Poly(A)-specific ribonuclease protein promotes the proliferation,invasion and migration of esophageal cancer cells

BACKGROUND Bioinformatics analysis showed that the expression of the poly(A)-specific ribonuclease(PARN)gene in gastric cancer,head and neck squamous cell carcinoma,melanoma,cervical cancer and lung squamous cell carcinoma tissues was significantly higher than that in normal tissues and was associated with high stage and poor prognosis.The expression of the PARN gene in esophageal cancer(EC)tissue is also significantly higher than that in normal tissues,but the effect of PARN on the proliferation,migration and invasion of EC cells remains unclear.AIM To investigate the relationship between PARN and the proliferation,migration and invasion of EC cells.METHODS The EC tissues of 91 patients after EC surgery and 63 paired precancerous healthy tissues were collected.PARN mRNA levels were measured using a tissue microarray,and the PARN expression level was evaluated using immunohistochemistry to analyze the relationship between PARN expression and clinicopathologic features as well as the survival and prognosis of patients.In addition,the effects of PARN gene knockout on tumor cell proliferation,invasion and migration were studied by using shRNA during the in vitro culture of EC cell lines Eca-109 and TE-1,and the effects of the PARN gene on tumor growth in vivo were verified by a xenotransplantation nude mice model.RESULTS The expression of PARN in EC tissues was higher than that in adjacent normal tissues,and the level of PARN expression was significantly positively correlated with lymphatic metastasis.Patients with high PARN levels had poor overall survival.BIM,IGFBP-5 and p21 levels were significantly increased in the PARN knockout group,while the expression levels of the antiapoptotic proteins Survivin and sTNF-R1 were significantly decreased in the apoptotic antibody array data.In addition,the expression levels of Akt,p-Akt,PIK3CA and CCND1 in the downstream signaling pathway regulating EC progression were significantly decreased.The culture of EC cell lines confirmed that the apoptosis rate of EC cells was significantly increased,the growth and proliferation of tumor cells were significantly inhibited,and the invasion and migration ability of tumor cells were significantly decreased after PARN gene knockout.In vivo experiments of BALB/c nude mice transfected with Eca-109 cells expressing control shRNA(sh-NC)and PARN shRNA(sh-PARN)showed that the tumor volume and weight of nude mice treated with sh-PARN were significantly decreased compared with those of nude mice treated with sh-NC,indicating that PARN knockdown significantly inhibited tumor growth in vivo.CONCLUSION PARN has antiapoptotic effects on EC cells and promotes their proliferation,invasion and migration,which is associated with the development of EC and poor patient prognosis.PARN may become a potential target for the diagnosis,prognosis prediction and treatment of EC.

Human ribonuclease 9,a member of ribonuclease A superfamily,specifically expressed in epididymis,is a novel sperm-binding protein

To explore the functions of human ribonuclease 9(RNase 9),we constructed a mammalian fusion expression vector pcDNA-hRNase9,prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences.According to the determined mature protein,recombinant human RNase 9 was prepared in E.coli.Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detected,and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated spermatozoa were analyzed via indirect immunofluorescence assay.The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA,but exhibited antibacterial activity,in a concentration/time dependent manner,against E.coli.Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis,but not present in other tissues examined,and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermatozoa.These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.

Arg462Gln and Asp541Glu polymorphisms in ribonuclease L and prostate cancer risk:a meta-analysis

