Inverse response of leukocyte heat shock proteins and DNA damage to exercise and heat

Elevated ambient temperature may exert an additional impact on the exercise-induced expression of heat shock proteins (HSP) and DNA damage in leukocytes. The protective functions of HSP include antioxidative and antiapoptotic effects and may prevent damage to DNA. Twelve athletes completed a continuous run (75% VO2max) on the treadmill, six at 28 degrees C and six at 18 degrees C room temperature. Leukocyte expression of HSP27 and inducible HSP70 was analyzed on mRNA- (RT-PCR) and protein-level (flow cytometry), while DNA damage was quantified by the comet assay. High ambient temperature induced an additional accumulation of HSP-mRNA and -protein in leukocytes compared with the exercise-induced expression at 18 degrees C. HSP27 showed a special heat sensitivity. Surprisingly, the increase of DNA damage was less pronounced after exercise at 28 degrees C compared to 18 degrees C although heat shock in vitro clearly induced DNA damage. The inverse relation between HSP and DNA damage may indicate functions of HSP which protect against exercise-induced DNA-damage in terms of thermotolerance or apoptosis.     

Conformationally-restricted analogues and partition coefficients of the 5-HT3 serotonin receptor ligands meta-chlorophenylbiguanide (mCPBG) and meta-chlorophenylguanidine (mCPG)

The present investigation examined two features of arylbiguanide and arylguanidine 5-HT(3) ligands: conformation and partition coefficients. Several conformationally-constrained analogues of mCPBG (2) and mCPG (11; K(i)=32 nM) were prepared and of these only 2-amino-5-chloro-3,4-dihydroquinazoline (14; K(i)=34 nM) retained high affinity. The partition coefficient of compound 11 (LogP(app)=-0.64) was less than that of its corresponding arylbiguanide 2 (LogP(app)=-0.38). The quinazoline structure may represent a pharmacologically-active conformation of these agents, and the arylbiguanides were found more lipid soluble than their arylguanidine counterparts at physiological pH.     

Divisome‐dependent subcellular localization of cell–cell joining protein SepJ in the filamentous cyanobacterium Anabaena

Heterocyst‐forming cyanobacteria are multicellular organisms that grow as filaments that can be hundreds of cells long. Septal junction complexes, of which SepJ is a possible component, appear to join the cells in the filament. SepJ is a cytoplasmic membrane protein that contains a long predicted periplasmic section and localizes not only to the cell poles in the intercellular septa but also to a position similar to a Z ring when cell division starts suggesting a relation with the divisome. Here, we created a mutant of Anabaena sp. strain PCC 7120 in which the essential divisome gene ftsZ is expressed from a synthetic NtcA‐dependent promoter, whose activity depends on the nitrogen source. In the presence of ammonium, low levels of FtsZ were produced, and the subcellular localization of SepJ, which was investigated by immunofluorescence, was impaired. Possible interactions of SepJ with itself and with divisome proteins FtsZ, FtsQ and FtsW were investigated using the bacterial two‐hybrid system. We found SepJ self‐interaction and a specific interaction with FtsQ, confirmed by co‐purification and involving parts of the SepJ and FtsQ periplasmic sections. Therefore, SepJ can form multimers, and in Anabaena, the divisome has a role beyond cell division, localizing a septal protein essential for multicellularity.     

Exploration of peptides bound to MHC class I molecules in melanoma

Advancements in high‐resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13 829 peptides were identified; 83–87% of these were 8–11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA‐type binding prediction for 10 078 9/10 mer peptides assigned 88–95% to a patient‐specific HLA subtype, revealing a disparity in strength of predicted binding. HLA‐B*27‐specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.     

Innate IFNs and plasmacytoid dendritic cells constrain Th2 cytokine responses to rhinovirus: a regulatory mechanism with relevance to asthma

Human rhinoviruses (RV) cause only minor illness in healthy individuals, but can have deleterious consequences in people with asthma. This study sought to examine normal homeostatic mechanisms regulating adaptive immunity to RV in healthy humans, focusing on effects of IFN-αβ and plasmacytoid dendritic cells (pDC) on Th2 immune responses. PBMC were isolated from 27 healthy individuals and cultured with RV16 for up to 5 d. In some experiments, IFN-αβ was neutralized using a decoy receptor that blocks IFN signaling, whereas specific dendritic cell subsets were depleted from cultures with immune-magnetic beads. RV16 induced robust expression of IFN-α, IFN-β, multiple IFN-stimulated genes, and T cell-polarizing factors within the first 24 h. At 5 d, the production of memory T cell-derived IFN-γ, IL-10, and IL-13, but not IL-17A, was significantly elevated. Neutralizing the effects of type-I IFN with the decoy receptor B18R led to a significant increase in IL-13 synthesis, but had no effect on IFN-γ synthesis. Depletion of pDC from RV-stimulated cultures markedly inhibited IFN-α secretion, and led to a significant increase in expression and production of the Th2 cytokines IL-5 (p = 0.02), IL-9 (p < 0.01), and IL-13 (p < 0.01), but had no effect on IFN-γ synthesis. Depletion of CD1c(+) dendritic cells did not alter cytokine synthesis. In healthy humans, pDC and the IFN-αβ they secrete selectively constrain Th2 cytokine synthesis following RV exposure in vitro. This important regulatory mechanism may be lost in asthma; deficient IFN-αβ synthesis and/or pDC dysfunction have the potential to contribute to asthma exacerbations during RV infections.     

