Constitutively hyposialylated human T-lymphocyte clones in the Tn-syndrome: binding characteristics of plant and animal lectins

Previously, 1,3-galactosyltransferase-deficient (Tn+) and normal (TF+) T-lymphocyte clones have been established from a patient suffering from Tn-syndrome [Thurnheret al. (1992)Eur J Immunol 22: 1835–42], Tn+ T lymphocytes express only Tn antigen (GalNAc1-O-R) while other O-glycan structures such as sialosyl-Tn (Neu5Ac2,6GalNAc1-O-R) or TF (Gal1-3GalNAc1-O-R) antigens are absent from these cells as shown by flow cytometry using specific mABs for TF and sialosyl-Tn antigen, respectively. Normal T lymphocytes express the TF antigen and derivatives thereof. The surface glycans of Tn+ and TF+ cells were then analysed by flow cytometry using the following sialic acid-binding lectins:Amaranthus caudatus (ACA),Maackia amurensis (MAA),Limax flavus (LFA),Sambucus nigra (SNA) andTriticum vulgare (WGA). Equal and weak binding of MAA and SNA to both TF+ and Tn+ cells was found. WGA, LFA and ACA bound more strongly to TF+ cells than to Tn+ cells. Binding of ACA to TF+ cells was enhanced after sialidase treatment. To investigate the possible biological consequences of hyposialylation, binding of three sialic acid-dependent adhesion molecules to Tn+ and TF+ cells was estimated using radiolabelled Fc-chimeras of sialoadhesin (Sn), myelin-associated glycoprotein (MAG) and CD22. Equal and strong binding of human CD22 to both TF+ and Tn+ cells was found. Whereas binding of Sn and MAG to TF+ cells was strong (100%), binding to Tn+ cells amounted only to 33% (Sn) and 19% (MAG). These results indicate that thein vivo interactions of T lymphocytes in the Tn syndrome with CD22 are not likely to be affected, whereas adhesion mediated by Sn or MAG could be strongly reduced.     

Circulating NOS3 Modulates Left Ventricular Remodeling following Reperfused Myocardial Infarction

Purpose

Nitric oxide (NO) is constitutively produced and released from the endothelium and several blood cell types by the isoform 3 of the NO synthase (NOS3). We have shown that NO protects against myocardial ischemia/reperfusion (I/R) injury and that depletion of circulating NOS3 increases within 24h of ischemia/reperfusion the size of myocardial infarction (MI) in chimeric mice devoid of circulating NOS3. In the current study we hypothesized that circulating NOS3 also affects remodeling of the left ventricle following reperfused MI.

Methods

To analyze the role of circulating NOS3 we transplanted bone marrow of NOS3^−/− and wild type (WT) mice into WT mice, producing chimerae expressing NOS3 only in vascular endothelium (BC−/EC+) or in both, blood cells and vascular endothelium (BC+/EC+). Both groups underwent 60 min of coronary occlusion in a closed-chest model of reperfused MI. During the 3 weeks post MI, structural and functional LV remodeling was serially assessed (24h, 4d, 1w, 2w and 3w) by echocardiography. At 72 hours post MI, gene expression of several extracellular matrix (ECM) modifying molecules was determined by quantitative RT-PCR analysis. At 3 weeks post MI, hemodynamics were obtained by pressure catheter, scar size and collagen content were quantified post mortem by Gomori’s One-step trichrome staining.

Results

Three weeks post MI, LV end-systolic (53.2±5.9μl;***p≤0.001;n = 5) and end-diastolic volumes (82.7±5.6μl;*p<0.05;n = 5) were significantly increased in BC−/EC+, along with decreased LV developed pressure (67.5±1.8mmHg;n = 18;***p≤0.001) and increased scar size/left ventricle (19.5±1.5%;n = 13;**p≤0.01) compared to BC+/EC+ (ESV:35.6±2.2μl; EDV:69.1±2.6μl n = 8; LVDP:83.2±3.2mmHg;n = 24;scar size/LV13.8±0.7%;n = 16). Myocardial scar of BC−/EC+ was characterized by increased total collagen content (20.2±0.8%;n = 13;***p≤0.001) compared to BC+/EC+ (15.9±0.5;n = 16), and increased collagen type I and III subtypes.

Conclusion

Circulating NOS3 ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction.     

