Energy-selective neutron radiography and tomography at FRM.

At the reactor FRM at Technical University of Munich energy-selective neutron radiography and tomography experiments were performed. For an energy separation of the neutrons from the primary beam a mechanical velocity selector was used. The radiography images show a different contrast of the investigated elements for neutron energies below and above their Bragg-cutoff energy. A comparison between the standard and energy-selective neutron tomography is presented. In spite of a reduction of the neutron intensity due to the velocity selector technique a realistic experimental time in order of some hours for the tomography experiment was achieved.     

Chemically synthesized solid phase oligosaccharide probes for carbohydrate-binding receptors. Interactions of the E-, L- and P-selectins with sialyl-Le(x) and O-sulphated forms linked to biotin or to polyacrylamide

There is a growing interest in chemically defined oligosaccharide reagents for identifying proteins that bind carbohydrates and determining the specificities of carbohydrate-binding proteins. Here, we compare three sets of chemically synthesized commercially available oligosaccharide conjugates as immobilized probes, for the binding signals that they elicit with known carbohydrate-binding receptors of the immune system, the E-, P- and L-selectins. The first set of conjugates is of oligosaccharides linked to biotin via a nine-carbon spacer. The second and third sets are multivalent derivatives in which the oligosaccharides are linked, via a three-carbon spacer to poly[N-(2-hydroxyethyl)acrylamide] (PAA) or to biotinylated PAA with an average of 20% substitution of the hydroxyethyl-amide groups by carbohydrate. The conjugates were immobilized on streptavidin-coated microwells if biotinylated, otherwise by drying in uncoated wells. The most robust binding curves, overall, were with the biotinylated PAA derivatives of the ligands immobilized on streptavidin wells. These reagents have permitted a reevaluation of selectin binding signals elicited by sialyl-Lewis(x) (SLe(x)) analogues having sulphate at position 6 of the galactose (6'SuSLe(x)) or of the N-acetylglucosamine (6SuSLe(x)). The results clarify the role of 6SuSLe(x), rather then 6'SuSLe(x), as a ligand for the selectins.     

The Histological Basis of Frank’s Sign

Frank’s sign is a diagonal crease of the ear lobe, supposedly related to cardiac pathology, and has strongly been associated with coronary artery atherosclerosis. A total of 45 consecutive adult patients referred for autopsy in a one-and-a-half-year period were extensively studied. Samples from both the ear lobes were obtained for histopathology, as well as cardiac samples from all four cardiac compartments. When compared patients with Frank’s sign and those without it had no statistical difference in age (p = 0.0575). There was however a statistically significant increased cardiac weight (p = 0.0005), left ventricular wall thickness (p = 0.0002), and right ventricular wall thickness (p = 0.0043). Histopathology obtained from the ear lobes revealed myoelastofibrosis in an arterial vessel, located at the base of the crease, diffuse fibrosis, and Wallerian-like degeneration, with eosinophilic inclusions in the peripheral nerves. These changes suggest a time-related progression of the crease-associated changes. Our data suggest a significant correlation between the morphological changes of the myocardium and the presence of the ear lobe creases, with arterial myoelastofibrosis, Wallerian-like degeneration in peripheral nerves and deep tissue fibrosis found in the base of the crease.     

Immunotoxic effects of short-term atrazine exposure in young male C57BL/6 mice.

The herbicide atrazine (ATR) is a very widely used pesticide; yet the immunotoxicological potential of ATR has not been studied extensively. Our objective was to examine the effect of ATR on selected immune parameters in juvenile mice. ATR (up to 250 mg/kg) was administered by oral gavage for 14 days to one-month-old male C57BL/6 mice. One day, one week, and seven weeks after the last ATR dose, mice were sacrificed, and blood, spleens, and thymuses were collected and processed for cell counting and flow cytometry. Thymus and spleen weights were decreased by ATR, with the thymus being more sensitive than the spleen; this effect was still present at seven days, but not at seven weeks after the last ATR dose. Similarly, organ cellularity was persistently decreased in the thymus and in the spleen, with the splenic, but not thymic cellularity still being depressed at seven weeks post ATR. Peripheral blood leukocyte counts were not affected by ATR. There were also alterations in the cell phenotypes in that ATR exposure decreased all phenotypes in the thymus, with the number of CD4(+)/CD8(+) being affected the least. At the higher doses, the decreases in the thymic T-cell populations were still present one week after the last ATR dose. In the spleen, the CD8(+) were increased and MHC-II(+) and CD19(+) cells were decreased one day after the last ATR dose. Also, ATR treatment decreased the number of splenic na?ve T helper and T cytotoxic cells, whereas it increased the percentage of highly activated cytotoxic/memory T cells. Interestingly, the proportion of mature splenic dendritic cells (DC; CD11c(high)), was also decreased and it persisted for at least one week, suggesting that ATR inhibited DC maturation. In the circulation, ATR exposure decreased CD4(+) lymphocytes at one day, whereas at seven days after the last ATR dose, in addition to the decrease in CD4(+) lymphocytes, the MHC-II(+) cells were also decreased at the 250 mg/kg dose. Thus, ATR exposure appears to be detrimental to the immune system of juvenile mice by decreasing cellularity and affecting lymphocyte distribution, with certain effects persisting long after exposure has been terminated.     

