during both operational modes were also collected. The implications of the information acquired are discussed, and the performance of the hairy roots (in terms of both growth and betalains production) in the bubble column reactor and previously investigated cultivation systems is compared.     

Chemical and biological folding contribute to temperature-sensitive DeltaF508 CFTR trafficking

Proteostasis (Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science 2008;319:916-919) refers to the biology that maintains the proteome in health and disease. Proteostasis is challenged by the most common mutant in cystic fibrosis, DeltaF508, a chloride channel [the cystic fibrosis transmembrane conductance regulator (CFTR)] that exhibits a temperature-sensitive phenotype for coupling to the coatomer complex II (COPII) transport machine for exit from the endoplasmic reticulum. Whether rescue of export of DeltaF508 CFTR at reduced temperature simply reflects energetic stabilization of the chemical fold defined by its primary sequence or requires a unique proteostasis environment is unknown. We now show that reduced temperature (30 degrees C) export of DeltaF508 does not occur in some cell types, despite efficient export of wild-type CFTR. We find that DeltaF508 export requires a local biological folding environment that is sensitive to heat/stress-inducible factors found in some cell types, suggesting that the energetic stabilization by reduced temperature is necessary, but not sufficient, for export of DeltaF508. Thus, the cell may require a proteostasis environment that is in part distinct from the wild-type pathway to restore DeltaF508 coupling to COPII. These results are discussed in the context of the energetics of the protein fold and the potential application of small molecules to achieve a proteostasis environment favoring export of a functional form of DeltaF508.     

In sito galanthamine extraction during the cultivation of Leucojum aestivum L. shoot culture in two‐phase bubble column cultivation system

Two‐phase bioreactor cultivation system was developed and applied for in sito recovery of extracellular galanthamine during the cultivation of Leucojum aestivum L. shoot culture in a modified column bioreactor system. The inclusion of an external circulation column with adsorbent resin Amberlite XAD‐4 as a second phase, on the 21st day of the beginning of cultivation resulted in 1.25 folds increase in biomass accumulation and maximal amounts of accumulated galanthamine of 6 mg/L (3.1 mg/L intracellular and 2.9 mg/L extracellular). It was demonstrated that the inclusion of a second phase at the cultivation of the L. aestivum shoot culture in a bubble column bioreactor with internal sections redirected the alkaloid metabolism to galanthamine synthesis and inhibits the synthesis of hemanthamine and lycorine type alkaloids. Our research demonstrated that the application of the two‐phase cultivation systems could be an important tool to increase the yields of valuable secondary metabolites in plant tissue culture‐based bioprocess.     

Some properties and protective activity of specific DLE against Salmonella cholerae suis infection

From a rabbit lymphoid tissue, twice immunized with a Salmonella ch. suis vaccine, was obtained a dialysable leucocyte extract (DLE) (m.w. 10 000Da; protein content 1.14 mg/ml; content of ribose 2.7 mg/ml; A₂₆₀/A₂₈₀ ratio 2.17 and pH 6.8). By gel filtration on Sephadex G-25, six peaks were obtained and activity was found in peak IV. The activity of the extract was determined by a dermo-application test (DAT) on 10 cows. The protective effect was tested by a challenge with Salmonella ch. suis and Salmonella dublin pathogen strains on white mice intraperitoneally treated with DLE. The DAT proved to be positive in 8 of the 10 cows. When applied on white mice, it induced a high specific protective effect against Salmonella ch. suis (70%), but not against Salmonella dublin infection.     

Long-term resistance to tomato spotted wilt virus in transgenic tobacco cultivars expressing the viral nucleoprotein gene: greenhouse and field tests

We examined the resistance phenotype of a large number of transgenic tobacco plants originating from 12 commercial (Nicotiana tabacum) cultivars expressing the sense form of the nucleoprotein (N) gene of L3, a Bulgarian isolate of tomato spotted wilt virus (TSWV). The analysis revealed that transgenic plants are completely protected against the homologous L3 isolate of TSWV irrespective of whether or not they contain detectable levels of translational product. The effectiveness of protection against the virus was investigated upon mechanical inoculation under greenhouse conditions and in field trials. Non-segregating resistant lines were selected and the inheritance of the resistance to TSWV was analysed in successive generations (R₃–R₆). Extensive tests under controlled conditions and two-year field trials proved that the resistance to TSWV is stable in different environments and is a stably inherited trait.