TNFα secretion of monocytes exposed to pulsed radiofrequency treatment: a possible working mechanism of PRF chronic pain management.

Pulsed radiofrequency treatment (PRF) is a promising new technique increasingly used in treatment of chronic pain. The molecular working mechanism of PRF is not exactly known and is currently being investigated. This study investigates a possible role of PRF-induced modulation of TNFα secretion by differentiated monocytes in chronic pain management. The results show no significant PRF-induced change in TNFα secretion of lipopolysaccharides (LPS)-stimulated monocytes. However, PRF does significantly increase TNFα secretion of differentiated monocytes that have not been stimulated with LPS. This may indicate a possible role of PRF treatment in increasing TNFα production of nonstimulated monocytes. More research is needed to determine whether this is truly a part of the working mechanism of PRF in chronic pain management and which other factors are involved.

Culture and time-lapse tracking of barley microspore-derived embryos.

Barley microspore embryogenesis represents an attractive system to study stress-induced cell differentiation and is a valuable tool for efficient plant breeding. In contrast to zygotic embryogenesis, all developmental stages are freely accessible at a large scale for observation, molecular analysis and manipulation techniques. In barley, there is a high percentage of microspores that become embryogenic after stress treatment in a mannitol solution. These microspores have the capacity to follow an embryogenic route in both liquid and solid cultures, yielding up to 10% of embryos. In this protocol, we describe three different culture systems for obtaining barley microspore-derived embryos, where embryos develop in liquid medium, on top of a solid medium layer or immobilized in a thin layer of agarose. While liquid culture systems allow the generation of large amounts of embryos for molecular analysis, solid culture systems are the ultimate tool for probing embryo development.

Lipidomics reveals multiple pathway effects of a multi-components preparation on lipid biochemistry in ApoE*3Leiden.CETP mice.

BACKGROUND: Causes and consequences of the complex changes in lipids occurring in the metabolic syndrome are only partly understood. Several interconnected processes are deteriorating, which implies that multi-target approaches might be more successful than strategies based on a limited number of surrogate markers. Preparations from Chinese Medicine (CM) systems have been handed down with documented clinical features similar as metabolic syndrome, which might help developing new intervention for metabolic syndrome. The progress in systems biology and specific animal models created possibilities to assess the effects of such preparations. Here we report the plasma and liver lipidomics results of the intervention effects of a preparation SUB885C in apolipoprotein E3 Leiden cholesteryl ester transfer protein (ApoE*3Leiden.CETP) mice. SUB885C was developed according to the principles of CM for treatment of metabolic syndrome. The cannabinoid receptor type 1 blocker rimonabant was included as a general control for the evaluation of weight and metabolic responses. METHODOLOGY/PRINCIPAL FINDINGS: ApoE*3Leiden.CETP mice with mild hypercholesterolemia were divided into SUB885C-, rimonabant- and non-treated control groups. SUB885C caused no weight loss, but significantly reduced plasma cholesterol (-49%, p<0.001), CETP levels (-31%, p<0.001), CETP activity (-74%, p<0.001) and increased HDL-C (39%, p<0.05). It influenced lipidomics classes of cholesterol esters and triglycerides the most. Rimonabant induced a weight loss (-9%, p<0.05), but only a moderate improvement of lipid profiles. In vitro, SUB885C extract caused adipolysis stimulation and adipogenesis inhibition in 3T3-L1 cells. CONCLUSIONS: SUB885C, a multi-components preparation, is able to produce anti-atherogenic changes in lipids of the ApoE*3Leiden.CETP mice, which are comparable to those obtained with compounds belonging to known drugs (e.g. rimonabant, atorvastatin, niacin). This study successfully illustrated the power of lipidomics in unraveling intervention effects and to help finding new targets or ingredients for lifestyle-related metabolic abnormality.

Time-lapse tracking of barley androgenesis reveals position-determined cell death within pro-embryos.

Following abiotic stress to induce barley (Hordeum vulgare L.) androgenesis, the development of 794 enlarged microspores in culture was monitored by time-lapse tracking. In total, 11% of the microspores tracked developed into embryo-like structures (type-I pathway), 36% formed multicellular structures (type-II pathway) and 53% of the microspores followed gametophytic divisions, accumulated starch and died in the first days of tracking (type-III pathway). Despite the microspore fate, enlarged microspores showed similar morphologies directly after stress treatment. Ultrastructural analysis, however, revealed two morphologically distinct cell types. Cells with a thin intine layer and an undifferentiated cytoplasm after stress treatment were associated with type-I and type-II pathways, whereas the presence of differentiated amyloplasts and a thick intine layer were associated with the type-III pathway. Tracking revealed that the first morphological change associated with embryogenic potential was a star-like morphology, which was a transitory stage between uninucleate vacuolated microspores after stress and the initiation of cell division. The difference between type-I and type-II pathways was observed during the time they displayed the star-like morphology. During the transition phase, embryo-like structures in the type-I pathway were always released out of the exine wall at the opposite side of the pollen germ pore, whereas in the type-II pathway multicellular structures were unable to break the exine and to release embryo-like structures. Moreover, by combining viability studies with cell tracking, we show that release of embryo-like structures was preceded by a decrease in viability of the cells positioned at the site of exine wall rupture. These cells were also positively stained by Sytox orange, a cell death indicator. Thereby, we demonstrate, for the first time, that a position-determined cell death process marks the transition from a multicellular structure into an embryo-like structure during barley androgenesis.

Glucosylation activity and complex formation of two classes of reversibly glycosylated polypeptides.

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. In plants, these proteins may function, for example, in cell wall synthesis and/or in synthesis of starch. We have isolated wheat (Triticum aestivum) and rice (Oryza sativa) Rgp cDNA clones to study the function of RGPs. Sequence comparisons showed the existence of two classes of RGP proteins, designated RGP1 and RGP2. Glucosylation activity of RGP1 and RGP2 from wheat and rice was studied. After separate expression of Rgp1 and Rgp2 in Escherichia coli or yeast (Saccharomyces cerevisiae), only RGP1 showed self-glucosylation. In Superose 12 fractions from wheat endosperm extract, a polypeptide with a molecular mass of about 40 kD is glucosylated by UDP-glucose. Transgenic tobacco (Nicotiana tabacum) plants, overexpressing either wheat Rgp1 or Rgp2, were generated. Subsequent glucosylation assays revealed that in RGP1-containing tobacco extracts as well as in RGP2-containing tobacco extracts UDP-glucose is incorporated, indicating that an RGP2-containing complex is active. Gel filtration experiments with wheat endosperm extracts and extracts from transgenic tobacco plants, overexpressing either wheat Rgp1 or Rgp2, showed the presence of RGP1 and RGP2 in high-molecular mass complexes. Yeast two-hybrid studies indicated that RGP1 and RGP2 form homo- and heterodimers. Screening of a cDNA library using the yeast two-hybrid system and purification of the complex by an antibody affinity column did not reveal the presence of other proteins in the RGP complexes. Taken together, these results suggest the presence of active RGP1 and RGP2 homo- and heteromultimers in wheat endosperm.