Interleukin-1α Activity in Necrotic Endothelial Cells Is Controlled by Caspase-1 Cleavage of Interleukin-1 Receptor-2: IMPLICATIONS FOR ALLOGRAFT REJECTION*

Inflammation is a key instigator of the immune responses that drive atherosclerosis and allograft rejection. IL-1α, a powerful cytokine that activates both innate and adaptive immunity, induces vessel inflammation after release from necrotic vascular smooth muscle cells (VSMCs). Similarly, IL-1α released from endothelial cells (ECs) damaged during transplant drives allograft rejection. However, IL-1α requires cleavage for full cytokine activity, and what controls cleavage in necrotic ECs is currently unknown. We find that ECs have very low levels of IL-1α activity upon necrosis. However, TNFα or IL-1 induces significant levels of active IL-1α in EC necrotic lysates without alteration in protein levels. Increased activity requires cleavage of IL-1α by calpain to the more active mature form. Immunofluorescence and proximity ligation assays show that IL-1α associates with interleukin-1 receptor-2, and this association is decreased by TNFα or IL-1 and requires caspase activity. Thus, TNFα or IL-1 treatment of ECs leads to caspase proteolytic activity that cleaves interleukin-1 receptor-2, allowing IL-1α dissociation and subsequent processing by calpain. Importantly, ECs could be primed by IL-1α from adjacent damaged VSMCs, and necrotic ECs could activate neighboring normal ECs and VSMCs, causing them to release inflammatory cytokines and up-regulate adhesion molecules, thus amplifying inflammation. These data unravel the molecular mechanisms and interplay between damaged ECs and VSMCs that lead to activation of IL-1α and, thus, initiation of adaptive responses that cause graft rejection.     

Distinctive roles of receptor‐interacting protein kinases 1 and 3 in caspase‐independent cell death of L929

Upon tumour necrosis factor alpha (TNFα) stimulation, cells respond actively by way of cell survival, apoptosis or programmed necrosis. The receptor‐interacting proteins 1 (RIP1) and 3 (RIP3) are responsible for TNFα‐mediated programmed necrosis. To delineate the differential contributions of RIP3 and RIP1 to programmed necrosis, L929 cells were stimulated with TNFα, carbobenzoxy‐valyl‐alanyl‐aspartyl‐[O‐methyl]‐fluoromethylketone (zVAD) or zVAD along with TNFα following RNA interference against RIP1 and RIP3, respectively. RIP1 silencing did not protect cells from TNFα‐mediated cell death, while RIP3 down‐regulation made them refractory to TNFα. The heat shock protein 90 inhibitor geldanamycin (GA) down‐regulated both RIP1 and RIP3 expression, which rendered cells resistant to zVAD/TNFα‐mediated cell death but not to TNFα‐mediated cell death alone. Therefore, the protective effect of GA on zVAD/TNFα‐stimulated necrosis might be attributed to RIP3, not RIP1, down‐regulation. Pretreatment of L929 cells with rapamycin mitigated zVAD‐mediated cell death, while the autophagy inhibitor chloroquine did not affect necrotic cell death. Meanwhile, necrotic cell death by zVAD and TNFα was caused by reactive oxygen species generation and effectively diminished by lipid‐soluble butylated hydroxyanisole. Taken together, the results indicate that RIP1 and RIP3 can independently mediate death signals being transduced by two different death stimuli, zVAD and TNFα. Copyright © 2013 John Wiley & Sons, Ltd.     

Physiological parameters correlated with Tomato Mosaic Virus inducing defensive response in Datura metel

Programed cell death resembles a real nature active defense in Datura metel against TMV after three days of virus infection. This adaptive plant immune response was quantitatively assessed against Tomato Mosaic Virus infection by the following physiological markers; Chlorophyll-a (mg/g), Chlorophyll-b (mg/g), total protein (mg/g), hydrogen peroxide H₂O₂ (μmol/100 mg), DNA (μg/100 mg), RNA (μg/100 mg), Salicylic acid (μg/g), and Comet Assays. Parameters were assessed for asymptomatic healthy and symptomatic infected detached leaves. The results indicated H₂O₂ and Chlorophyll-a as the most potential parameters. Chlorophyll-a was considered the only significant predictor variant for the H₂O₂ dependent variant with a P value of 0.001 and R-square of 0.900. The plant immune response was measured within three days of virus infection using the cutoff value of H₂O₂ (⩽1.095 μmol/100 mg) and (⩽3.201 units) for the tail moment in the Comet Assay. Their percentage changes were 255.12% and 522.40% respectively which reflects the stress of virus infection in the plant. Moreover, H₂O₂ showed 100% specificity and sensitivity in the symptomatic infected group using the receiver-operating characteristic (ROC). All tested parameters in the symptomatic infected group had significant correlations with twenty-five positive and thirty-one negative correlations where the P value was <0.05 and 0.01. Chlorophyll-a parameter had a crucial role of highly significant correlation between total protein and salicylic acid. Contrarily, this correlation with tail moment unit was (r = −0.930, P < 0.01) where the P value was <0.01. The strongest significant negative correlation was between Chlorophyll-a and H₂O₂ at P < 0.01, while moderate negative significant correlation was seen for Chlorophyll-b where the P value < 0.05. The present study discloses the secret of the three days of rapid transient production of activated oxygen species (AOS) that was enough for having potential quantitative physiological parameters for defensive plant response toward the virus.     

