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...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.展开更多
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 ...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°C 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.展开更多
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 carc...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.展开更多
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 assignment...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.展开更多
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-medi...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.展开更多
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 m...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.展开更多
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 mol...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展开更多
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, whic...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.展开更多
Balancing the risks and benefits of organophosphate pesticides(OPs)on human and environmental health relies partly on their accurate measurement.A highly sensitive fluorescence anti-quenching multi-residue bio-barcode...Balancing the risks and benefits of organophosphate pesticides(OPs)on human and environmental health relies partly on their accurate measurement.A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs(triazophos,parathion,and chlorpyrifos)in apples,turnips,cabbages,and rice.Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs.DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification.The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore.The resulting fluorescence signal enables multiplexed quantification of triazophos,parathion,and chlorpyrifos residues over the concentration range of 0.01-25,0.01-50,and 0.1-50 ng/mL with limits of detection of 0.014,0.011,and 0.126 ng/mL,respectively.The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%,which correlate well with results obtained by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The proposed bio-barcode immunoassay is stable,reproducible and reliable,and is able to detect low residual levels of multi-residue OPs in agricultural products.展开更多
Members of the Pathogenesis Related(PR)10 protein family have been identified in a variety of plant species and a wide range of functions ranging from defense to growth and development has been attributed to them.PR10...Members of the Pathogenesis Related(PR)10 protein family have been identified in a variety of plant species and a wide range of functions ranging from defense to growth and development has been attributed to them.PR10 protein possesses ribonuclease(RNase)activity,interacts with phytohormones,involved in hormone-mediated signalling,afforded protection against various phytopathogenic fungi,bacteria,and viruses particularly in response to biotic and abiotic stresses.The resistance mechanism of PR10 protein may include activation of defense signalling pathways through possible interacting proteins involved in mediating responses to pathogens,degradation of RNA of the invading pathogens.Moreover,several morphological changes have been shown to accompany the enhanced abiotic stress tolerance.In this review,the possible mechanism of action of PR10 protein against biotic and abiotic stress has been discussed.Furthermore,our findings also confirmed that the in vivo Nitric oxide(NO)is essential for most of environmental abiotic stresses and disease resistance against pathogen infection.The proper level of NO may be necessary and beneficial,not only in plant response to the environmental abiotic stress,but also to biotic stress.The updated information on this interesting group of proteins will be useful in future research to develop multiple stress tolerance in plants.展开更多
Early stage expression of PR10 combined with phytoalexins contributed to Verticillium wilt resistance in cotton. In order to analysis the activities of PR10 proteins during pathogens’ infection, we cloned a Verticill...Early stage expression of PR10 combined with phytoalexins contributed to Verticillium wilt resistance in cotton. In order to analysis the activities of PR10 proteins during pathogens’ infection, we cloned a Verticillium-induced PR10 (GbPR10-1) gene from cotton (Gossypium barbadense) and compared its expression patterns and domains with other PR10 proteins. Bioinformatics indicated that GbPR10-1 showed the lowest similarity with other 12 different PR10 genes in cotton (Upland and sea-island cotton). Expression profiles showed that GbPR10-1 gene instantly up-regulated after infection by V. dahliae in the sea-island cotton plants. GbPR10-1 was also induced by environmental stimulus including heat, submergence and salt, and ethylene but not by ABA and salicylic acid. The GbPR10-1 protein expressed in E. coli BL21 demonstrated that it had a low ribonuclease-like activity in vitro, and could inhibit V. dahliae hyphae growth but not its spores. Comparison analysis of GbPR10-1 (from resistant species) and GhPR10-1 (from susceptible species) responding to V. dahliae infection, only GbPR10-1 gene was strongly induced in the sea-island cotton plants (incompatible response), indicating that PR10-1 genes was linked to resistance signal. In summary, the earlier activation of GbPR10-1 gene, as the index of resistance response, would be aid to block展开更多
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 expr...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.展开更多
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...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.展开更多
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...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.展开更多
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,a...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.展开更多
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 o...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.展开更多
Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)...Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)variants of PARN-like ribonuclease domain-containing exonuclease 1(PNLDC1)have been reported to experience infertility with nonobstructive azoospermia.The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia(OAT)in a patient from a Chinese Han family.Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant(NM_173516.2,c.l42C>T,p.Gln48Ter)in PNLDC1.Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype,including microcephaly,head tapering,and globozoospermia.Consistently,peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome.Furthermore,the disomy rate of chromosomes in the patient’s spermatozoa was significantly increased compared with that of a fertile control sample.We reported an LOF variant of the PNLDC1 gene responsible for OAT.展开更多
Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression.A key endoribonuclease in Gram-negative bacteria is RNase E.To ensure an ap...Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression.A key endoribonuclease in Gram-negative bacteria is RNase E.To ensure an appropriate supply of RNase E,some bacteria,such as Escherichia coli,feedback-regulate RNase E expression via the rne 5′-untranslated region(5′UTR)in cis.However,the mechanisms involved in the control of RNase E in other bacteria largely remain unknown.Cyanobacteria rely on solar light as an energy source for photosynthesis,despite the inherent ultraviolet(UV)irradiation.In this study,we first investigated globally the changes in gene expression in the cyanobacterium Synechocystis sp.PCC ^(6)803 after a brief exposure to UV.Among the 407 responding genes 2 h after UV exposure was a prominent upregulation of rne mRNA level.Moreover,the enzymatic activity of RNase E rapidly increased as well,although the protein stability decreased.This unique response was underpinned by the increased accumulation of full-length rne mRNA caused by the stabilization of its 5′UTR and suppression of premature transcriptional termination,but not by an increased transcription rate.Mapping of RNA 3′ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5′UTR,indicating autoregulation.These observations suggest that RNase E in cyanobacteria contributes to reshaping the transcriptome during the UV stress response and that its required activity level is secured at the RNA level despite the enhanced turnover of the protein.展开更多
基金The authors would like to thank Mr Shou-Xin Zhang and other members of the Research Center,Yuhuangding Hospital(Yantai,China)for technical assistance.