Objective：The association between ribonuclease L（RNASEL）gene polymorphisms and prostate cancer risk has been widely reported,but the results of these studies remained controversial and underpowered.We performed a meta-analysis of 28 studies to evaluate the association between Arg462Gln and Asp541Glu polymorphisms in the RNASEL gene and prostate cancer risk.Methods：Odds ratios（ORs）with 95%confidence intervals（CIs） were estimated to assess the association between RNASEL polymorphisms and prostate cancer risk.Results：A significantly increased prostate cancer risk was found for the Arg462Gln polymorphism in Africans（Gln/Gln vs Arg/Arg：OR=2.50,95%CI=1.28-4.87;Gln/Gln vs Gln/Arg＋Arg/Arg：OR=2.54,95%CI=1.30-4.95）,but not in Europeans and Asians.Additionally,the Asp541Glu polymorphism was associated with increased total prostate cancer risk（Glu-allele vs Asp-allele：OR=1.04,95%CI=1.01-1.07;Glu/Glu vs Asp/Asp：OR=1.22,95%CI= 1.03-1.46;Glu/Glu vs Glu/Asp＋Asp/Asp：OR=1.09,95%CI=1.02-1.16）.In the stratified analysis for the Asp541Glu polymorphism,there was a significantly increased prostate cancer risk in Africans and Europeans,and in hospital-based prostate cancer cases.Conclusion：The meta-analysis results showed evidence that RNASEL Arg462Gln and Asp541Glu polymorphisms are associated with prostate cancer risk and could be low-penetrance prostate cancer susceptibility biomarkers.

Antiviral Effect of Ribonuclease from <i>Bacillus pumilus</i>against Phytopathogenic Rna-Viruses

Background: Viruses can cause different diseases in plants. To prevent viral infections, plants are treated with chemical compounds and antiviral agents. Chemical antiviral agents usually have narrow specificity, which limits their wide application. Alternative antiviral strategy is associated with the use of microbial enzymes, which are less toxic and are readily decomposed without accumulation of harmful substances. The aim of this work is to study the effect of Bacillus pumilus ribonuclease on various phytopathogenic viruses with specific focus on the ability of enzyme to eliminate them from plant explants in vitro. Materials and methods: Extracellular ribonuclease of B. pumilus is tested as an antiviral agent. To study the antiviral effect of RNase, depending on concentration and the time of application several plant-virus model systems are used. Virus detection is conducted by serological testing and RT-PCR. Results: Bacillus pumilus ribonuclease possesses antiviral activity against plant Rna-viruses RCMV (red clover mottle virus), PVX (Potato Virus X) and AMV (Alfalfa Mosaic Virus). The maximum inhibitory effect against actively replicating viruses is observed when plants are treated with the enzyme in the concentration of 100 ug/ml prior to infection. In case of local necrosis ribonuclease in the concentration of 1 ug/ml completely inhibits the development of RCMV virus on bean plants. The enzyme is able to penetrate plants and inhibit the development of viral infection, inhibiting effect for untreated surfaces decreased on average for 20%. It is also found that B. pumilus ribonuclease protects apical explants of sprouts of potato tubers from PVM and PVS viruses. Conclusion: B. pumilus ribonuclease possesses antiviral activity against plant Rna-viruses and produces viruses-free plants in the apical meristem culture.

Biological implications of 2-chlorocyclohexa-2,5-diene-1,4-dione toward ribonuclease A

2-Chlorocyclohexa-2,5-diene-1,4-dione (CBQ) or 2-chloro1,4-benzquinone is one of the common metabolites of polycyclic aromatic hydrocarbons generated through industrial processes. This report describes the biological effects of CBQ toward ribonuclease A (RNase). We also investigated the inhibition of RNase modifications and the reactivity of CBQ toward selected amino acids. The study was carried out by incubating RNase or amino acids with CBQ in a concentration- and a time-dependent manner at 37&degC and pH 7.0. SDS-PAGE results showed oligomerization as well as polymeric aggregation of RNase when incubated with CBQ as early as in 10 min. CBQ-induced RNase modifications were inhibited in the presence of NADH or ascorbic acid. CBQ reactivity toward selected amino acids was also evaluated by determining the second-order rate constants for the reactions of CBQ with selected amino acids. It was found that the reactivity toward CBQ decreased in the order of lysine > threonine > serine >> aspartate > cysteine.

