The effect of different carbon and nitrogen sources on the activity of glutamine synthetase and glutamate dehydrogenase in lupine embryonic axes

Embryos of yellow lupine ( Lupinus luteus L. cv. Jantar), deprived of cotyledons, were incubated for 72 h in media containing various combinations of saccharose, ammonia, nitrate, glutamine and asparagine. Induction of glutamine synthetase (GS) was observed in embryos in media containing saccharose while the activity of this enzyme was inhibited by glutamine, asparagine and ammonia in the absence of sugar. The above mentioned nutritional factors had an opposite effect on the activity of glutamate dehydrogenase (GDH). Changes in glutamate dehydrogenase activity were preceded by reverse changes in the activity of glutamine synthetase. The possibility of GDH repression by GS in lupine embryos is discussed.     

The protection,stabilization and reactivation of estradiol receptors in human myometrial cytosol

Protection of estradiol receptor binding sites in human mymetrial cytosol is achieved though the agency of dithiothreitol (Cleland's reagent) (lmM) at 4°C and ? 20°C storage temperatures. Prolonged storage of cytosol at 4° C without the protective reagent results in substantial loss of binding activity. This is partially restored after Cleland's reagent (lmM) addition. However, cytosol inactivated by heating at 60°C for 30 min cannot be reconstituted in this way.     

Integrated proteomic and metabolomic analyses revealed molecular adjustments in Populus × canescens colonized with the ectomycorrhizal fungus Paxillus involutus,which limited plant host growth

Ectomycorrhizae (ECMs) are a highly context-dependent interactions that are not always beneficial for the plant host, sometimes leading to a decrease in plant growth. However, the molecular status of these plants remains unknown. We studied Populus × canescens microcuttings characterized by impaired growth in response to colonization by a Paxillus involutus strain via integrative proteomics–metabolomics analyses. The analysed strain was characterized by low compatibility and formed only mantles, not a Hartig net, in the majority of root tips. The increased abundance of photosynthetic proteins and foliar carbohydrates co-occurred with signals of intensified resource exchange via the stems of colonized plants. In the roots, intensified C metabolism resulted in the biosynthesis of secondary C compounds unavailable to the fungal partner but also C skeletons necessary to increase insufficient N uptake from the hyphae. The stress response was also detected in colonized plants but was similar to that reported previously during mutualistic ECM interactions. In colonized poplar plants, mechanisms to prevent imbalanced C/N trade-offs were activated. Root metabolism strongly depended on features of the whole plant, especially the foliar C/N budget. However, despite ECM-triggered growth impairment and the foliar nutrient status, the fungal partner was recognized to be a symbiotic partner.     

ROS production and antioxidative system activity in embryonic axes of <Emphasis Type="Italic">Quercus robur</Emphasis> seeds under different desiccation rate conditions

The seeds of pedunculate oak (Quercus robur L.) were subjected to slow (S) and rapid (R) desiccation at desiccation rates of 0.16 and 0.39% H₂O per hour, respectively. Till ca. 40% water content (WC) the germination capacity of seeds in the S and R variants was high (ca. 100%). Between 40 and 28% WC, germination capacity declined to 20 and 50% in S and R variants, respectively. The decrease in seed viability was accompanied by a significant increase of electrolyte leakage from embryonic axes (28% for S and 15% for R variants). In the embryonic axes of seeds subjected to slow desiccation, malondialdehyde (MDA) and free fatty acid (FFA) contents were significantly higher than those in R variants, indicating greater membrane damage due to lipid peroxidation. The production of ROS (H₂O₂ and O₂^·−) was significantly higher in S than in R variants. The low molecular weight antioxidants α-tocopherol, ascorbic acid (ASA), and phenolic compounds indicated different reactions in response to desiccation stress. ASA levels decreased during desiccation to a similar degree in both the S and R variants. A significant decrease of total phenols was observed in R variant, which coincided with a significant increase of guaiacol peroxidase (POX) activity. α-Tocopherol content was significantly higher in the embryonic axes of seeds subjected to rapid drying. The activities of the enzymatic scavengers APX and GR had similar runs and were slightly higher in R variant. The activities of POX and SOD were significantly higher in the embryonic axes of seeds subjected to rapid drying. These results show that rapid dehydration of Q. robur seeds leads to the greater mobilization of antioxidant system in embryonic axes, particularly increased levels of α-tocopherol and POX and SOD activities, in the first stages of water loss. This mobilization has a greater impact on maintenance of higher viability of seeds after drying to lower level of WC.     

The cell cycle- and insulin-signaling-inhibiting miRNA expression pattern of very small embryonic-like stem cells contributes to their quiescent state

Murine Oct4⁺, very small embryonic-like stem cells (VSELs), are a quiescent stem cell population that requires a supportive co-culture layer to proliferate and/or to differentiate in vitro. Gene expression studies have revealed that the quiescence of these cells is due to changes in expression of parentally imprinted genes, including genes involved in cell cycle regulation and insulin and insulin-like growth factor signaling (IIS). To investigate the role of microRNAs (miRNAs) in VSEL quiescence, we performed miRNA studies in highly purified VSELs and observed a unique miRNA expression pattern in these cells. Specifically, we observed significant differences in the expression of certain miRNA species (relative to a reference cell population), including (i) miRNA-25_1 and miRNA-19 b, whose downregulation has the effect of upregulating cell cycle checkpoint genes and (ii) miRNA-675-3 p and miRNA-675-5 p, miRNA-292-5 p, miRNA-184, and miRNA-125 b, whose upregulation attenuates IIS. These observations are important for understanding the biology of these cells and for developing efficient ex vivo expansion strategies for VSELs isolated from adult tissues.     

