Age-related loss of stress-induced nuclear proteasome activation is due to low PARP-1 activity

Changes in protein turnover are among the dominant metabolic changes during aging. Of special importance is the maintenance of nuclear protein homeostasis to ensure a coordinated cellular metabolism. Therefore, in the nucleus a special PARP-1-mediated mechanism of proteasomal activation exists to ensure a rapid degradation of oxidized nuclear proteins. It was already demonstrated earlier that the cytosolic proteasomal system declines dramatically with aging, whereas the nuclear proteasome remains less affected. We demonstrate here that the stress-mediated proteasomal activation in the nucleus declines during replicative senescence of human fibroblasts. Furthermore, we clearly show that this decline in the PARP-1-mediated proteasomal activation is due to a decline in the expression and activity of PARP-1 in senescent fibroblasts. In a final study we show that this process also happens in vivo, because the protein expression level of PARP-1 is significantly lower in the skin of aged donors compared to that of young ones. Therefore, we conclude that the rate-limiting factor in poly(ADP-ribose)-mediated proteasomal activation in oxidative stress is PARP-1 and not the nuclear proteasome itself.     

Mining the soluble chloroplast proteome by affinity chromatography

Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO(2), they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions.     

New models for the wing extension in pterosaurs

All powered flying animals have to face the same energetic problems: operating the wings during steady flight with muscles that require constant energy input and neural control to work. Accordingly the extant flying vertebrates have apparently found very similar solutions to parts of these issues – the biomechanical automatism built in their skeletal, muscular and connective tissue system. Based on these extant analogues (birds and bats) two new models are presented here for the mechanism of the distal wing extension in pterosaurs, an extinct group of flying vertebrates. The elongate fourth finger which solely supported their extensive flight membrane was a long lever arm that experienced significant loads and for which a reduction in muscle mass through automatisation would have been strongly beneficial. In the first model we hypothesize the presence of a propatagial ligament or ligamentous system which, as a result of the elbow extension, automatically performs and maintains the extension of the wing finger during flight and prohibits the hyperextension of the elbow. The second model has a co-operating bird-like propatagial ligamentous system and bat-like tendinous extensor muscle system on the forearm of the hypothetical pterosaur. Both models provide strong benefits to an animal with powered flight: (1) reduction of muscles and weight in the distal wing; (2) prevention of hyper extension of the elbow against drag; (3) automating wing extension and thereby reducing metabolic costs required to operate the pterosaurian locomotor apparatus. These models, although hypothetical, fit with the existing fossil evidence and lay down a basis for further biomechanical and/or aerodynamical investigations.     

The detection of age-, gender-, and region-specific changes in mouse brain tocopherol levels via the application of different validated HPLC methods

The aging process clearly increases the demand for antioxidant protection, especially in the brain, involving that provided by α-tocopherol (αT). However, little is known about the age-related changes in brain αT levels and the influencing effect of gender on it, in human or murine samples as well. Accordingly, the aim of the current study was to detect age-, gender- and region-specific changes in αT concentrations in mouse brain tissue and to assess the influencing effect of plasma αT levels on it. Female and male C57BL/6 mice at the ages of 6, 16 and 66 weeks (n?=?9 in each group) were applied. αT levels were determined with high performance liquid chromatography (HPLC) from the striatum, cortex, hippocampus, cerebellum, brainstem and from plasma samples. A detailed validation process was carried out for the applied HPLC method as well. The results demonstrated that brain αT levels significantly increased in the striatum, cortex, and hippocampus with aging in both genders, but in a more pronounced way in females with an increasing magnitude of this difference. In case of the cerebellum, a moderate elevation could be detected only in females, whereas in case of the brainstem there was no significant change in αT level. With regard to plasma samples, no clear trend could be identified. The current study is the first to present age-dependent gender-specific changes in αT level in certain brain regions of the C57Bl/6 mouse strain, and may provide meaningful information for future therapeutic studies targeting aging-related processes.     

Biomimetic potential of some methacrylate‐based copolymers: A comparative study

Preparation of new biocompatible materials for bone recovery has consistently gained interest in the last few decades. Special attention was given to polymers that contain negatively charged groups, such as phosphate, carboxyl, and sulfonic groups toward calcification. This present paper work demonstrates that other functional groups present also potential application in bone pathology. New copolymers of 2‐hydroxyethyl methacrylate with diallyldimethylammonium chloride (DADMAC), glycidyl methacrylate (GlyMA), methacrylic acid (MAA), 2‐methacryloyloxymethyl acetoacetate (MOEAA), 2‐methacryloyloxyethyltriethylammonium chloride (MOETAC), and tetrahydrofurfuryl methacrylate (THFMA) were obtained. The copolymers were characterized by FTIR, swelling potential, and they were submitted to in vitro tests for calcification and cytotoxicity evaluation. GlyMA and MOETAC‐containing copolymers show promising results for further in vivo mineralization tests, as a potential alternative to the classical bone grafts, in bone tissue engineering. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 966–973, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Gene mining in halophytes: functional identification of stress tolerance genes in Lepidium crassifolium

Extremophile plants are valuable sources of genes conferring tolerance traits, which can be explored to improve stress tolerance of crops. Lepidium crassifolium is a halophytic relative of the model plant Arabidopsis thaliana, and displays tolerance to salt, osmotic and oxidative stresses. We have employed the modified Conditional cDNA Overexpression System to transfer a cDNA library from L. crassifolium to the glycophyte A. thaliana. By screening for salt, osmotic and oxidative stress tolerance through in vitro growth assays and non‐destructive chlorophyll fluorescence imaging, 20 Arabidopsis lines were identified with superior performance under restrictive conditions. Several cDNA inserts were cloned and confirmed to be responsible for the enhanced tolerance by analysing independent transgenic lines. Examples include full‐length cDNAs encoding proteins with high homologies to GDSL‐lipase/esterase or acyl CoA‐binding protein or proteins without known function, which could confer tolerance to one or several stress conditions. Our results confirm that random gene transfer from stress tolerant to sensitive plant species is a valuable tool to discover novel genes with potential for biotechnological applications.     

