Gamma radiation is known to induce cell death in several organs. This damage is associated with endonuclease-mediated DNA fragmentation; however, the enzyme that produces the latter and is likely to cause cell death is unknown. To determine whether the most abundant cytotoxic endonuclease DNase I mediates γ-radiation-induced tissue injury, we used DNase I knockout mice and zinc chelate of 3,5-diisopropylsalicylic acid (Zn-DIPS), which, as we show, has DNase I inhibiting activity in vitro. The study demonstrated for the first time that inactivation or inhibition of DNase I ameliorates radiation injury to the white pulp of spleen, intestine villi and bone marrow as measured using a quantitative TUNEL assay. The spleen and intestine of DNase I knockout mice were additionally protected from radiation by Zn-DIPS, perhaps due to the broad radioprotective effect of the zinc ions. Surprisingly, the main DNase I-producing tissues such as the salivary glands, pancreas and kidney showed no effect of DNase I inactivation. Another unexpected observation was that even without irradiation, DNA fragmentation and cell death were significantly lower in the intestine of DNase I knockout mice than in wild-type mice. This points to the physiological role of DNase I in normal cell death in the intestinal epithelium. In conclusion, our results suggested that DNase I-mediated mechanism of DNA damage and subsequent tissue injury are essential in γ-radiation-induced cell death in radiosensitive organs.
Objective-The present study investigates the role of Src and Syk tyrosine kinases in signaling by G-protein coupled and platelet adhesion receptors. Methods and Results-Using SykϪ/Ϫ platelets or the Src kinase inhibitor PP2, we demonstrate a critical role for Src and Syk kinases in mediating lamellipodia formation on VWF, collagen, CRP, fibrinogen, and fibronectin. In all cases, the spreading defect was overcome by addition of thrombin. Conversely, platelet aggregation and ␣ IIb  3 activation induced by thrombin was similar to controls, arguing against a functional role for Src and Syk in ␣ IIb  3 activation. Unexpectedly, CRP potentiated integrin ␣ IIb  3 activation and platelet aggregation induced by subthreshold concentrations of thrombin in Syk Ϫ/Ϫ platelets or in the presence of the Src kinase inhibitor PP2. Potentiation in the presence of PP2 was lost in the absence of FcR␥-chain or GPVI confirming that it was mediated through the immunoglobulin receptor. Further delineation of this PP2-resistant synergy revealed that PAR4 could trigger the enhanced response in combination with CRP. Conclusions-We show that Syk is critical for lamellipodia formation on a range of immobilized proteins but that this can be overcome by addition of thrombin. Further, we reveal a novel role for GPVI in supporting thrombin-induced activation, independent of Syk and Src kinases. Key Words: platelets Ⅲ GPVI signaling Ⅲ Src kinases Ⅲ Syk Ⅲ thrombin T he tyrosine kinase Syk contains two SH2 domains and a C-terminal kinase domain and is expressed in cells of the hematopoietic lineage. The absence of Syk results in the abrogated development of B cells, functional defects in immune receptors, and perinatal lethality. 1,2 Interestingly, mice deficient in Syk, or the Syk substrate SLP-76, also show a failure to separate emerging blood vessels from those of the emerging lymphatic system, thereby triggering embryonic hemorrhaging. 3 In platelets, Syk is recognized for its role in mediating signaling of the collagen receptor complex, GPVI-FcR␥ chain, by binding to the immunoreceptor tyrosine-based activation motif (ITAM) within the intracellular portion of the FcR␥ chain. Signaling by this receptor complex parallels that by immune receptors, such as the T-and B-cell antigen receptors and Fc receptors. Reviewed by Watson et al, 4,5 the two Src kinases Fyn and Lyn, are believed to initially phosphorylate the ITAM of the FcR␥ chain. Syk then binds to the dually phosphorylated tyrosine residues via its SH2 domains, resulting in autophosphorylation of Syk and sequential activation and phosphorylation of several adapter proteins, including LAT, SLP-76, and a number of signaling proteins, including Tec kinases and PI-3 kinase. This ultimately leads to phosphorylation of phospholipase C (PLC) ␥ isoforms and a rise in intracellular calcium.Syk is also implicated in signaling by other classes of adhesion and G protein-coupled receptors in platelets. For example, Syk plays a critical role in signaling by the major platelet integrin, ␣ IIb  3 . Thi...
