Induction of shikonin formation by agar in Lithospermum erythrorhizon cell suspension cultures

Cultured cells of Lithospermum erythrorhizon capable of producing red naphthoquinone (shikonin) derivatives on Linsmaier-Skoog agar medium ceased s     

Acceleration of fungal spore production by embedding a hydrophobic polymer net in a nutrient agar plate

A simple and novel procedure for the acceleration of fungal spore production was developed. A net of hydrophobic polymer such as polypropylene (PP) and polytetrafluoroethylene (PTFE) was embedded in a nutrient agar plate, and effect of the polymer net on spore production by 6 fungal strains, such as Aspergillus terreus, Penicillium multicolor, and Trichoderma virens were estimated. The effect of hydrophobic polymer net was insufficient in a liquid-surface immobilization (LSI) system with fungal cells immobilized on a ballooned microsphere layer formed on a liquid medium surface. On the other hand, the embedding of a PTFE net in an agar plate remarkably enhanced the spore production in all 6 strains tested to produce 2.0–8.5 × 10⁷ spores/cm²-agar plate surface. Especially, the spore production by A. terreus ATCC 20542 in the presence of a PTFE net was 7.7 times as much than that in no net. Positive correlations between the hydrophobicity of net and the spore production were observed in all 6 strains (R², 0.653–0.999).     

Differential performance of marubakaido apple rootstock shoots grown in culture media containing different agar brands: dynamic rheological analysis

Shoots of the marubakaido apple rootstock grown in culture medium containing BBL agar presented significantly lower multiplication rate (MR) compared to MRs found for shoots grown in medium containing A-7002, A-7921, Select, and Phytagar as gelling agents. In addition, significant hyperhydricity was found for shoots grown in Phytagar and A-7921 agar-containing media. Analysis of elastic (G′) and viscous (G″) modulus showed that for all of the five agar brands used in this study, G′ was always much higher, i.e., typically one order of magnitude higher than G″, which characterizes a strong gel. G′ changed randomly with time for all of the agar brands studied, except for BBL, which presented progressive decline in G′ throughout the culture cycle. Examination of G′, within the same week, showed that Select agar always had the smallest G′, while Phytagar always had the highest G′. Analysis of the loss tangent (tan δ = G″/G′), a better indicator for gel behavior compared to G′ isolated, showed that tan δ for Select and Phytagar were always between tan δ values found for A-7002 and BBL. In addition, analysis of tan δ also indicated that BBL and Select agars showed a significantly weaker gel network, compared to Phytagar, A-7002 and A-7921 agars after the third week of culture. When seen together, these results indicate that shoot performance for the marubakaido rootstock is not related to agar gel strength. In addition, the high hyperhydricity rate found for shoots grown on agars A-7921 and Phytagar could not be related to agar gel strength, as well. Analysis of HPSEC profiles indicated that the best performance, i.e., multiplication rate, of marubakaido shoots in agars A-7002 and A-7921 is likely to be related to their lower polydispersity and/or smaller amount of high molecular weight fractions, compared to BBL, Phytagar, and Select agars.     

Identification of an agar constituent responsible for hydric control in micropropagation of radiata pine

Tissue cultured Pinus radiata grown on media containing agar as the gelling agent display toxic symptoms and poor long-term shoot survival, however it does have the attribute of hydric control, through a mechanism which, until now, has not been elucidated. Gelrite as an alternative gelling agent is clearly non-toxic but results in hyperhydric (vitrified) tissues. In an effort to overcome these problems, the controlling mechanism found in agar was examined. Hydric control was shown to be effected by a non-gelling, cold-water soluble constituent of a commercial agar, rather than by physical properties of the gel. It could be separated from low molecular weight components of the agar responsible for the toxic symptoms by dialysis. It was identified as being an agaroid-type xylogalactan bearing pyruvate and sulphate substituents. Improved management of gelling agents in culture medium has contributed substantially to a thirty fold increase in propagation rates.     

