Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on ...Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on electrooculogram.The potential pathogenic mechanism involves mutations in the BEST1 gene,which encodes Ca2+-activated Cl−channels in the retinal pigment epithelium(RPE),resulting in degeneration of RPE and photoreceptor.In this study,the complete clinical characteristics of two Chinese ARB families were summarized.Methods:Pacific Biosciences(PacBio)single-molecule real-time(SMRT)sequencing was performed on the probands to screen for disease-causing gene mutations,and Sanger sequencing was applied to validate variants in the patients and their family members.Results:Two novel mutations,c.202T>C(chr11:61722628,p.Y68H)and c.867+97G>A,in the BEST1 gene were identified in the two Chinese ARB families.The novel missense mutation BEST1 c.202T>C(p.Y68H)resulted in the substitution of tyrosine with histidine in the N-terminal region of transmembrane domain 2 of bestrophin-1.Another novel variant,BEST1 c.867+97G>A(chr11:61725867),located in intron 7,might be considered a regulatory variant that changes allele-specific binding affinity based on motifs of important transcriptional regulators.Conclusion:Our findings represent the first use of third-generation sequencing(TGS)to identify novel BEST1 mutations in patients with ARB,indicating that TGS can be a more accurate and efficient tool for identifying mutations in specific genes.The novel variants identified further broaden the mutation spectrum of BEST1 in the Chinese population.展开更多
Background: Omicron JN.1 has become the dominant SARS-CoV-2 variant in recent months. JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation...Background: Omicron JN.1 has become the dominant SARS-CoV-2 variant in recent months. JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation. The immune evasion capability of JN.1 is a subject of scientific investigation. The US CDC used SGTF of TaqPath COVID-19 Combo Kit RT-qPCR as proxy indicator of JN.1 infections for evaluation of the effectiveness of updated monovalent XBB.1.5 COVID-19 vaccines against JN.1 and recommended that all persons aged ≥ 6 months should receive an updated COVID-19 vaccine dose. Objective: Recommend Sanger sequencing instead of proxy indicator to diagnose JN.1 infections to generate the data based on which guidelines are made to direct vaccination policies. Methods: The RNA in nasopharyngeal swab specimens from patients with clinical respiratory infection was subjected to nested RT-PCR, targeting a 398-base segment of the N-gene and a 445-base segment of the RBD of SARS-CoV-2 for amplification. The nested PCR amplicons were sequenced. The DNA sequences were analyzed for amino acid mutations. Results: The N-gene sequence showed R203K, G204R and Q229K, the 3 mutations associated with Omicron BA.2.86 (+JN.1). The RBD sequence showed 24 of the 26 known amino acid mutations, including the hallmark L455S mutation for JN.1 and the V483del for BA.2.86 lineage. Conclusions: Sanger sequencing of a 445-base segment of the SARS-CoV-2 RBD is useful for accurate determination of emerging variants. The CDC may consider using Sanger sequencing of the RBD to diagnose JN.1 infections for statistical analysis in making vaccination policies.展开更多
The first internal transcribed spacer(ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced wi...The first internal transcribed spacer(ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced with automated fluorescent sequencing. The sequences of ITS1 ranged from 193 bp to 218 bp in size and G/C content varied from 69.3% to 72.7%. In pairwise comparisons among the five taxa, sequence site divergence ranged from 1.5% to 10.6%. Phylogenetic analysis of ITS1 sequences using Wagner parsimony generated a single well resolved tree, which revealed that Oryza rufipogon was much more closely related to cultivated rice species than to the other two wild species. Oryza granulata was less closely related to either cultivated rice species or the other two wild species, and might be a unique and isolated taxon in the genus Oryza. The phylogenetic relationships of the three wild rice species and two cultivated rice subspecies inferred from ITS1 sequences is highly concordant with those based on the molecular evidence from isozyme, chloroplast DNA (cpDNA), mitochondrial DNA(mtDNA) and nuclear DNA (nDNA) of the genus Oryza .展开更多
