Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides

Cytotoxic CD8(+) T cells recognize the antigenic peptides presented by class I major histocompatibility complex (MHC) molecules. These T cells have key roles in infectious diseases, autoimmunity and tumor immunology, but there is currently no unbiased method for the reliable identification of their target antigens. This is because of the low affinities of antigen-specific T cell receptors (TCR) to their target MHC-peptide complexes, the polyspecificity of these TCRs and the requirement that these TCRs recognize protein antigens that have been processed by antigen-presenting cells (APCs). Here we describe a technology for the unbiased identification of the antigenic peptides presented by MHC class I molecules. The technology uses plasmid-encoded combinatorial peptide libraries and a single-cell detection system. We validated this approach using a well-characterized influenza-virus–specific TCR, MHC and peptide combination. Single APCs carrying antigenic peptides can be detected among several million APCs that carry irrelevant peptides. The identified peptide sequences showed a converging pattern of mimotopes that revealed the parent influenza antigen. This technique should be generally applicable to the identification of disease-relevant T cell antigens.     

Neuroprotective effects of Argon are mediated via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1 in retinal ganglion cells

Retinal ischemia and reperfusion injuries (R‐IRI) damage neuronal tissue permanently. Recently, we demonstrated that Argon exerts anti‐apoptotic and protective properties. The molecular mechanism remains unclear. We hypothesized that Argon inhalation exert neuroprotective effects in rats retinal ganglion cells (RGC) via an ERK‐1/2 dependent regulation of heat‐shock proteins. Inhalation of Argon (75 Vol%) was performed after R‐IRI on the rats′ left eyes for 1 h immediately or with delay. Retinal tissue was harvested after 24 h to analyze mRNA and protein expression of heat‐shock proteins ?70, ?90 and heme‐oxygenase‐1, mitogen‐activated protein kinases (p38, JNK, ERK‐1/2) and histological changes. To analyze ERK dependent effects, the ERK inhibitor PD98059 was applicated prior to Argon inhalation. RGC count was analyzed 7 days after injury. Statistics were performed using anova . Argon significantly reduced the R‐IRI‐affected heat‐shock protein expression (p < 0.05). While Argon significantly induced ERK‐1/2 expression (p < 0.001), inhibition of ERK‐1/2 before Argon inhalation resulted in significantly lower vital RGCs (p < 0.01) and increase in heme‐oxygenase‐1 (p < 0.05). R‐IRI‐induced RGC loss was reduced by Argon inhalation (p < 0.001). Immunohistochemistry suggested ERK‐1/2 activation in Müller cells. We conclude, that Argon treatment protects R‐IRI‐induced apoptotic loss of RGC via an ERK‐1/2 dependent regulation of heme‐oxygenase‐1.

     

Isolation and Identification of Aspergillus fumigatus Mycotoxins on Growth Medium and Some Building Materials

Genotoxic and cytotoxic compounds were isolated and purified from the culture medium of an indoor air mold, Aspergillus fumigatus. One of these compounds was identified as gliotoxin, a known fungal secondary metabolite. Growth of A. fumigatus and gliotoxin production on some building materials were also studied. Strong growth of the mold and the presence of gliotoxin were detected on spruce wood, gypsum board, and chipboard under saturation conditions.     

Identification and characterization of the avian erythroblastosis virus erbB gene product as a membrane glycoprotein

Avian erythroblastosis virus causes erythroid leukemia and sarcomas in chickens. The viral oncogene responsible for these diseases, erb, is divided into two regions known as erbA and erbB, and recent evidence suggests that it is the erbB gene that is responsible for the transforming activity. From rats bearing avian erythroblastosis virus-induced sarcomas, we have obtained antisera which are specific for the erb gene products. Using such antisera, we have been able to characterize the erbB gene product as a 68,000 molecular weight protein. Pulse-chase and cell-free in vitro translation experiments show that the initial product is a 62,500 dalton protein which is initially modified to a 66,000 dalton protein, and then further modified to a 68,000 dalton form. These modifications could be shown to be associated with glycosylation and phosphorylation. Cell fractionation experiments revealed that the 66,000 and 68,000 dalton proteins were located in cell membrane fractions, and immunofluorescence results showed the erbB gene product to be expressed on the cell surface.     

Innate immune collectin surfactant protein D simultaneously binds both neutrophil extracellular traps and carbohydrate ligands and promotes bacterial trapping

Neutrophils release DNA-based extracellular traps to capture and kill bacteria. The mechanism(s) and proteins that promote neutrophil extracellular trap (NET)-mediated bacterial trapping are not clearly established. Surfactant protein D (SP-D) is an innate immune collectin present in many mucosal surfaces. We hypothesized that SP-D can bind both the pathogens and NETs to augment NET-mediated bacterial trapping. To test this hypothesis, we used LPS and Pseudomonas aeruginosa pneumonia mouse models and performed in vivo and ex vivo assays. In this study, we show that NETs are produced by the neutrophils recruited to the airways in response to the bacterial ligand. Notably, NETs are detected as short fragments of DNA-protein complexes in the airways as opposed to the long stringlike structures seen in ex vivo cultures. SP-D recognizes both the short NET fragments and the long NET DNA structures. SP-D-NET copurification studies further show that SP-D can simultaneously recognize NETs and carbohydrate ligands in vivo. Similar to the LPS model, soluble DNA-protein complexes and increased amounts of SP-D are detected in the murine model of P. aeruginosa pneumonia. We then tested the effect of SP-D on NET-mediated trapping of P. aeruginosa by means of Western blots, fluorescence microscopy, and scanning electron microscopy. Results of these experiments show that SP-D microagglutinates P. aeruginosa and allows an efficient bacterial trapping by NETs. Collectively, these findings provide a unique biological relevance for SP-D-DNA interactions and places SP-D as an important innate immune protein that promotes bacterial trapping by NETs during neutrophil-mediated host defense.     

