不同染色技术和双色荧光原位杂交技术对宽翅菘蓝（Isatis violascens Bunge）的细胞遗传学分析

为了解十字花科菘蓝属植物宽翅菘蓝（宽翅菘蓝,Isatis violascens Bunge）的染色体结构,本文利用45SrDNA和5SrDNA双色荧光原位杂交、银染技术和CMA3对宽翅菘蓝进行分子细胞遗传学研究。45SrDNA和5SrDNA荧光原位杂交结果显示宽翅菘蓝染色体上有1对45SrDNA信号和1对5SrDNA信号;银染结果显示其中期染色体有1对银染点;CMA3染色结果发现宽翅菘蓝中期染色体存在1对CMA3信号。     

不同染色技术和双色荧光原位杂交技术对宽翅菘蓝 （Isatis violascens Bunge）的细胞遗传学分析

为了解十字花科菘蓝属植物宽翅菘蓝(宽翅菘蓝,Isatis violascens Bunge)的染色体结构,本文利用45SrDNA和5SrDNA双色荧光原位杂交、银染技术和CMA3对宽翅菘蓝进行分子细胞遗传学研究。45SrDNA和5SrDNA荧光原位杂交结果显示宽翅菘蓝染色体上有1对45SrDNA信号和1对5SrDNA信号;银染结果显示其中期染色体有1对银染点;CMA3染色结果发现宽翅菘蓝中期染色体存在1对CMA3信号。     

纯合等臂假双着丝粒X染色体致性腺发育不良1例病例报告与文献复习

目的提高对纯合等臂假双着丝粒X染色体特纳综合征的鉴别诊断和临床特征的认识。方法用染色体核型分析技术,对1例14岁女性发育迟缓、先天子宫及双侧卵巢缺如患者的外周血细胞做细胞遗传学检查;用荧光原位杂交(FISH)和染色体微阵列芯片技术(chromosome microarray assay, CMA)进行验证。结果:发现该患者染色体核型为纯合等臂假双着丝粒X染色体;FISH检测在间期细胞中确认有3个X染色体着丝粒,在中期细胞中确认了其中2个着丝粒位于同一条染色体;CMA检测确认了Xpter-Xq22.1的片段重复及Xq22.1-Xqter的片段缺失。结论确诊患者为特纳综合征;纯合等臂双着丝粒X染色体的TS患者比较罕见,染色体核型分析、FISH及CMA技术的合理组合应用,有助于鉴别诊断并可能指导早期治疗。     

秸秆还田土壤改良培肥基质和复合菌剂配施对土壤生态的影响

通过田间试验,研究水旱轮作（冬小麦-夏水稻）中水稻秸秆全量还田条件下土壤改良培肥基质和复合菌剂配施对小麦土壤养分、土壤物理结构和土壤生物学性质及土壤微生物区系的影响,为快速土壤培肥、提高中低产农田产量提供实践基础和技术支持。试验于江苏省盐城市滨海县黄河湾项目基地进行,共设置五个处理：①土壤改良培肥基质+复合菌剂+常规化肥（MOS+CMA+CF）;②复合菌剂+常规化肥（CMA+CF）;③土壤改良培肥基质+常规化肥（MOS+CF）;④常规化肥（CF）;⑤不施肥对照（CK）。对小麦返青期（S1）、拔节孕穗期（S2）和成熟期（S3）分别进行土壤理化性质、土壤酶活性、微生物量碳氮和微生物区系分析。结果表明,与CK相比,MOS+CMA+CF处理能够在短时期内提高土壤速效养分含量（其中速效氮提高了23.59%、速效磷提高了40.74%、速效钾提高了43.78%）,降低土壤容重,提高土壤孔隙度,显著提高土壤微生物量氮含量和土壤脲酶活性;同时,该处理还能在小麦返青期和拔节孕穗期增加土壤细菌和真菌多样性,提高微生物丰度,最终提高了小麦产量（与CK和CF相比,产量分别提高了149.29%和24.93%）。CMA+CF能够显著提高土壤纤维素酶活及土壤微生物量碳含量,表现出有较好的秸秆降解能力;并且在提高土壤理化指标含量、提高脲酶酶活、提高微生物量氮含量和小麦产量方面仅次于联合处理。由于MOS富含有机质,MOS+CF处理能够维持并提高土壤有机质含量、改善土壤物理环境。总之,短期内,MOS+CMA+CF处理提升土壤肥力,提高小麦产量的效果是最显著的;但CMA+CF处理在加速秸秆养分还田、改善土壤理化状况,增强微生物活性和丰富以及提高小麦产量方面也表现出优势,且复合菌剂经济方便有效,具有很好的田间技术推广价值。     

