Objective:Glucose-6-phosphate isomerase(GPI)deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants.This disorder exhibits wide heterogeneity in its clinical manifestations and mole...Objective:Glucose-6-phosphate isomerase(GPI)deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants.This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics,often posing challenges for precise diagnoses using conventional methods.To this end,this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family.Methods:The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis.Novel compound heterozygous variants of the GPI gene,c.174C>A(p.Asn58Lys)and c.1538G>T(p.Trp513Leu),were identified using whole-exome and Sanger sequencing.The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure.Results:By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study,we found that most variants were located in exons 3,4,12,and 18,with a few localized in exons 8,9,and 14.This study identified novel compound heterozygous variants associated with GPI deficiency.These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids.Conclusion:Early family-based sequencing analyses,especially for patients with congenital anemia,can help increase diagnostic accuracy for GPI deficiency,improve child healthcare,and enable genetic counseling.展开更多
PDI is a molecular chaperone and plays an important role in Endoplasmic Reticulum quality control (ERQC).PDI participates in the refolding of the misfolded/unfolded proteins to maintain cellular homeostasis under diff...PDI is a molecular chaperone and plays an important role in Endoplasmic Reticulum quality control (ERQC).PDI participates in the refolding of the misfolded/unfolded proteins to maintain cellular homeostasis under differentstresses. However, bioinformatic characteristics and potential functions of PDIs in diatom Phaeodactylumtricornutum (Pt) are still unknown so far. Hence, the genome-wide characteristics of PtPDI proteins in P. tricornutumwere first studied via bioinformatic and transcriptomic methods. 42 PtPDI genes were identified from thegenome of P. tricornutum. The motif, protein structure, classification, number of introns, phylogenetic relationship,and the expression level of 42 PtPDI genes under the tunicamycin stress were analyzed. A pair of tandemduplicated genes (PtPDI15 and PtPDI18) was observed in P. tricornutum. The 42 PtPDIs with different genecharacteristics were divided into three independent clades, indicating different evolutional relationships and functionsof these PtPDIs. The 14 up-regulated PtPDI genes under the tunicamycin treatment might have a positiveeffect on the ER quality control of the unfolded/misfolded proteins, while the 7 down-regulated PtPDIs mightnegatively affect the ERQC. The characteristics of all 42 PtPDIs and their proposed working model here providea comprehensive understanding of the PtPDIs gene family. The differential expression of 21 PtPDIs will be usefulfor further functional study in the ERQC.展开更多
Objective Protein disulfide isomerase A2(PDIA2),a member of the protein disulfide isomerase family,plays a key role in the folding of nascent proteins in the endoplasmic reticulum by forming disulfide bonds,together w...Objective Protein disulfide isomerase A2(PDIA2),a member of the protein disulfide isomerase family,plays a key role in the folding of nascent proteins in the endoplasmic reticulum by forming disulfide bonds,together with enzymes such as thiol isomerase,oxidase,and reductase.This study investigated the clinical significance and potential functions of PDIA2 in glioma.Methods The expression of PDIA2 in gliomas was explored using The Cancer Genome Atlas and Gene Expression Omnibus databases.We analyzed the clinical characteristics of glioma patients and the prognostic and diagnostic value of PDIA2 expression.Kaplan-Meier and Cox regression analyses were used to examine the effect of PDIA2 expression on overall survival,progression-free interval,and disease-specific survival.Furthermore,we performed Gene Set Enrichment Analysis and immune infiltration analysis to investigate the functions of PDIA2.PDIA2 mRNA and protein expression was evaluated in cell lines and glioma tissues.Results PDIA2 was expressed at low levels in glioma patients.Kaplan-Meier survival analysis showed that glioma patients with low PDIA2 levels had a worse prognosis than those with high PDIA2 levels.Receiver operating characteristic curve analysis indicated the diagnostic and prognostic ability of PDIA2(area under the curve=0.918).Pathways associated with PD1,PI3K/AKT,cancer immunotherapy via PD1 blockade,Fceri-mediated NF-kB activation,FOXM1,and DNA repair were enriched in glioma patients with low levels of PDIA2.PDIA2 expression levels were negatively correlated with immune cell infiltrate levels.Conclusion PDIA2 levels are significantly downregulated in glioma.PDIA2 expression may be a potential biomarker for the diagnosis and prognosis of glioma patients.展开更多
Objective: Tumor heterogeneity renders identification of suitable biomarkers of gastric cancer(GC)challenging. Here, we aimed to identify prognostic genes of GC using computational analysis.Methods: We first used micr...Objective: Tumor heterogeneity renders identification of suitable biomarkers of gastric cancer(GC)challenging. Here, we aimed to identify prognostic genes of GC using computational analysis.Methods: We first used microarray technology to profile gene expression of GC and paired nontumor tissues from 198 patients. Based on these profiles and patients’ clinical information, we next identified prognostic genes using novel computational approaches. Phosphoglucose isomerase, also known as glucose-6-phosphate isomerase(GPI), which ranked first among 27 candidate genes, was further investigated by a new analytical tool namely enviro-geno-pheno-state(E-GPS) analysis. Suitability of GPI as a prognostic marker, and its relationship with physiological processes such as metabolism, epithelial-mesenchymal transition(EMT), as well as drug sensitivity were evaluated using both our own and independent public datasets.Results: We found that higher expression of GPI in GC correlated with prolonged survival of patients.Particularly, a combination of CDH2 and GPI expression effectively stratified the outcomes of patients with TNM stage Ⅱ/Ⅲ. Down-regulation of GPI in tumor tissues correlated well with depressed glucose metabolism and fatty acid synthesis, as well as enhanced fatty acid oxidation and creatine metabolism, indicating that GPI represents a suitable marker for increased probability of EMT in GC cells.Conclusions: Our findings strongly suggest that GPI acts as a novel biomarker candidate for GC prognosis,allowing greatly enhanced clinical management of GC patients. The potential metabolic rewiring correlated with GPI also provides new insights into studying the relationship between cancer metabolism and patient survival.展开更多
L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillu...L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillus plantarum was isolated from the number of lactic acid bacteria from pickled vegetables. The crude L-arabinose isomerase activity of Lactobacillus plantarum WU14 with high D-tagatose yield was 13.95 U/mL under the optimal temperature 60°C, pH 7.17 and substrate concentration 0.8 mol/L, and the conversion rate of 56.12% could be gained after 28 hours. Protein structure and specific of L-Arabinose Isomerase of Lactobacillus plantarum WU14 were researched. The results showed that L-arabinose isomerase is mainly composed of alpha helix and random coil. Then the recombinant L-AI gene was inserted into the food-grade expression vector pRNA48 and expressed in L. lactis NZ9000 successfully. The target protein expression reached the maximum amount when the induced concentration of nisin reaches 30 ng/mL after 12 h. And the crude enzyme activity of recombinant bacteria reached 6.21 U/mL under 60°C. Otherwise the optimal conversion rate recombinant of L. lactis NZ9000/pRNA48-L-AI can reach 39.21% under the temperature of 50°C, pH 7.17 and D-galactose concentration was 0.6 mol/L.展开更多
Phosphoglucose isomerase (PGI) is a key enzyme in early glycolysis, which catalyzes the reversible isomerization of glucose 6-phosphate (G6Ph) to fructose 6-phosphate. We have constructed an Escherichia coli K12 strai...Phosphoglucose isomerase (PGI) is a key enzyme in early glycolysis, which catalyzes the reversible isomerization of glucose 6-phosphate (G6Ph) to fructose 6-phosphate. We have constructed an Escherichia coli K12 strain with a deleted pgi gene (Δpgi) and shown that this strain in comparison with the parental strain 1) accumulates higher amount of G6Ph, 2) grows slowly, and 3) exhibits higher spontaneous mutation frequency to rifampicin resistance (Rifr), when grown on high glucose minimal medium. Intriguingly, the spontaneous mutation rate to Rifr was inversely related to the degree of E. coli chromosomal DNA modification with sugar derivatives. We measured higher concentrations of Amadori products, fluorophores (360 nm excitation/440 nm emission) and carboxymethyl residues in the chromosomal DNA of the E. coli parental strain than in DNA of the isogenic Δpgi strain. To explain this apparent paradox we hypothesized that PGI might be implicated in repair of G6Ph-derived lesions in DNA. In favor of our hypothesis, we further demonstrate that protein extract from the E. coli PGI proficient strain but not from the PGI deficient strain catalyzes the release of G6Ph from G6Ph-modified single stranded DNA oligonucleotide and from its hybrid duplex with a complementary peptide nucleic acid.展开更多
The phosphomannose isomerase (PMI) gene from Saccharomyces cerevisiae acted as selectable marker and mannose acted as selective agent for the production of transgenic plants of rice (Oryza sativa L.) via Agrobacte...The phosphomannose isomerase (PMI) gene from Saccharomyces cerevisiae acted as selectable marker and mannose acted as selective agent for the production of transgenic plants of rice (Oryza sativa L.) via Agrobacterium-mediated transformation. The concentration of mannose during the selection was stepwise increased, 5 g L-1 mannose combined with 15 g L-1 sucrose and 500 mg L-1 cefotaxime was used in the initial selection stage, then the concentration of mannose was increased to 11 g L-1, the highest transformation rate was 20.0%. The integration of PMI gene was confirmed by PCR, and the result of RT-PCR assay proved that the intron of PMI gene can be excised correctly during RNA splicing. 13- Glucuronidase (GUS) activity analysis confirmed the expression of GUS gene. All those means the PMI gene from yeast can be used as a selectable marker in rice transformation.展开更多
Protein disulphide isomerase (PDI) is an oxidoreductase enzyme abundant in the endoplasmic reticulum (ER). In plants, PDIs have been shown to assist the folding and deposition of seed storage proteins during the bioge...Protein disulphide isomerase (PDI) is an oxidoreductase enzyme abundant in the endoplasmic reticulum (ER). In plants, PDIs have been shown to assist the folding and deposition of seed storage proteins during the biogenesis of protein bodies in the endosperm. Cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv. Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species were reported in our previous publications. Promoter sequences of three homoeologous genes encoding typical PDI, located on chromosome group 4 of bread wheat, and PDI promoter sequence analysis of Triticum urartu, Aegilops speltoides and Aegilops tauschii had also been reported previously. In this study, we report the isolation and sequencing of a ~700 bp region, comprising ~600 bp of the putative promoter region and 88 bp of the first exon of the typical PDI gene, in five accessions each from Triticum urartu (AA), Aegilops speltoides (BB) and Aegilops tauschii (DD). Sequence analysis indicated large variation among sequences belonging to the different genomes, while close similarity was found within each species and with the corresponding homoeologous PDI sequences of Triticum aestivum cv. CS (AABBDD) resulting in an overall high conservation of the sequence conferring endosperm-specific expression.展开更多
Establishing a transgenic plant largely relies on a selectable marker gene that can confer antibiotic or herbicide resistance to plant cells.The existence of such selectable marker genes in genetically modified foods ...Establishing a transgenic plant largely relies on a selectable marker gene that can confer antibiotic or herbicide resistance to plant cells.The existence of such selectable marker genes in genetically modified foods has long been criticized.Plant cells generally exhibit too low an activity of phosphomannose isomerase(PMI)to grow with mannose as a sole carbon source.In this study,we characterized PMI from the green microalga Chlorococcum sp.and assessed its feasibility as a selectable marker for plant biotechnology.Chlorococcum sp.PMI(ChlPMI)was shown to be closely related to higher plants but more distant to bacterial counterparts.Overexpression of ChlPMI in tomato induced callus and shoot formation in media containing mannose(6 g/L)and had an average transformation rate of 3.9%.Based on this transformation system,a polycistronic gene cluster containing crtB,HpBHY,CrBKT and SlLCYB(BBBB)was co-expressed in a different tomato cultivar.Six putative transformants were achieved with a transformation rate of 1.4%,which produced significant amounts of astaxanthin due to the expression of the BBBB genes.Taken together,these findings indicate that we have established an additional tool for plant biotechnology that may be suitable for genetically modifying foods safely.展开更多
Objective: To investigate whether glucose-6-phosphate isomerase (G6PI) antigen and anti-G6PI antibodies could be applied for the clinical diagnostic markers of rheumatoid arthritis (RA) and its associations with RA ac...Objective: To investigate whether glucose-6-phosphate isomerase (G6PI) antigen and anti-G6PI antibodies could be applied for the clinical diagnostic markers of rheumatoid arthritis (RA) and its associations with RA activity states. Methods: The levels of G6PI antigens and anti-G6PI Abs in sera from 176 RA patients in different states, 35 non-RA patients and 100 healthy donors and in synovia fluids from 33 patients and 11 non-RA patients were measured by ELISA. Results: The sensitivity and specificity of G6PI antigens in the RA patients were 75.0% and 93.3%, respectively. The levels of serum G6PI antigens in 176 RA patients were significantly higher than non-RA patients and the health controls. Especially, there was a significant difference between the active phase and the inactive phase in G6PI antigens levels. The levels of G6PI antigens in synovia fluid were also significantly higher in RA groups than in non-RA patients. With the values of the anti-G6PI Abs in sera, there were no marked differences among RA, non-RA patients and health controls. Also, there was no significant difference between the active phase and the inactive phase in RA patients. However, there were no significant differences of G6PI and anti-G6PI between RA patients and health controls in synovial fluid. Conclusions: G6PI is highly correlated with the activity states of RA, and could be applied for a clinical biomarker with high sensitivity and specificity for the diagnosis of RA.展开更多
Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to spee...Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to speed up protein structure determination. Phase problem is a bottleneck in macromolecular structure determination and also in model building which is a time-consuming task. The simplest approach to phasing macromolecular crystal structures is the use of a SAD signal. SAD data can be collected using the in-house copper (1.54 A) wavelength source. Data collected using copper wavelength with the incorporation of anomalously scattering heavy metal atoms may serve as a powerful tool for structural biologists to solve novel protein structures as well where synchrotron beam line is not available. A short soak of protein crystals in heavy metal solution or by incorporating heavy atoms into the protein drop while crystallizing the protein (co-crystallization) leads to incorporation of these heavy metal ions into the ordered solvent shell around the protein surface. The present work aims to determine whether cerium ion can be successfully incorporated into the protein crystal through quick-soaking method while maintaining the isomorphism. The study also aims in understanding whether this metal ion can be used for phasing purpose. The intensity data are collected and analyzed for anomalous signal, substructure solution and the binding sites.展开更多
Newly synthesized membrane and secretory proteins in cells undergo folding in the endoplasmic reticulum with the introduction of disulfide bonds and acquire the correct three-dimensional structure. Disulfide bonds are...Newly synthesized membrane and secretory proteins in cells undergo folding in the endoplasmic reticulum with the introduction of disulfide bonds and acquire the correct three-dimensional structure. Disulfide bonds are especially important for protein folding. It has been thought that formation of protein disulfide bonds in eukaryotes is mainly carried out by an enzyme called protein disulfide isomerase. Proteins, bearing the C-terminus of amino acids sequences with His-Asp-Glu-Leu (HDEL) sequence in yeast, in the endoplasmic reticulum (ER), which is a eukaryotic cellular organelle involved in protein synthesis, processing, and transport, have been considered to recycle between ER and Golgi apparatus. The proposal for this recycling model derives from the study of an HDEL-tagged fusion protein. Here, the localization and oligosaccharide modification of protein disulfide isomerase were investigated in yeast, and showed the first direct evidence that this intrinsic ER protein transports from ER to Golgi. Results suggest that this native protein is also accessible to post-ER enzymes, and yet accumulates in the ER.展开更多
Carotenoid isomerase(CRTISO)is a key enzyme that catalyzes the conversion of cis-lycopene to alltrans lycopene. In this study, we isolated and characterized the CRTISO gene from Lycium chinense(Lc CRTISO) for the firs...Carotenoid isomerase(CRTISO)is a key enzyme that catalyzes the conversion of cis-lycopene to alltrans lycopene. In this study, we isolated and characterized the CRTISO gene from Lycium chinense(Lc CRTISO) for the first time. The open reading frame of Lc CRTISO was 1 815 bp encoding a protein of 604 amino acids with a molecular mass of 66.24 k Da. Amino acid sequence analysis revealed that the Lc CRTISO had a high level of similarity to other CRTISO. Phylogenetic analysis displayed that Lc CRTISO kept a closer relationship with the CRTISO of plants than with those of other species. Semi-quantitative PCR analysis indicated that Lc CRTISO gene was expressed in all tissues tested with the highest expression in maturing fruits. The overexpression of Lc CRTISO gene in transgenic tobacco resulted in an increase of total carotenoids in the leaves with β-carotene and lutein being the predominants. The results obtained here clearly suggested that the Lc CRTISO gene was a promising candidate for carotenoid production.展开更多
文摘Objective:Glucose-6-phosphate isomerase(GPI)deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants.This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics,often posing challenges for precise diagnoses using conventional methods.To this end,this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family.Methods:The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis.Novel compound heterozygous variants of the GPI gene,c.174C>A(p.Asn58Lys)and c.1538G>T(p.Trp513Leu),were identified using whole-exome and Sanger sequencing.The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure.Results:By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study,we found that most variants were located in exons 3,4,12,and 18,with a few localized in exons 8,9,and 14.This study identified novel compound heterozygous variants associated with GPI deficiency.These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids.Conclusion:Early family-based sequencing analyses,especially for patients with congenital anemia,can help increase diagnostic accuracy for GPI deficiency,improve child healthcare,and enable genetic counseling.
基金the funding of Educational and Scientific Research Projects for Young and Middle-Aged Teachers in Fujian Province(Grant Number:2022JAT220693)Natural Science Foundation of Guangdong Province(Grant Number:2022A1515012141)+2 种基金the Program for University Innovation Team of Guangdong Province(Grant Number:2022KCXTD008)National Natural Science Foundation of China(92158201 and 42376001)the Innovation and Entrepreneurship Project of Shantou(201112176541391).
文摘PDI is a molecular chaperone and plays an important role in Endoplasmic Reticulum quality control (ERQC).PDI participates in the refolding of the misfolded/unfolded proteins to maintain cellular homeostasis under differentstresses. However, bioinformatic characteristics and potential functions of PDIs in diatom Phaeodactylumtricornutum (Pt) are still unknown so far. Hence, the genome-wide characteristics of PtPDI proteins in P. tricornutumwere first studied via bioinformatic and transcriptomic methods. 42 PtPDI genes were identified from thegenome of P. tricornutum. The motif, protein structure, classification, number of introns, phylogenetic relationship,and the expression level of 42 PtPDI genes under the tunicamycin stress were analyzed. A pair of tandemduplicated genes (PtPDI15 and PtPDI18) was observed in P. tricornutum. The 42 PtPDIs with different genecharacteristics were divided into three independent clades, indicating different evolutional relationships and functionsof these PtPDIs. The 14 up-regulated PtPDI genes under the tunicamycin treatment might have a positiveeffect on the ER quality control of the unfolded/misfolded proteins, while the 7 down-regulated PtPDIs mightnegatively affect the ERQC. The characteristics of all 42 PtPDIs and their proposed working model here providea comprehensive understanding of the PtPDIs gene family. The differential expression of 21 PtPDIs will be usefulfor further functional study in the ERQC.
基金the Natural Science Foundation of Southwest Medical University(No.2016XNYD217,No.2018-ZRQN-032 and No.2016LZXNYD-G03)the National Natural Science Foundation of China(No.82072780)Sichuan Science and Technology Program(No.2022YFS0630).
文摘Objective Protein disulfide isomerase A2(PDIA2),a member of the protein disulfide isomerase family,plays a key role in the folding of nascent proteins in the endoplasmic reticulum by forming disulfide bonds,together with enzymes such as thiol isomerase,oxidase,and reductase.This study investigated the clinical significance and potential functions of PDIA2 in glioma.Methods The expression of PDIA2 in gliomas was explored using The Cancer Genome Atlas and Gene Expression Omnibus databases.We analyzed the clinical characteristics of glioma patients and the prognostic and diagnostic value of PDIA2 expression.Kaplan-Meier and Cox regression analyses were used to examine the effect of PDIA2 expression on overall survival,progression-free interval,and disease-specific survival.Furthermore,we performed Gene Set Enrichment Analysis and immune infiltration analysis to investigate the functions of PDIA2.PDIA2 mRNA and protein expression was evaluated in cell lines and glioma tissues.Results PDIA2 was expressed at low levels in glioma patients.Kaplan-Meier survival analysis showed that glioma patients with low PDIA2 levels had a worse prognosis than those with high PDIA2 levels.Receiver operating characteristic curve analysis indicated the diagnostic and prognostic ability of PDIA2(area under the curve=0.918).Pathways associated with PD1,PI3K/AKT,cancer immunotherapy via PD1 blockade,Fceri-mediated NF-kB activation,FOXM1,and DNA repair were enriched in glioma patients with low levels of PDIA2.PDIA2 expression levels were negatively correlated with immune cell infiltrate levels.Conclusion PDIA2 levels are significantly downregulated in glioma.PDIA2 expression may be a potential biomarker for the diagnosis and prognosis of glioma patients.
基金supported by grants from the Ministry of Science and Technology of the People’s Republic of China (No. SS2014AA020603)Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (No. ZYLX201701)+3 种基金Beijing Municipal Science and Technology Commission (No. D1311 00005313010)the National Natural Science Foundation of China (No. 31520103905)the National High Technology Research and Development Program (“863” Program) of China (No. 2015AA020108)the Zhi-Yuan chair professorship start-up grant WF220103010 from Shanghai Jiao Tong University
文摘Objective: Tumor heterogeneity renders identification of suitable biomarkers of gastric cancer(GC)challenging. Here, we aimed to identify prognostic genes of GC using computational analysis.Methods: We first used microarray technology to profile gene expression of GC and paired nontumor tissues from 198 patients. Based on these profiles and patients’ clinical information, we next identified prognostic genes using novel computational approaches. Phosphoglucose isomerase, also known as glucose-6-phosphate isomerase(GPI), which ranked first among 27 candidate genes, was further investigated by a new analytical tool namely enviro-geno-pheno-state(E-GPS) analysis. Suitability of GPI as a prognostic marker, and its relationship with physiological processes such as metabolism, epithelial-mesenchymal transition(EMT), as well as drug sensitivity were evaluated using both our own and independent public datasets.Results: We found that higher expression of GPI in GC correlated with prolonged survival of patients.Particularly, a combination of CDH2 and GPI expression effectively stratified the outcomes of patients with TNM stage Ⅱ/Ⅲ. Down-regulation of GPI in tumor tissues correlated well with depressed glucose metabolism and fatty acid synthesis, as well as enhanced fatty acid oxidation and creatine metabolism, indicating that GPI represents a suitable marker for increased probability of EMT in GC cells.Conclusions: Our findings strongly suggest that GPI acts as a novel biomarker candidate for GC prognosis,allowing greatly enhanced clinical management of GC patients. The potential metabolic rewiring correlated with GPI also provides new insights into studying the relationship between cancer metabolism and patient survival.
文摘L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillus plantarum was isolated from the number of lactic acid bacteria from pickled vegetables. The crude L-arabinose isomerase activity of Lactobacillus plantarum WU14 with high D-tagatose yield was 13.95 U/mL under the optimal temperature 60°C, pH 7.17 and substrate concentration 0.8 mol/L, and the conversion rate of 56.12% could be gained after 28 hours. Protein structure and specific of L-Arabinose Isomerase of Lactobacillus plantarum WU14 were researched. The results showed that L-arabinose isomerase is mainly composed of alpha helix and random coil. Then the recombinant L-AI gene was inserted into the food-grade expression vector pRNA48 and expressed in L. lactis NZ9000 successfully. The target protein expression reached the maximum amount when the induced concentration of nisin reaches 30 ng/mL after 12 h. And the crude enzyme activity of recombinant bacteria reached 6.21 U/mL under 60°C. Otherwise the optimal conversion rate recombinant of L. lactis NZ9000/pRNA48-L-AI can reach 39.21% under the temperature of 50°C, pH 7.17 and D-galactose concentration was 0.6 mol/L.
文摘Phosphoglucose isomerase (PGI) is a key enzyme in early glycolysis, which catalyzes the reversible isomerization of glucose 6-phosphate (G6Ph) to fructose 6-phosphate. We have constructed an Escherichia coli K12 strain with a deleted pgi gene (Δpgi) and shown that this strain in comparison with the parental strain 1) accumulates higher amount of G6Ph, 2) grows slowly, and 3) exhibits higher spontaneous mutation frequency to rifampicin resistance (Rifr), when grown on high glucose minimal medium. Intriguingly, the spontaneous mutation rate to Rifr was inversely related to the degree of E. coli chromosomal DNA modification with sugar derivatives. We measured higher concentrations of Amadori products, fluorophores (360 nm excitation/440 nm emission) and carboxymethyl residues in the chromosomal DNA of the E. coli parental strain than in DNA of the isogenic Δpgi strain. To explain this apparent paradox we hypothesized that PGI might be implicated in repair of G6Ph-derived lesions in DNA. In favor of our hypothesis, we further demonstrate that protein extract from the E. coli PGI proficient strain but not from the PGI deficient strain catalyzes the release of G6Ph from G6Ph-modified single stranded DNA oligonucleotide and from its hybrid duplex with a complementary peptide nucleic acid.
基金the Genetically Modified Organisms Breeding Major Projects, China (2011ZX08003-003)
文摘The phosphomannose isomerase (PMI) gene from Saccharomyces cerevisiae acted as selectable marker and mannose acted as selective agent for the production of transgenic plants of rice (Oryza sativa L.) via Agrobacterium-mediated transformation. The concentration of mannose during the selection was stepwise increased, 5 g L-1 mannose combined with 15 g L-1 sucrose and 500 mg L-1 cefotaxime was used in the initial selection stage, then the concentration of mannose was increased to 11 g L-1, the highest transformation rate was 20.0%. The integration of PMI gene was confirmed by PCR, and the result of RT-PCR assay proved that the intron of PMI gene can be excised correctly during RNA splicing. 13- Glucuronidase (GUS) activity analysis confirmed the expression of GUS gene. All those means the PMI gene from yeast can be used as a selectable marker in rice transformation.
文摘Protein disulphide isomerase (PDI) is an oxidoreductase enzyme abundant in the endoplasmic reticulum (ER). In plants, PDIs have been shown to assist the folding and deposition of seed storage proteins during the biogenesis of protein bodies in the endosperm. Cloning and characterization of the complete set of genes encoding PDI and PDI like proteins in bread wheat (Triticum aestivum cv. Chinese Spring) and the comparison of their sequence, structure and expression with homologous genes from other plant species were reported in our previous publications. Promoter sequences of three homoeologous genes encoding typical PDI, located on chromosome group 4 of bread wheat, and PDI promoter sequence analysis of Triticum urartu, Aegilops speltoides and Aegilops tauschii had also been reported previously. In this study, we report the isolation and sequencing of a ~700 bp region, comprising ~600 bp of the putative promoter region and 88 bp of the first exon of the typical PDI gene, in five accessions each from Triticum urartu (AA), Aegilops speltoides (BB) and Aegilops tauschii (DD). Sequence analysis indicated large variation among sequences belonging to the different genomes, while close similarity was found within each species and with the corresponding homoeologous PDI sequences of Triticum aestivum cv. CS (AABBDD) resulting in an overall high conservation of the sequence conferring endosperm-specific expression.
基金supported by a grant from Yunnan high talents program(Y33D331),Yunnan Province,China.
文摘Establishing a transgenic plant largely relies on a selectable marker gene that can confer antibiotic or herbicide resistance to plant cells.The existence of such selectable marker genes in genetically modified foods has long been criticized.Plant cells generally exhibit too low an activity of phosphomannose isomerase(PMI)to grow with mannose as a sole carbon source.In this study,we characterized PMI from the green microalga Chlorococcum sp.and assessed its feasibility as a selectable marker for plant biotechnology.Chlorococcum sp.PMI(ChlPMI)was shown to be closely related to higher plants but more distant to bacterial counterparts.Overexpression of ChlPMI in tomato induced callus and shoot formation in media containing mannose(6 g/L)and had an average transformation rate of 3.9%.Based on this transformation system,a polycistronic gene cluster containing crtB,HpBHY,CrBKT and SlLCYB(BBBB)was co-expressed in a different tomato cultivar.Six putative transformants were achieved with a transformation rate of 1.4%,which produced significant amounts of astaxanthin due to the expression of the BBBB genes.Taken together,these findings indicate that we have established an additional tool for plant biotechnology that may be suitable for genetically modifying foods safely.
文摘Objective: To investigate whether glucose-6-phosphate isomerase (G6PI) antigen and anti-G6PI antibodies could be applied for the clinical diagnostic markers of rheumatoid arthritis (RA) and its associations with RA activity states. Methods: The levels of G6PI antigens and anti-G6PI Abs in sera from 176 RA patients in different states, 35 non-RA patients and 100 healthy donors and in synovia fluids from 33 patients and 11 non-RA patients were measured by ELISA. Results: The sensitivity and specificity of G6PI antigens in the RA patients were 75.0% and 93.3%, respectively. The levels of serum G6PI antigens in 176 RA patients were significantly higher than non-RA patients and the health controls. Especially, there was a significant difference between the active phase and the inactive phase in G6PI antigens levels. The levels of G6PI antigens in synovia fluid were also significantly higher in RA groups than in non-RA patients. With the values of the anti-G6PI Abs in sera, there were no marked differences among RA, non-RA patients and health controls. Also, there was no significant difference between the active phase and the inactive phase in RA patients. However, there were no significant differences of G6PI and anti-G6PI between RA patients and health controls in synovial fluid. Conclusions: G6PI is highly correlated with the activity states of RA, and could be applied for a clinical biomarker with high sensitivity and specificity for the diagnosis of RA.
文摘Current structural genomics projects aim to solve a large number of selected protein structures as fast as possible. High degree of automation and standardization is required at every step of the whole process to speed up protein structure determination. Phase problem is a bottleneck in macromolecular structure determination and also in model building which is a time-consuming task. The simplest approach to phasing macromolecular crystal structures is the use of a SAD signal. SAD data can be collected using the in-house copper (1.54 A) wavelength source. Data collected using copper wavelength with the incorporation of anomalously scattering heavy metal atoms may serve as a powerful tool for structural biologists to solve novel protein structures as well where synchrotron beam line is not available. A short soak of protein crystals in heavy metal solution or by incorporating heavy atoms into the protein drop while crystallizing the protein (co-crystallization) leads to incorporation of these heavy metal ions into the ordered solvent shell around the protein surface. The present work aims to determine whether cerium ion can be successfully incorporated into the protein crystal through quick-soaking method while maintaining the isomorphism. The study also aims in understanding whether this metal ion can be used for phasing purpose. The intensity data are collected and analyzed for anomalous signal, substructure solution and the binding sites.
文摘Newly synthesized membrane and secretory proteins in cells undergo folding in the endoplasmic reticulum with the introduction of disulfide bonds and acquire the correct three-dimensional structure. Disulfide bonds are especially important for protein folding. It has been thought that formation of protein disulfide bonds in eukaryotes is mainly carried out by an enzyme called protein disulfide isomerase. Proteins, bearing the C-terminus of amino acids sequences with His-Asp-Glu-Leu (HDEL) sequence in yeast, in the endoplasmic reticulum (ER), which is a eukaryotic cellular organelle involved in protein synthesis, processing, and transport, have been considered to recycle between ER and Golgi apparatus. The proposal for this recycling model derives from the study of an HDEL-tagged fusion protein. Here, the localization and oligosaccharide modification of protein disulfide isomerase were investigated in yeast, and showed the first direct evidence that this intrinsic ER protein transports from ER to Golgi. Results suggest that this native protein is also accessible to post-ER enzymes, and yet accumulates in the ER.
基金This work was supported by the National Natural Science Foundation of China (No.20336010) and the State Key Basic Research and Development Plan of China (No.2003CB716000).
文摘有 D 木糖的 Thermus thermophilus 木糖 isomerase (TtXI ) 的复杂模型被构造,并且分子的动力学(MD ) 模拟被 NAMD2.5 为 10 ns 在 300 和 360 K 执行。旋转(Rg ) 的半径,子单元相互作用,和残余灵活性被分析。结果表演残余 6069, 142148, 169172,和 332340 在 300 和 360 K 有高灵活性。有在 360 K 的更高的灵活性的残余能主要在 300 K 比那被划分成二个组:一个人在由残余组成的 helix-loop-helix 区域定位 5580 在里面催化领域;其它在子单元连接。在 360 K 的催化领域的 Rg 显示出 0.16 吗?高,比那,在小 C 终端的 300 K,而是 Rg,领域没有明显的差别。结果显示催化领域的那提高的 Rg 可以导致 TtXI 的活跃地点的强烈运动并且支持 D 木糖 isomization 反应。八张氢契约和五离子对与 300 K 相比在 360 K 在子单元接口被减少,那可以是为在在在 TtXI 的高温度的活动的刚硬和增加的减少的主要原因。也,解释 TtXI E372G 异种的冷改编的现象的帮助以前报导了的这结果。我们的结果揭示在温度和 TtXI 的结构灵活性之间的关系,并且在与多重子单元理解 thermophile 蛋白质的 thermostability 起一个重要作用。
基金Supported by the National Natural Science Foundation of China(No.31271419 and No.31271793)the National Science and Technology Key Project of China on GMO Cultivation for New Varieties(No.2014ZX08003-002B)
文摘Carotenoid isomerase(CRTISO)is a key enzyme that catalyzes the conversion of cis-lycopene to alltrans lycopene. In this study, we isolated and characterized the CRTISO gene from Lycium chinense(Lc CRTISO) for the first time. The open reading frame of Lc CRTISO was 1 815 bp encoding a protein of 604 amino acids with a molecular mass of 66.24 k Da. Amino acid sequence analysis revealed that the Lc CRTISO had a high level of similarity to other CRTISO. Phylogenetic analysis displayed that Lc CRTISO kept a closer relationship with the CRTISO of plants than with those of other species. Semi-quantitative PCR analysis indicated that Lc CRTISO gene was expressed in all tissues tested with the highest expression in maturing fruits. The overexpression of Lc CRTISO gene in transgenic tobacco resulted in an increase of total carotenoids in the leaves with β-carotene and lutein being the predominants. The results obtained here clearly suggested that the Lc CRTISO gene was a promising candidate for carotenoid production.