Geochemical baseline determination and pollution assessment of heavy metals in urban soils of Karachi,Pakistan

Karachi is one of the most populated urban agglomerations in the world. No categorical study has yet discussed the geochemical baseline concentrations of metals in the urban soil of Karachi. The main objectives of this study were to establish geochemical baseline values and to assess the pollution status of different heavy metals. Geochemical baseline concentrations of heavy metals were estimated using the cumulative frequency distribution (CDF) curves. The estimated baseline concentrations of Pb, Cr, Cu, Zn and Fe were 56.23, 12.9, 36.31, 123.03 and 11,776 mg kg⁻¹, respectively. The pollution status of heavy metals in urban soils was evaluated using different quantitative indices (enrichment factor–EF, Geo-accumulation Index–I_geo, and pollution index–PI). Enrichments factors of the selected heavy metals determined by using Fe as a normalizer showed that metal contamination was the product of anthropogenic activities. The urban soils of Karachi were found to have a moderate to moderately severe enrichment with Pb, whereas Cr and Cu has moderate and Zn has minor enrichment. I_geo results indicated moderate soil contamination by Pb at some of the sampling locations. PI for Pb, Cr, Cu and Zn was found in the range of 0.04–3.42, 0.19–1.55, 0.27–2.45 and 0.32–1.57, respectively. Large variations in PI values of Pb revealed that soil in those areas of the city which are influenced by intensive anthropogenic activities have exceptionally high concentrations of Pb. The findings of this study would contribute to the environmental database of the soil of the region and would also facilitate both at the local and the international scales, in a more accurate global environmental monitoring, which will eventually facilitate the development of management and remediation strategies for heavy metal contaminated urban soil.     

Modulation of spiking of neurons of the cat anterior hypothalamus induced by experimental shifts in the osmolarity of the blood plasma

We examined changes in the impulse activity (IA) generated by neurons of the anterior hypothalamus (including the preoptic region) resulting from infusions of hyper-and hypotonic NaCl solutions (3.0 and 0.2%, respectively; hyper-and hypoosmotic stimulations, respectively); the infused volumes did not exceed 200 μl. The effects of hyper-and hypoosmotic stimulations were studied in detail in 83 and 88 neurons, respectively. In 31.2% (26 cells) and 29.6% (26 cells) of the neurons of the above groups, these stimulations evoked changes in the IA frequency greater than +40 or −40% of the mean background IA frequency. In approximately 50% of the responding neurons in each group, such shifts in the IA frequency were observed in the course of infusions of test solutions (5 sec long) and within the subsequent 5 min after termination of the infusion. In another 50% of the neurons, changes in the IA frequency occurred within the afterperiod (30 sec long); these shifts could develop exclusively within the latter interval, or these changes accompanied an initial early reaction. In general, activating responses dominated (they were observed in 65% of the cases where test stimulations of both modalities were used). The possible aspects of the involvement of neurons of the anterior hypothalamus in the control of the water/salt balance in the organism are discussed. Neirofiziologiya/Neurophysiology, Vol. 38, No. 1, pp. 40–45, January–February, 2006.     

Radio frequency radiation causes no nonthermal damage in enzymes and living cells

The ability of radio frequency radiation (RFR) to exert irreversible nonthermal (i.e., not caused by accompanying heat) effects on biologics has been widely debated due to a relative paucity of comprehensive critical details in published reports dealing with this issue. In this study, we used rigorous control over experimental conditions to determine whether continuous RFR nonthermally affects commercially important enzymes and live bacterial and human cells using three most commonly used frequencies in current RF identification technology, namely 2.45 GHz, 915 MHz, and 13.56 MHz. Diverse biological samples were exposed to RFR under deliberately harsh conditions to increase the likelihood of observing such effects should they exist. Enzymatic activities of horseradish peroxidase and β‐galactosidase in aqueous solution exhibited no statistically discernable consequences of even very intense RFR. Likewise, with putative thermal effects excluded, the viabilities of bacteria (both gram‐positive and gram‐negative) and of human cells were not detectably compromised by such an RFR exposure. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010