文摘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.
文摘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°C 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.
文摘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.
基金the National Key Basic Research SpecialFunds of China (No.G19990 75 6 0 7) the National KeyScience and Technology Program of China (No.96 -90 0 - 0 9- 0 3) +1 种基金 THSJZ of Tsinghua UniversityP.R.China
文摘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.
基金supported by the National Natural Science Foundation of China(31570842 to W.C.)the National Young Thousand Talents Programthe Sichuan Province Thousand Talents Program in China
文摘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.
基金supported by the program of key medical department of Jiangsu Province(No.XK17 200904)
文摘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.
基金Project supported in part by the National Natural Science Foundation of China.
文摘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
文摘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.
基金supported by the Central Public Interest Scientific Institution Basal Research Fund for the Chinese Academy of Agricultural Sciences(Grant No.:Y2021PT05)National Institute of Environmental Health Science Superfund Research Program(Grant No.:P42 ES004699)+1 种基金National Academy of Sciences(Subaward No.:2000009144)Ningbo Innovation Project for Agro-Products Quality and Safety(Grant No.:2019CXGC007).
文摘Balancing the risks and benefits of organophosphate pesticides(OPs)on human and environmental health relies partly on their accurate measurement.A highly sensitive fluorescence anti-quenching multi-residue bio-barcode immunoassay was developed to detect OPs(triazophos,parathion,and chlorpyrifos)in apples,turnips,cabbages,and rice.Gold nanoparticles were functionalized with monoclonal antibodies against the tested OPs.DNA oligonucleotides were complementarily hybridized with an RNA fluorescent label for signal amplification.The detection signals were generated by DNA-RNA hybridization and ribonuclease H dissociation of the fluorophore.The resulting fluorescence signal enables multiplexed quantification of triazophos,parathion,and chlorpyrifos residues over the concentration range of 0.01-25,0.01-50,and 0.1-50 ng/mL with limits of detection of 0.014,0.011,and 0.126 ng/mL,respectively.The mean recovery ranged between 80.3% and 110.8% with relative standard deviations of 7.3%-17.6%,which correlate well with results obtained by liquid chromatography-tandem mass spectrometry(LC-MS/MS).The proposed bio-barcode immunoassay is stable,reproducible and reliable,and is able to detect low residual levels of multi-residue OPs in agricultural products.
基金The work supported by the grant Ministry of Education of the Czech Republic with co-financing from the European Union(grant“KOROLID”,CZ.02.1.01/0.0/0.0/15_003/0000336)the Czech Academy of Sciences(RVO:60077344).
文摘Members of the Pathogenesis Related(PR)10 protein family have been identified in a variety of plant species and a wide range of functions ranging from defense to growth and development has been attributed to them.PR10 protein possesses ribonuclease(RNase)activity,interacts with phytohormones,involved in hormone-mediated signalling,afforded protection against various phytopathogenic fungi,bacteria,and viruses particularly in response to biotic and abiotic stresses.The resistance mechanism of PR10 protein may include activation of defense signalling pathways through possible interacting proteins involved in mediating responses to pathogens,degradation of RNA of the invading pathogens.Moreover,several morphological changes have been shown to accompany the enhanced abiotic stress tolerance.In this review,the possible mechanism of action of PR10 protein against biotic and abiotic stress has been discussed.Furthermore,our findings also confirmed that the in vivo Nitric oxide(NO)is essential for most of environmental abiotic stresses and disease resistance against pathogen infection.The proper level of NO may be necessary and beneficial,not only in plant response to the environmental abiotic stress,but also to biotic stress.The updated information on this interesting group of proteins will be useful in future research to develop multiple stress tolerance in plants.
