Role of the chaperonin CCT/TRiC complex in G protein betagamma-dimer assembly

Gbetagamma dimer formation occurs early in the assembly of heterotrimeric G proteins. On nondenaturing (native) gels, in vitro translated, (35)S-labeled Ggamma subunits traveled primarily according to their pI and apparently were not associated with other proteins. In contrast, in vitro translated, (35)S-labeled Gbeta subunits traveled at a high apparent molecular mass (approximately 700 kDa) and co-migrated with the chaperonin CCT complex (also called TRiC). Different FLAG-Gbeta isoforms coprecipitated CCT/TRiC to a variable extent, and this correlated with the ability of the different Gbeta subunits to efficiently form dimers with Ggamma. When translated Ggamma was added to translated Gbeta, a new band of low apparent molecular mass (approximately 50 kDa) was observed, which was labeled by either (35)S-labeled Gbeta or Ggamma, indicating that it is a dimer. Formation of the Gbetagamma dimer was ATP-dependent and inhibited by either adenosine 5'-O-(thiotriphosphate) or aluminum fluoride in the presence of Mg(2+). This inhibition led to increased association of Gbeta with CCT/TRiC. Although Ggamma did not bind CCT/TRiC, addition of Ggamma to previously synthesized Gbeta caused its release from the CCT/TRiC complex. We conclude that the chaperonin CCT/TRiC complex binds to and folds Gbeta subunits and that CCT/TRiC mediates Gbetagamma dimer formation by an ATP-dependent reaction.     

Genetic Variants in Inflammation-Related Genes Are Associated with Radiation-Induced Toxicity Following Treatment for Non-Small Cell Lung Cancer

Treatment of non-small cell lung cancer (NSCLC) with radiotherapy or chemoradiotherapy is often accompanied by the development of esophagitis and pneumonitis. Identifying patients who might be at increased risk for normal tissue toxicity would help in determination of the optimal radiation dose to avoid these events. We profiled 59 single nucleotide polymorphisms (SNPs) from 37 inflammation-related genes in 173 NSCLC patients with stage IIIA/IIIB (dry) disease who were treated with definitive radiation or chemoradiation. For esophagitis risk, nine SNPs were associated with a 1.5- to 4-fold increase in risk, including three PTGS2 (COX2) variants: rs20417 (HR:1.93, 95% CI:1.10–3.39), rs5275 (HR:1.58, 95% CI:1.09–2.27), and rs689470 (HR:3.38, 95% CI:1.09–10.49). Significantly increased risk of pneumonitis was observed for patients with genetic variation in the proinflammatory genes IL1A, IL8, TNF, TNFRSF1B, and MIF. In contrast, NOS3:rs1799983 displayed a protective effect with a 45% reduction in pneumonitis risk (HR:0.55, 95% CI:0.31–0.96). Pneumonitis risk was also modulated by polymorphisms in anti-inflammatory genes, including genetic variation in IL13. rs20541 and rs180925 each resulted in increased risk (HR:2.95, 95% CI:1.14–7.63 and HR:3.23, 95% CI:1.03–10.18, respectively). The cumulative effect of these SNPs on risk was dose-dependent, as evidenced by a significantly increased risk of either toxicity with an increasing number of risk genotypes (P<0.001). These results suggest that genetic variations among inflammation pathway genes may modulate the development of radiation-induced toxicity and, ultimately, help in identifying patients who are at an increased likelihood for such events.     

Diferrocenes containing thiadiazole connectivities

2,5-Diferrocenyl-1,3,4-thiadiazole, 2,5-Fc₂-^cC₂N₂S, (3) has been synthesized by a two-fold Negishi ferrocenylation of dibromothiadiazole (1) with FcZnCl (2) (Fc = Fe(η⁵-C₅H₄)(η⁵-C₅H₅)) in presence of [Pd(Ph₃P)₄] as catalyst. Additional spacer units between the ferrocenyls and the ^cC₂N₂S core could be introduced by using the Sonogashira C,C cross-coupling protocol. Reaction of 2,5-Br₂-^cC₂N₂S (1) or 2,5-(C₆H₄-4′-I)₂-^cC₂N₂S (6) with FcCCH (4) using [PdCl₂(Ph₃P)₂] and [CuI] as catalyst produced the appropriate organometallics 2,5-(FcCC)₂-^cC₂N₂S (5) or 2,5-(C₆H₄-4′-CCFc)₂-^cC₂N₂S (7). The electronic and structural properties of 3, 5, and 7 were investigated with UV-Vis spectroscopy and single crystal X-ray diffraction (3). Complex 3 adopts a solid state structure with none of the ferrocenyl substituents being coplanar with the thiadiazole ring. Cyclic, square wave, linear sweep voltammetry and in-situ NIR spectro-electrochemistry highlight the electrochemical properties of 3. In dichloromethane (0.1 mol L⁻¹ [N(ⁿBu)₄][B(C₆F₅)₄]), compound 3 displays two well resolved electrochemical reversible one-electron events with formal reduction potentials of 0.192 and 0.338 V versus FcH/FcH⁺. In contrast, in presence of [N(ⁿBu)₄][PF₆], the thiadiazoles 3 (E⁰ = 0.22 V), 5 (E⁰ = 0.18 V) and 7 (E⁰ = 0.09 V) show simultaneously oxidation of the two ferrocenyl termini versus FcH/FcH⁺. Spectro-electrochemical studies, performed in a dichloromethane solution of 0.2 mol L⁻¹ [N(ⁿBu)₄][B(C₆F₅)₄], also show that 3 can successively be oxidized via 3⁺ to 3²⁺. A weak IVCT absorption (ε ca. 300 L mol⁻¹ cm⁻¹) at 1560 nm was found and is consistent with appreciable interactions between neutral ferrocenyl and positively charged ferrocenium mixed valent intermediates. Mixed-valent compound 3⁺ corresponds to a class II molecule according to Robin and Day.     