Further evidence that paroxysmal nocturnal haemoglobinuria is a disorder of defective cell membrane lipid rafts

The glycolipid glycosylphosphatidylinositol anchor (GPI-A) plays an important role in lipid raft formation, which is required for proper expression on the cell surface of two inhibitors of the complement cascade, CD55 and CD59. The absence of these markers from the surface of blood cells, including erythrocytes, makes the cells susceptible to complement lysis, as seen in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). However, the explanation for why PNH-affected hematopoietic stem/progenitor cells (HSPCs) expand over time in BM is still unclear. Here, we propose an explanation for this phenomenon and provide evidence that a defect in lipid raft formation in HSPCs leads to defective CXCR4- and VLA-4-mediated retention of these cells in BM. In support of this possibility, BM-isolated CD34⁺ cells from PNH patients show a defect in the incorporation of CXCR4 and VLA-4 into membrane lipid rafts, respond weakly to SDF-1 stimulation, and show defective adhesion to fibronectin. Similar data were obtained with the GPI-A⁻ Jurkat cell line. Moreover, we also report that chimeric mice transplanted with CD55^−/− CD59^−/− BM cells but with proper GPI-A expression do not expand over time in transplanted hosts. On the basis of these findings, we propose that a defect in lipid raft formation in PNH-mutated HSPCs makes these cells more mobile, so that they expand and out-compete normal HSPCs from their BM niches over time.     

Pannexin-1 channel “fuels” by releasing ATP from bone marrow cells a state of sterile inflammation required for optimal mobilization and homing of hematopoietic stem cells

An efficient harvest of hematopoietic stem/progenitor cells (HSPCs) after pharmacological mobilization from the bone marrow (BM) into peripheral blood (PB) and subsequent proper homing and engraftment of these cells are crucial for clinical outcomes from hematopoietic transplants. Since extracellular adenosine triphosphate (eATP) plays an important role in both processes as an activator of sterile inflammation in the bone marrow microenvironment, we focused on the role of Pannexin-1 channel in the secretion of ATP to trigger both egress of HSPCs out of BM into PB as well as in reverse process that is their homing to BM niches after transplantation into myeloablated recipient. We employed a specific blocking peptide against Pannexin-1 channel and noticed decreased mobilization efficiency of HSPCs as well as other types of BM-residing stem cells including mesenchymal stroma cells (MSCs), endothelial progenitors (EPCs), and very small embryonic-like stem cells (VSELs). To explain better a role of Pannexin-1, we report that eATP activated Nlrp3 inflammasome in Gr-1⁺ and CD11b⁺ cells enriched for granulocytes and monocytes. This led to release of danger-associated molecular pattern molecules (DAMPs) and mitochondrial DNA (miDNA) that activate complement cascade (ComC) required for optimal egress of HSPCs from BM. On the other hand, Pannexin-1 channel blockage in transplant recipient mice leads to a defect in homing and engraftment of HSPCs. Based on this, Pannexin-1 channel as a source of eATP plays an important role in HSPCs trafficking.

     

The endoplasmic reticulum‐associated protein,OS‐9, behaves as a lectin in targeting the immature calcium‐sensing receptor

The mechanisms responsible for the processing and quality control of the calcium‐sensing receptor (CaSR) in the endoplasmic reticulum (ER) are largely unknown. In a yeast two‐hybrid screen of the CaSR C‐terminal tail (residues 865–1078), we identified osteosarcoma‐9 (OS‐9) protein as a binding partner. OS‐9 is an ER‐resident lectin that targets misfolded glycoproteins to the ER‐associated degradation (ERAD) pathway through recognition of specific N‐glycans by its mannose‐6‐phosphate receptor homology (MRH) domain. We show by confocal microscopy that the CaSR and OS‐9 co‐localize in the ER in COS‐1 cells. In immunoprecipitation studies with co‐expressed OS‐9 and CaSR, OS‐9 specifically bound the immature form of wild‐type CaSR in the ER. OS‐9 also bound the immature forms of a CaSR C‐terminal deletion mutant and a C677A mutant that remains trapped in the ER, although binding to neither mutant was favored over wild‐type receptor. OS‐9 binding to immature CaSR required the MRH domain of OS‐9 indicating that OS‐9 acts as a lectin most likely to target misfolded CaSR to ERAD. Our results also identify two distinct binding interactions between OS‐9 and the CaSR, one involving both C‐terminal domains of the two proteins and the other involving both N‐terminal domains. This suggests the possibility of more than one functional interaction between OS‐9 and the CaSR. When we investigated the functional consequences of altered OS‐9 expression, neither knockdown nor overexpression of OS‐9 was found to have a significant effect on CaSR cell surface expression or CaSR‐mediated ERK1/2 phosphorylation.