Signaling by IL-31 and functional consequences

Cytokines are key to control cellular communication. Interleukin-31 (IL-31) was recently discovered as a new member of the IL-6 family of cytokines. IL-31 signals through a heterodimeric receptor composed of OSMR and IL-31RA, a complex that stimulates the JAK-STAT, the RAS/ERK and the PI3K/AKT signal transduction pathways. The available data suggests that IL-31 is important for both innate and adaptive immunity in tissues that are in close contact with the environment, i.e. the skin, the airways and the lung, and the lining of the intestine. Enhanced expression of IL-31 is associated with a number of diseases, including pruritic diseases such as atopic dermatitis, but also in allergy and inflammatory bowel disease. In these tissues IL-31 coordinates the interaction of different immune cells, including T-cells, mast cells, and eosinophils, with epithelial cells. In this review we have summarized the available data on IL-31 and its receptor, their expression pattern and how they are regulated. We describe the current state of knowledge of the involvement of IL-31 in diseases, both in humans and in mouse models. From these studies it is becoming clear that IL-31 plays an important role in the proper functioning of the skin and of airway and intestinal epithelia. The findings available suggest that IL-31 might be an interesting target for directed drug therapy.     

The oncogenic serine/threonine kinase Pim-1 directly phosphorylates and activates the G2/M specific phosphatase Cdc25C

The proto-oncogene Pim-1 encodes a serine-threonine kinase which is a downstream effector of cytokine signaling and can enhance cell cycle progression by altering the activity of several cell cycle regulators among them the G1 specific inhibitor p21(Waf), the phosphatase Cdc25A and the kinase C-TAK1. Here, we demonstrate by using biochemical assays that Pim-1 can interact with the phosphatase Cdc25C and is able to directly phosphorylate the N-terminal region of the protein. Cdc25C is functionally related to Cdc25A but acts specifically at the G2/M cell cycle transition point and can be inactivated by C-TAK1-mediated phosphorylation. Immuno-fluorescence experiments showed that Pim-1 and Cdc25C co-localize in the cytoplasm of both epithelial and myeloid cells. We find that phosphorylation by Pim-1 enhances the phosphatase activity of Cdc25C and in transfected cells that are arrested in G2/M by bleomycin, Pim-1 can enhance progression into G1. Therefore, we propose that Pim-1 activates Cdc25C by a direct phosphorylation and can thereby assume the function of a positive cell cycle regulator at the G2/M transition.     

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Determination of nitrie in human blood by combination of a specific sample preparation with high-performance anion-exchange chromatography and electrochemical detection

All photometric or HPLC methods described to date have been unable to detect nitrite, a reliable marker of NO synthase activity, in human blood because of its rapid metabolism within the erythrocytes. We now elaborate on method to prevent nitrite degradation during sample preparation which in combination with high-performance anion-exchange chromatography and electrochemical detection allows a sensitive measurement of nitrite. A linear current response in the concentration range of 10–1000 nmol/l nitrite was observed yielding a correlation coefficient of 0.99. In addition, the combination of the electrochemical with a UV detector allowed us to simultaneously quantify nitrate one analytical run, which is the end product of NO/nitrite metabolism. Basal levels for nitrate and nitrite in human blood were determined with 25±4 μmol/l and 578±116 nmol/l (n=8), respectively and thus were in the same concentration range as expected from NO measurement in saline perfused isolated organs or cultured endothelial cells. Therefore, the presented method may be used to assess activity of endothelial constitutive NO synthase in humans under physiological and pathophysiological conditions.     

Aphyly: identifying the flotsam and jetsam of systematics

Many taxon names in any classification will be composed of taxa that have yet to be demonstrated as monophyletic, that is, characterized by synapomorphies. Such taxa might be called aphyletic, the flotsam and jetsam in systematics, simply meaning they require taxonomic revision. The term aphyly is, however, the same as, if not identical to, Hennig's “Restkörper” and Bernardi's merophyly. None of these terms gained common usage. We outline Hennig's use of “Restkörper” and Bernardi's use of merophyly and compare it to aphyly. In our view, application of aphyly would avoid the oft made assumption that when a monophyletic group is discovered from within an already known and named taxon, then the species left behind are rendered paraphyletic. By identifying the flotsam and jetsam in systematics, we can focus on taxa in need of attention and avoid making phylogenetic faux pas with respect to their phylogenetic status.     

Species-specific probes and real-time PCR as a tool for fast detection and differentiation of 15 bacteria relevant in intensive care medicine

Detection of bacterial DNA from laboratory-prepared specimens such as water, urine, and blood has the potential to improve diagnostic tools in microbiology. A novel real-time PCR-based assay was developed and its performance and robustness were evaluated for a panel of spiked suspensions of 15 clinically relevant bacteria. As low as ten colony forming units (CFU)/100 μl were detectable. No cross-reactivity was observed, except for Staphylococcus aureus and Staphylococcus epidermidis. Nevertheless, S. aureus and S. epidermidis were reliably differentiated by melting curve analysis. The protocol was also validated with three groups containing a mixture of five spiked bacteria each, with the result of reliable differentiation. This novel assay allows an exact identification of 15 microbes relevant in intensive care medicine, including mixed infections, in a one run experiment in less than 4 h.