Quantitative Determination of Geranyl Diphosphate Levels in Cultured Human Cells

Geranyl diphosphate (GPP), a 10-carbon isoprenoid, is a key intermediate in the isoprenoid biosynthetic pathway. This pathway, in addition to leading to sterol synthesis, results in the synthesis of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP), which serve as substrates for protein isoprenylation reactions. Basal levels of GPP in mammalian cells previously have been undetectable. Here we present a novel, sensitive, nonradioactive method which allows for measurement of GPP in mammalian cells. This methodology involves extraction of isoprenoids from cultured cells followed by enzymatic conjugation of GPP to a fluorescent dansylated-peptide via farnesyl transferase and quantification with high-performance liquid chromatography (HPLC). The lower limit of detection of GPP is 5 pg, or 0.015 pmol. Basal levels of GPP were determined in three human multiple myeloma cell lines (RPMI-8226, U266, H929). Treatment of cells with inhibitors of the isoprenoid biosynthetic pathway results in marked changes in GPP levels: the HMG-CoA reductase inhibitor lovastatin decreases GPP levels by over 50%, while the FPP synthase inhibitor zoledronic acid increases GPP levels 16- to 107-fold. This method also allows for the simultaneous measurement of GPP, FPP, and GGPP, thus leading to improved understanding of the pathway in a multitude of biological systems. Furthermore, as drugs targeting this pathway are developed, their biological activity can be more directly linked to effects on isoprenoid levels.     

Die Versatzrichtlinie des BVÖ: ein Leitfaden für die zeitgemäße Planung eines Versatzsystems

Several cases demonstrated that a common understanding between mine operators and the relevant authorities concerning the possible application of backfill is missing. Based on this conclusion, the Bergmännischer Verband Österreichs in form of the members of its Committee on Underground Mining decided to develop a procedural standard dealing with the issues of the design and operation of a backfill system. The objective of this paper is to assist the users of this procedural standard by providing additional explanations and examples of application.     

Vitamin C and E Supplementation Effects in Professional Soccer Players Under Regular Training

Exercise training is known to induce an increase in free radical production potentially leading to enhanced muscle injury. Vitamins C and E are well known antioxidants that may prevent muscle cell damage. The purpose of this study was to determine the effects of these supplemental antioxidant vitamins on markers of oxidative stress, muscle damage and performance of elite soccer players. Ten male young soccer players were divided into two groups. Supplementation group (n = 5) received vitamins C and E supplementation daily during the pre-competitive season (S group), while the placebo group (PL group, n = 5) received a pill containing maltodextrin. Both groups performed the same training load during the three-month pre-season training period. Erythrocyte antioxidant enzymes glutathione reductase, catalase and plasma carbonyl derivatives did not show any significant variation among the experimental groups. Similarly, fitness level markers did not differ among the experimental groups. However, S group demonstrated lower lipid peroxidation and muscle damage levels (p < 0.05) compared to PL group at the final phase of pre-competitive season. In conclusion, our data demonstrated that vitamin C and E supplementation in soccer players may reduce lipid peroxidation and muscle damage during high intensity efforts, but did not enhance performance.     

Stereochemistry-dependent inhibition of RAS farnesylation by farnesyl phosphonic acids

This investigation compares the effects of three farnesyl pyrophosphate analogs on selected aspects of isoprenoid metabolism. E,E-α-Hydroxyfarnesylphosphonate was prepared by an improved variation on a literature synthesis, which also gave access to the new Z,E-α-hydroxyfarnesyl- and α-hydroxygeranylphosphonates. A striking find is that only E,E-α-hydroxyfarnesylphosphonate induces alteration of RAS processing in intact human-derived leukemia cells and inhibits farnesyl protein transferase in enzyme assays, while the Z,E-α-farnesyl- and geranylphosphonates are inactive. The inhibitory activity of E,E-α-hydroxyfarnesylphosphonate is greater in enzyme than intact cell assays. This active compound does not significantly inhibit geranylgeranyl protein transferase I or squalene synthase, nor does it diminish cholesterol synthesis. These results indicate that the length of the terpenoid chain and olefin stereochemistry allow selective inhibition of critical enzymes of terpenoid metabolism. Discrimination was observed between inhibition of farnesyl protein transferase and squalene synthase by E,E-α-hydroxyfarnesylphosphonate, even though both enzymes utilize farnesyl pyrophosphate as their natural substrate.     

可持续冷喂高精度生产的COMBEX预成型机

介绍COMBEX预成型机的组成、工作原理和优越性。COMBEX预成型机中齿轮泵技术的运用对挤出效率及产品精度提供了重要的保障。与传统柱塞系统相比，COMBEX机器具有过程持续、冷喂生产、最少占用空间、无滞留空气、高稳定性及精度、全电动、准确的温度控制及操作简单等优越性.     

