Reduced cellular Ca availability enhances TDP-43 cleavage by apoptotic caspases

Accumulation of transactive response DNA binding protein (TDP-43) fragments in motor neurons is a post mortem hallmark of different neurodegenerative diseases. TDP-43 fragments are the products of the apoptotic caspases-3 and -7. Either excessive or insufficient cellular Ca²⁺ availability is associated with activation of apoptotic caspases. However, as far as we know, it is not described whether activation of caspases, due to restricted intracellular Ca²⁺, affects TDP-43 cleavage. Here we show that in various cell lineages with restricted Ca²⁺ availability, TDP-43 is initially cleaved by caspases-3 and -7 and then, also by caspases-6 and -8 once activated by caspase-3. Furthermore, we disclose the existence of a TDP-43 caspase-mediated fragment of 15 kDa, in addition to the well-known fragments of 35 and 25 kDa. Interestingly, with respect to the other two fragments this novel fragment is the major product of caspase activity on murine TDP-43 whereas in human cell lines the opposite occurs. This outcome should be considered when murine models are used to investigate TDP-43 proteinopathies.     

Cytotoxicity of mitochondria-targeted resveratrol derivatives: Interactions with respiratory chain complexes and ATP synthase

We recently reported that mitochondria-targeted derivatives of resveratrol are cytotoxic in vitro, selectively inducing mostly necrotic death of fast-growing and tumoral cells when supplied in the low μM range (N. Sassi et al., Curr. Pharm. Des. 2014). Cytotoxicity is due to H₂O₂ produced upon accumulation of the compounds into mitochondria. We investigate here the mechanisms underlying ROS generation and mitochondrial depolarization caused by these agents. We find that they interact with the respiratory chain, especially complexes I and III, causing superoxide production. “Capping” free hydroxyls with acetyl or methyl groups increases their effectiveness as respiratory chain inhibitors, promoters of ROS generation and cytotoxic agents. Exposure to the compounds also induces an increase in the occurrence of short transient [Ca^2 +] “spikes” in the cells. This increase is unrelated to ROS production, and it is not the cause of cell death. These molecules furthermore inhibit the F₀F₁ ATPase. When added to oligomycin-treated cells, the acetylated/methylated ones cause a recovery of the cellular oxygen consumption rates depressed by oligomycin. Since a protonophoric futile cycle which might account for the uncoupling effect is impossible, we speculate that the compounds may cause the transformation of the ATP synthase and/or respiratory chain complex(es) into a conduit for uncoupled proton translocation. Only in the presence of excess oligomycin the most effective derivatives appear to induce the mitochondrial permeability transition (MPT) within the cells. This may be considered to provide circumstantial support for the idea that the ATP synthase is the molecular substrate for the MPT pore.     

Fusogenic potential of sperm membrane lipids: nature's wisdom to accomplish targeted gene delivery

The membrane-membrane fusion during fertilization of oocyte by spermatozoa is believed to be mainly mediated by so called "fusion proteins". In the present study we have tried to demonstrate that beside the proteins, lipid components of membrane may play an important role in fusion of oocyte with spermatozoa. Conventional membrane-membrane fusion assays were used as means to demonstrate fusogenic potential of human sperm membrane lipids. The liposomes (spermatosomes) made of the lipids isolated from sperm membrane were found to undergo strong membrane-membrane fusion as evident from fluorescence dequenching and resonance energy transfer assays. Furthermore, the fusion of these liposomes with living cells (J774 A.1 macrophage cell line) was demonstrated to result in an effective transfer of a water-soluble fluorescent probe (calcein) to cytosol of the target cell. Lastly, the liposomes were demonstrated to behave like efficient vehicles for the in vivo cytosolic delivery of the antigens to target cells resulting in elicitation of antigen specific CD8(+) T cell responses.     

靴隼雕繁殖习性初报

国内关于靴隼雕(Hieraaetus pennata)资料缺乏,仅有几例新纪录报道,无繁殖信息。本文报道2010~2016年间靴隼雕在新疆南部和北部的繁殖与分布状况。靴隼雕巢址选择开阔的地带,营巢于大树上(n=7),巢距地面高度在7~12 m,巢直径约74~102 cm。窝卵数2~3枚,孵卵期37~40 d,育雏期48~58 d,繁殖期持续4~5个月。育雏前期与后期亲鸟的行为变化较大。其巢区附近野生动物资源丰富,食物以鸟类为主,主要是水鸟的幼鸟(体重小于300 g);哺乳类包括草兔(Lepus capensis)、大耳猬(Hemiechinus auritus)、鼹形田鼠(Ellobius talpinus)等。在新疆和硕利用红外相机监测32 d,共收获77 894张图片。育雏期人工观察16 d,约248 h,同时拍下行为照片及录像作为辅助记录。育雏前期,雌鸟的陪护时间占88.43%,雄鸟只有3.26%。亲鸟育雏期的活动节律、投食次数呈现单峰型;续巢频次则为双峰型。在中亚,靴隼雕有明显"东扩"之趋势。通过野外观察靴隼雕的行为,了解繁殖状况,积累基础资料,对其种群保护和管理具有意义。     