木薯花叶病毒AC4蛋白与AtPARN互作研究

木薯(Manihot esculenta Crantz)作为重要的热带作物,是全球六大粮食作物之一,并为全球近七亿人口提供主粮,然而病毒侵染引起的病害在全球范围内严重威胁木薯产业的发展。木薯花叶病毒病(cassava mosaic disease,CMD)是最有威胁的病害之一,造成严重的经济损失。斯里兰卡木薯花叶病毒(SriLankancassavamosaicvirus,SLCMV)是引发木薯花叶病的病原物之一,SLCMV是典型的双组分双生病毒,其基因组由DNA-A和DNA-B两个环状组分组成。无义介导的mRNA降解(nonsense-mediatedmRNAdecay,NMD)是真核细胞mRNA降解的主要途径之一,也是真核生物普遍存在的抗病毒防御机制。越来越多的研究显示,NMD不仅是真核生物重要的mRNA数量、质量调控机制,还与转录后基因沉默(post transcriptional gene silencing,PTGS)同样能降解病毒RNA,是真核生物普遍存在的抗病毒防御机制。NMD的mRNA衰减过程包括PTC的识别、脱腺苷酸、脱帽和最后的核酸外切酶降解4个过程,UPF1、PARN、DCP2和XRN4分别是上述4个过程中的关键蛋白。病毒是专性寄生生物,必须逃避或耐受寄主细胞的降解,才能成功感染。聚腺苷酸特异性核糖核酸酶[poly(A)-specific ribonuclease,PARN]是NMD信号通路的一个关键因子。目前,关于SLCMV抵御寄主NMD的分子机制尚不明确。本研究采用酵母双杂交(yeast two-hybrid system)和荧光双分子互补(bimolecular fluorescence complementation,BiFC)试验证明斯里兰卡木薯花叶病毒(SLCMV)编码的沉默抑制子AC4与拟南芥PARN相互作用。据此推测SLCMV AC4蛋白可能通过与AtPARN相互作用而抑制寄主NMD的病毒防御功能帮助病毒逃避或耐受寄主细胞的降解。研究结果为阐明木薯花叶病毒调控寄主NMD抗病毒防御功能的分子机理奠定基础。

Regnase-1, a rapid response ribonuclease regulating nflammation and stress responses

RNA-binding proteins （RBPs） are central players in post-transcriptional regulation and immune homeostasis. The ribonuclease and RBP Regnase-1 exerts critical roles in both immune cells and non-immune cells. Its expression is rapidly induced under diverse conditions including microbial infections, treatment with inflammatory cytokines and chemical or mechanical stimulation. Regnase-1 activation is transient and is subject to negative feedback mechanisms including proteasome-mediated degradation or mucosa-associated lymphoid tissue 1 （MALT1） mediated cleavage. The major function of Regnase-1 is promoting mRNA decay via its ribonuclease activity by specifically targeting a subset of genes in different cell types. In monocytes, Regnase-1 downregulates IL-6 and IL-12B mRNAs, thus mitigating inflammation, whereas in T cells, it restricts T-cell activation by targeting c-Rel, Ox40 and 11-2 transcripts. In cancer cells, Regnase-1 promotes apoptosis by inhibiting anti-apoptotic genes including Bcl2L1, Bcl2A1, RelB and Bcl3. Together with up-frameshift protein-1 （UPF1）, Regnase-1 specifically cleaves mRNAs that are active during translation by recognizing a stem-loop （SL） structure within the 3＇UTRs of these genes in endoplasmic reticulum-bound ribosomes. Through this mechanism, Regnase-1 rapidly shapes mRNA profiles and associated protein expression, restricts inflammation and maintains immune homeostasis. Dysregulation of Regnase-1 has been described in a multitude of pathological states including autoimmune diseases, cancer and cardiovascular diseases. Here, we provide a comprehensive update on the function, regulation and molecular mechanisms of Regnase-1, and we propose that Regnase-1 may function as a master rapid response gene for cellular adaption triggered by microenvironmental changes.