Influence of Nanoparticles and Drop Size Distributions on the Rheology of w/o Pickering Emulsions

The rheological behavior of particle/oil suspensions and w/o Pickering emulsions consisting of water, 1‐dodecene and different fumed silica nanoparticles was investigated. The particles varied in hydrophobicity and specific surface area. The influence of particle concentration and water content on rheology was determined and the emulsion drop size distributions were examined. Emulsions with different drop sizes were created by either varying the particle concentration or the water content. It was found that the particles in the continuous oil phase and not the drop size distribution seem to be the major influencing factor on the Pickering emulsion rheology.     

In vitro assessment of the pulmonary toxicity and gastric availability of lead-rich particles from a lead recycling plant

Epidemiological studies in urban areas have linked increasing respiratory and cardiovascular pathologies with atmospheric particulate matter (PM) from anthropic activities. However, the biological fate of metal-rich PM industrial emissions in urban areas of developed countries remains understudied. Lead toxicity and bioaccessibility assessments were therefore performed on emissions from a lead recycling plant, using complementary chemical acellular tests and toxicological assays, as a function of PM size (PM(10-2.5), PM(2.5-1) and PM(1)) and origin (furnace, refining and channeled emissions). Process PM displayed differences in metal content, granulometry, and percentage of inhalable fraction as a function of their origin. Lead gastric bioaccessibility was relatively low (maximum 25%) versus previous studies; although, because of high total lead concentrations, significant metal quantities were solubilized in simulated gastrointestinal fluids. Regardless of origin, the finest PM(1) particles induced the most significant pro-inflammatory response in human bronchial epithelial cells. Moreover, this biological response correlated with pro-oxidant potential assay results, suggesting some biological predictive value for acellular tests. Pulmonary effects from lead-rich PM could be driven by thiol complexation with either lead ions or directly on the particulate surface. Finally, health concern of PM was discussed on the basis of pro-inflammatory effects, accellular test results, and PM size distribution.     

Pleiotropic Effects of a Schweinfurthin on Isoprenoid Homeostasis

The schweinfurthins, a family of natural products derived from the isoprenoid biosynthetic pathway (IBP), have marked growth inhibitory activity. However, the biochemical basis for the schweinfurthins cellular effects has remained ill-defined. Here, the effects of the synthetic schweinfurthin, 3-deoxyschweinfurthin (3dSB) on multiple aspects of isoprenoid homeostasis are explored. Cytotoxicity assays demonstrate a synergistic interaction between 3dSB and the HMG-CoA reductase inhibitor lovastatin but not with other IBP inhibitors in a variety of human cancer cell lines. The cytotoxic effects of 3dSB were enhanced in cells incubated in lipid-depleted serum. 3dSB was found to enhance the lovastatin-induced decrease in protein prenylation. In addition, 3dSB decreases intracellular farnesyl pyrophosphate and geranylgeranyl pyrophosphate levels in both established cell lines and primary cells. To determine whether 3dSB alters the regulation of expression of genes involved in isoprenoid homeostasis, real-time PCR studies were performed in human cell lines cultured in either lipid-replete or -deplete conditions. These studies demonstrate that 3dSB abrogates lovastatin-induced upregulation of sterol regulatory element-containing genes and lovastatin-induced downregulation of ABCA1. In aggregate, these studies are the first to demonstrate that a schweinfurthin exerts pleiotropic effects on isoprenoid homeostasis.     

Model studies on reactions of plant phenols with whey proteins

Whey proteins were modified by reaction with selected phenolic compounds (ferulic-, chlorogenic-, caffeic- and gallic acid) and related substances (quinic acid and p-quinone) as well as with extracts from coffee, tea, potato and pear at pH 9. The derivatives formed were characterized in terms of their physicochemical and digestion properties. The derivatization was accompanied by a reaction at the lysine and tryptophan side chains, whereby their content was decreased in comparison to that in the control whey proteins. Moreover, the solubility of the derivatives decreased over a broad pH range and the derivatization influenced the hydrophobe-hydrophile character of the whey proteins. The isoelectric points were shifted to lower pH values in the order of reactivity as follows: gallic acid > p-quinone > caffeic acid > chlorogenic acid. The other derivatives showed no or few changes compared to the control whey proteins. The formation of high molecular fractions was documented with SDS-PAGE. Especially the derivatives of chlorogenic-, caffeic-, gallic acid and p-quinone showed an increase in molecular weight of beta-lactoglobulin fraction from 18,300 to 20,000 Da. A dimer formation in molecular range 40,000 was also registered. MALDI-TOF-MS was applied to characterize the binding of the individual phenolic compounds or their oxidation products to the whey protein fractions, alpha-lactalbumin and beta-lactoglobulin. In vitro experiments showed that the digestion of the derivatized whey proteins with the enzymes of the gastrointestinal tract (trypsin, chymotrypsin, pepsin and pancreatin) was adversely effected. Similar results with regard to physicochemical characterization and digestion properties of the whey proteins treated with the applied extracts from plant beverages, fruit and vegetable were also documented. Coffee and tee were comparatively the most reactive extracts.