Objective: To investigate the p21WAF1 /CIP1gene DNA sequence change and their relationship with the phenotype of human osteosarcoma. Methods: p21WAF1 /CIP1gene DNA of 36 osteosarcoma spec- ...Objective: To investigate the p21WAF1 /CIP1gene DNA sequence change and their relationship with the phenotype of human osteosarcoma. Methods: p21WAF1 /CIP1gene DNA of 36 osteosarcoma spec- imens was examined by using polymerase chain reaction-single strand conformation polymorphism (PCR- SSCP) method. The PCR products were sequenced directly. Results: In p21WAF1 /CIP1 gene exon3 of 36 cases of human osteosarcoma, the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 17 cases with the incidence being 44.4%. In 10 normal blood samples, DNA sequence analysis showed the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 8 cases with the incidence being 80%. Conclusion: The novel location of p21WAF1 /CIP1gene polymorphism of osteosarcoma, but not mutation was de?ned, and this location might provide the meaningful reference for the further research of p21WAF1/CIP1 gene.p2lWAF1/CIP1基因DNA序列分析及其与骨肉瘤表型的关系展开更多
[Objective] The study aimed at cloning and analyzing the insulin-like growth factor-1 （IGF-1） gene from liver of Hubei white swine. [Method] The total RNA was extracted by using Trizol from the liver of Hubei white ...[Objective] The study aimed at cloning and analyzing the insulin-like growth factor-1 （IGF-1） gene from liver of Hubei white swine. [Method] The total RNA was extracted by using Trizol from the liver of Hubei white swine and used as template to amplify IGF-1 gene cDNA by RT-PCR. The cDNA product was cloned into pCRII vector, screened with blue-white colonies, digested with double enzymes and sequenced. [Result] The sequencing result indicated that the IGF-1 gene consisted of 607 nucleotides, containing 5＇-untranslated region at nucleotides 1-145, a complete ORF at nucleotides 146-538 encoding 130 amino acids, and 3＇-untranslated region at nucleotides 539-607. It shared 100% homology with the porcine IGF-1 gene reported by Muller et al. [Conclusion] The successful cloning and sequencing of the Hubei white swine IGF-1 gene confirmed that IGF-I gene was highly conserved, which provided technical basis for the use of transgenic technology for breeding of Hubei white swine.展开更多
Previous studies have found that deficiency in nuclear receptor-related factor 1(Nurr1),which participates in the development,differentiation,survival,and degeneration of dopaminergic neurons,is associated with Parkin...Previous studies have found that deficiency in nuclear receptor-related factor 1(Nurr1),which participates in the development,differentiation,survival,and degeneration of dopaminergic neurons,is associated with Parkinson s disease,but the mechanism of action is perplexing.Here,we first asce rtained the repercussion of knocking down Nurr1 by pe rforming liquid chromatography coupled with tandem mass spectrometry.We found that 231 genes were highly expressed in dopaminergic neurons with Nurr1 deficiency,14 of which were linked to the Parkinson’s disease pathway based on Kyoto Encyclopedia of Genes and Genomes analysis.To better understand how Nurr1 deficiency autonomously invokes the decline of dopaminergic neurons and elicits Parkinson’s disease symptoms,we performed single-nuclei RNA sequencing in a Nurr1 LV-shRNA mouse model.The results revealed cellular heterogeneity in the substantia nigra and a number of activated genes,the preponderance of which encode components of the major histocompatibility Ⅱ complex.Cd74,H2-Ab1,H2-Aα,H2-Eb1,Lyz2,Mrc1,Slc6α3,Slc47α1,Ms4α4b,and Ptprc2 were the top 10 diffe rentially expressed genes.Immunofluorescence staining showed that,after Nurr1knockdown,the number of CD74-immunoreactive cells in mouse brain tissue was markedly increased.In addition,Cd74 expression was increased in a mouse model of Parkinson’s disease induced by treatment with 6-hydroxydopamine.Ta ken togethe r,our res ults suggest that Nurr1 deficiency results in an increase in Cd74 expression,thereby leading to the destruction of dopaminergic neuro ns.These findings provide a potential therapeutic target for the treatment of Parkinson’s disease.展开更多
