Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8(+) T cells sensitizing them to apoptotic cell death

The life-long homeostasis of memory CD8(+) T cells as well as persistent viral infections have been shown to facilitate the accumulation of highly differentiated CD8(+) CD28(-) T cells, a phenomenon that has been associated with an impaired immune function in humans. However, the molecular mechanisms regulating homeostasis of CD8(+) CD28(-) T cells have not yet been elucidated. In this study, we demonstrate that the miR-23～24～27 cluster is up-regulated during post-thymic CD8(+) T-cell differentiation in humans. The increased expression of miR-24 in CD8(+) CD28(-) T cells is associated with decreased expression of the histone variant H2AX, a protein that plays a key role in the DNA damage response (DDR). Following treatment with the classic chemotherapeutic agent etoposide, a topoisomerase II inhibitor, apoptosis was increased in CD8(+) CD28(-) when compared to CD8(+) CD28(+) T cells and correlated with an impaired DDR in this cell type. The reduced capacity of CD8(+) CD28(-) T cell to repair DNA was characterized by the automated fluorimetric analysis of DNA unwinding (FADU) assay as well as by decreased phosphorylation of H2AX at Ser139, of ATM at Ser1981, and of p53 at Ser15. Interleukin (IL)-15 could prevent etoposide-mediated apoptosis of CD8(+) CD28(-) T cells, suggesting a role for IL-15 in the survival and the age-dependent accumulation of CD8(+) CD28(-) T cells in humans.     

Regulation by kinetin and abcisic acid of correlative senescence in relation to grain maturation,source-sink relationship and yield of rice (Oryza sativa L.)

When kinetin was applied to the source organ (flag leaf) of rice (Oryza sativa L. cv. Ratna), foliar senescence was delayed and grain yield per plant (as evidenced by grain weight, grain/straw weight ratio and 1,000 grain growth) was increased through the increase of sink activity (increase in dry weight of the grains/plant), duration of sink capacity as well as photosynthetic ability of the glumes (as determined by the chlorophyll content of the glumes of the developing grains). However, application of kinetin to the sink organs (fruits), promoted senescence of the source but increased the yield by increasing the sink capacity and 1,000 grain growth mostly at the earlier stage of reproductive development. Lower sterility percentage was associated with higher grain yield of the plant by kinetin treatments. ABA applied either to the source or the sink promoted leaf senescence and reduced the grain yield by reducing the sink activity, harvest index, sink capacity duration and increasing the sterility percentage. Thousand grain dry weight at harvest did not vary significantly amongst the treatments. It was concluded that nutrient drainage was associated with the correlative influence of fruit on the monocarpic senescence of rice plant and that a competetion for differential allocation of cytokinin and ABA in the source and sink organs initiates this senescence syndrome.     

Change in conformation with reduction of alpha-helix content causes loss of neutrophil binding activity in fully cytotoxic Shiga toxin 1

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.     

Carboxy group derivatization for enhanced electron‐transfer dissociation mass spectrometric analysis of phosphopeptides

A novel strategy based on carboxy group derivatization is presented for specific characterization of phosphopeptides. By tagging the carboxy group with 1‐(2‐pyrimidyl) piperazine (PP), the ion charge states of phosphopeptides can be largely enhanced, showing great advantages for sequencing phosphorylated peptides with electron‐transfer dissociation MS. Besides, after PP‐derivatization, most non‐specific bindings can be avoided by eliminating the interaction between the carboxy group and TiO₂, greatly improving the specificity of TiO₂‐based phosphopeptide enrichment strategy. Moreover, being tagged with a hydrophobic group, the retention time of phosphopeptides in RPLC can be prolonged, overcoming the difficulty of separating phosphopeptides in RPLC‐based approach. Together with several other advantages, such as ease of handling, rapid reaction time, broad applicability and good reproducibility, this PP‐derivatization method is promising for high‐throughput phosphoproteome research.     