Low-dose radiation exposure and atherosclerosis in ApoE⁻/⁻ mice

The hypothesis that single low-dose exposures (0.025-0.5 Gy) to low-LET radiation given at either high (about 150 mGy/min) or low (1 mGy/min) dose rate would promote aortic atherosclerosis was tested in female C57BL/6J mice genetically predisposed to this disease (ApoE?/?). Mice were exposed either at an early stage of disease (2 months of age) and examined 3 or 6 months later or at a late stage of disease (8 months of age) and examined 2 or 4 months later. Changes in aortic lesion frequency, size and severity as well as total serum cholesterol levels and the uptake of lesion lipids by lesion-associated macrophages were assessed. Statistically significant changes in each of these measures were observed, depending on dose, dose rate and disease stage. In all cases, the results were distinctly non-linear with dose, with maximum effects tending to occur at 25 or 50 mGy. In general, low doses given at low dose rate during either early- or late-stage disease were protective, slowing the progression of the disease by one or more of these measures. Most effects appeared and persisted for months after the single exposures, but some were ultimately transitory. In contrast to exposure at low dose rate, high-dose-rate exposure during early-stage disease produced both protective and detrimental effects, suggesting that low doses may influence this disease by more than one mechanism and that dose rate is an important parameter. These results contrast with the known, generally detrimental effects of high doses on the progression of this disease in the same mice and in humans, suggesting that a linear extrapolation of the known increased risk from high doses to low doses is not appropriate.     

A scaffold-free in vitro model for osteogenesis of human mesenchymal stem cells

For studying cellular processes three-dimensional (3D) in vitro models are of a high importance. For tissue engineering approaches osseous differentiation is performed on 3D scaffolds, but material depending influences promote cellular processes like adhesion, proliferation and differentiation. To investigate developmental processes of mesenchymal stem cells without cell-substrate interactions, self-contained in vitro models mimicking physiological condition are required. However, with respect to scientific investigations and pharmaceutical tests, it is essential that these tissue models are well characterised and are of a high reproducibility. In order to establish an appropriate in vitro model for bone formation, different protocols are compared and optimised regarding their aggregate formation efficiency, homogeneity of the aggregates, the viability and their ability to induce differentiation into the osteogenic lineage. The protocols for the generation of 3D cell models are based on rotation culture, hanging drop technique, and the cultivation in non adhesive culture vessels (single vessels as well as 96 well plates). To conclude, the cultivation of hMSCs in 96 well non adhesive plates facilitates an easy way to cultivate homogenous cellular aggregates with high performance efficiency in parallel. The size can be controlled by the initial cell density per well and within this spheroids, bone formation has been induced.     

Reproductive cycle of the elephant

The combination of a few factors, including poor captive reproduction, secession of importation from the wild and advances in hormone detection and ultrasonography, has contributed to the current knowledge on the elephant reproductive cycle. Several reproductive features in elephants differ markedly from other mammals. These include the urogenital tract anatomy, length and structure of the reproductive cycle, the formation of multiple corpora lutea and the type and secretion pattern of reproductive hormones. Being 13-18 weeks in length, the elephant estrous cycle is the longest amongst all studied non-seasonal mammals to date. Progesterone increases 1-3 days after ovulation, indicating the start of the luteal phase, which lasts 6-12 weeks. This is followed by a 4- to 6-week follicular phase that is concluded by two, precisely spaced and timed, LH surges. In general, the first, anovulatory LH surge occurs exactly 19-21 days before the second, ovulatory surge. Normally, a single follicle is ovulated. However, beside a corpus luteum (CL) forming on the site of ovulation, multiple accessory CLs can be found on the ovaries. Unlike many other species, the predominant progestagen secreted by luteal tissues is not progesterone, but rather its 5-alpha-reduced metabolites. The currently known aspects of the unique estrous cycle in Asian and African elephants, covering estrous behavior, circulating hormones, ultrasonography and anatomy of the reproductive organs as well as hormonal manipulation treatment possibilities, will be reviewed here.     