目的:探讨精氨酸-甘氨酸-天冬氨酸(RGD)修饰对肿瘤抑素19肽(T-19)抗肝癌活性的影响,比较分析T-19及RGD修饰的T-19(RGD-T-19)对肝癌SK-Hep-1细胞增殖、侵袭和迁移能力的影响。方法:用Fmoc固相法合成T-19及RGD-T-19,用高效液相色谱仪和质...目的:探讨精氨酸-甘氨酸-天冬氨酸(RGD)修饰对肿瘤抑素19肽(T-19)抗肝癌活性的影响,比较分析T-19及RGD修饰的T-19(RGD-T-19)对肝癌SK-Hep-1细胞增殖、侵袭和迁移能力的影响。方法:用Fmoc固相法合成T-19及RGD-T-19,用高效液相色谱仪和质谱进行分离、鉴定。常规培养SK-Hep-1细胞,用0、50、100、150、200、250μg/mL的T-19及RGD-T-19分别处理细胞,分为0μg/mL(对照)组、50μg/mL组、100μg/mL组、150μg/mL组、200μg/mL组、250μg/mL组。CCK-8法、克隆形成实验、划痕愈合实验和Tanswell小室实验、WB法和q PCR法分别检测SK-Hep-1细胞的增殖、迁移、侵袭能力,以及环氧合酶-2(COX-2)、基质金属蛋白酶-2(MMP-2)、MMP-9、组织基质金属蛋白酶抑制剂-1(TIMP-1)、TIMP-2蛋白和MMP-1、MMP-2 mRNA的表达。结果:经质谱鉴定,用Fmoc固相法合成的T-19及RGD-T-19纯度高。T-19和RGD-T-19均能显著抑制SK-Hep-1细胞的增殖、迁移、侵袭能力,抑制COX-2蛋白、MMP-2和MMP-9蛋白及mRNA的表达、促进TIMP-1、TIMP-2蛋白的表达(P <0.05, P <0.01, P <0.001),RGD-T-19的抑制或促进效应均明显强于T-19(均P <0.05)。结论:利用Fmoc固相法合成了纯度高、活性好的T-19及RGD-T-19,两种肽均能抑制SK-Hep-1细胞增殖、侵袭和迁移能力,RGD-T-19作用明显强于T-19。展开更多
Objective To study the alternative expression and sequence of human elongation factor-1δ （human EF-1δ p31） during malignant transformation of human bronchial epithelial cells induced by cadmium chloride （CdCl2） ...Objective To study the alternative expression and sequence of human elongation factor-1δ （human EF-1δ p31） during malignant transformation of human bronchial epithelial cells induced by cadmium chloride （CdCl2） and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells （16HBE） induced by CdCl2 at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-18 mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2 was elevated （P〈0.01 or P〈0.05）. Compared with their corresponding non-transformed ceils, the overexpression level of EF-15 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed ceils and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2, but is not correlated with DNA mutations.展开更多
AIM: To evaluate the expression of special AT-rich sequence-binding protein 1 (SATB1 ) gene in colorectal cancer and its role in colorectal cancer cell proliferation and invasion.METHODS: Immunohistochemistry was used...AIM: To evaluate the expression of special AT-rich sequence-binding protein 1 (SATB1 ) gene in colorectal cancer and its role in colorectal cancer cell proliferation and invasion.METHODS: Immunohistochemistry was used to detect the protein expression of SATB1 in 30 colorectal cancer (CRC) tissue samples and pair-matched adjacent nontumor samples. Cell growth was investigated after enhancing expression of SATB1. Wound-healing assay and Transwell assay were used to investigate the impact of SATB1 on migratory and invasive abilities of SW480 cells in vitro . Nude mice that received subcutaneous implantation or lateral tail vein were used to study the effects of SATB1 on tumor growth or metastasis in vivo . RESULTS: SATB1 was over-expressed in CRC tissues and CRC cell lines. SATB1 promotes cell proliferation and cell cycle progression in CRC SW480 cells. SATB1 over-expression could promote cell growth in vivo . In addition, SATB1 could significantly raise the ability of cell migration and invasion in vitro and promote the ability of tumor metastasis in vivo . SATB1 could up-regulate matrix metalloproteases 2, 9, cyclin D1 and vimentin, meanwhile SATB1 could down-regulate E-cadherin in CRC. CONCLUSION: SATB1 acts as a potential growth and metastasis promoter in CRC. SATB1 may be useful as a therapeutic target for CRC.展开更多
The epidemiology of HIV-1 varies in different areas of the world, and it is possible that this complexity may leave unique footprints in the viral genome. Thus, we attempted to find significant patterns in global HIV-...The epidemiology of HIV-1 varies in different areas of the world, and it is possible that this complexity may leave unique footprints in the viral genome. Thus, we attempted to find significant patterns in global HIV-1 genome sequences. By applying the rule inference algorithm RIPPER (Repeated Incremental Pruning to Produce Error Reduction) to multiple sequence alignments of Env sequences from four classes of compiled datasets, we generated four sets of signature patterns. We found that these patterns were able to distinguish southeastern Asian from non- southeastern Asian sequences with 97.5% accuracy, Chinese from non-Chinese sequences with 98.3% accuracy, African from non-African sequences with 88.4% accuracy, and southern African from non-southern African sequences with 91.2% accuracy. These patterns showed different associations