雷帕霉素介导的caspase 9同源二聚化调控人类多能干细胞自杀

人诱导多能干细胞（human induced pluripotent stem cells，hiPSCs）在再生医学领域具有广阔的应用前景。然而，多能干细胞（pluripotent stem cells，PSCs）具有肿瘤化风险，成为其临床应用最主要的安全性问题。雷帕霉素是一种安全和广泛使用的免疫抑制药物，通过诱导FKBP12与FRB片段的异源二聚起作用。为了保障hiPSCs治疗的安全性，本研究将雷帕霉素诱导的caspase 9（riC9）基因插入到AAVS1安全位点，构建了含有EF1α启动子、FRB-FKBP-Caspase9（CARD结构域）融合蛋白和嘌呤霉素抗性基因的供体，并与sgRNA/Cas9载体共转染hiPSCs。用嘌呤霉素筛选2周后，收集单个克隆进行基因和表型分析。最后，用雷帕霉素诱导caspase9同源二聚化，激活工程细胞的凋亡。通过对筛选获得的5个hiPSCs克隆鉴定，表明供体DNA准确敲入内源性AAVS1位点。hiPSCs保持正常的多潜能状态和增殖能力。雷帕霉素可诱导caspase 9同源二聚化，并激活细胞凋亡程序。本研究通过药物精确调控caspase 9的同源二聚化来启动细胞自杀，实现了可控的hiPSCs存活，这为保证hiPSCs治疗的安全性提供了新的策略。     

大鼠脑缺血再灌注后Cathepsin D和caspase．9表达的动态变化

目的：探讨大鼠缺血再灌注后溶酶体组织蛋白酶D（Cathepsin DCD）、caspase-9在不同时段蛋白质及mRNA表达变化。方法：将60只SD大鼠随机分为三组：正常组（10只）,假手术组（10只）,脑缺血再灌注组（40只）,线栓法制备大脑中动脉梗死模型（MCAO）,免疫组化及RT—PCR法分别检测CathepsinD、caspase-9的蛋白和mRNA表达。结果：与正常组和假手术组比较,模型组大鼠脑缺血再灌注损伤后6h Cathepsin D的蛋白和mRNA表达明显增强（P〈0．05）,24h达高峰,48h仍保存高水平。caspase．9蛋白和mRNA6h开始明显升高,12h达高峰,此后缓慢下降,但48h组仍显著高于对照组（P〈0．05）,结论：Cathepsin D、caspase．9在大鼠脑缺血再灌注后表达增强,溶酶体可能参与了脑缺血再灌注损伤后神经细胞凋亡的过程。     

"杀手"蛋白酶caspase

细胞凋亡(apoptosis)或称程序性细胞死亡(Programmed cell death,PCD)是多细胞生物正常发育、分化过程中进行细胞删除的一种基本机制,与组织自稳、衰老及细胞损伤密切相关。凋亡异常可引起人类难治性疾病,如Parkinson’s 和Huntington’s 病,AIDS、Alzheimer’s病、免疫缺陷、自身免疫紊乱、缺血性心血管疾病、神经系统疾病、秃发、白血病、淋巴瘤及其他癌症,并与肿瘤治疗抗性有关。虽然细胞凋亡时许多生化事件已被确认,但凋亡诱发、调控的分子生化机制仍不明     

Miscellaneous mistletoe notes, 1–9

Three new species,Cladocolea biflora Kuijt,Struthanthus condensatus Kuijt, andS. lojae Kuijt are described and six new combinations,Cladocolea diversifolia (Benth.) Kuijt,C. lenticellata (Diels) Kuijt,Dendrophthora virgata (Trel.) Kuijt,Korthalsella chilensis (Hook. & Arn.) Kuijt,Phthirusa retroflexa (Ruiz & Pavon) Kuijt, andStruthanthus flexilis (Rusby) Kuijt are proposed. Nomenclature of two more neotropical Loranthaceae,Psittacanthus ramiflorus (DC.) G. Don andStruthanthus interruptus (H.B.K.) Blume is revised.     

Involvement of caspase activation and mitochondrial stress in taxol-induced apoptosis of Epstein–Barr virus-infected Akata cells

Taxol (paclitaxel) is one of the most potent antimicrotubule agents currently used in cancer chemoprevention and treatment. However, the effects of taxol on the induction of apoptosis in Epstein–Barr virus (EBV)-infected cells are unknown. This study investigated the mechanisms of taxol on cell cycle arrest and apoptosis induction using the EBV-infected cell line, Akata. Taxol treatment sensitively and dose-independently induced growth inhibition, cytotoxicity, and apoptosis in the cells, which was demonstrated by the decreased level of tritium incorporation and cell viability, the increased number of positively stained cells in the trypan blue staining and TUNEL assay, the increased population of cells in the sub-G₀/G₁ phase in flow cytometric analysis, and ladder formation of the genomic DNA. Treatment with z-VAD-fmk almost completely protected the cells from taxol-induced apoptosis indicating that the taxol-induced apoptosis of Akata cells is caspase-dependent. In addition, taxol-induced apoptosis is proposed to be associated with a lower mitochondrial membrane potential and G₂/M arrest. However, the tubulin expression level doses not appear to be a direct mediator of taxol-induced apoptosis in cells. The presence of EBV in these cells was not related to the sensitivity of the cells to the induction of apoptosis by taxol. Overall, these results demonstrate that taxol induces apoptosis in EBV-infected Akata cells in a dose-independent manner, and that caspase activation and mitochondrial stress are involved in the induction.     

CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri

Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR–Cas systems, such as the Streptococcus pyogenes CRISPR–Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR–Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR–Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR–Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR–Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR–Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR–Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria.     

Notes on some African hepatic genera 6–9

The present study deals with African species ofSyzygiella Spruce,Allisoniella Hodgs. andGymnomitriaceae Klinggr. Three species ofSyzygiella Spruce,S. geminifolia (Mitt.) Steph.,S. concreta (Gott.) Spruce and one indeterminable species with orbicular leaves (known only in sterile condition) occur in Africa.Allisoniella nigra (Rodw.) Schust. represents a genus hitherto unreported for the African flora. The present knowledge ofGymnomitriaceae Klinggr. in subsaharan Africa is summarised; five species ofMarsupella Dum., one ofGymnomitrion Corda and five ofHerzogobryum Grolle are reported. Finally, some additions to the generaIsotachis Mitt.,Anastrophyllum (Spruce) Steph. andLophozia (Dum.) Dum. are presented.     

半胱天冬酶（caspase）

半胱天冬酶是ＩＣＥ／ＣＥＤ－３蛋白酶家族的总称,在细胞凋亡中起着关键的作用,迄今已鉴定的人类半胱天冬梅共有１１种,其底物可分为４类,半胱天冬酶的激活机制已初见端倪,有非自然抑制剂和自然抑制剂,抑制半胱天冬酶活性作为神经变性疾病的治疗途径已开始探索。     

胱天蛋白酶（caspase）的前结构域

胱天蛋白酶 (ｃａｓｐａｓｅ)是白细胞介素 1β转化酶(ｉｎｔｅｒｌｅｕｋｉｎ 1βｅｎｚｙｍｅ ,ＩＣＥ)家族的总称 ,Ｃａｓｐａｓｅ(ｃｙｓｔｅｉｎｅａｓｐａｒｔａｔｅ ｓｐｅｃｉａｌｐｒｏｔｅａｓｅｓ)的含义是该类蛋白酶的活性部位为极为保守的半胱氨酸 (ｃｙｓｔｅｉｎｅ)残基 (取第一个字母“ｃ”) ,又特异性切割底物的天冬氨酸 ,用“ａｓｐａｓｅ”表示 ,简称ｃａｓｐａｓｅ ,该酶在细胞凋亡过程中起关键作用 ,是目前研究的热点。现已发现的ｃａｓｐａｓｅ有 14种 ,它们均以无活性的酶原的形式存在 ,包括一个Ｎ末端前结构域 (ｐ…     

昆虫中分离与鉴定的caspase及其作用

天冬氨酸特异性的半胱氨酸蛋白酶(caspase)家族是执行细胞凋亡的主要酶类,对caspase结构及生物学功能的研究有助于更深入的研究细胞凋亡的分子机制。Caspase具有高度保守性,它们具有相似的氨基酸序列、结构和底物特异性。且具有QACRG的五肽活性位点,该活性位点是caspase家族的典型结构。昆虫caspase在caspase依赖型的细胞凋亡中起关键作用,文章介绍和评述昆虫中已经分离、鉴定的caspase及其功能。     

Sox9-related signaling controls zebrafish juvenile ovary–testis transformation

In almost all vertebrates, the downstream of the sox9 signaling axis is well conserved for testis differentiation. The upstream genes of this pathway vary from species to species during evolution. Yet, little is known about how these signaling cascades are regulated and what cellular processes are dominant in ovary–testis transformation in juvenile zebrafish. In this study, we find that the transforming gonads undergo activation of sox9a-expressing stromal cells with increased deposition of extracellular matrix and formation of degenerative compartments. This leads to follicle disassembly, oocyte degeneration, follicle cell-cyp19a1a-amh conversions, and, eventually, formation of the testis cord. In vitro primary culture of juvenile ovary tissue in gonadotropins increases cytoplasmic accumulation of sox9a and p-Erk1/2, and induces mesenchymal morphology. MAPK inhibitors (MKI), a mixture of PD98059 and U0216, eliminate the cytoplasmic distribution but do not eradicate nuclear localization of sox9a and p-Erk1/2. Nuclear p53 are relatively increased in MKI-treated cells that exhibit less spreading and reduced proliferation. Despite uniform nuclear condensation, only a fraction of cells displayed the apoptotic phenotype. These results suggest that high levels of cytoplasmic sox9a and p-Erk1/2 activity activate stromal cells and enhance the production of extracellular matrix required for testis cord formation, whereas deregulation and translocation of sox9a and p-Erk1/2 induce follicle disassembly and incomplete apoptosis associated with nuclear p53. Together with the established FSH/cAMP/MAPK/AMH pathway in mammalian granulosa and Sertoli cells, we demonstrated that the sox9 axis signaling that determines testis formation in mammals also induces zebrafish ovary–testis transition, and adds to its conserved role in sex reversal.