INCREASED PROINFLAMMATORY CYTOKINES PRODUCTION BY ERGOTAMINE IN MALE BALB/c MICE

The ergopeptine alkaloid ergotamine (ET) mimics the effects of ergopeptine alkaloids found in endophyte-infected (E+) fescue forage considered causative for fescue toxicosis. Altered immune capacity, compromised intake and thermoregulation, and inflammatory changes are observed in fescue toxicosis. Taken together, these suggest the cytokine pattern may be altered by ergot alkaloids. Thus, the objective of this study was to determine whether major splenocyte-derived cytokines-interleukin 2 (IL-2), interleukin 4 (IL-4), interferon gamma (IFN-gamma)-and macrophage-derived cytokines-interleukin 1, (IL-1), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha)-were affected by ergotamine. Two sets of male BALB/c mice (n = 5/treatment) were treated with ergotamine tartrate (sc) for 10 d at doses of 0 (control), 0.4, 2, 10, or 50 mg/kg body weight. Twentyfour hours after the last treatment, splenocytes (S) were isolated from one set of animals and macrophages (MO) from the other set for determination of IL-2, IL-4, INF-gamma, and IL 1beta, IL-6, TNF-alpha, respectively. Following activation with 5 mug/ml concanavalin A (Con A) (S) and 10 mug/ml lipopolysaccharide (LPS) (MO), cells were incubated for 48 and 24 h, respectively, and supernatants were collected and assayed for respective cytokines by enzyme-linked immunosorbent assay (ELISA). Additionally, differential white blood cell (WBC) counts were performed and the neutrophil (N) :lymphocyte (L) ratio calculated. Ergotamine treatment significantly increased IL-6 levels at the 2.0 mg/kg dose and greater and TNF-alpha at the highest dose. There was no treatment effect on IL-1 , IL-2, IL-4, and IFN-gamma. Also, no effect was observed upon total and differential WBC counts as well as N:L ratio. Ergotamine affected the proinflammatory cytokine IL-6, and this increase may contribute to fescue tosicosis.     

cAbl tyrosine kinase mediates reactive oxygen species- and caveolin-dependent AT1 receptor signaling in vascular smooth muscle: role in vascular hypertrophy

Important output signals of the angiotensin subtype 1 receptor (AT1R) in vascular smooth muscle cells (VSMCs) are mediated by angiotensin II (Ang II)-stimulated transactivation of the epidermal growth factor receptor (EGF-R), which is critical for vascular hypertrophy. Ang II-induced EGF-R transactivation is mediated through cSrc, a proximal target of reactive oxygen species (ROS) derived from NAD(P)H oxidase (NOX) and is dependent on AT(1)R trafficking through caveolin1 (Cav1)-enriched lipid rafts. Underlying molecular mechanisms are incompletely understood. The nonreceptor tyrosine kinase, proto-oncogene cAbl is a substrate of Src and is a major mediator for ROS-dependent tyrosine phosphorylation of Cav1. We thus hypothesized that cAbl is important for ROS-, cSrc-, and Cav1-dependent growth-related AT1R signal transduction. Here we show that Ang II induces tyrosine phosphorylation of cAbl in rat VSMCs and mouse aorta, and that Ang II promotes association of cAbl with AT(1)R, both of which are Src-dependent. Pretreatment of rat VSMCs with the NOX inhibitor diphenylene iodonium or the antioxidants N-acetylcysteine or ebselen significantly inhibited Ang II-induced cAbl phosphorylation. Cell fractionation shows that both EGF-Rs and cAbl are found basally in Cav1-enriched membrane fractions. Knockdown of cAbl protein using small interference RNA inhibits Ang II-stimulated: (1) trafficking of AT1R into, and EGF-R out of, Cav1-enriched lipid rafts; (2) EGF-R transactivation; (3) appearance of the transactivated EGF-R and phospho-Cav1 at focal adhesions; and (4) vascular hypertrophy. These studies provide a novel role of cAbl in the spatial and temporal organization of growth-related AT1R signaling in VSMCs and suggest that cAbl may be generally important in signaling of G-protein coupled receptors.