with subtypes and with amino acid positions. In addition, some signature patterns were characteristic of the geographic area from which the sample was taken. Amino acid features corresponding to the phylogenetic clustering of HIV-1 sequences were consistent with some of the deduced patterns. Using a combination of patterns inferred from subtypes B, C, and all subtypes chimeric with CRF01_AE worldwide, we found that signature patterns of subtype C were extremely common in some sampled countries (for example, Zambia in southern Africa), which may hint at the origin of this HIV-1 subtype and the need to pay special attention to this area of Africa. Signature patterns of subtype B sequences were associated with different countries. Even more, there are distinct patterns at single position 21 with glycine, leucine and isoleucine corresponding to subtype C, B and all possible recombination forms chimeric with CRF01_AE, which also indicate distinct geographic features. Our method widens the scope of inference of signature from geographic, genetic, and genomic viewpoints. These findings may provide a valuable reference for epidemiological research or vaccine design.展开更多
文摘Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on electrooculogram.The potential pathogenic mechanism involves mutations in the BEST1 gene,which encodes Ca2+-activated Cl−channels in the retinal pigment epithelium(RPE),resulting in degeneration of RPE and photoreceptor.In this study,the complete clinical characteristics of two Chinese ARB families were summarized.Methods:Pacific Biosciences(PacBio)single-molecule real-time(SMRT)sequencing was performed on the probands to screen for disease-causing gene mutations,and Sanger sequencing was applied to validate variants in the patients and their family members.Results:Two novel mutations,c.202T>C(chr11:61722628,p.Y68H)and c.867+97G>A,in the BEST1 gene were identified in the two Chinese ARB families.The novel missense mutation BEST1 c.202T>C(p.Y68H)resulted in the substitution of tyrosine with histidine in the N-terminal region of transmembrane domain 2 of bestrophin-1.Another novel variant,BEST1 c.867+97G>A(chr11:61725867),located in intron 7,might be considered a regulatory variant that changes allele-specific binding affinity based on motifs of important transcriptional regulators.Conclusion:Our findings represent the first use of third-generation sequencing(TGS)to identify novel BEST1 mutations in patients with ARB,indicating that TGS can be a more accurate and efficient tool for identifying mutations in specific genes.The novel variants identified further broaden the mutation spectrum of BEST1 in the Chinese population.
文摘Background: Omicron JN.1 has become the dominant SARS-CoV-2 variant in recent months. JN.1 has the highest number of amino acid mutations in its receptor binding domain (RBD) and has acquired a hallmark L455S mutation. The immune evasion capability of JN.1 is a subject of scientific investigation. The US CDC used SGTF of TaqPath COVID-19 Combo Kit RT-qPCR as proxy indicator of JN.1 infections for evaluation of the effectiveness of updated monovalent XBB.1.5 COVID-19 vaccines against JN.1 and recommended that all persons aged ≥ 6 months should receive an updated COVID-19 vaccine dose. Objective: Recommend Sanger sequencing instead of proxy indicator to diagnose JN.1 infections to generate the data based on which guidelines are made to direct vaccination policies. Methods: The RNA in nasopharyngeal swab specimens from patients with clinical respiratory infection was subjected to nested RT-PCR, targeting a 398-base segment of the N-gene and a 445-base segment of the RBD of SARS-CoV-2 for amplification. The nested PCR amplicons were sequenced. The DNA sequences were analyzed for amino acid mutations. Results: The N-gene sequence showed R203K, G204R and Q229K, the 3 mutations associated with Omicron BA.2.86 (+JN.1). The RBD sequence showed 24 of the 26 known amino acid mutations, including the hallmark L455S mutation for JN.1 and the V483del for BA.2.86 lineage. Conclusions: Sanger sequencing of a 445-base segment of the SARS-CoV-2 RBD is useful for accurate determination of emerging variants. The CDC may consider using Sanger sequencing of the RBD to diagnose JN.1 infections for statistical analysis in making vaccination policies.