Extension of the molecular analysis to the promoter region of the iduronate 2-sulfatase gene reveals genomic alterations in mucopolysaccharidosis type II patients with normal coding sequence

Hunter disease or mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by the deficit of the enzyme iduronate-2-sulfatase (IDS), involved in the catabolism of the glycosaminoglycans heparan and dermatan sulfate. Our aim was to search for molecular defects in the promoter region of the IDS gene in patients with previous biochemical diagnosis of MPS II and after we sequenced the whole IDS coding region and the exon/intron boundaries without detecting any pathogenic mutations. Screening of the promoter region of four patients detected in two of them a 178 bp deletion and in the other two a single nucleotide substitution 818 bp upstream of the coding region. The latter had never been described before in MPS II patients and it turned out to be a polymorphism. Our experience suggests that MPS II patients with no mutations detected in the IDS coding region should be screened in the promoter region of the gene. Findings will hopefully help to clarify the relationship between genotype and phenotype and will be useful for the correct molecular diagnosis of Hunter patients and the identification of female carriers, the latter particularly important for genetic counseling.     

Identification of human low-density lipoprotein receptor as a novel target gene regulated by liver X receptor alpha

Liver X receptor alpha (LXRalpha) is a member of the nuclear receptor superfamily that is activated by oxysterols, and plays a pivotal role in regulating the metabolism, transport and uptake of cholesterol. Here, we demonstrate that LXRalpha also regulates the low-density lipoprotein receptor (LDLR) gene, which mediates the endocytic uptake of LDL cholesterol in the liver. An LXR agonist induced the expression of LDLR in cultured hepatoblastoma cells. Moreover, the LDLR promoter contained an LXR response element that was recognized by LXRalpha/RXRalpha (retinoid X receptor alpha) heterodimers in hepatoblastoma cells. These results suggest a novel pathway whereby LXRalpha might modulate cholesterol metabolism.     

Isolation,purification and characterization of naturally derived Crocetin beta-d-glucosyl ester from Crocus sativus L. against breast cancer and its binding chemistry with ER-alpha/HDAC2

Saffron plant (Crocus sativus L.) is being used as a source of saffron spice and medicine to cure or prevent different types of diseases including cancers. We report the isolation, characterization of bioactive small molecule ([crocetin (β-d-glucosyl) ester] from the leaf biowastes of saffron plant of Kashmir, India. MTTC assay and Bio-autography aided approach were used to assess anti-oxidant activity and anti-cancer properties of crocin (s) against DPPH free radical and breast cancer cell line respectively. Crocetin beta-d-glucosyl ester restrained proliferation of human breast adeno-carcinoma cell model (MCF-7) without significantly affecting normal cell line (L-6). Further studies involving molecular mechanics generalized born surface area and molecular docking showed that crocetin beta-d-glucosyl ester exhibits strong affinity for estrogen receptor alpha and histone deacetylase 2 (crucial receptors involved in breast cancer signalling) as evidenced by the negative docking score and binding free energy (BFE) values. Therefore, crocetin beta-d-glucosyl ester from Crocus sativus biowastes showed antiproliferative effect possibly by inhibiting estrogen receptor alpha and HDAC2 mediated signalling cascade.     

MicroRNA-567 inhibits cell proliferation and induces cell apoptosis in A549 NSCLC cells by regulating cyclin-dependent kinase 8

MicroRNA-567 (miR-567) plays a decisive role in cancers whereas its role in non-small cell lung cancer (NSCLC) is still unexplored. This study was therefore planned to explore the regulatory function of miR-567 in A549 NSCLC cells and investigate its possible molecular mechanism that may help in NSCLC treatment. In the current study, miR-567 expression was examined by quantitative real time-polymerase chain reaction (qRT-PCR) in different NSCLC cell lines in addition to normal cell line. A549 NSCLC cells were transfected by miR-567 mimic, miR-567 inhibitor, and negative control siRNA. Cell proliferation was evaluated by MTT and 5-bromo-2′deoxyuridine assays. Cell cycle distribution and apoptosis were studied by flow cytometry. Bioinformatics analysis programs were used to expect the putative target of miR-567. The expression of cyclin-dependent kinase 8 (CDK8) gene at mRNA and protein levels were evaluated by using qRT-PCR and western blotting. Our results found that miR-567 expressions decreased in all the studied NSCLC cells as compared to the normal cell line. A549 cell proliferation was suppressed by miR-567 upregulation while cell apoptosis was promoted. Also, miR-567 upregulation induced cell cycle arrest at sub-G1 and S phases. CDK8 was expected as a target gene of miR-567. MiR-567 upregulation decreased CDK8 mRNA and protein expression while the downregulation of miR-567 increased CDK8 gene expression. These findings revealed that miR-567 may be a tumor suppressor in A549 NSCLC cells through regulating CDK8 gene expression and may serve as a novel therapeutic target for NSCLC treatment.