Trypanosoma cruzi: colony formation and clonal growth in agar

Trypanosoma cruzi exhibited colonial growth when incorporated into 0.5–0.6% agar. Colonies were established from a single organism and clones readily derived. The plating efficiencies were variable depending on the original inoculum but were consistently over 50% when 10⁴ to 10⁵ parasites were added. The use of this technique for evaluation of an antitrypanosomal agent, nifurtimox, was demonstrated, making possible large-scale testing of potential antitrypanosomal agents and assessment of microbicidal and microbio-static drug levels.     

The influence of gelling agent purity and ion additions on in vitro tomato pollen germinability and pollen tube growth on semi-solid substrates

The physicochemical properties and inherent ion content of the gelling agents used for the preparation of semi-solid substrates significantly affect the germination of tomato pollen in vitro. The addition of Ca, K and Mg to semi-solid, agar-based, substrates improved the germination of tomato pollen when the inherent Ca content of the agar was low, but was without effect or even inhibitory when the Ca level was high. However, Κ and/or Mg addition was beneficial irrespective of the agar source. When agarose replaced agar and K, Mg and Ca were added individually or in combination, pollen germination and pollen tube growth were affected differently by each ion but were optimal only in the presence of all three ions, reflecting the absence of these ions in agarose. An involvement of Na was also implicated since reduction of the inherently high Na content of agar by washing improved germination, with or without the addition of Κ, Mg and Ca. Since >3 mM Ca in the semi-solid substrate impairs tomato pollen germination, the gelling agent must be of high purity, which in the case of agar may entail washing, followed by the addition of K, Mg and Ca. The adoption of such a medium would permit the standardization of semi-solid substrates for in vitro tomato pollen germination studies.     

New insights into deleterious impacts of in vivo glycation on albumin antioxidant activities

Background

Albumin constitutes the most abundant circulating antioxidant and prevents oxidative damages. However, in diabetes, this plasmatic protein is exposed to several oxidative modifications, which impact on albumin antioxidant properties.

Methods

Most studies dealing on albumin antioxidant activities were conducted on in vitro modified protein. Here we tried to decipher whether reduced antioxidant properties of albumin could be evidenced in vivo. For this, we compared the antioxidant properties of albumin purified from diabetic patients to in vitro models of glycated albumin.

Results

Both in vivo and in vitro glycated albumins displayed impaired antioxidant activities in the free radical-induced hemolysis test. Surprisingly, the ORAC method (Oxygen Radical Antioxidant Capacity) showed an enhanced antioxidant activity for glycated albumin. Faced with this paradox, we investigated antioxidant and anti-inflammatory activities of our albumin preparations on cultured cells (macrophages and adipocytes). Reduced cellular metabolism and enhanced intracellular oxidative stress were measured in cells treated with albumin from diabetics. NF-kB –mediated gene induction was higher in macrophages treated with both type of glycated albumin compared with cells treated with native albumin. Anti inflammatory activity of native albumin is significantly impaired after in vitro glycation and albumin purified from diabetics significantly enhanced IL6 secretion by adipocytes. Expression of receptor for advanced glycation products is significantly enhanced in glycated albumin-treated cells.

Conclusions and general significance

Our results bring new evidences on the deleterious impairments of albumin important functions after glycation and emphasize the importance of in vivo model of glycation in studies relied to diabetes pathology.     

New insights in the management of patients with obstructive sleep apnea

Sleep and Biological Rhythms - The prevalence of patients with obstructive sleep apnea (OSA) is high and increasing. Controlling OSA is an important issue for a medical domain. OSA adversely...     

New insights of epithelial-mesenchymal transition in cancer metastasis

Epithelial-mesenchymal transition (EMT) is a key step during embryonic morphogenesis, heart development, chronic degenerative fibrosis, and cancer metastasis. Several distinct traits have been conveyed by EMT, including cell motility, invasiveness, resistance to apoptosis, and some properties of stem cells. Many signal pathways have contributed to the induction of EMT, such as transforming growth factor-β, Wnt, Hedgehog, Notch, and nuclear factor-κB. Over the last few years, increasing evidence has shown that EMT plays an essential role in tumor progression and metastasis. Understanding the molecular mechanism of EMT has a great effect in unraveling the metastatic cascade and may lead to novel interventions for metastatic disease.     

Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.     

Hydrophobic plastics films synthesis by mean of agaroid acylation with lauroyl chloride in the N,N-dimethylacetamide homogeneous system

The synthesis of hydrophobic plastic films was performed by acylation of agaroids with lauroyl chloride in the N,N-dimethylacetamide homogeneous system. All the plastic films were characterized by FT-IR spectroscopy and their degrees of substitution (DS) was deduced from their ¹H-NMR spectra. In addition, thermomechanical feature of plastic films were analyzed and compared to those obtained from other kinds of hydrophobic plastic films. Latin square design of experiments helped us to determine optimized experimental conditions and identify the most important factors. Hence, mild conditions of acylation (90∘C, 1 eq/OH of lauroyl chloride, 1 eq/OH of 4-dimethylaminopyridine, 5,min) led to the production of highly substituted plastic films (DS = 3.62; maximum 4) with a high weight yield (211%) displaying mechanical properties close to polyethylene low density.     

Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution

Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation, and prefractionation of protein interactions in solution independent of isoelectric point. We demonstrate that this assay is compatible with immunochemical methods and mass spectrometry. The assay was used to investigate interactions with several potential substrates for calreticulin, a chaperone that is involved in different biological aspects through interaction with other proteins. The current analytical assays used to investigate these interactions are mainly spectroscopic aggregation assays or solid phase assays that do not provide a direct visualization of the stable protein complex but rather provide an indirect measure of interactions. Therefore, no interaction studies between calreticulin and substrates in solution have been investigated previously. The results presented here indicate that calreticulin has a preference for substrates with a quaternary structure and primarily β-sheets in their secondary structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis.     

New insights into the interaction of ribosomal protein L1 with RNA

The RNA-binding ability of ribosomal protein L1 is of profound interest, since L1 has a dual function as a ribosomal structural protein that binds rRNA and as a translational repressor that binds its own mRNA. Here, we report the crystal structure at 2.6 A resolution of ribosomal protein L1 from the bacterium Thermus thermophilus in complex with a 38 nt fragment of L1 mRNA from Methanoccocus vannielii. The conformation of RNA-bound T.thermophilus L1 differs dramatically from that of the isolated protein. Analysis of four copies of the L1-mRNA complex in the crystal has shown that domain II of the protein does not contribute to mRNA-specific binding. A detailed comparison of the protein-RNA interactions in the L1-mRNA and L1-rRNA complexes identified amino acid residues of L1 crucial for recognition of its specific targets on the both RNAs. Incorporation of the structure of bacterial L1 into a model of the Escherichia coli ribosome revealed two additional contact regions for L1 on the 23S rRNA that were not identified in previous ribosome models.     

Lipopolysaccharide aggregates in native agarose gels detected by reversible negative staining with imidazole and zinc salts

We investigated the use of imidazole and zinc salts for the detection of lipopolysaccharide (LPS) aggregates separated by native agarose gel electrophoresis (NAGE). As a result, a new staining procedure was established by which as little as 1.5 μg of Escherichia coli O55:B5 LPS aggregates was detected by means of inducing a clear, transparent pattern, contrasted against an opaque background. E. coli O55:B5 LPS preparations treated with nucleases and proteinase K proved that the reverse-stained LPS pattern is not related to any potential artifacts caused by unrelated biomolecules (e.g., nucleic acids, proteins). After this, we showed that the procedure is applicable to two-dimensional LPS separation using NAGE/SDS-PAGE, while at the same time confirming that real polydisperse LPS aggregates are represented by the stained profile. Also, we demonstrated the general applicability of this stain to the detection of different NAGE-separated LPS aggregates (e.g., from E. coli 026:B6, E. coli 0111:B4, Salmonella minnesota Re595). Finally, using lysozyme as a model protein, we found that imidazole–zinc may be combined with Coomassie brilliant blue R-250 into a double-staining process to enable the use of NAGE for investigating the interaction of cationic proteins and LPS aggregates and protein or LPS concentration effects on protein–LPS binding.