Centrosomes and cilia in human disease

Centrioles are microtubule-derived structures that are essential for the formation of centrosomes, cilia and flagella. The centrosome is the major microtubule organiser in animal cells, participating in a variety of processes, from cell polarisation to cell division, whereas cilia and flagella contribute to several mechanisms in eukaryotic cells, from motility to sensing. Although it was suggested more than a century ago that these microtubule-derived structures are involved in human disease, the molecular bases of this association have only recently been discovered. Surprisingly, there is very little overlap between the genes affected in the different diseases, suggesting that there are tissue-specific requirements for these microtubule-derived structures. Knowledge of these requirements and disease mechanisms has opened new avenues for therapeutical strategies. Here, we give an overview of recent developments in this field, focusing on cancer, diseases of brain development and ciliopathies.     

The positional sterile (ps) mutation affects cuticular transpiration and wax biosynthesis of tomato fruits

Cuticular waxes are known to play a pivotal role in limiting transpirational water loss across primary plant surfaces. The astomatous tomato fruit is an ideal model system that permits the functional characterization of intact cuticular membranes and therefore allows direct correlation of their permeance for water with their qualitative and quantitative composition. The recessive positional sterile (ps) mutation, which occurred spontaneously in tomato (Solanum lycopersicum L.), is characterized by floral organ fusion and positional sterility. Because of a striking phenotypical similarity with the lecer6 wax mutant of tomato, which is defective in very-long-chain fatty acid elongation, ps mutant fruits were analyzed for their cuticular wax and cutin composition. We also examined their cuticular permeance for water following the developmental course of fruit ripening. Wild type and ps mutant fruits showed considerable differences in their cuticular permeance for water, while exhibiting similar quantitative wax accumulation. The ps mutant fruits showed a five- to eightfold increase in water loss per unit time and surface area when compared to the corresponding wild type fruits. The cuticular waxes of ps mutant fruits were characterized by an almost complete absence of n-alkanes and aldehydes, with a concomitant increase in triterpenoids and sterol derivatives. We also noted the occurrence of alkyl esters not present in the wild type. Quantitative and qualitative cutin monomer composition remained largely unaffected. The significant differences in the cuticular wax composition of ps mutant fruits induced a distinct increase of cuticular water permeance. The fruit wax compositional phenotype indicates the ps mutation is responsible for effectively blocking the decarbonylation pathway of wax biosynthesis in epidermal cells of tomato fruits.     

Functional diversity of leaf nitrogen concentrations drives grassland carbon fluxes

Little is known about the role of plant functional diversity for ecosystem‐level carbon (C) fluxes. To fill this knowledge gap, we translocated monoliths hosting communities with four and 16 sown species from a long‐term grassland biodiversity experiment (‘The Jena Experiment’) into a controlled environment facility for ecosystem research (Ecotron). This allowed quantifying the effects of plant diversity on ecosystem C fluxes as well as three parameters of C uptake efficiency (water and nitrogen use efficiencies and apparent quantum yield). By combining data on ecosystem C fluxes with vegetation structure and functional trait‐based predictors, we found that increasing plant species and functional diversity led to higher gross and net ecosystem C uptake rates. Path analyses and light response curves unravelled the diversity of leaf nitrogen concentration in the canopy as a key functional predictor of C fluxes, either directly or indirectly via LAI and aboveground biomass.     

Facial EMG Responses to Emotional Expressions Are Related to Emotion Perception Ability

Although most people can identify facial expressions of emotions well, they still differ in this ability. According to embodied simulation theories understanding emotions of others is fostered by involuntarily mimicking the perceived expressions, causing a “reactivation” of the corresponding mental state. Some studies suggest automatic facial mimicry during expression viewing; however, findings on the relationship between mimicry and emotion perception abilities are equivocal. The present study investigated individual differences in emotion perception and its relationship to facial muscle responses - recorded with electromyogram (EMG) - in response to emotional facial expressions. N° = °269 participants completed multiple tasks measuring face and emotion perception. EMG recordings were taken from a subsample (N° = °110) in an independent emotion classification task of short videos displaying six emotions. Confirmatory factor analyses of the m. corrugator supercilii in response to angry, happy, sad, and neutral expressions showed that individual differences in corrugator activity can be separated into a general response to all faces and an emotion-related response. Structural equation modeling revealed a substantial relationship between the emotion-related response and emotion perception ability, providing evidence for the role of facial muscle activation in emotion perception from an individual differences perspective.