RIF1 Is Essential for 53BP1-Dependent Nonhomologous End Joining and Suppression of DNA Double-Strand Break Resection

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Highlights? RIF1 is essential for 53BP1-dependent CSR and fusion of dysfunctional telomeres ? BRCA1 antagonizes RIF1 in S phase to prevent error-prone repair by toxic NHEJ ? N-terminal phospho-SQ/TQ domain of 53BP1 interacts with and recruits RIF1 to DSBs ? RIF1 and 53BP1 promote NHEJ in G1 by blocking 5′ end resection of DSBs     

Loss of periostin occurs in aging adipose tissue of mice and its genetic ablation impairs adipose tissue lipid metabolism

Remodeling of the extracellular matrix is a key component of the metabolic adaptations of adipose tissue in response to dietary and physiological challenges. Disruption of its integrity is a well‐known aspect of adipose tissue dysfunction, for instance, during aging and obesity. Adipocyte regeneration from a tissue‐resident pool of mesenchymal stem cells is part of normal tissue homeostasis. Among the pathophysiological consequences of adipogenic stem cell aging, characteristic changes in the secretory phenotype, which includes matrix‐modifying proteins, have been described. Here, we show that the expression of the matricellular protein periostin, a component of the extracellular matrix produced and secreted by adipose tissue‐resident interstitial cells, is markedly decreased in aged brown and white adipose tissue depots. Using a mouse model, we demonstrate that the adaptation of adipose tissue to adrenergic stimulation and high‐fat diet feeding is impaired in animals with systemic ablation of the gene encoding for periostin. Our data suggest that loss of periostin attenuates lipid metabolism in adipose tissue, thus recapitulating one aspect of age‐related metabolic dysfunction. In human white adipose tissue, periostin expression showed an unexpected positive correlation with age of study participants. This correlation, however, was no longer evident after adjusting for BMI or plasma lipid and liver function biomarkers. These findings taken together suggest that age‐related alterations of the adipose tissue extracellular matrix may contribute to the development of metabolic disease by negatively affecting nutrient homeostasis.     

Activity of cell wall-associated enzymes in ripening olive fruit

Enzymatically active cell wall isolaled from olive (Olea europaea) fruit was employed Hi investigate some hydrolytic enzymes bound to the cell wall and the changes in these during ripening. Seven glycosidases. β-glucosidase (EC 3.2.1.21) α-galactosidase (EC 3.2.1.22). β-galactosidase (EC 3.2.1.23). α-arabinosidase (EC 3.2.1.55), α-mannosidase (EC 3.2.1,24). β-xylosidase (EC 3.2.1.37) and β-N-acetylglucosamidase (EC 3.2.1.30). as well as Cx-cellulase (EC 3.2.1.4) and endo-polygalacturonase (EC 3.2.1.15). were identified in the cell wall preparation, at four stages of ripeness (mature green. changing colour, black and black-ripe). Activities of all these cell wall-associated enzymes fionicallv and covalently linked) were determined either by cell wall incubation with artificial substrate or after extraction from the cell wall with buffers of high salt concentration (Cx-cellulase). and were compared to those of forms solubilized from acetone powders with 500 nM citrate buffer (cytoplasmic and/or apoplastic plus ionically hound to cell wall) In general, the activities of low ionic strength buffer-soluble enzymes were found to be much higher than those of the bound enzymes. The bound enzymes are present in the fruit at the green colour stage, whereas the activities of the soluble enzymes only increased from the changing colour stage onwards. The tenacity of binding of enzymes to the wall was investigated by treating the walls with high salt and measuring residual activity. The nature of the ionic and covalent binding and the changes during ripening were also established for wall-hound glycosidase During ripening there was a marked change in the percentages of covalently- and tonically linked activities of β-glucosidase and β-galaclosidase: al the changing colour stages about 75–80% of the bound active in was present in high ionic strength buffer while al the black-ripe stage it was only 15–20. A possible role for these cell wall degradative enzymes in olive softening is discussed.     

High levels of genetic structure and striking phenotypic variability in a sexually dimorphic suckermouth catfish from the African Highveld

Uncovering biological diversity to more accurately understand diversity patterns, and ultimately the processes driving diversification, is important not only from an evolutionary perspective but also a conservation perspective. This is particularly pertinent in Africa's rivers in which overall diversity, as well as how it arose, is poorly understood in comparison with lacustrine environments. Here we investigate population divergence in the sexually dimorphic suckermouth catfish species Chiloglanis anoterus (Crass, 1960) from the African Highveld, in which we observe striking variability in exaggerated male caudal fins across its range. As this trait is likely to be indirect evidence for sexual selection by female choice, a mechanism that has been shown to increase species diversity in different taxa, we used an integrated approach to test if current diversity in this species is underestimated. Results based on phylogenetic inference, population genetics and geometric morphometrics indicate that the recognized species C. anoterus represents five distinct lineages that may be considered confirmed candidate species. We suggest that diversification in these highland catfish has been facilitated through geographical isolation in upper river catchments, and that sexual selection through female choice has probably driven variation in male caudal fin morphology. In contrast to the relatively large range size of the currently recognized species (C. anoterus), our findings highlight highly restricted ranges of the lineages identified here, indicating that these highland habitats may harbour higher levels of endemic diversity than previously thought.