二苯乙烯苷通过上调XIAP抑制人脐静脉内皮细胞凋亡

目的:二苯乙烯苷(2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside,TSG)具有抗炎、抗氧化、抗动脉粥样硬化(atherosclerosis,As)等作用。课题组前期研究表明TSG对过氧化氢(H2O2)诱导损伤的内皮细胞具有保护作用,并抑制内皮细胞的凋亡,但机制尚未完全明确。本研究目的在于探讨TSG是否通过影响X连锁凋亡抑制蛋白(X-linked inhibitor of apoptosis protein,XIAP)、Caspase-9的表达来抑制细胞凋亡。方法:体外培养人脐静脉内皮细胞,实验分为正常对照组、模型组(300μmol·L-1H2O2)、TSG预处理组(10μmol·L-1TSG+300μmol·L-1H2O2)、Embelin与TSG联合处理组(30μmol·L-1Embelin+10μmol·L-1TSG+300μmol·L-1H2O2)、TSG单独处理组(10μmol·L-1TSG)、Embelin组(30μmol·L-1Embelin)。采用MTT法检测细胞增殖率,Hoechst 33258染色观察凋亡细胞核形态,RT-PCR和Western blot检测XIAP、Caspase-9的表达。结果:与空白对照组相比,H2O2组内皮细胞增殖率降低,核损伤明显,XIAP表达显著性下降,Caspase-9表达显著增加(P0.01);与H2O2组比较,经TSG预处理后,细胞增殖率增加,核损伤减轻,XIAP的表达上升,Caspase-9表达减少,差异均有显著性(P0.01)。与TSG预处理组比较,用XIAP阻断剂Embelin与TSG联合处理后,内皮细胞活力下降,XIAP表达显著降低,Caspase-9表达增加(P0.01)。结论:TSG具有抑制H2O2诱导的人脐静脉内皮细胞凋亡作用,其机制与增加XIAP的表达,抑制Caspase-9的表达有关。     

Study of complex structural variations of X-linked deafness-2 based on single-molecule sequencing

X-linked deafness-2 (DFNX2) is cochlear incomplete partition type III (IP-III), one of inner ear malformations characterized by an abnormally wide opening in the bone separating the basal turn of the cochlea from the internal auditory canal, fixation of the stapes and cerebrospinal fluid (CSF) gusher upon stapedectomy or cochleostomy. The causative gene of DFNX2 was POU3F4. To investigate the genetic causes of DFNX2 and compare the efficiency of different sequencing methods, 12 unrelated patients were enrolled in the present study. Targeted next-generation sequencing (NGS) and long-read sequencing were used to analyze the genetic etiology of DFNX2. Six variants of POU3F4 were identified in this cohort by NGS. Three patients with a negative diagnosis based on NGS were enrolled in further long-read sequencing. Two of them were all found to carry structural variations (SVs) on chromosome X, consisting of an 870-kb deletion (DEL) at upstream of POU3F4 and an 8-Mb inversion (INV). The 870-kb DEL may have been due to non-homologous end joining (NHEJ), while non-allelic homologous recombination (NAHR) within a single chromatid may have accounted for the 8-Mb INV. Common POU3F4 mutations in DFNX2 included point mutations, small insertions and deletions (INDELs), and exon mutations, which can be detected by Sanger sequencing and NGS. Single-molecule long-read sequencing constitutes an additional and valuable method for accurate detection of pathogenic SVs in IP-III patients with negative NGS results.     

ABCD1 mutations and phenotype distribution in Chinese patients with X-linked adrenoleukodystrophy

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder resulting from mutations within the ABCD1 gene. Adrenomyeloneuropathy (AMN) and childhood cerebral ALD (CCALD) are most common phenotypes in the Western ALD patients. Here we performed mutation analysis of ABCD1 in 10 Chinese ALD families and identified 8 mutations, including one novel deletion (c.1477_1488 + 11del23) and 7 known mutations. Mutations c.1772G>A and c.1816T>C were first reported in the Chinese patients. Mutations c.1661G>A and c.1679C>T were demonstrated to be de novo mutations. The dinucleotide deletion 1415_16delAG, described as a mutational hotspot in different ethnic groups, was identified in two families. In addition, we performed a retrospective nation-wide mutation study of X-linked ALD in China based on a literature review. The retrospective study further confirmed the hypothesis that exon 6 is a potential mutation cluster region in the Asian populations. Furthermore, it suggested that CCALD is the most common phenotype in China.     

Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: a role for NF-κB-STAT3-directed gene expression

Mitochondrial DNA depleted (ρ⁰) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ⁺ HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ⁰ cells compared to ρ⁺ HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ⁺ HSF, but this response was substantially decreased in ρ⁰ HSF. Suppression of the IKK–NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2–STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ⁺ HSF. Inhibitory antibodies against IL6, the main activator of JAK2–STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ⁰ HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6–JAK2–STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis.     

The phosphatonins and the regulation of phosphate transport and vitamin D metabolism

Phosphate homeostasis is preserved during variations in phosphate intake by short-term intrinsic renal and intestinal adaptations in transport processes, and by more long-term hormonal mechanisms, which regulate the efficiency of phosphate transport in the kidney and intestine. Recently, several phosphaturic peptides such as fibroblast growth factor 23 (FGF-23), secreted frizzled-related protein-4 (sFRP-4), extracellular phosphoglycoprotein (MEPE) and fibroblast growth factor 7 (FGF-7) have been shown to play a pathogenic role in several hypophosphatemic disorders such as tumor-induced osteomalacia (TIO), autosomal dominant hypophosphatemic rickets (ADHR), X-linked hypophosphatemic rickets (XLH), the McCune-Albright syndrome (MAS) and fibrous dysplasia (FD). These proteins induce phosphaturia and hypophosphatemia in vivo, and inhibit sodium-dependent renal phosphate transport in cultured renal epithelial cells. Interestingly, despite the induction of hypophosphatemia by FGF-23 and sFRP-4 in vivo, serum 1, 25-dihydroxyvitamin D (1alpha,25(OH)(2)D) concentrations are decreased or remain inappropriately normal, suggesting an inhibitory effect of these proteins on 25-hydroxyvitamin D 1alpha-hydroxylase activity. In FGF-23 knockout mice, 25-hydroxyvitamin D 1alpha-hydroxylase expression is increased and elevated serum 1alpha,25(OH)(2)D levels cause significant hypercalcemia and hyperphosphatemia. MEPE, however, increases circulating 1alpha,25(OH)(2)D. Circulating or local concentrations of these peptides/proteins may regulate 25-hydroxyvitamin D 1alpha-hydroxylase activity in renal tissues under physiologic circumstances.     

High prevalence of multiple paternity within fruits in natural populations of Silene latifolia, as revealed by microsatellite DNA analysis

Data on multiple paternity within broods has been gathered in several animal species, and comparable data in plants would be of great importance to understand the evolution of reproductive traits in a common framework. In this study, we first isolated and characterized six microsatellite loci from the dioecious plant Silene latifolia (Caryophyllaceae). The polymorphism of the loci was assessed in 60 individual females from four different populations. Two of the investigated loci showed a pattern of inheritance consistent with X-linkage. These microsatellite loci were highly polymorphic and therefore useful tools for parentage analysis. We then used four of the markers to determine paternity within naturally pollinated fruits in four European populations. This study revealed widespread multiple paternity in all populations investigated. The minimum number of fathers per fruit varied from one to nine, with population means ranging from 3.4 to 4.9. The number of fathers per fruit was not significantly correlated with offspring sex ratios. High prevalence of multiple paternity within fruits strongly suggest that pollen competition is likely to occur in this species. This may substantially impact male reproductive success and possibly contribute to increase female and offspring fitness, either through postpollination selection or increased genetic diversity. Wide variation in outcrossing rates may be an overlooked aspect of plant mating systems.     