文摘Early stage expression of PR10 combined with phytoalexins contributed to Verticillium wilt resistance in cotton. In order to analysis the activities of PR10 proteins during pathogens’ infection, we cloned a Verticillium-induced PR10 (GbPR10-1) gene from cotton (Gossypium barbadense) and compared its expression patterns and domains with other PR10 proteins. Bioinformatics indicated that GbPR10-1 showed the lowest similarity with other 12 different PR10 genes in cotton (Upland and sea-island cotton). Expression profiles showed that GbPR10-1 gene instantly up-regulated after infection by V. dahliae in the sea-island cotton plants. GbPR10-1 was also induced by environmental stimulus including heat, submergence and salt, and ethylene but not by ABA and salicylic acid. The GbPR10-1 protein expressed in E. coli BL21 demonstrated that it had a low ribonuclease-like activity in vitro, and could inhibit V. dahliae hyphae growth but not its spores. Comparison analysis of GbPR10-1 (from resistant species) and GhPR10-1 (from susceptible species) responding to V. dahliae infection, only GbPR10-1 gene was strongly induced in the sea-island cotton plants (incompatible response), indicating that PR10-1 genes was linked to resistance signal. In summary, the earlier activation of GbPR10-1 gene, as the index of resistance response, would be aid to block
基金This work was supported by the Distinguished Professorship Program of Jiangsu Province to YF the National Natural Science Foundation of China （81641164+2 种基金 81600386 81471539 and 30801350） and the Natural Science Foundation of Jiangsu Province （BK20141236）. EWH is supported by NIH grants RO1CA135362 and R21AI112763. We apologize to the many scientists who made contributions to the field but have not been cited because of space limitations.
文摘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.
基金supported by the National Natural Science Foundation of China (31721004)the National Postdoctoral Program for Innovative Talents (BX20180142)+2 种基金the China Postdoctoral Science Foundation (2018M640496)the Natural Science Foundation of Jiangsu Province (BK20190520)the Fundamental Research Funds for the Central Universities (KYXK202010)。
文摘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.
文摘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.
基金the Yunnan Applied Basic Research Project(2017FD148)the State Key Basic Research and Development Plan in Kunming Institute of Zoology(GREKF17-02,to DTL)the National Natural Science Foundation of China(31760619,to XPW).
文摘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.
基金supported by Special Funds of Department Education,Liaoning Province (No.20272280)。
文摘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.
基金supported by grants from the National Key Research and Development Program of China(2022YFC2702604)the National Natural Science Foundation of China(82171608,82201773,and 81971447)+1 种基金the China Postdoctoral Science Foundation(2022M711119)the Scientific Research Foundation of the Health Committee of Hunan Province(B202301039323 and B202301039518).
文摘Male infertility is a major reproductive disorder,which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality.To date,five male patients with biallelic loss-of-function(LOF)variants of PARN-like ribonuclease domain-containing exonuclease 1(PNLDC1)have been reported to experience infertility with nonobstructive azoospermia.The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia(OAT)in a patient from a Chinese Han family.Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant(NM_173516.2,c.l42C>T,p.Gln48Ter)in PNLDC1.Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype,including microcephaly,head tapering,and globozoospermia.Consistently,peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome.Furthermore,the disomy rate of chromosomes in the patient’s spermatozoa was significantly increased compared with that of a fertile control sample.We reported an LOF variant of the PNLDC1 gene responsible for OAT.
基金supported by Grants-in-Aid 25850056,S2306,and 20K05793 from the Ministry of Education,Culture,Sports,Science and Technology of Japan,the Advanced Low Carbon Technology Research and Development Program(ALCA)of the Japan Science and Technology Agency(JST)(to S.W.)the German Ministry of Education and Research(BMBF),grant no.031L0164B“RNAProNet”(to W.R.H.)+1 种基金the German Science Foundation(DFG)research training group MeInBio 322977937/GRK2344(to A.W.and W.R.H.)grant STE 1119/4-2(to C.S.).
文摘Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression.A key endoribonuclease in Gram-negative bacteria is RNase E.To ensure an appropriate supply of RNase E,some bacteria,such as Escherichia coli,feedback-regulate RNase E expression via the rne 5′-untranslated region(5′UTR)in cis.However,the mechanisms involved in the control of RNase E in other bacteria largely remain unknown.Cyanobacteria rely on solar light as an energy source for photosynthesis,despite the inherent ultraviolet(UV)irradiation.In this study,we first investigated globally the changes in gene expression in the cyanobacterium Synechocystis sp.PCC ^(6)803 after a brief exposure to UV.Among the 407 responding genes 2 h after UV exposure was a prominent upregulation of rne mRNA level.Moreover,the enzymatic activity of RNase E rapidly increased as well,although the protein stability decreased.This unique response was underpinned by the increased accumulation of full-length rne mRNA caused by the stabilization of its 5′UTR and suppression of premature transcriptional termination,but not by an increased transcription rate.Mapping of RNA 3′ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5′UTR,indicating autoregulation.These observations suggest that RNase E in cyanobacteria contributes to reshaping the transcriptome during the UV stress response and that its required activity level is secured at the RNA level despite the enhanced turnover of the protein.