The UBAP1 subunit of ESCRT-I interacts with ubiquitin via a SOUBA domain

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Highlights? ESCRT-I subunit UBAP1 is essential for degradation of antiviral protein tetherin ? UBAP1 has a domain consisting of a solenoid of overlapping UBAs (SOUBA) ? Each of the three UBAs in the SOUBA binds monoubiquitin     

Regulation of enzymatic lipid peroxidation: the interplay of peroxidizing and peroxide reducing enzymes

For a long time lipid peroxidation has only been considered a deleterious process leading to disruption of biomembranes and thus, to cellular dysfunction. However, when restricted to a certain cellular compartment and tightly regulated, lipid peroxidation may have beneficial effects. Early on during evolution of living organisms special lipid peroxidizing enzymes, called lipoxygenases, appeared and they have been conserved during phylogenesis of plants and animals. In fact, a diverse family of lipoxygenase isoforms has evolved starting from a putative ancient precursor. As with other enzymes, lipoxygenases are regulated on various levels of gene expression and there are endogenous antagonists controlling their cellular activity. Among the currently known mammalian lipoxygenase isoforms only 12/15-lipoxygenases are capable of directly oxygenating ester lipids even when they are bound to membranes and lipoproteins. Thus, these enzymes represent the pro-oxidative part in the cellular metabolism of complex hydroperoxy ester lipids. Its metabolic counterplayer, representing the antioxidative part, appears to be the phospholipid hydroperoxide glutathione peroxidase. This enzyme is unique among glutathione peroxidases because of its capability of reducing ester lipid hydroperoxides. Thus, 12/15-lipoxygenase and phospholipid hydroperoxide glutathione peroxidase constitute a pair of antagonizing enzymes in the metabolism of hydroperoxy ester lipids, and a balanced regulation of the two proteins appears to be of major cell physiological importance. This review is aimed at summarizing the recent developments in the enzymology and molecular biology of 12/15-lipoxygenase and phospholipid hydroperoxide glutathione peroxidase, with emphasis on cytokine-dependent regulation and their regulatory interplay.     

On the use of ultrasounds to quantify the longitudinal threshold force to detach osteoblastic cells from a conditioned glass substrate

Cell adhesion on a biomaterial is an important phase of the cell-material interactions and the quality of this phase governs the success of the biomaterial integration. Understanding of the phenomena of cell adhesion and in particular understanding of cell adhesion on biomaterials is of crucial importance for the development of new biomaterials with excellent biocompatibility. One of the physical quantitative indexes to evaluate the quality of cell-material adhesion is its strength. Determining the strength of adhesive bonds requires applying external forces to the cells. Thus, a few methods have been developed to evaluate the strength of cell-material adhesion (micropipette, microplates, microcantilever, ...). These methods apply shear forces on adherent cells. The aim of our work is the development of a new ultrasonic characterization method of cellular adhesion on substrates. With our method, longitudinal acoustic waves are applied on cell culture to impose a longitudinal strain on cells. Only the cells subjected to a sufficient level of strain will be detached from the substrate. The idea is to correlate cell detachment rate to the longitudinal strain threshold supported by cells. From this result, we can deduce the critical force just sufficient to detach the cell. This global method can be adapted for different cell types and for different substrates. This method can provide an evaluation of the effect of functionalization on substrates. The technique is investigated for the 200 kHz ultrasound frequency. An insonificator adapted to the use of cell culture boxes was developed and calibrated. Tests were carried out on a glass substrate with or without biological conditioning. We used the MC3T3-E1 osteoblastic cell line. Our results to date provide the value of the necessary force to detach with reproducibility osteoblastic cells from glass.     

银杏内生菌Chaetomium globosum ZY-22次生代谢产物分离鉴定

采用柱层析方法从银杏叶内生真菌Chaetomium globosum ZY-22的培养菌丝体提取物中分离得到脑苷脂B(1)、脑苷脂C(2)、尿囊素(3)、9(11)-去氢麦角甾醇过氧化物(4)以及4,6,8,22-四烯-3-酮-麦角甾烷(5)和球毛壳甲素(6)共6个次生代谢物;经波谱分析确定了6个化合物的结构,其中脑苷脂B、脑苷脂C和尿囊素是首次从内生真菌中得到;海虾致死试验结果显示,化合物1～6在10 μg/mL浓度下对丰年虾的致死率分别为1.6%、4.2%、7.4%、16.9%、12.8%、83.6%、表明球毛壳甲素对海虾表现出很强的毒性作用.