环境和生物技术

美国CMA召开的半年会议讨论了关于未来生物技术对环境的影响的一些预测。Monsano公司的H.Schneiderman博士和哈佛大学的医学院遗传系主任P.Leder博士在这方面曾做过大量的工作。 Robert Nicholas(议会科学技术委员会总顾问)谈了国会这方面的观点。他说,人们对生物技术的兴趣越来越大。他还说,为了对“基因治疗”中涉及科学、法律和道德的一些     

不同培养基对兰科药用植物手参原球茎共生真菌的分离效果

兰科植物手参Gymnadenia conopsea作为国家二级重点保护野生植物具有重要的药用价值。目前手参还未实现人工栽培,但其种子的真菌共生萌发已获成功。为明确除促萌发真菌外,还有哪些土著真菌参与了手参种子的萌发过程,本研究在自然条件下采用促萌发真菌伴播手参种子,获得了种子萌发形成的原球茎,进而对比了6种常见的培养基PDA (马铃薯葡萄糖琼脂培养基)、MMN (改良Melin-Norkrans培养基)、FIM (真菌分离培养基)、MEA (麦芽浸膏琼脂培养基)、CAM (胡萝卜葡萄糖琼脂培养基)和CMA (玉米粉琼脂培养基)对手参原球茎共生真菌分离效果的影响。共从6种培养基上分离获得了75个菌株,其中MMN、CAM、PDA、FIM、MEA、CMA培养基依次分离得到20株、16株、15株、11株、8株、5株菌。此外,真菌的多样性分析结果表明,MMN培养基的Chao 1、Shannon-Wiener和Simpson多样性指数最高,CAM和PDA培养基次之,CMA培养基最低。综上所述,真菌分离效果最好的是MMN培养基,其次是CAM和PDA培养基,而FIM和MEA培养基对真菌的分离效果影响不大,CMA培养基的分离效果最差。本研究结果可为其他兰科植物原球茎共生真菌的分离提供借鉴,所获得的菌株也有望进一步应用于功能菌剂的研发。     

“Becn1-BCL2-Klotho轴”——自噬抗衰老新机制

正细胞自噬(autophagy)是胞内物质周转的重要环节;一般指营养缺乏、缺氧等应激时,部分胞浆内物质被双层膜自噬小泡包裹、送入溶酶体降解,并循环利用以维系细胞稳态的过程。同时,根据被包裹物质运送至溶酶体腔内的方式,自噬可分为巨自噬(macroautophagy)、微自噬(microautophagy)、分子伴侣介导的自噬(chaperone-mediated autophagy,CMA);近年来,根据被自     

IgE介导牛乳蛋白过敏婴幼儿肠道菌群改变初探

目的 应用高通量测序技术探讨IgE介导的牛乳蛋白过敏(cow′s milk protein allergy, CMA)婴幼儿肠道菌群结构和组成的变化。方法 回顾性分析2012年6月-2013年6月入组食物过敏患儿,选择出IgE介导的CMA(IgE-CMA)患儿4例,非IgE介导的CMA(non-IgE-CMA)患儿4例,采集粪便样本进行454焦磷酸测序分析,血清样本进行细胞因子检测。结果 与non-IgE-CMA患儿相比较,IgE-CMA患儿肠道菌群多样性Shannon指数(t=-2.996,P=0.010)和Simpson指数(t=-1.786,P=0.036)显著改变,菌群多样性下降,菌群丰富度指数ACE(t=0.406,P=0.021)显著升高。β-多样性PCA分析可显著区分两组,提示IgE-CMA患儿肠道菌群发生显著改变。LEfSe组成差异分析发现肠道菌群中硬壁菌门和拟杆菌门的改变与CMA的不同表型无关,IgE-CMA婴儿中链球菌、肠球菌、鞘氨醇单胞菌、双歧杆菌、罗思氏菌属、红球菌和梭菌属第Ⅺ簇等减少,而厌氧杆菌和狭义梭菌属增加,可用于鉴别诊断CMA表型。结论 IgE可决定CM...     