Fg12 ribonuclease secretion contributes to Fusarium graminearum virulence and induces plant cell death

Filamentous fungal pathogens secrete effectors that modulate host immunity and facilitate infection. Fusarium graminearum is an important plant pathogen responsible for various devastating diseases. However, little is known about the function of effector proteins secreted by F. graminearum. Herein, we identified several effector candidates in the F. graminearum secretome. Among them, the secreted ribonuclease Fg12 was highly upregulated during the early stages of F. graminearum infection in soybean;its deletion compromised the virulence of F. graminearum. Transient expression of Fg12 in Nicotiana benthamiana induced cell death in a light-dependent manner. Fg12 possessed ribonuclease(RNase) activity, degrading total RNA. The enzymatic activity of Fg12 was required for its cell death-promoting effects. Importantly, the ability of Fg12 to induce cell death was independent of BAK1/SOBIR1, and treatment of soybean with recombinant Fg12 protein induced resistance to various pathogens, including F. graminearum and Phytophthora sojae. Overall, our results provide evidence that RNase effectors not only contribute to pathogen virulence but also induce plant cell death.

Expression of self-incompatibility ribonucleases of Antirrhinum in Escherichia coli

Self-incompatibility is an intraspecific reproductive barrier to prevent self-fertilization inthe flowering plants. In many species, self-incompatibility is controlled by a single S locus with multiple alleles. So far, the only gene known in the S locus of the Solanaceae, Scrophulariaceae and Rosaceae encodes a class of ribonucleases, called self-incompatibility ribonucleases (S RNases), which have been shown to mediate stylar expression of self-incompatible reaction. As the first step to investigate their three-dimensional structure, we successfully expressed three biologically active S RNases of Antirrihnum (S2, S4 and S5) in Escherichia coli (E. coli). Their functional expressions caused no detrimental effect on host bacteria growth and provided a basis for a large scale preparation of S RNase proteins. Possible reasons for non-lethality of S RNases on E. coliare discussed.

Adaptive evolutionary expansion of the ribonuclease 6 in Rodentia

Ribonuclease 6(RNase6 or RNase K6)is a protein that belongs to a superfamily thought to be the sole verte­brate-specific enzyme known for a wide range of physiological functions,including digestion,cytotoxicity,an­giogenesis,male reproduction and host defense.In our study,51 functional genes and 11 pseudogenes were identified from 27 Rodentia species.Intriguingly,in the 3 main lineages of rodents there were multiple RNas­e6s identified in all species of Ctenohystrica,whereas only a single RNase6 was observed in other Rodentia spe­cies examined except for 2 species in the mouse-related clade.The evolutionary scenario of“birth(gene dupli­cation)and death(gene deactivation)”and gene sorting have been demonstrated in Ctenohystrica.In addition,bursts of positive selection,diversification of isoelectric point and positive net charge have been identified in Ctenohystrica,especially at two key sites that are involved in antimicrobial function.Site Trp30 has undergone positive selection and Ile45 has changed into other residues in Group B and Group C of the Ctenohystrica.Our results demonstrated a complex and intriguing evolutionary pattern of rodent RNase6,and indicated that func­tional modification may have occurred,which establishes an important theoretical foundation for future func­tional assays in rodent RNase6.

Structural insights into the CRISPR-Cas-associated ribonuclease activity of Staphylococcus epidermidis Csm3 and Csm6

The Clustered Regularly Interspaced Short _Palindromic Repeats （CRISPR）-CRISPR-associated （Cas） system is an adaptive immune system in bacteria and archaea that resists exogenous invasion through nucleic acid-mediated cleavage. In the type III-A system, the Csm complex contains five effectors and a CRISPR RNA, which edits both single stranded RNA and double stranded DNA. It has recently been demonstrated that cyclic oligoadenylates （cOAs）, which are synthesized by the Csm complex, act as second messengers that bind and activate Csm6. Here, we report the crystal structures of Staphylococcus epiderrnidis Csm3 （SeCsm3） and an N-terminally truncated Csm6 （SeCsm6AN） at 2.26 and 2.0 A, respectively. The structure of SeCsm3 highly resembled previously reported Csm3 structures from other species; however, it provided novel observations allowing further enzyme characterization. The homodimeric SeCsm6AN folds into a compact structure. The dimerization of the HEPN domain leads to the formation of the ribonuclease active site, which is consistent with the reported Csm6 structures. Altogether, our studies provide a struc- tural view of the ribonuclease activity mediated by Csm3 and Csm6 of the type III-A CRISPR-Cas system.