Gamma radiation‐induced cell death is associated with and mediated by enzymatic DNA fragmentation. A nuclease that produces this fragmentation of DNA is unknown. To determine whether deoxyribonuclease I (DNase I), a major cytotoxic/apoptotic nuclease expressed in all cells and tissues, can mediate gamma radiation‐induced tissue injury, we used DNase I knockout (KO) mice and a zinc chelate of 3,5‐diisopropylsalicylic acid (Zn‐DIPS) that, as we show, has DNase I inhibiting activity in vitro. Our study demonstrated that inactivation or inhibition of DNase I strongly suppressed radiation injury (8 Gy, LD50/30) to white pulp of spleen, intestine villi, and bone marrow as measured using quantitative TUNEL assay. Spleen and intestine of DNase I KO mice were additionally protected from radiation by Zn‐DIPS due to the broad radioprotective efficacy of zinc. It is interesting that main DNase I‐producing tissues such as salivary gland, pancreas, and kidney showed no effect of DNase I inactivation. These results suggest that DNase I‐mediated mechanism of DNA damage and tissue injury has important regulatory role in radiation‐mediated cell death in radiosensitive organs. The study was supported by NIH/NIDDK and VA Merit Review grants to A.G.B.
Platelet glycoprotein (GP) Ib-IX is a key receptor in arterial platelet adhesion and thrombus formation. A growing body of evidence suggests the inherent adhesive nature of platelets contribute to pathological events such as inflammation and atherosclerosis. However, the molecular mechanisms by which platelets might participate in these processes remain unknown. In the current study, we used a mouse model of platelet receptor GPIb-IX deficiency (mBSS), an equivalent to the human Bernard-Soulier syndrome, to determine whether the development of atherosclerotic lesions is influenced by normal platelet adhesion. Our hypothesis is that mBSS platelets might have a protective role in the development of atherosclerosis given their abnormal function and lack of a critical platelet adhesion receptor. To test this hypothesis we first generated congenic mBSS mice by backcrossing for 10 generations with wild-type C57BL/6J mice. C57BL/6J-GPIb−/− mice were then crossed to the well-characterized murine model of hypercholesterolemia, LDL-receptor deficient animals (C57BL/6J-LDLrtm1Her). Following genotypic screening and second generation breeding we established a colony of double knockout animals (C57BL/6J-LDLrtm1 Her-GPIb−/−) lacking both the gene for the LDL receptor and platelet GPIbα. Experimental animals were divided into 3 groups:double knockouts (C57BL/6J-LDLrtm1Her-GpIb−/−);animals only missing the LDL receptor gene (C57BL/6J-LDLrtm1Her); andwild-type controls (C57BL/6J). At 5 weeks of age mice were weaned and placed on a Western style atherogenic diet composed of 0.2% cholesterol. We have examined aortas following exposure to this high fat diet for 18 and 24 weeks. Six mice from each group were euthanized and the aortas were dissected. The hearts were perfused with 10 ml of phosphate-buffered saline and aortas were opened longitudinally from the heart to the iliac bifurcation. The specimens were stained with Sudan IV to identify the gross presence of fatty streaks in the intimal layer of aortas. Digital images of the stained aortas were captured. All double knockout animals and LDL receptor-deficient mice developed extensive atherosclerotic lesions throughout the aorta with especially pronounced lesions in the area of the aortic arch and arterial branching points. No lesions were observed in control C57BL/6J aortas. Sudan IV-positive lesions were counted visually and they ranged from 21–23 in double knockout aortas and 16–21 in LDL receptor deficient mice. The lesions were more pronounced at earlier time points in male animals. We also observed a correlation between the development of advanced aortic lesions and clinically sound manifestations, such as skin trophic changes and limb self-amputation in some animals. Overall, these findings do not support our initial hypothesis. In fact, we observed that double knockout mice appear to have exaggerated development of atherosclerotic lesions. This finding has led us to a biochemical analysis of mBSS platelets where we have observed phosphorylated resting platelet proteins not present in resting normal mouse platelets. Thus, we conclude in this model of atherosclerosis the BSS platelet may be functioning as a “pseudo-activated” form of a platelet and might be the molecular basis for the apparent support rather than inhibition of arterial atherogenesis.
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