Patterns of biosynthesis and accumulation of hydrocarbons and contact sex pheromone in the female german cockroach,Blattella germanica

De novo synthesis of contact female sex pheromone and hydrocarbons in Blattella germanica was examined using short in vivo incubations. Accumulation of pheromone on the epicuticular surface and the internal pheromone titer were related to age-specific changes in hydrocarbon synthesis and accumulation in normal and allatectomized females. The incorporation of radiolabel from [1-¹⁴C]propionate into the cuticular methyl ketone pheromone fraction was positively related to corpora allata activity during two gonotrophic cycles. During peak pheromone production the total internal lipid fraction contained greater titers of pheromone than the cuticular surface, and it too exhibited a cycle internally, preceding the rise in external pheromone. This suggests that synthesis and accumulation of pheromone internally are followed by transport of pheromone to the epicuticular surface where it accumulates. Radiolabel was incorporated efficiently into both cuticular and internal hydrocarbons after the imaginal molt and until the peak of pheromone synthesis, but it declined to lower levels before ovulation and throughout pregnancy. The internal hydrocarbon titer decreased 58% after oviposition, suggesting deposition in the egg case. It remained relatively unchanged during pregnancy and increased again during the second gonotrophic cycle. In allatectomized females, hydrocarbon synthesis was reduced relative to control females until oviposition in the latter. However, subsequent rates of hydrocarbon synthesis in allatectomized females (without oothecae) exceeded the rates in sham-operated females (with oothecae). In the absence of ovarian uptake of hydrocarbons, the internal titer increased without the decline found in control females at oviposition. As internal hydrocarbons increased, so did cuticular hydrocarbons and both internal and cuticular methyl ketone pheromones. These patterns corresponded well with feeding patterns in sham-operated and allatectomized females, suggesting that pheromone production is normally regulated by stage-specific feeding-induced hydrocarbon synthesis (precursor accumulation internally) and juvenile hormoneinduced conversion of hydrocarbon to pheromone. They also suggest that both the cuticle and the ovaries might be target sites for hydrocarbon and possibly methyl ketone deposition. © 1994 Wiley-Liss, Inc.     

Regulatory T cells inhibit dendritic cells by lymphocyte activation gene-3 engagement of MHC class II

Lymphocyte activation gene-3 (LAG-3) is a CD4-related transmembrane protein expressed by regulatory T cells that binds MHC II on APCs. It is shown in this study that during Treg:DC interactions, LAG-3 engagement with MHC class II inhibits DC activation. MHC II cross-linking by agonistic Abs induces an ITAM-mediated inhibitory signaling pathway, involving FcgammaRgamma and ERK-mediated recruitment of SHP-1 that suppresses dendritic cell maturation and immunostimulatory capacity. These data reveal a novel ITAM-mediated inhibitory signaling pathway in DCs triggered by MHC II engagement of LAG-3, providing a molecular mechanism in which regulatory T cells may suppress via modulating DC function.     

Some epidemiological and epizootiological aspects of Lyme borreliosis in Slovakia with the emphasis on the problems of serological diagnostics

The paper analyses the results of serological examinations of domestic, farm and free-living animals from different regions of Slovakia, Southern Moravia, Southern Bohemia and Southern Poland using ELISA, indirect hemagglutination assay (IHA) and Western blot (WB). In Slovakia, significantly higher seroprevalence was recorded in dogs (33.5%) than in horses (26.5%), cattle (22.5%), sheep (16.6%) and rodents (17.8%) by using a mixture of Borrelia garinii, B. afzelii, B. burgdorferi sensu stricto (s.s.) antigens in ELISA. Seroprevalence in horses was significantly higher than in sheep and rodents, and seroprevalence in cattle was significantly higher than in rodents. By using IHA in free-living species, the highest seropositivity rates were detected in fallow deer (40.7%) compared with moufflons (16.6%), pheasants (8.0%) and pigeons (1.2%). When testing sera of horses, dogs and cattle from Slovakia by using different Slovak B. burgdorferi sensu lato (s.l.) isolates as antigens in ELISA, significantly higher seroprevalence of anti-Borrelia IgG antibodies and consistency of positive and negative findings was detected in comparison when American isolates were used. In WB analyses using the Eurocarpathian antigens, dog sera from Eastern Slovakia and Southern Moravia showed statistically insignificant differences in sensitivity and consistency of positive and negative findings. By using different methods and antigens in the same group of dog sera, significant differences in seroprevalence were only found in IHA with a mixture of Euroamerican B.b.s.l and WB CB26 B.b.s.s. In addition to other factors, the complexity of the standardization of the assay system with regard to the genetic and geographical heterogeneity of B. burgdorferi s.l. isolates used as antigens is also discussed.