Fourier Transformed Infrared Spectra of the Unfolding of Ribonuclease A in Guanidinium Chloride

The unfolding of proteins during denaturation by guanidine or urea has been extensively studied. However, the methods hitherto employed usually provide only a limited amount of information on gross changes of such molecular properties as shape, size, or the exposure of buried aromatic residues. CD studies are hampered by high absorption of the denaturants commonly employed in the far ultraviolet region. Recent development in Fourier

Thermal Transition of Ribonuclease A Observed Using Proton Nuclear Magnetic Resonance

The thermal transition of bovine pancreatic ribonuclease A (RNase A) was investigated using proton nuclear magnetic resonance (NMR). Significant resonance overlap in the large native protein limits accurate assignments in the 1H NMR spectrum. This study proposes extending the investigation of large proteins by dynamic analysis. Comparison of the traditional method and the correlation coefficient method suggests successful application of spectrum image analysis in dynamic protein studies by NMR.

Anti-oxidative effect of ribonuclease inhibitor by site-directed mutagenesis and expression in Pichia pastoris

Human placental ribonuclease inhibitor(hRI)is an acidic protein of Mr∼50kDa with unusually high contents of leucine and cysteine residues.It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease.hRI has 32 cysteine residues,and the oxidative formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates hRI.The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence.In the present aork,two molecules of alanine substituting for Cys328 and Cys329 were performed by site-directed mutagenesis.The site-mutated RI cDNA was constructed into plasmid pPIC9K and then transformed Pichia pastoris GS115 by electroporation.After colony screening,the bacterium was cultured and the product was purified with affinity chromatography.The affinity of the recombinant human RI with double site mutation was examined for RNase A and its anti-oxidative effect.Results indicated that there were not many changes in the affinity for RNase A detected when compared with the wild type of RI.But the capacity of anti-oxidative effect increased by 7~9 times.The enhancement in anti-oxidative effect might be attributed to preventing the formation of disulfide bond between Cys328 and Cys329 and the three dimensional structure of RI was thereby maintained.

核糖核酸酶A家族典型成员在肿瘤发生中的作用

人体中核糖核酸酶A(ribonuclease A,RNaseA)家族包含8个典型成员(RNase 1至RNase 8)。已有研究显示,除RNase 8外,该家族其它典型成员影响了胰腺癌、结直肠癌、膀胱癌、乳腺癌和皮肤癌等多种肿瘤的发生发展。在肿瘤发生过程中,特定RNase表达量及糖基化修饰会发生显著改变,是肿瘤诊断的潜在标志物;它们能以多种机制参与肿瘤发生、生长和转移等过程,有望成为肿瘤治疗的靶点;而部分成员则具有杀伤肿瘤细胞、抑制肿瘤发展的功能,存在临床开发成肿瘤治疗药物的可能。具体而言,RNase 1通过核糖核酸酶活性依赖的细胞毒性和细胞外RNA降解功能,发挥直接杀伤肿瘤细胞或降低局部炎症而抑制肿瘤生长的作用;RNase 1还能结合并激活促红细胞生成素,产生肝细胞癌受体相互作用蛋白A4 (erythropoietin-producing hepatocellular carcinoma receptor-interacting protein A4, EphA4)信号通路,促进乳腺癌的发生。RNase 2和RNase 3是嗜酸性粒细胞颗粒蛋白质的重要成分,依赖于阳离子性及核糖核酸酶活性在抗肿瘤免疫防御中发挥重要作用。RNase 4和RNase 5则通过诱导血管生成、加快肿瘤细胞增殖和抑制肿瘤细胞凋亡等方式,促进肿瘤发生发展。其中RNase 5发挥作用的分子机制包括促进47 S前体rRNA转录和激活促肿瘤生长的信号通路,以及产生tRNA衍生的应激诱导的RNA(tRNA-derived stress-induced RNA,tiRNA)等。虽然RNase 6和RNase 7与肿瘤的发生存在相关性,但其具体作用及机制的研究仍较少。本文总结了RNase A家族典型成员与肿瘤的相关性和作用机制,并对其临床应用前景进行了展望,以期为肿瘤治疗药物的开发提供新思路。