原子力显微技术在细胞生物学中的应用

对近年来原子力显微技术(AFM)在细胞生物学中的应用大致归纳为几个方面进行了简单介绍,还指出了细胞表面结构难于识别、细胞内部结构难以原位观察等AFM应用于细胞生物学中的难题,并提出了“形状探针”的概念以及超薄切片的思路以解决这些难题。AFM在细胞生物学中的应用研究还远远不足,需要更多的科学工作者加入其中。     

Role of chaperone-mediated autophagy in degrading Huntington＇s disease-associated huntingtin protein

Mutant N-terminal huntingtin （Htt） protein resulting from Huntington＇s disease （HD） with expanded polyglutamine accumulates and forms aggregates in vulnerable neurons. Both ubiquitin proteasomai and autophagic pathways con- tribute to the degradation of mutant Htt. Here, we focus on the involvement of chaperone-mediated autophagy （CMA）, a selective form of autophagy in the clearance of Htt. Selective catabolism in CMA is conferred by the presence of a KFERQ-Iike targeting motif in the substrates, by which molecular chaperones recognize the hydrophobic surfaces of the misfolded substrates, and transfer them to the lysosomal membrane protein type-2A, LAMP-2A. The substrates are taken into the lysosomes through LAMP-2A and are rapidly degraded by the lysosomal enzymes. Taken together, we summarize the recent evidence to elucidate that Htt is also a potential substrate of CMA. We propose that the manipulation of CMA could be a therapeutic strat- egy for HD.     

Chaperone-mediated autophagy prevents cellular transformation by regulating MYC proteasomal degradation

Chaperone-mediated autophagy (CMA), a selective form of protein lysosomal degradation, is maximally activated in stress situations to ensure maintenance of cellular homeostasis. CMA activity decreases with age and in several human chronic disorders, but in contrast, in most cancer cells, CMA is upregulated and required for tumor growth. However, the role of CMA in malignant transformation remains unknown. In this study, we demonstrate that CMA inhibition in fibroblasts augments the efficiency of MYC/c-Myc-driven cellular transformation. CMA blockage contributes to the increase of total and nuclear MYC, leading to enhancement of cell proliferation and colony formation. Impaired CMA functionality accentuates tumorigenesis-related metabolic changes observed upon MYC-transformation. Although not a direct CMA substrate, we have found that CMA regulates cellular MYC levels by controlling its proteasomal degradation. CMA promotes MYC ubiquitination and degradation by regulating the degradation of C330027C09Rik/KIAA1524/CIP2A (referred to hereafter as CIP2A), responsible for MYC stabilization. Ubiquitination and proteasomal degradation of MYC requires dephosphorylation at Ser62, and CIP2A inhibits the phosphatase responsible for this dephosphorylation. Failure to degrade CIP2A upon CMA blockage leads to increased levels of phosphorylated MYC (Ser62) and to stabilization of this oncogene. We demonstrate that this phosphorylation is essential for the CMA-mediated effect, since specific mutation of this site (Ser62 to Ala62) is enough to normalize MYC levels in CMA-incompetent cells. Altogether these data demonstrate that CMA mitigates MYC oncogenic activity by promoting its proteasomal degradation and reveal a novel tumor suppressive role for CMA in nontumorigenic cells.     