基金The project supported by the Grant from Presidentof Chinese Academy of Sciences
文摘The first internal transcribed spacer(ITS1) of nuclear ribosomal DNA of three wild rice species and two subspecies of cultivated rice, which are distributed in China, was amplified using PCR technique and sequenced with automated fluorescent sequencing. The sequences of ITS1 ranged from 193 bp to 218 bp in size and G/C content varied from 69.3% to 72.7%. In pairwise comparisons among the five taxa, sequence site divergence ranged from 1.5% to 10.6%. Phylogenetic analysis of ITS1 sequences using Wagner parsimony generated a single well resolved tree, which revealed that Oryza rufipogon was much more closely related to cultivated rice species than to the other two wild species. Oryza granulata was less closely related to either cultivated rice species or the other two wild species, and might be a unique and isolated taxon in the genus Oryza. The phylogenetic relationships of the three wild rice species and two cultivated rice subspecies inferred from ITS1 sequences is highly concordant with those based on the molecular evidence from isozyme, chloroplast DNA (cpDNA), mitochondrial DNA(mtDNA) and nuclear DNA (nDNA) of the genus Oryza .
文摘Objective: To investigate the p21WAF1 /CIP1gene DNA sequence change and their relationship with the phenotype of human osteosarcoma. Methods: p21WAF1 /CIP1gene DNA of 36 osteosarcoma spec- imens was examined by using polymerase chain reaction-single strand conformation polymorphism (PCR- SSCP) method. The PCR products were sequenced directly. Results: In p21WAF1 /CIP1 gene exon3 of 36 cases of human osteosarcoma, the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 17 cases with the incidence being 44.4%. In 10 normal blood samples, DNA sequence analysis showed the change of C→T in the p21WAF1 /CIP1gene CDNA sequence of position 609th occurred in 8 cases with the incidence being 80%. Conclusion: The novel location of p21WAF1 /CIP1gene polymorphism of osteosarcoma, but not mutation was de?ned, and this location might provide the meaningful reference for the further research of p21WAF1/CIP1 gene.p2lWAF1/CIP1基因DNA序列分析及其与骨肉瘤表型的关系
基金Supported by Projects for Transgenic Research (2008ZX08006-002,2008ZX08006-003,2008ZX08010-003,2008ZX08011-004)Hubei Key Laboratory Project (2011ZD127)~~
文摘[Objective] The study aimed at cloning and analyzing the insulin-like growth factor-1 （IGF-1） gene from liver of Hubei white swine. [Method] The total RNA was extracted by using Trizol from the liver of Hubei white swine and used as template to amplify IGF-1 gene cDNA by RT-PCR. The cDNA product was cloned into pCRII vector, screened with blue-white colonies, digested with double enzymes and sequenced. [Result] The sequencing result indicated that the IGF-1 gene consisted of 607 nucleotides, containing 5＇-untranslated region at nucleotides 1-145, a complete ORF at nucleotides 146-538 encoding 130 amino acids, and 3＇-untranslated region at nucleotides 539-607. It shared 100% homology with the porcine IGF-1 gene reported by Muller et al. [Conclusion] The successful cloning and sequencing of the Hubei white swine IGF-1 gene confirmed that IGF-I gene was highly conserved, which provided technical basis for the use of transgenic technology for breeding of Hubei white swine.
基金supported by the National Natural Science Foundation of China,No. 81971006 (to DSG)。
文摘Previous studies have found that deficiency in nuclear receptor-related factor 1(Nurr1),which participates in the development,differentiation,survival,and degeneration of dopaminergic neurons,is associated with Parkinson s disease,but the mechanism of action is perplexing.Here,we first asce rtained the repercussion of knocking down Nurr1 by pe rforming liquid chromatography coupled with tandem mass spectrometry.We found that 231 genes were highly expressed in dopaminergic neurons with Nurr1 deficiency,14 of which were linked to the Parkinson’s disease pathway based on Kyoto Encyclopedia of Genes and Genomes analysis.To better understand how Nurr1 deficiency autonomously invokes the decline of dopaminergic neurons and elicits Parkinson’s disease symptoms,we performed single-nuclei RNA sequencing in a Nurr1 LV-shRNA mouse model.The results revealed cellular heterogeneity in the substantia nigra and a number of activated genes,the preponderance of which encode components of the major histocompatibility Ⅱ complex.Cd74,H2-Ab1,H2-Aα,H2-Eb1,Lyz2,Mrc1,Slc6α3,Slc47α1,Ms4α4b,and Ptprc2 were the top 10 diffe rentially expressed genes.Immunofluorescence staining showed that,after Nurr1knockdown,the number of CD74-immunoreactive cells in mouse brain tissue was markedly increased.In addition,Cd74 expression was increased in a mouse model of Parkinson’s disease induced by treatment with 6-hydroxydopamine.Ta ken togethe r,our res ults suggest that Nurr1 deficiency results in an increase in Cd74 expression,thereby leading to the destruction of dopaminergic neuro ns.These findings provide a potential therapeutic target for the treatment of Parkinson’s disease.
文摘目的从轮状病毒阳性的牛粪便标本中,分离出一株G6P[1]型牛轮状病毒(Bovine Rotavirus,BRV),对其进行培养和鉴定。方法用PBS溶液重悬粪便标本并离心,将其上清过滤除菌和胰酶处理后,利用MA104细胞进行分离培养;通过逆转录-聚合酶链式反应(Reverse Transcription-polymerase Chain Reaction,RT-PCR)对样本VP4和VP7基因进行扩增和测序,与GenBank上的参考序列进行同源性分析,构建进化树,分析确定其G/P基因分型。通过聚丙烯酰胺凝胶电泳法(Polyacrylamine Gel Electrophoresis,PAGE)、噬斑实验和电镜(Transmission Electron Microscope,TEM)等技术对分离到的病毒进行鉴定和纯化。绘制病毒生长动力学曲线。结果分离培养了1株BRV毒株,将其命名BLL。VP7和VP4基因测序结果显示此毒株为G6P[1]型轮状病毒。PAGE胶结果显示分离株电泳型为长型,条带呈现A组轮状病毒排列电泳图谱;通过噬斑实验将毒株进行了纯化。电镜检测到典型的轮状病毒颗粒。病毒生长动力学曲线可发现病毒在感染后6 h已经开始复制。结论本研究成功分离到G6P[1]型牛型轮状病毒,为研究G6P[1]型轮状病毒的病原学特征提供实验基础和技术参考。
文摘目的:探讨精氨酸-甘氨酸-天冬氨酸(RGD)修饰对肿瘤抑素19肽(T-19)抗肝癌活性的影响,比较分析T-19及RGD修饰的T-19(RGD-T-19)对肝癌SK-Hep-1细胞增殖、侵袭和迁移能力的影响。方法:用Fmoc固相法合成T-19及RGD-T-19,用高效液相色谱仪和质谱进行分离、鉴定。常规培养SK-Hep-1细胞,用0、50、100、150、200、250μg/mL的T-19及RGD-T-19分别处理细胞,分为0μg/mL(对照)组、50μg/mL组、100μg/mL组、150μg/mL组、200μg/mL组、250μg/mL组。CCK-8法、克隆形成实验、划痕愈合实验和Tanswell小室实验、WB法和q PCR法分别检测SK-Hep-1细胞的增殖、迁移、侵袭能力,以及环氧合酶-2(COX-2)、基质金属蛋白酶-2(MMP-2)、MMP-9、组织基质金属蛋白酶抑制剂-1(TIMP-1)、TIMP-2蛋白和MMP-1、MMP-2 mRNA的表达。结果:经质谱鉴定,用Fmoc固相法合成的T-19及RGD-T-19纯度高。T-19和RGD-T-19均能显著抑制SK-Hep-1细胞的增殖、迁移、侵袭能力,抑制COX-2蛋白、MMP-2和MMP-9蛋白及mRNA的表达、促进TIMP-1、TIMP-2蛋白的表达(P <0.05, P <0.01, P <0.001),RGD-T-19的抑制或促进效应均明显强于T-19(均P <0.05)。结论:利用Fmoc固相法合成了纯度高、活性好的T-19及RGD-T-19,两种肽均能抑制SK-Hep-1细胞增殖、侵袭和迁移能力,RGD-T-19作用明显强于T-19。
基金Supported by the National Natural Science Foundation of China (No. 30771781)the Natural Science Foundation of Guangdong Province (No.06022672)
文摘Objective To study the alternative expression and sequence of human elongation factor-1δ （human EF-1δ p31） during malignant transformation of human bronchial epithelial cells induced by cadmium chloride （CdCl2） and its possible mechanism. Methods Total RNA was isolated at different stages of transformed human bronchial epithelial cells （16HBE） induced by CdCl2 at a concentration of 5.0 μM. Special primers and probe for human EF-1δ p31 were designed and expression of human EF-18 mRNA from different cell lines was detected with fluorescent quantitative PCR technique. EF-18 cDNA from different cell lines was purified and cloned into pMD 18-T vector followed by confirming and sequencing analysis. Results The expressions of human EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2 was elevated （P〈0.01 or P〈0.05）. Compared with their corresponding non-transformed ceils, the overexpression level of EF-15 p31 was averagely increased 2.9 folds in Cd-pretransformed cells, 4.3 folds in Cd-transformed ceils and 7.2 folds in Cd-tumorigenic cells. No change was found in the sequence of overexpressed EF-1δ p31 at different stages of 16HBE cells transformed by CdCl2. Conclusion Overexpression of human EF-1δ p31 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2, but is not correlated with DNA mutations.
基金Supported by The National Natural Science Foundation of China, No. 81101580
文摘AIM: To evaluate the expression of special AT-rich sequence-binding protein 1 (SATB1 ) gene in colorectal cancer and its role in colorectal cancer cell proliferation and invasion.METHODS: Immunohistochemistry was used to detect the protein expression of SATB1 in 30 colorectal cancer (CRC) tissue samples and pair-matched adjacent nontumor samples. Cell growth was investigated after enhancing expression of SATB1. Wound-healing assay and Transwell assay were used to investigate the impact of SATB1 on migratory and invasive abilities of SW480 cells in vitro . Nude mice that received subcutaneous implantation or lateral tail vein were used to study the effects of SATB1 on tumor growth or metastasis in vivo . RESULTS: SATB1 was over-expressed in CRC tissues and CRC cell lines. SATB1 promotes cell proliferation and cell cycle progression in CRC SW480 cells. SATB1 over-expression could promote cell growth in vivo . In addition, SATB1 could significantly raise the ability of cell migration and invasion in vitro and promote the ability of tumor metastasis in vivo . SATB1 could up-regulate matrix metalloproteases 2, 9, cyclin D1 and vimentin, meanwhile SATB1 could down-regulate E-cadherin in CRC. CONCLUSION: SATB1 acts as a potential growth and metastasis promoter in CRC. SATB1 may be useful as a therapeutic target for CRC.
基金the funding by the Chinese Key National Science and Technology Program in the 12th Five-Year Period, grant 2012ZX10001006-002
文摘The epidemiology of HIV-1 varies in different areas of the world, and it is possible that this complexity may leave unique footprints in the viral genome. Thus, we attempted to find significant patterns in global HIV-1 genome sequences. By applying the rule inference algorithm RIPPER (Repeated Incremental Pruning to Produce Error Reduction) to multiple sequence alignments of Env sequences from four classes of compiled datasets, we generated four sets of signature patterns. We found that these patterns were able to distinguish southeastern Asian from non- southeastern Asian sequences with 97.5% accuracy, Chinese from non-Chinese sequences with 98.3% accuracy, African from non-African sequences with 88.4% accuracy, and southern African from non-southern African sequences with 91.2% accuracy. These patterns showed different associations with subtypes and with amino acid positions. In addition, some signature patterns were characteristic of the geographic area from which the sample was taken. Amino acid features corresponding to the phylogenetic clustering of HIV-1 sequences were consistent with some of the deduced patterns. Using a combination of patterns inferred from subtypes B, C, and all subtypes chimeric with CRF01_AE worldwide, we found that signature patterns of subtype C were extremely common in some sampled countries (for example, Zambia in southern Africa), which may hint at the origin of this HIV-1 subtype and the need to pay special attention to this area of Africa. Signature patterns of subtype B sequences were associated with different countries. Even more, there are distinct patterns at single position 21 with glycine, leucine and isoleucine corresponding to subtype C, B and all possible recombination forms chimeric with CRF01_AE, which also indicate distinct geographic features. Our method widens the scope of inference of signature from geographic, genetic, and genomic viewpoints. These findings may provide a valuable reference for epidemiological research or vaccine design.