核糖核酸酶A超家族:不仅仅是一组降解RNA的酶

核糖核酸酶A超家族(ribonuclease A superfamily;RNase A superfamily),也称脊椎动物分泌型核糖核酸酶超家族(vertebrate secreted ribonucleases superfamily),是二十世纪蛋白质结构、酶学和分子进化领域研究最多最广泛的核糖核酸酶家族。自上世纪初期从牛胰腺中分离鉴定第一个成员以来,已从哺乳动物、两栖动物、爬行动物、鸟和鱼等几百种动物中鉴定了几千个成员。早期对该家族成员的研究不仅促进了蛋白质化学技术的发展,而且为现代生物学研究奠定了基础。目前已知人的核糖核酸酶A超家族成员包括8个典型成员(RNase 1~RNase 8)和5个非典型成员(RNase 9~RNase 13)。功能方面,曾一度以为该家族成员只具有降解核糖核酸的能力。随着血管生成素(angiogenin;RNase 5)、嗜酸性粒细胞衍生神经毒素(eosinophils-derived neurotoxin, EDN;RNase 2)、嗜酸性粒细胞阳离子蛋白(eosinophils cationic protein, ECP;RNase 3)的发现,人们意识到该家族成员除了消化核糖核酸外,还有依赖酶活性和不依赖酶活性的其他功能,包括宿主防御、免疫调节、血管生成和肿瘤抑制等,但仍了解不够全面。本文回顾了核糖核酸酶A超家族的研究历程,探讨了未来研究方向,特别呼吁要系统研究其除降解核糖核酸外的其他生理病理功能,希望能为该领域的研究提供思路。

人核糖核酸酶A家族生物学功能的结构基础

人核糖核酸酶A(ribonuclease A, RNaseA)家族成员有13个,分别为RNase1-RNase13,它们具有很高的序列相似性,大多含有6~8个半胱氨酸并形成分子内二硫键,以维持特有的空间结构。其中,RNase1-RNase8具有多种生物活性,可概括为3类:涉及核糖核酸转录后的剪切、修饰和降解;具有抗细菌、抗真菌和抗病毒活性;以及机体免疫调节作用。而RNase9-RNase13不具有核糖核酸酶活性。因此,本文将重点对RNaseA家族成员RNase1-RNase8的结构与功能研究进行综述,重点概述决定RNaseA生物学功能的结构特征,以期指导以RNaseA为基础的抗微生物药物开发及RNaseA在机体免疫中的功能研究。