Effects of Small Molecules on Chaperone-Mediated Autophagy

Autophagy, including macroautophagy (MA), chaperone-mediated autophagy (CMA), crinophagy, pexophagy and microautophagy, are processes by which cells select internal components such as proteins, secretory vesicles, organelles, or foreign bodies, and deliver them to lysosomes for degradation. MA and CMA are activated during conditions of serum withdrawal in cell culture and during short-term (MA) and prolonged (CMA) starvation in organisms. Although MA and CMA are activated under similar conditions, they are regulated by different mechanisms. We used pulse/chase analysis under conditions in which most intracellular proteolysis is due to CMA to test a variety of compounds for effects on CMA. We show that inhibitors of MA such as 3-methyladenine, wortmannin, and LY294002 have no effect on CMA. Protein degradation by MA is sensitive to microtubule inhibitors such as colcemide and vinblastine, but protein degradation by CMA is not. Activators of MA such as rapamycin also have no effect on CMA. We demonstrate that CMA, like MA, is inhibited by protein synthesis inhibitors anisomycin and cycloheximide. CMA is also partially inhibited when the P38 mitogen activated protein kinase is blocked. Finally we demonstrate that the glucose-6-phophate dehydrogenase inhibitor, 6-aminonicotinamide, and heat shock protein of 90 kilodaltons inhibitor, geldanamycin, have the ability to activate CMA.     

Chaperone-mediated autophagy in health and disease

Chaperone-mediated autophagy (CMA) is a lysosomal pathway that participates in the degradation of cytosolic proteins. CMA is activated by starvation and in response to stressors that result in protein damage. The selectivity intrinsic to CMA allows for removal of damaged proteins without disturbing nearby functional ones. CMA works in a coordinated manner with other autophagic pathways, which can compensate for each other. Interest in CMA has recently grown because of the connections established between this autophagic pathway and human pathologies. Here we review the unique properties of CMA compared to other autophagic pathways and its relevance in health and disease.     

Effects of small molecules on chaperone-mediated autophagy

Autophagy, including macroautophagy (MA), chaperone-mediated autophagy (CMA), crinophagy, pexophagy and microautophagy, are processes by which cells select internal components such as proteins, secretory vesicles, organelles, or foreign bodies, and deliver them to lysosomes for degradation. MA and CMA are activated during conditions of serum withdrawal in cell culture and during short-term and prolonged starvation in organisms, respectively. Although MA and CMA are activated under similar conditions, they are regulated by different mechanisms. We used pulse/chase analysis under conditions in which most intracellular proteolysis is due to CMA to test a variety of compounds for effects on this process. We show that inhibitors of MA such as 3-methyladenine, wortmannin, and LY294002 have no effect on CMA. Protein degradation by MA is sensitive to microtubule inhibitors such as colcemide and vinblastine, but protein degradation by CMA is not. Activators of MA such as rapamycin also have no effect on CMA. We demonstrate that CMA, like MA, is inhibited by protein synthesis inhibitors anisomycin and cycloheximide. CMA is also partially inhibited when the p38 mitogen activated protein kinase is blocked. Finally we demonstrate that the glucose-6-phophate dehydrogenase inhibitor, 6-aminonicotinamide, and heat shock protein of 90 kilodaltons inhibitor, geldanamycin, have the ability to activate CMA.     

Establishment of a novel fluorescence-based method to evaluate chaperone-mediated autophagy in a single neuron

Background

Chaperone-mediated autophagy (CMA) is a selective autophagy-lysosome protein degradation pathway. The role of CMA in normal neuronal functions and in neural disease pathogenesis remains unclear, in part because there is no available method to monitor CMA activity at the single-cell level.

Methodology/Principal Findings

We sought to establish a single-cell monitoring method by visualizing translocation of CMA substrates from the cytosol to lysosomes using the HaloTag (HT) system. GAPDH, a CMA substrate, was fused to HT (GAPDH-HT); this protein accumulated in the lysosomes of HeLa cells and cultured cerebellar Purkinje cells (PCs) after labeling with fluorescent dye-conjugated HT ligand. Lysosomal accumulation was enhanced by treatments that activate CMA and prevented by siRNA-mediated knockdown of LAMP2A, a lysosomal receptor for CMA, and by treatments that inactivate CMA. These results suggest that lysosomal accumulation of GAPDH-HT reflects CMA activity. Using this method, we revealed that mutant γPKC, which causes spinocerebellar ataxia type 14, decreased CMA activity in cultured PCs.

Conclusion/Significance

In the present study, we established a novel fluorescent-based method to evaluate CMA activity in a single neuron. This novel method should be useful and valuable for evaluating the role of CMA in various neuronal functions and neural disease pathogenesis.     