Proteomic identification of elevated saliva kallikrein levels in the mdx-4cv mouse model of Duchenne muscular dystrophy

Dystrophinopathies are multi-system disorders that affect the skeletal musculature, the cardio-respiratory system and the central nervous system. The systematic screening of suitable biofluids for released or altered proteins promises new insights into the highly complex pathophysiology of X-linked muscular dystrophy. However, standard detection approaches using antibody-based assays often fail to reproducibly detect low-abundance protein isoforms in dilute biological fluids. In contrast, mass spectrometric screening approaches enable the proteome-wide identification of minor protein changes in biofluids. This report describes the findings from the comparative proteomic analysis of whole saliva samples from wild type versus the established mdx-4cv mouse model of highly progressive muscular dystrophy, focusing on the kallikrein protein family. Kallikrein-1 (Klk1) and 13 Klk1-related peptidases were identified in saliva and serum from normal mice. Comparative proteomics revealed elevated saliva levels of the Klk1-related peptidases Klk1-b1, Klk1-b5 and Klk-b22, as well as an increased Klk-1 concentration, which agrees with higher Klk-1 levels in serum from mdx-4cv mice. This indicates altered cellular signaling, extracellular matrix remodeling and an altered immune response in the mdx-4cv mouse, and establishes liquid biopsy procedures as suitable bioanalytical tools for the systematic survey of complex pathobiochemical changes in animal models of muscular dystrophy.     

Bax/Bak-dependent,Drp1-independent Targeting of X-linked Inhibitor of Apoptosis Protein (XIAP) into Inner Mitochondrial Compartments Counteracts Smac/DIABLO-dependent Effector Caspase Activation

Efficient apoptosis requires Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP), which releases death-promoting proteins cytochrome c and Smac to the cytosol, which activate apoptosis and inhibit X-linked inhibitor of apoptosis protein (XIAP) suppression of executioner caspases, respectively. We recently identified that in response to Bcl-2 homology domain 3 (BH3)-only proteins and mitochondrial depolarization, XIAP can permeabilize and enter mitochondria. Consequently, XIAP E3 ligase activity recruits endolysosomes into mitochondria, resulting in Smac degradation. Here, we explored mitochondrial XIAP action within the intrinsic apoptosis signaling pathway. Mechanistically, we demonstrate that mitochondrial XIAP entry requires Bax or Bak and is antagonized by pro-survival Bcl-2 proteins. Moreover, intramitochondrial Smac degradation by XIAP occurs independently of Drp1-regulated cytochrome c release. Importantly, mitochondrial XIAP actions are activated cell-intrinsically by typical apoptosis inducers TNF and staurosporine, and XIAP overexpression reduces the lag time between the administration of an apoptotic stimuli and the onset of mitochondrial permeabilization. To elucidate the role of mitochondrial XIAP action during apoptosis, we integrated our findings within a mathematical model of intrinsic apoptosis signaling. Simulations suggest that moderate increases of XIAP, combined with mitochondrial XIAP preconditioning, would reduce MOMP signaling. To test this scenario, we pre-activated XIAP at mitochondria via mitochondrial depolarization or by artificially targeting XIAP to the intermembrane space. Both approaches resulted in suppression of TNF-mediated caspase activation. Taken together, we propose that XIAP enters mitochondria through a novel mode of mitochondrial permeabilization and through Smac degradation can compete with canonical MOMP to act as an anti-apoptotic tuning mechanism, reducing the mitochondrial contribution to the cellular apoptosis capacity.     

Screening for possible miRNA–mRNA associations in a colon cancer cell line

MicroRNAs (miRNAs) are small non-coding RNAs mediating the regulation of gene expression in various biological contexts, including carcinogenesis. Here, we screened putative associations between 34, 45, and 103 miRNAs and 164, 391, and 81 mRNAs via Argonaute1 (Ago1) or Ago2 immunoprecipitation (IP) experiments in a colon cancer cell line. We used a combination of RIP Seq analysis. RNAs that were co-immunoprecipitated with Ago1 or Ago2 were used for massively parallel small RNA and mRNA sequencing. The detected miRNAs and mRNAs were further associated with one another based on in silico target predictions. Analysis of the putative associations indicated that, although Ago1 and Ago2 shared a similar repertory of miRNAs, the mRNAs possibly regulated by those miRNAs seemed different. The mRNAs detected with Ago1 IP were indicated to be frequently associated with genes having constitutive cellular functions, regulated by a smaller number of miRNAs, and appeared to receive more stringent translational regulation. In contrast, putative miRNA-mRNA associations detected with Ago2 IP appeared to be related to signal transduction genes, which had a larger number of possible miRNA binding sites. We then conducted a similar analysis using the colon cancer cells cultured under hypoxia and identified potential hypoxia-induced miRNA-mRNA associations, which included several well-characterized cancer-related genes as novel putative miRNA targets.