核糖核酸酶A超家族抗菌活性评价

抗菌肽是机体重要的免疫防御分子,具有广谱杀菌活性。核糖核酸酶A是脊椎动物特异性的分泌型蛋白质,在人基因组中包含8个经典成员(RNase1-8)。它们作为一类重要的抗菌肽,广泛分布于机体需要抵抗外界病原微生物的组织中,除了特有的生物学功能外,均具有一定的抗菌活性。然而,目前各实验室对它们抗菌活性的报道并不一致,有必要开展横向比较分析。为此,我们表达纯化了人核糖核酸酶A超家族8个成员的重组蛋白质,并在同一实验条件下以半致死浓度评估了它们对革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(金黄色葡萄球菌)的抗菌活性。结果显示,RNase1-8重组蛋白质对大肠杆菌半数致死浓度分别为:0.081,0.046,0.008,0.250,2.028,0.072,0.001μmol·L~(-1)和1.1416μmol·L~(-1);对金黄色葡萄球菌半数致死浓度分别为:3.427,1.856,2.211,5.188,8.274,4.356,2.502μmol·L~(-1)和9.916μmol·L~(-1)。该结果提示,RNase1-8对大肠杆菌的抗菌活性存在明显差异,其中RNase3和RNase7活性最强,且均显著高于各自对金黄色葡萄球菌的抗菌活性;8个成员对金黄色葡萄球菌的抗菌活性无明显差异。据此我们认为,核糖核酸酶A超家族对革兰氏阴性菌具有较好的杀伤活性,可用于革兰氏阴性菌抗菌产品的开发。

粟酒裂殖酵母N-糖酰胺酶在大肠杆菌中的表达、纯化及活性分析

根据GenBank中公布的粟酒裂殖酵母(Schizosaccharomyces pombe)N-糖酰胺酶(Pnglp)cDNA序列,设计并合成一对特异性引物,利用RT-PCR技术从粟酒裂殖酵母中克隆出糖酰胺酶cDNA。将得到的基因克隆到表达载体pET-15b中。重组质粒转入大肠杆菌BL21(DE3)中,经诱导表达和纯化提取后,进行酶活测定。实验结果表明,该酶的分子量约为39 kD,纯化后的重组N-糖酰胺酶可以对变性处理的糖蛋白进行糖链的切除,且这种作用需要还原剂DTT的辅助作用;N-糖酰胺酶只对错误折叠的糖蛋白有作用,对天然的糖蛋白没有作用。等量粟酒裂殖酵母Png1p在不同温度、pH、DTT浓度和底物变性温度下对等量核糖核酸酶B(RNase B)的脱糖基化检测发现,重组酶的最适反应温度30℃,最适反应pH为7.0,需要的最适DTT浓度为10 mmol/L,底物在100℃处理10 min时酶的脱糖基化率最高。

转染人核糖核酸酶抑制因子基因对B16黑色瘤细胞及肿瘤转移的影响(英文)

人核糖核酸酶抑制因子 (humanribonucleaseinhibitor ,RI)是一种细胞质中分子质量为 5 0ku的酸性糖蛋白 .RI能抑制核糖核酸酶A (RNaseA)的活性 ,RNaseA与血管生成因子 (angiogenin ,Ang)的氨基酸有着高度保守的同源序列 .Ang是RNaseA超家族的一员 ,RI通过与RNaseA和Ang的紧密结合而抑制其活性 .血管生成及新血管的形成 ,是肿瘤发生和转移的必要条件 .所以抗血管生成将是一种很有希望的对抑制肿瘤生长和转移的有效方法 .实验显示RI能有效地抑制肿瘤诱导血管的生成 .RI由含有许多亮氨酸重复序列的多肽组成 .含有这样重复序列的 10 0多种蛋白质显示了广泛的功能 ,包括细胞周期调节 ,DNA修复 ,对细胞外基质相互作用以及抑制酶活性等 .RI被认为是胚胎发育 ,创伤愈合及肿瘤发生中新血管形成的一种调节因子 .RI定位于染色体的 11p15 5 ,与ras基因邻近 ,在肿瘤病人中经常存在染色体 11p15 5部位的变异和异常 .RI可能与细胞的生长和分化有关 ,因此 ,RI可能还具有尚未知的生物学作用 .为了进一步了解RI的潜在功能以及探讨RI与肿瘤侵润、转移的关系 ,将人的核糖核酸酶抑制因子基因的cDNA通过逆转录包装细胞PA317,并转染到B16小鼠黑色瘤细胞中 ,用转染空载体和未转染的B16细胞作为对照 .通过PCR ,RT PCR 。