Metformin activates chaperone-mediated autophagy and improves disease pathologies in an Alzheimer disease mouse model

Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative dis-eases.However,the mechanisms that regulate CMA are not fully understood.Here,using unbiased drug screening approaches,we discover Metformin,a drug that is commonly the first medication prescribed for type 2 diabetes,can induce CMA.We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/β signaling that leads to phos-phorylation of Ser85 of the key mediator of CMA,Hsc70,and its activation.Notably,we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/β-dependent manner.The inhibition of CMA-mediated degradation of APP enhan-ces its cytotoxicity.Importantly,we find that in the APP/PS1 mouse model of Alzheimer's disease (AD),activation of CMA by Hsc70 overexpression or Met-formin potently reduces the accumulated brain Aβ pla-que levels and reverses the molecular and behavioral AD phenotypes.Our study elucidates a novel mecha-nism of CMA regulation via Metformin-TAK1-IKKα/β-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases,such as AD,where such therapeutic intervention could be beneficial.     

Pathophysiology of chaperone-mediated autophagy

In contrast to the classically described “in bulk” lysosomal degradation, the first evidence for selective degradation of cytosolic proteins in lysosomes was presented more than 20 years ago. Throughout this time, we have gained a better understanding about this process, now known as chaperone-mediated autophagy (CMA). The identification of new substrates for CMA and novel components, in both the cytosol and the lysosomes, along with better insights on how CMA is regulated, have all helped to shape the possible physiological roles of CMA. We review here different intracellular functions of CMA that arise from its unique characteristics when compared to other forms of autophagy. In view of these functions, we discuss the relevance of the changes in CMA activity in aging and in different pathological conditions.     

Identification of N(omega)-carboxymethylarginine, a new advanced glycation endproduct in serum proteins of diabetic patients: possibility of a new marker of aging and diabetes

A new advanced glycation end product (AGE), N(omega)-carboxymethyl-arginine (CMA), was found in acid-soluble skin collagen of a newborn bovine prepared by in vitro glycation with 1 M glucose incubation at 37 degrees C for about 30 days [ 1 ]. CMA production was increased with incubation time in parallel, and after 30 days incubation the yield was 100 times higher than that of pentosidine [ 1 ]. This result suggested the importance of CMA as a major AGE in collagen. We have detected and measured the CMA level in human serum proteins by electrospray ionization/liquid chromatography/mass spectrometry (ESI/LC/MS), using CMA standard concentration curve. In this report, we first show the existence of CMA in vivo, and its serum level is significantly elevated in diabetic serum proteins, compared to age-matched control serum proteins. These results provide strong evidence that CMA is a new diagnostic marker of glycation in diabetes.     

Cloning and Expression of Genes Required for Coronamic Acid (2-Ethyl-1-Aminocyclopropane 1-Carboxylic Acid), an Intermediate in the Biosynthesis of the Phytotoxin Coronatine

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. Tn5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two characterized intermediates in the COR biosynthetic pathway. In the present study, additional Tn5 insertions were generated to more precisely define the region required for CMA biosynthesis. A new analytical method for CMA detection which involves derivatization with phenylisothiocyanate and detection by high-performance liquid chromatography (HPLC) was developed. This method was used to analyze and quantify the production of CMA by selected derivatives of P. syringae pv. glycinea which contained mutagenized or cloned regions from the CMA biosynthetic region. pMU2, a clone containing a 6.45-kb insert from the CMA region, genetically complemented mutants which required CMA for COR production. When pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produced, indicating that pMU2 does not contain the complete CMA biosynthetic gene cluster. However, when two plasmid constructs designated pMU234 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPLC. The biological activity of the CMA produced by P. syringae pv. glycinea 18a/90 derivatives was demonstrated by the production of COR in cosynthesis experiments in which 18a/90 transconjugants were cocultivated with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae B1; however, these two constructs did not enable Escherichia coli K-12 to synthesize CMA. The production of CMA in P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic acid biosynthesis and raises interesting questions regarding the evolutionary origin of the COR biosynthetic pathway.