Investigation of various structures of DNA molecules (Ⅲ)——Coil-globe transition of λ-DNA induced by cationic surfactant

The structure transition of λ-DNA induced by cationic surfactant cellar media was investigated by using CD, SEM and AFM. The experimental data of CD revealed that λ-DNA can be induced from B-form to a collapsed structure with the addition of the cationic surfactant CTAB to the system. The condensed process of λ-DNA from coil state to small globular state (diameter about 1.25 μm) and finally big globular state (diameter about 5.4 μm) was observed by using SEM and AFM.     

P-selectin and CD63 use different mechanisms for delivery to Weibel-Palade bodies

The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the trans-Golgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC).     

N-acetyl-l-methionine is a superior protectant of human serum albumin against photo-oxidation and reactive oxygen species compared to N-acetyl-l-tryptophan

Background

Sodium octanoate (Oct) and N-acetyl-l-tryptophan (N-AcTrp) are widely used as stabilizers during pasteurization and storage of albumin products. However, exposure to light photo-degrades N-AcTrp with the formation of potentially toxic compounds. Therefore, we have examined the usefulness of N-acetyl-l-methionine (N-AcMet) in comparison with N-AcTrp for long-term stability, including photo stability, of albumin products.

Methods

Recombinant human serum albumin (rHSA) with and without additives was photo-irradiated for 4 weeks. The capability of the different stabilizers to scavenge reactive oxygen species (ROS) was examined by ESR spectrometry. Carbonyl contents were assessed by a spectrophotometric method using fluoresceinamine and Western blotting, whereas the structure of rHSA was examined by SDS-PAGE, far-UV circular dichroism and differential scanning calorimetry. Binding was determined by ultrafiltration.

Results

N-AcMet was found to be a superior ROS scavenger both before and after photo-irradiation. The number of carbonyl groups formed was lowest in the presence of N-AcMet. According to SDS-PAGE, N-AcMet stabilizes the monomeric form of rHSA, whereas N-AcTrp induces degradation of rHSA during photo-irradiation. The decrease in α-helical content of rHSA was the smallest in the presence of Oct, without or with N-AcMet. Photo-irradiation did not affect the denaturation temperature or calorimetric enthalpy of rHSA, when N-AcMet was present.

Conclusion

The weakly bound N-AcMet is a superior protectant of albumin, because it is a better ROS-protector and structural stabilizer than N-AcTrp, and it is probable and also useful for other protein preparations.

General significance

N-AcMet is an effective stabilizer of albumin during photo-irradiation, while N-Ac-Trp promotes photo-oxidative damage to albumin.     

Combined deletion of SCD1 from adipose tissue and liver does not protect mice from obesity

Stearoyl-CoA desaturase 1 (SCD1) catalyzes the synthesis of monounsaturated fatty acids (MUFA) from saturated FA. Mice with whole-body or skin-specific deletion of SCD1 are resistant to obesity. Here, we show that mice lacking SCD1 in adipose and/or liver are not protected from either genetic- (agouti; A(y)/a) or diet-induced obesity (DIO) despite a robust reduction in SCD1 MUFA products in both subcutaneous and epididymal white adipose tissue. Adipose SCD1 deletion had no effect on glucose or insulin tolerance or on hepatic triglyceride (TG) accumulation. Interestingly, lack of SCD1 from liver lowered the MUFA levels of adipose tissue and vice versa, as reflected by the changes in FA composition. Simultaneous deletion of SCD1 from liver and adipose resulted in a synergistic lowering of tissue MUFA levels, especially in the A(y)/a model in which glucose tolerance was also improved. Lastly, we found that liver and plasma TG show nearly identical genotype-dependent differences in FA composition, indicating that FA composition of plasma TG is predictive for hepatic SCD1 activity and TG FA composition. The current study suggests that SCD1 deletion from adipose and/or liver is insufficient to elicit protection from obesity, but it supports the existence of extensive lipid cross-talk between liver and adipose tissue.     

CAY10593 inhibits the human P2X7 receptor independently of phospholipase D1 stimulation

The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the ‘low affinity’ IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium⁺ uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself.