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.     

Lipid Binding Defects and Perturbed Synaptogenic Activity of a Collybistin R290H Mutant That Causes Epilepsy and Intellectual Disability

Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects—or synaptopathies—are at the basis of many neurological and psychiatric disorders. In key areas of the mammalian brain, such as the hippocampus or the basolateral amygdala, the clustering of the scaffolding protein Gephyrin and of γ-aminobutyric acid type A receptors at inhibitory neuronal synapses is critically dependent upon the brain-specific guanine nucleotide exchange factor Collybistin (Cb). Accordingly, it was discovered recently that an R290H missense mutation in the diffuse B-cell lymphoma homology domain of Cb, which carries the guanine nucleotide exchange factor activity, leads to epilepsy and intellectual disability in human patients. In the present study, we determined the mechanism by which the Cb^R290H mutation perturbs inhibitory synapse formation and causes brain dysfunction. Based on a combination of biochemical, cell biological, and molecular dynamics simulation approaches, we demonstrate that the R290H mutation alters the strength of intramolecular interactions between the diffuse B-cell lymphoma homology domain and the pleckstrin homology domain of Cb. This defect reduces the phosphatidylinositol 3-phosphate binding affinity of Cb, which limits its normal synaptogenic activity. Our data indicate that impairment of the membrane lipid binding activity of Cb and a consequent defect in inhibitory synapse maturation represent a likely molecular pathomechanism of epilepsy and mental retardation in humans.     

Immunopathological Changes in the Brain of Immunosuppressed Mice Experimentally Infected with Toxocara canis

Toxocariasis is a soil-transmitted helminthozoonosis due to infection of humans by larvae of Toxocara canis. The disease could produce cognitive and behavioral disturbances especially in children. Meanwhile, in our modern era, the incidence of immunosuppression has been progressively increasing due to increased incidence of malignancy as well as increased use of immunosuppressive agents. The present study aimed at comparing some of the pathological and immunological alterations in the brain of normal and immunosuppressed mice experimentally infected with T. canis. Therefore, 180 Swiss albino mice were divided into 4 groups including normal (control) group, immunocompetent T. canis-infected group, immunosuppressed group (control), and immunosuppressed infected group. Infected mice were subjected to larval counts in the brain, and the brains from all mice were assessed for histopathological changes, astrogliosis, and IL-5 mRNA expression levels in brain tissues. The results showed that under immunosuppression, there were significant increase in brain larval counts, significant enhancement of reactive gliosis, and significant reduction in IL-5 mRNA expression. All these changes were maximal in the chronic stage of infection. In conclusion, the immunopathological alterations in the brains of infected animals were progressive over time, and were exaggerated under the effect of immunosuppression as did the intensity of cerebral infection.     

Fish recolonization of a lowland river with non-buffered storm water discharges but with abated pollution from a large municipality

Water pollution from industrial Metro Łódź (ML), Poland, made the Ner River almost fishless in its middle-lower course for most of the 19th and 20th century. The new sewage treatment plant of ML and reduction of industry have caused pollution abatement there since the 1990s. As a result, the Ner became repopulated, which was shown by fish samples collected along its course in 2000–2012. Multivariate statistical methods helped distinguish unpolluted (I and II, in the upper course), and recovered (III, IV and V, in the middle-lower course) sections of the river. Historical and present data indicated that section III (downstream of ML) recovered least, both before and during the study. Section V (outflow one) recovered most and its fish fauna (almost exclusively native) now displays high and stable biomass, abundance and species richness, including those of obligatory riverine species. Non-native Prussian carp's dominance followed the river degradation gradient, i.e. was highest in section III, and in section V declined to almost absence. This study shows that the revival of native fish fauna seems to be a method of restricting the dominance of this highly tolerant species. Despite the abatement, storm events are very harmful to fish (mostly in section III), because the Ner discharge may then increase manifold and all storm water is drained by the ML combined sewer system to the Ner in several hours. Other stressors are numerous dams and desorption of pollutants from sediment in the middle Ner, and perhaps pollutant inflow from agriculture or local urban areas. Some moderation of storm impact on water entering the Ner from ML by constructing buffer reservoirs would probably cause further fish recovery in section III.     

荧光显微成像在医学细胞生物学实验教学中的应用探索

医学细胞生物学是医学院校本科生的重要基础学科之一,其实验课程教学工作的改革与创新具有重要意义。本文深入分析了医学细胞生物学实验课程现状,认为课程普遍存在目的不明确、内容不新颖、缺乏整体性、临床联系少等问题。荧光显微成像技术是细胞生物学研究中的关键技术,利用这一技术设计具有整体性的科研实验对于培养医学生操作能力和科研素养具有重要意义。顺铂(cisplatin)为当前肿瘤联合化疗中最常用的药物之一,由于其抗癌机制涉及细胞增殖、细胞凋亡、细胞骨架等一系列细胞生物学知识,且与临床联系紧密,非常适合用于综合性实验设计。实验方案拟使用BrdU染色、Hoechst33258染色及鬼笔环肽染色法,分别检测人肺癌细胞系A549顺铂处理后细胞增殖、细胞凋亡及细胞骨架重排情况。实验完成后,鼓励学生通过查阅文献得出综合的实验结论。希望能够通过科研与教学相融合的方式,更好的激发医学生的学习热情,提高医学生的科研素质,培养全方位的医学人才。     

The tRNA A76 Hydroxyl Groups Control Partitioning of the tRNA-dependent Pre- and Post-transfer Editing Pathways in Class I tRNA Synthetase

Aminoacyl-tRNA synthetases catalyze ATP-dependent covalent coupling of cognate amino acids and tRNAs for ribosomal protein synthesis. Escherichia coli isoleucyl-tRNA synthetase (IleRS) exploits both the tRNA-dependent pre- and post-transfer editing pathways to minimize errors in translation. However, the molecular mechanisms by which tRNA^Ile organizes the synthetic site to enhance pre-transfer editing, an idiosyncratic feature of IleRS, remains elusive. Here we show that tRNA^Ile affects both the synthetic and editing reactions localized within the IleRS synthetic site. In a complex with cognate tRNA, IleRS exhibits a 10-fold faster aminoacyl-AMP hydrolysis and a 10-fold drop in amino acid affinity relative to the free enzyme. Remarkably, the specificity against non-cognate valine was not improved by the presence of tRNA in either of these processes. Instead, amino acid specificity is determined by the protein component per se, whereas the tRNA promotes catalytic performance of the synthetic site, bringing about less error-prone and kinetically optimized isoleucyl-tRNA^Ile synthesis under cellular conditions. Finally, the extent to which tRNA^Ile modulates activation and pre-transfer editing is independent of the intactness of its 3′-end. This finding decouples aminoacylation and pre-transfer editing within the IleRS synthetic site and further demonstrates that the A76 hydroxyl groups participate in post-transfer editing only. The data are consistent with a model whereby the 3′-end of the tRNA remains free to sample different positions within the IleRS·tRNA complex, whereas the fine-tuning of the synthetic site is attained via conformational rearrangement of the enzyme through the interactions with the remaining parts of the tRNA body.